# Patent

## Title
Modified polynucleotides for the production of cosmetic proteins and peptides

## Abstract

The invention relates to cosmetic mRNAs encoding elastin, and methods of using such mRNAs.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. §371 U.S. National Stage Entry of International Application No. PCT/US2013/030068 filed Mar. 9, 2013, which claims the priority of U.S. Provisional Patent Application No. 61/618,862, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Biologics, U.S. Provisional Patent Application No. 61/681,645, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Biologics, U.S. Provisional Patent Application No. 61/737,130, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Biologics, U.S. Provisional Patent Application No. 61/618,866, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Antibodies, U.S. Provisional Patent Application No. 61/681,647, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Antibodies, U.S. Provisional Patent Application No. 61/737,134, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Antibodies, U.S. Provisional Patent Application No. 61/618,868, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Vaccines, U.S. Provisional Patent Application No. 61/681,648, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Vaccines, U.S. Provisional Patent Application No. 61/737,135, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Vaccines, U.S. Provisional Patent Application No. 61/618,870, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides, U.S. Provisional Patent Application No. 61/681,649, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides, U.S. Provisional Patent Application No. 61/737,139, filed Dec. 14, 2012, Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides, U.S. Provisional Patent Application No. 61/618,873, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Secreted Proteins, U.S. Provisional Patent Application No. 61/681,650, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Secreted Proteins, U.S. Provisional Patent Application No. 61/737,147, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Secreted Proteins, U.S. Provisional Patent Application No. 61/618,878, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Plasma Membrane Proteins, U.S. Provisional Patent Application No. 61/681,654, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Plasma Membrane Proteins, U.S. Provisional Patent Application No. 61/737,152, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Plasma Membrane Proteins, U.S. Provisional Patent Application No. 61/618,885, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins, U.S. Provisional Patent Application No. 61/681,658, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins, U.S. Provisional Patent Application No. 61/737,155, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins, U.S. Provisional Patent Application No. 61/618,896, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins, U.S. Provisional Patent Application No. 61/668,157, filed Jul. 5, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins, U.S. Provisional Patent Application No. 61/681,661, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins, U.S. Provisional Patent Application No. 61/737,160, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins, U.S. Provisional Patent Application No. 61/618,911, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Nuclear Proteins, U.S. Provisional Patent Application No. 61/681,667, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Nuclear Proteins, U.S. Provisional Patent Application No. 61/737,168, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Nuclear Proteins, U.S. Provisional Patent Application No. 61/618,922, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Proteins, U.S. Provisional Patent Application No. 61/681,675, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Proteins, U.S. Provisional Patent Application No. 61/737,174, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Proteins, U.S. Provisional Patent Application No. 61/618,935, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No. 61/681,687, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No. 61/737,184, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No. 61/618,945, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No. 61/681,696, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No. 61/737,191, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No. 61/618,953, filed Apr. 2, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No. 61/681,704, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No. 61/737,203, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No. 61/681,720, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides, U.S. Provisional Patent Application No. 61/737,213, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides, U.S. Provisional Patent Application No. 61/681,742, filed, Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides, U.S. Provisional Patent Application No. 61/618,961, filed Apr. 2, 2012, entitled Dosing Methods for Modified mRNA, U.S. Provisional Patent Application No. 61/648,286, filed May 17, 2012, entitled Dosing Methods for Modified mRNA, U.S. Provisional Patent Application No. 61/618,957, filed Apr. 2, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S. Provisional Patent Application No. 61/648,244, filed May 17, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S. Provisional Patent Application No. 61/681,712, filed Aug. 10, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S. Provisional Patent Application No. 61/696,381, filed Sep. 4, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S. Provisional Patent Application No. 61/709,303, filed Oct. 3, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S. Provisional Patent Application No. 61/712,490, filed Oct. 11, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, the contents of each of which are herein incorporated by reference in its entirety.
This application is related to U.S. Provisional Patent Application No. 61/737,224, filed Dec. 14, 2012, entitled Terminally Optimized Modified RNAs, International Application No PCT/US2012/069610, filed Dec. 14, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, International Publication No. PCT/US2012/58519, filed Oct. 3, 2012, entitled Modified Nucleosides, Nucleotides, and Nucleic Acids, and Uses Thereof, the contents of each of which are herein incorporated by reference in its entirety.
The instant application is also related to co-pending applications, each filed concurrently with International Application No. PCT/US2013/030066 on Mar. 9, 2013, entitled Modified Polynucleotides for the Production of Biologics and Proteins Associated with Human Disease; entitled Modified Polynucleotides for the Production of Secreted Proteins; entitled Modified Polynucleotides for the Production of Membrane Proteins; entitled Modified Polynucleotides for the Production of Proteins; entitled Modified Polynucleotides for the Production of Nuclear Proteins; entitled Modified Polynucleotides for the Production of Proteins; entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease; entitled Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides, entitled Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides, the contents of each of which are herein incorporated by reference in its entirety.


REFERENCE TO THE SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file entitled MNC1_SL.txt, created on Sep. 30, 2014 which is 17,037,615 bytes in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention relates to compositions, methods, processes, kits and devices for the design, preparation, manufacture and/or formulation of cosmetic polynucleotides, cosmetic primary constructs and cosmetic modified mRNA molecules (mmRNA).
BACKGROUND OF THE INVENTION
Expression and/or levels of cosmetic polypeptides and other biomolecules may change with age, injury or exposure to environmental factors. For example, cosmetic polypeptides such as collagen, elastin, growth factors and repair enzymes may decline with age and matrix metalloproteinase may increase with age increases collagen breakdown causing undesirable changes in aesthetics. Genetic therapy can provide for a targeted approach for the improvement, treatment and maintenance of the cell, tissue and/or organism related to cosmetic appearance.
To this end, the inventors have shown that certain modified mRNA sequences have the potential as therapeutics with benefits beyond just evading, avoiding or diminishing the immune response. Such studies are detailed in published co-pending applications International Application PCT/US2011/046861 filed Aug. 5, 2011 and PCT/US2011/054636 filed Oct. 3, 2011, International Application number PCT/US2011/054617 filed Oct. 3, 2011, the contents of which are incorporated herein by reference in their entirety.
The present invention addresses this need by providing nucleic acid based compounds or cosmetic polynucleotides which encode a cosmetic polypeptide of interest (e.g., modified mRNA or mmRNA) and which have structural and/or chemical features that avoid one or more of the problems in the art.
SUMMARY OF THE INVENTION
Described herein are compositions, methods, processes, kits and devices for the design, preparation, manufacture and/or formulation of modified mRNA (mmRNA) molecules encoding at least one cosmetic polypeptide of interest.
The present invention provides a method of treating a disease, disorder and/or condition in a subject in need thereof by increasing the level of at least one cosmetic polypeptide of interest comprising administering to said subject an isolated polynucleotide selected from the group consisting of SEQ ID NOs 884-1611 and 5691-5707 comprising, a first region of linked nucleosides, said first region encoding the cosmetic polypeptide of interest, a first flanking region located at the 5′ terminus of said first region comprising a sequence of linked nucleosides selected from the group consisting of the native 5′ untranslated region (UTR) of any of SEQ ID NOs: 884-1611 and 5691-5707, SEQ ID NO: 1-4 and functional variants thereof and a second flanking region located at the 3′ terminus of said first region comprising a sequence of linked nucleosides selected from the group consisting of the native 3′ UTR of any of SEQ ID NOs: 884-1611 and 5691-5707, SEQ ID NOs: 5-21 and functional variants thereof and a 3′ tailing sequence of linked nucleosides. The isolated polynucleotide may further be substantially purified.
The present invention also provides a method of altering, modifying and/or changing the appearance of a member of the integumentary system of a subject comprising contacting said member with an isolated polynucleotide comprising administering to said subject an isolated polynucleotide selected from the group consisting of SEQ ID NOs 884-1611 and 5691-5707 comprising, a first region of linked nucleosides, said first region encoding the cosmetic polypeptide of interest, a first flanking region located at the 5′ terminus of said first region comprising a sequence of linked nucleosides selected from the group consisting of the native 5′ untranslated region (UTR) of any of SEQ ID NOs: 884-1611 and 5691-5707 SEQ ID NO: 1-4 and functional variants thereof and a second flanking region located at the 3′ terminus of said first region comprising a sequence of linked nucleosides selected from the group consisting of the native 3′ UTR of any of SEQ ID NOs: 884-1611 and 5691-5707, SEQ ID NOs: 5-21 and functional variants thereof and a 3′ tailing sequence of linked nucleosides. The isolated polynucleotide may further be substantially purified.
The isolated polynucleotides of the present invention may alter, modify and/or change the appearance of a member of the integumenary system of a subject such as, but not limited, to skin, hair and nails. In a further embodiment, the subject may suffer from at least one disease, disorder and/or condition.
Further, the first region of the isolated polynucleotides of the present may comprise two stop codons. In one embodiment, the first region further comprises a first stop codon “TGA” and a second stop codon selected from the group consisting of “TAA,” “TGA” and “TAG.”
The 3′ tailing sequence of the isolated polynucleotides of the present invention may further include linked nucleosides such as, but not limited to, a poly-A tail of approximately 130 nucleotides and a poly A-G quartet.
The first flanking region of the isolated polynucleotide may further comprise at least one 5′ terminal cap such as, but not limited to, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
The nucleotides of the isolated polynucleotides of the present invention may comprise at least two modifications and a translatable region. The modification may be on at least one nucleoside and/or the backbone of said nucleotides, on both a nucleoside and a backbone linkage or on a sugar of the nucleoside. The modification may comprise replacing at least one phosphodiester linkage with a phosphorothioate linkage. The modification may include, but are not limited to, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. The backbone linkage may be modified by the replacement of one or more oxygen atoms. The nucleobase modified may be selected from, but is not limited to, cytosine, guanine, adenine, thymine and uracil.
The isolated polypeptides of the present invention may be used to treat a disease, disorder and/or condition and/or may alter, modify or change the appearance of a member of the integumentary system of a subject suffering from a disease, disorder and/or condition such as, but not limited to, acne vulgaris, acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, actinic keratosis, acne vulgaris, acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, acute urticaria, allergic contact dermatitis, alopecia areata, angioedema, athlete's foot, atopic dermatitis, autoeczematization, baby acne, balding, bastomycosis, blackheads, birthmarks and other skin pigmentation problems, boils, bruises, bug bites and stings, burns, cellulitis, chiggers, chloracne, cholinergic or stress uricara, chronic urticara, cold type urticara, confluent and reticulated papillomatosis, corns, cysts, dandruff, dermatitis herpetiformis, dermatographism, dyshidrotic eczema, diaper rash, dry skin, dyshidrosis, ectodermal dysplasia such as, hyprohidrotic ectodermal dysplasia and X-linked hyprohidrotic ectodermal dysplasia, eczema, epidermaodysplasia verruciformis, erythema nodosum, excoriated acne, exercise-induced anaphylasis folliculitis, excess skin oil, folliculitis, freckles, frostbite, fungal nails, hair density, hair growth rate, halogen acne, hair loss, heat rash, hematoma, herpes simplex infections (non-genital), hidradenitis suppurativa, hives, hyperhidrosis, hyperpigmentation, hypohidrotic ectodermal dysplasia, hypopigmentation, impetigo, ingrown hair, heat type urticara, ingrown toenail, infantile acne or neonatal acne, itch, irritant contact dermatitis, jock itch, keloid, keratosis pilaris, lichen planus, lichen sclerosus, lupus miliaris disseminatus faciei, melasma, moles, molluscum contagiosum, nail growth rate, nail health, neurodermatitis, nummular eczema, occupational acne, oil acne, onychomycosis, physical urticara, pilonidal cyst, pityriasis rosea, pityriasis versicolor, poison ivy, pomade acne, pseudofolliculitis barbae or acne keloidalis nuchae, psoriasis, psoriatic arthritis, pressure or delayed pressue urticara, puncture wounds such as cuts and scrapes, rash, rare or water type urticara, rhinoplasty, ringworm, rosacea, rothmund-thomson syndrome, sagging of the skin, scabis, scars, seborrhea, seborrheic dermatitis, shingles, skin cancer, skin tag, solar type urticara, spider bite, stretch marks, sunburn, tar acne, tropical acne, thinning of skin, thrush, tinea versicolor, transient acantholytic dermatosis, tycoon's cap or acne necrotica miliaris, uneven skin tone, varicose veins, venous eczema, vibratory angioedema, vitiligo, warts, Weber-Christian disease, wrinkles, x-linked hypohidrotic ectodermal dysplasia, xerotic eczema, yeast infection and general signs of aging.
The isolated polynucleotides of the present invention may be formulated. The formulation may comprise a lipid which may include, but is not limited to, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, PLGA, PEG, PEG-DMG, PEGylated lipids and mixtures thereof.
The isolated polynucleotides of the present invention may be administered at a total daily dose of between 1 ug and 150 ug. The administration may be by injection, topical administration, ophthalmic administration and intranasal administration. The injection may include injections such as, but not limited to, intradermal, subcutaneous and intramuscular. The topical administration may be, but is not limited to, a cream, lotion, ointment, gel, spray, solution and the like. The topical administration may further include a penetration enhancer such as, but not limited to, surfactants, fatty acids, bile salts, chelating agents, non-chelating non-surfactants, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, fatty acids and/or salts in combination with bile acids and/or salts, sodium salt in combination with lauric acid, capric acid and UDCA, and the like. The topical administration may also include a fragrance, a colorant, a sunscreen, an antibacterial and/or a moisturizer.
The isolated polynucleotides of the present invention may be administered to at least one site such as, but not limited to, forehead, scalp, hair follicles, hair, upper eyelids, lower eyelids, eyebrows, eyelashes, infraorbital area, periorbital areas, temple, nose, nose bridge, cheeks, tongue, nasolabial folds, lips, periobicular areas, jaw line, ears, neck, breast, forearm, upper arm, palm, hand, finger, nails, back, abdomen, sides, buttocks, thigh, calf, feet, toes and the like.
The present invention provides a cosmetic composition comprising an isolated polynucleotide selected from the group consisting of SEQ ID NOs 884-1611 and 5691-5707 comprising, a first region of linked nucleosides, said first region encoding the cosmetic polypeptide of interest, a first flanking region located at the 5′ terminus of said first region comprising a sequence of linked nucleosides selected from the group consisting of the native 5′ untranslated region (UTR) of any of SEQ ID NOs: 884-1611 and 5691-5707, SEQ ID NO: 1-4 and functional variants thereof and a second flanking region located at the 3′ terminus of said first region comprising a sequence of linked nucleosides selected from the group consisting of the native 3′ UTR of any of SEQ ID NOs: 884-1611 and 5691-5707, SEQ ID NOs: 5-21 and functional variants thereof and a 3′ tailing sequence of linked nucleosides. The isolated polynucleotide may further be substantially purified.
The cosmetic composition may further include a pharmaceutical acceptable excipient such as, but not limited to, a solvent, aqueous solvent, non-aqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, lipid, lipidoids liposome, lipid nanoparticle, core-shell nanoparticles, polymer, lipoplex, peptide, protein, cell, hyaluronidase, and mixtures thereof.
The cosmetic composition may further include a lipid such as, but not limited to, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, PEG, PEG-DMG, PEGylated lipids, PLGA and mixtures thereof.
The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.
FIG. 1 is a schematic of a cosmetic primary construct of the present invention.
FIG. 2 illustrates lipid structures in the prior art useful in the present invention. Shown are the structures for 98N12-5 (TETA5-LAP), DLin-DMA, DLin-K-DMA (2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA and C12-200.
FIG. 3 is a representative plasmid useful in the IVT reactions taught herein. The plasmid contains Insert 64818, designed by the instant inventors.
FIG. 4 is a gel profile of modified mRNA encapsulated in PLGA microspheres.



DETAILED DESCRIPTION
It is of great interest in the fields of therapeutics, diagnostics, reagents and for biological assays to be able to deliver a nucleic acid, e.g., a ribonucleic acid (RNA) inside a cell, whether in vitro, in vivo, in situ or ex vivo, such as to cause intracellular translation of the nucleic acid and production of an encoded polypeptide of interest. Of particular importance is the delivery and function of a non-integrative polynucleotide.
Described herein are compositions (including pharmaceutical compositions) and methods for the design, preparation, manufacture and/or formulation of polynucleotides encoding one or more cosmetic polypeptides of interest. Also provided are systems, processes, devices and kits for the selection, design and/or utilization of the polynucleotides encoding the cosmetic polypeptides of interest described herein.
According to the present invention, these cosmetic polynucleotides are preferably modified as to avoid the deficiencies of other polypeptide-encoding molecules of the art. Hence these polynucleotides are referred to as modified mRNA or mmRNA.
The use of modified polynucleotides in the fields of antibodies, viruses, veterinary applications and a variety of in vivo settings has been explored by the inventors and these studies are disclosed in for example, co-pending and co-owned U.S. provisional patent application Ser. Nos. 61/470,451 filed Mar. 31, 2011 teaching in vivo applications of mmRNA; 61/517,784 filed on Apr. 26, 2011 teaching engineered nucleic acids for the production of antibody polypeptides; 61/519,158 filed May 17, 2011 teaching veterinary applications of mmRNA technology; 61/533,537 filed on Sep. 12, 2011 teaching antimicrobial applications of mmRNA technology; 61/533,554 filed on Sep. 12, 2011 teaching viral applications of mmRNA technology, 61/542,533 filed on Oct. 3, 2011 teaching various chemical modifications for use in mmRNA technology; 61/570,690 filed on Dec. 14, 2011 teaching mobile devices for use in making or using mmRNA technology; 61/570,708 filed on Dec. 14, 2011 teaching the use of mmRNA in acute care situations; 61/576,651 filed on Dec. 16, 2011 teaching terminal modification architecture for mmRNA; 61/576,705 filed on Dec. 16, 2011 teaching delivery methods using lipidoids for mmRNA; 61/578,271 filed on Dec. 21, 2011 teaching methods to increase the viability of organs or tissues using mmRNA; 61/581,322 filed on Dec. 29, 2011 teaching mmRNA encoding cell penetrating peptides; 61/581,352 filed on Dec. 29, 2011 teaching the incorporation of cytotoxic nucleosides in mmRNA and 61/631,729 filed on Jan. 10, 2012 teaching methods of using mmRNA for crossing the blood brain barrier; all of which are herein incorporated by reference in their entirety.
Provided herein, in part, are cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encoding cosmetic polypeptides of interest which have been designed to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretion efficiency (when applicable), accessibility to circulation, protein half-life and/or modulation of a cell's status, function and/or activity.
I. Compositions of the Invention (mmRNA)
The present invention provides nucleic acid molecules, specifically cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA which encode one or more cosmetic polypeptides of interest. The term “nucleic acid,” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof.
In preferred embodiments, the nucleic acid molecule is a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any cosmetic polynucleotide which encodes a cosmetic polypeptide of interest and which is capable of being translated to produce the encoded cosmetic polypeptide of interest in vitro, in vivo, in situ or ex vivo.
Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. Building on this wild type modular structure, the present invention expands the scope of functionality of traditional mRNA molecules by providing cosmetic polynucleotides or cosmetic primary RNA constructs which maintain a modular organization, but which comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide including, in some embodiments, the lack of a substantial induction of the innate immune response of a cell into which the cosmetic polynucleotide is introduced. As such, modified mRNA molecules of the present invention are termed “mmRNA.” As used herein, a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the cosmetic polynucleotide.
mmRNA Architecture
The mmRNA of the present invention are distinguished from wild type mRNA in their functional and/or structural design features which serve to, as evidenced herein, overcome existing problems of effective polypeptide production using nucleic acid-based therapeutics.
FIG. 1 shows a representative polynucleotide primary construct 100 of the present invention. As used herein, the term “primary construct” or “primary mRNA construct” refers to a cosmetic polynucleotide transcript which encodes one or more cosmetic polypeptides of interest and which retains sufficient structural and/or chemical features to allow the cosmetic polypeptide of interest encoded therein to be translated. Cosmetic primary constructs may be cosmetic polynucleotides of the invention. When structurally or chemically modified, the cosmetic primary construct may be referred to as a cosmetic mmRNA.
Returning to FIG. 1, the cosmetic primary construct 100 here contains a first region of linked nucleotides 102 that is flanked by a first flanking region 104 and a second flaking region 106. As used herein, the “first region” may be referred to as a “coding region” or “region encoding” or simply the “first region.” This first region may include, but is not limited to, the encoded cosmetic polypeptide of interest. The cosmetic polypeptide of interest may comprise at its 5′ terminus one or more signal sequences encoded by a signal sequence region 103. The flanking region 104 may comprise a region of linked nucleotides comprising one or more complete or incomplete 5′ UTRs sequences. The flanking region 104 may also comprise a 5′ terminal cap 108. The second flanking region 106 may comprise a region of linked nucleotides comprising one or more complete or incomplete 3′ UTRs. The flanking region 106 may also comprise a 3′ tailing sequence 110.
Bridging the 5′ terminus of the first region 102 and the first flanking region 104 is a first operational region 105. Traditionally this operational region comprises a start codon. The operational region may alternatively comprise any translation initiation sequence or signal including a start codon.
Bridging the 3′ terminus of the first region 102 and the second flanking region 106 is a second operational region 107. Traditionally this operational region comprises a stop codon. The operational region may alternatively comprise any translation initiation sequence or signal including a stop codon. According to the present invention, multiple serial stop codons may also be used. In one embodiment, the operation region of the present invention may comprise two stop codons. The first stop codon may be “TGA” and the second stop codon may be selected from the group consisting of “TAA,” “TGA” and “TAG.”
Generally, the shortest length of the first region of the cosmetic primary construct of the present invention can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the length may be sufficient to encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids. Examples of dipeptides that the polynucleotide sequences can encode or include, but are not limited to, carnosine and anserine.
Generally, the length of the first region encoding the cosmetic polypeptide of interest of the present invention is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). As used herein, the “first region” may be referred to as a “coding region” or “region encoding” or simply the “first region.”
In some embodiments, the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).
According to the present invention, the first and second flanking regions may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).
According to the present invention, the tailing sequence may range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA binding protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of polyA binding protein. PolyA binding protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.
According to the present invention, the capping region may comprise a single cap or a series of nucleotides forming the cap. In this embodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the cap is absent.
According to the present invention, the first and second operational regions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a start and/or stop codon, one or more signal and/or restriction sequences.
Cyclic mmRNA
According to the present invention, a cosmetic primary construct or cosmetic mmRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5′-end binding proteins. The mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed 5′-/3′-linkage may be intramolecular or intermolecular.
In the first route, the 5′-end and the 3′-end of the nucleic acid may contain chemically reactive groups that, when close together, form a new covalent linkage between the 5′-end and the 3′-end of the molecule. The 5′-end may contain an NHS-ester reactive group and the 3′-end may contain a 3′-amino-terminated nucleotide such that in an organic solvent the 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNA molecule will undergo a nucleophilic attack on the 5′-NHS-ester moiety forming a new 5′-/3′-amide bond.
In the second route, T4 RNA ligase may be used to enzymatically link a 5′-phosphorylated nucleic acid molecule to the 3′-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage. In an example reaction, 1 μg of a nucleic acid molecule is incubated at 37° C. for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) according to the manufacturer's protocol. The ligation reaction may occur in the presence of a split oligonucleotide capable of base-pairing with both the 5′- and 3′-region in juxtaposition to assist the enzymatic ligation reaction.
In the third route, either the 5′- or 3′-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5′-end of a nucleic acid molecule to the 3′-end of a nucleic acid molecule. The ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37° C.
mmRNA Multimers
According to the present invention, multiple distinct cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA may be linked together through the 3′-end using nucleotides which are modified at the 3′-terminus. Chemical conjugation may be used to control the stoichiometry of delivery into cells. For example, the glyoxylate cycle enzymes, isocitrate lyase and malate synthase, may be supplied into HepG2 cells at a 1:1 ratio to alter cellular fatty acid metabolism. This ratio may be controlled by chemically linking cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA using a 3′-azido terminated nucleotide on one cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA species and a C5-ethynyl or alkynyl-containing nucleotide on the opposite cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA species. The modified nucleotide is added post-transcriptionally using terminal transferase (New England Biolabs, Ipswich, Mass.) according to the manufacturer's protocol. After the addition of the 3′-modified nucleotide, the two cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA species may be combined in an aqueous solution, in the presence or absence of copper, to form a new covalent linkage via a click chemistry mechanism as described in the literature.
In another example, more than two cosmetic polynucleotides may be linked together using a functionalized linker molecule. For example, a functionalized saccharide molecule may be chemically modified to contain multiple chemical reactive groups (SH—, NH2—, N3, etc. . . . ) to react with the cognate moiety on a 3′-functionalized cosmetic mRNA molecule (i.e., a 3′-maleimide ester, 3′-NHS-ester, alkynyl). The number of reactive groups on the modified saccharide can be controlled in a stoichiometric fashion to directly control the stoichiometric ratio of conjugated cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA.
mmRNA Conjugates and Combinations
In order to further enhance cosmetic protein production, cosmetic primary constructs or cosmetic mmRNA of the present invention can be designed to be conjugated to other polynucleotides, cosmetic polypeptides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.
Conjugation may result in increased stability and/or half life and may be particularly useful in targeting the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA to specific sites in the cell, tissue or organism.
According to the present invention, the cosmetic mmRNA or cosmetic primary constructs may be administered with, or further encode one or more of RNAi agents, siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like.
Bifunctional mmRNA
In one embodiment of the invention are bifunctional cosmetic polynucleotides (e.g., bifunctional cosmetic primary constructs or cosmetic bifunctional mmRNA). As the name implies, bifunctional cosmetic polynucleotides are those having or capable of at least two functions. These molecules may also by convention be referred to as multi-functional.
The multiple functionalities of bifunctional cosmetic polynucleotides may be encoded by the RNA (the function may not manifest until the encoded product is translated) or may be a property of the polynucleotide itself. It may be structural or chemical. Bifunctional modified cosmetic polynucleotides may comprise a function that is covalently or electrostatically associated with the polynucleotides. Further, the two functions may be provided in the context of a complex of a mmRNA and another molecule.
Bifunctional cosmetic polynucleotides may encode cosmetic peptides which are anti-proliferative. These peptides may be linear, cyclic, constrained or random coil. They may function as aptamers, signaling molecules, ligands or mimics or mimetics thereof. Anti-proliferative peptides may, as translated, be from 3 to 50 amino acids in length. They may be 5-40, 10-30, or approximately 15 amino acids long. They may be single chain, multichain or branched and may form complexes, aggregates or any multi-unit structure once translated.
Noncoding Cosmetic Polynucleotides and Primary Constructs
As described herein, provided are cosmetic polynucleotides and cosmetic primary constructs having sequences that are partially or substantially not translatable, e.g., having a noncoding region. Such noncoding region may be the “first region” of the cosmetic primary construct. Alternatively, the noncoding region may be a region other than the first region. Such molecules are generally not translated, but can exert an effect on protein production by one or more of binding to and sequestering one or more translational machinery components such as a ribosomal protein or a transfer RNA (tRNA), thereby effectively reducing protein expression in the cell or modulating one or more pathways or cascades in a cell which in turn alters protein levels. The cosmetic polynucleotide and/or cosmetic primary construct may contain or encode one or more long noncoding RNA (lncRNA, or lincRNA) or portion thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).
Cosmetic Polypeptides of Interest
According to the present invention, the cosmetic primary construct is designed to encode one or more cosmetic polypeptides of interest or fragments thereof. A cosmetic polypeptide of interest may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or variants of any of the aforementioned. As used herein, the term “cosmetic polypeptides of interest” refers to any polypeptide which is selected to be encoded in the cosmetic primary construct of the present invention. As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.
In some embodiments “variant mimics” are provided. As used herein, the term “variant mimic” is one which contains one or more amino acids which would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic, e.g., phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.
“Homology” as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
By “homologs” as it applies to polypeptide sequences means the corresponding sequence of other species having substantial identity to a second sequence of a second species.
“Analogs” is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.
The present invention contemplates several types of compositions which are polypeptide based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives. The term “derivative” is used synonymously with the term “variant” but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule.
As such, mmRNA encoding cosmetic polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the cosmetic polypeptide sequences disclosed herein, are included within the scope of this invention. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the invention (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.
“Substitutional variants” when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
As used herein the term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
“Insertional variants” when referring to polypeptides are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. “Immediately adjacent” to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.
“Deletional variants” when referring to polypeptides are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
“Covalent derivatives” when referring to polypeptides include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and/or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
Certain post-translational modifications are the result of the action of recombinant host cells on the expressed cosmetic polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the cosmetic polypeptides produced in accordance with the present invention.
Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).
“Features” when referring to polypeptides are defined as distinct amino acid sequence-based components of a molecule. Features of the polypeptides encoded by the mmRNA of the present invention include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.
As used herein when referring to polypeptides the term “surface manifestation” refers to a polypeptide based component of a protein appearing on an outermost surface.
As used herein when referring to polypeptides the term “local conformational shape” means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.
As used herein when referring to polypeptides the term “fold” refers to the resultant conformation of an amino acid sequence upon energy minimization. A fold may occur at the secondary or tertiary level of the folding process. Examples of secondary level folds include beta sheets and alpha helices. Examples of tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.
As used herein the term “turn” as it relates to protein conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
As used herein when referring to polypeptides the term “loop” refers to a structural feature of a polypeptide which may serve to reverse the direction of the backbone of a peptide or polypeptide. Where the loop is found in a polypeptide and only alters the direction of the backbone, it may comprise four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997). Loops may be open or closed. Closed loops or “cyclic” loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the bridging moieties. Such bridging moieties may comprise a cysteine-cysteine bridge (Cys-Cys) typical in polypeptides having disulfide bridges or alternatively bridging moieties may be non-protein based such as the dibromozylyl agents used herein.
As used herein when referring to polypeptides the term “half-loop” refers to a portion of an identified loop having at least half the number of amino acid resides as the loop from which it is derived. It is understood that loops may not always contain an even number of amino acid residues. Therefore, in those cases where a loop contains or is identified to comprise an odd number of amino acids, a half-loop of the odd-numbered loop will comprise the whole number portion or next whole number portion of the loop (number of amino acids of the loop/2+/−0.5 amino acids). For example, a loop identified as a 7 amino acid loop could produce half-loops of 3 amino acids or 4 amino acids (7/2=3.5+/−0.5 being 3 or 4).
As used herein when referring to polypeptides the term “domain” refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).
As used herein when referring to polypeptides the term “half-domain” means a portion of an identified domain having at least half the number of amino acid resides as the domain from which it is derived. It is understood that domains may not always contain an even number of amino acid residues. Therefore, in those cases where a domain contains or is identified to comprise an odd number of amino acids, a half-domain of the odd-numbered domain will comprise the whole number portion or next whole number portion of the domain (number of amino acids of the domain/2+/−0.5 amino acids). For example, a domain identified as a 7 amino acid domain could produce half-domains of 3 amino acids or 4 amino acids (7/2=3.5+/−0.5 being 3 or 4). It is also understood that sub-domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).
As used herein when referring to polypeptides the terms “site” as it pertains to amino acid based embodiments is used synonymously with “amino acid residue” and “amino acid side chain.” A site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention.
As used herein the terms “termini” or “terminus” when referring to polypeptides refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions. The polypeptide based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.
Once any of the features have been identified or defined as a desired component of a polypeptide to be encoded by the cosmetic primary construct or cosmetic mmRNA of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.
Modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
According to the present invention, the cosmetic polypeptides may comprise a consensus sequence which is discovered through rounds of experimentation. As used herein a “consensus” sequence is a single sequence which represents a collective population of sequences allowing for variability at one or more sites.
As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of cosmetic polypeptides of interest of this invention. For example, provided herein is any protein fragment (meaning a cosmetic polypeptide sequence at least one amino acid residue shorter than a reference cosmetic polypeptide sequence but otherwise identical) of a reference cosmetic protein 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length. In another example, any cosmetic protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the invention. In certain embodiments, a polypeptide to be utilized in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
Encoded Cosmetic Polypeptides
The cosmetic primary constructs or cosmetic mmRNA of the present invention may be designed to encode cosmetic polypeptides of interest such as cosmetic peptides and proteins.
In one embodiment cosmetic primary constructs or cosmetic mmRNA of the present invention may encode variant polypeptides which have a certain identity with a reference cosmetic polypeptide sequence. As used herein, a “reference cosmetic polypeptide sequence” refers to a starting cosmetic polypeptide sequence. Reference sequences may be wild type sequences or any sequence to which reference is made in the design of another sequence. A “reference polypeptide sequence” may, e.g., be any one of SEQ ID NOs: 884-1611 or 5691-5707 as disclosed herein, e.g., any of SEQ ID NOs 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 5674, 5675, 5676, 5677, 5678, 5679, 5680, 5681, 5682, 5683, 5684, 5685, 5686, 5687, 5688, 5689, 5690, 5691, 5692, 5693, 5694, 5695, 5696, 5697, 5698, 5699, 5700, 5701, 5702, 5703, 5704, 5705, 5706, 5707, 5708, 5709, 5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717, 5718, 5719, 5720, 5721, 5722, 5723, 5724, 5725, 5726, 5727, 5728, 5729, 5730, 5731, 5732, 5733, 5734, 5735, 5736, 5737, 5738, 5739, 5740, 5741, 5742, 5743, 5744, 5745, 5746, 5747, 5748, 5749, 5750, 5751, 5752, 5753, 5754.
The term “identity” as known in the art, refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).
In some embodiments, the polypeptide variant may have the same or a similar activity as the reference cosmetic polypeptide. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to a reference cosmetic polypeptide. Generally, variants of a particular cosmetic polynucleotide or cosmetic polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference cosmetic polynucleotide or cosmetic polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schïffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402.) Other tools are described herein, specifically in the definition of “Identity.”
Default parameters in the BLAST algorithm include, for example, an expect threshold of 10, Word size of 28, Match/Mismatch Scores 1, -2, Gap costs Linear. Any filter can be applied as well as a selection for species specific repeats, e.g., Homo sapiens. 
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may restore and/or enhance a biochemical and/or physiological process that will have a positive effect on a subject's outward appearance. In another embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may reduce a biochemical and/or physiological process that will have a negative effect on a subject's outward appearance. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may improve the production of molecules which can enhance the health and/or longevity of such cells. The biochemical and/or physiological process may affect cells such as skin, fat, muscle, connective tissue, nerve cells found in the epidermis, dermis and subcutaneous layers including nail root or nail bed, nail matrix and nail plate, and scalp, hair follicles and hair strands, as well as muscles found under the subcutaneous fat layer and the tongue; cells found in the eye including the iris and stroma covering the iris.
Cosmetic Proteins or Cosmetic Peptides
The cosmetic primary constructs or cosmetic mmRNA disclosed herein, may encode one or more validated or “in testing” cosmetic proteins or cosmetic peptides. As used herein, a “cosmetic protein” or a “cosmetic peptide” refers to a protein or peptide that upon contact or administration can enhance, alter, modify, and/or change the phenotype or aesthetic presentation of a cell, tissue, system, and/or organism.
According to the present invention, one or more cosmetic proteins or cosmetic peptides currently being marketed or in development may be encoded by the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the present invention. While not wishing to be bound by theory, it is believed that incorporation into the cosmetic primary constructs or cosmetic mmRNA of the invention will result in improved therapeutic efficacy due at least in part to the specificity, purity and selectivity of the construct designs.
Cosmetic proteins and peptides encoded in the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention may be utilized to treat diseases, disorders, and/or conditions in many therapeutic areas such as, but not limited to, those effecting the ingumentary system; hereditary diseases, disorders and/or conditions; genetic mutations; genetic disease, disorder and/or conditions; and diseases, disorders and/or conditions caused by fungus and/or yeast.
The cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may alter a biological and/or physiolocial process to reduce skin sagging, increase skin thickness, increase skin volume, reduce the number of wrinkles, the length of wrinkles and/or the depth of wrinkles, increase skin tightness, firmness, tone and/or elasticity, increase skin hydration and ability to retain moisture, water flow and osmotic balance. In another embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may alter a biological and/or physiolocial process may increase the levels of skin lipids; increase the extracellular matrix and/or adhesion and communication polypeptides; increase skin energy production; utilization and conservation; improve oxygen utilization; improve skin cell life; improve skin cell immunity defense, heat shock/stress response, antioxidant defense capacity to neutralize free radicals, and/or toxic defense; improve the protection and recovery from ultraviolet rays; improve skin cell communication and skin cell innervations; improve cell cohesion/adhesion; improve calcium mineral and other mineral metabolism; improve cell turnover; and improve cell circadian rhythms.
In one embodiment, the use of the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may result in improved skin texture, smoothness, softness, radiance, glow, reduced discoloration and/or unevenness of skin color including, but not limited to, redness, hyperpigmentation and hypopigmentation, improved blood vessel health, improved dermalepidermal junction, increased lipolysis to reduce cellulite (or dimpling), reduced pore size, decreased or increased contraction of muscles, increased size of skin cells.
In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may change the cells of the ingumentary system by producing permanent or temporary color and pigmentation changes including, but not limited to, fluorescence, iridescence, phosphorescence, reflectance, refraction, photoluminescence, chemiluminescence and/or bioluminescence.
Flanking Regions: Untranslated Regions (UTRs)
Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5′UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3′UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA of the present invention to enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
5′ UTR and Translation Initiation
Natural 5′UTRs bear features which play roles in for translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5′UTR also have been known to form secondary structures which are involved in elongation factor binding.
By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and cosmetic protein production of the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention. For example, introduction of 5′ UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could be used to enhance expression of a nucleic acid molecule, such as a mmRNA, in hepatic cell lines or liver. Likewise, use of 5′ UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A/B/C/D).
Other non-UTR sequences may be incorporated into the 5′ (or 3′ UTR) UTRs. For example, introns or portions of introns sequences may be incorporated into the flanking regions of the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention. Incorporation of intronic sequences may increase protein production as well as mRNA levels.
3′ UTR and the AU Rich Elements
3′ UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3′ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.
Introduction, removal or modification of 3′ UTR AU rich elements (AREs) can be used to modulate the stability of cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention. When engineering specific cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA, one or more copies of an ARE can be introduced to make cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.
Incorporating microRNA Binding Sites
microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bind to the 3′UTR of nucleic acid molecules and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. The cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention may comprise one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond to any known microRNA such as those taught in US Publication US2005/0261218 and US Publication US2005/0059005, the contents of which are incorporated herein by reference in their entirety.
A microRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence. A microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA. In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1. In some embodiments, a microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked byan adenine (A) opposed to microRNA position 1. See for example, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P, Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105; each of which is herein incorporated by reference in their entirety. The bases of the microRNA seed have complete complementarity with the target sequence. By engineering microRNA target sequences into the 3′UTR of cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention one can target the molecule for degradation or reduced translation, provided the microRNA in question is available. This process will reduce the hazard of off target effects upon nucleic acid molecule delivery. Identification of microRNA, microRNA target regions, and their expression patterns and role in biology have been reported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; each of which is herein incorporated by reference in their entirety).
For example, if the nucleic acid molecule is an mRNA and is not intended to be delivered to the liver but ends up there, then miR-122, a microRNA abundant in liver, can inhibit the expression of the gene of interest if one or multiple target sites of miR-122 are engineered into the 3′UTR of the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA. Introduction of one or multiple binding sites for different microRNA can be engineered to further decrease the longevity, stability, and protein translation of a cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA. As used herein, the term “microRNA site” refers to a microRNA target site or a microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that “binding” may follow traditional Watson-Crick hybridization rules or may reflect any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.
Conversely, for the purposes of the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the present invention, microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues. For example, miR-122 binding sites may be removed to improve protein expression in the liver. Regulation of expression in multiple tissues can be accomplished through introduction or removal or one or several microRNA binding sites.
Examples of tissues where microRNA are known to regulate mRNA, and thereby protein expression, include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulate complex biological processes such as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176; herein incorporated by reference in its entirety). In the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the present invention, binding sites for microRNAs that are involved in such processes may be removed or introduced, in order to tailor the expression of the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA expression to biologically relevant cell types or to the context of relevant biological processes. A listing of MicroRNA, miR sequences and miR binding sites is listed in Table 9 of U.S. Provisional Application No. 61/753,661 filed Jan. 17, 2013, in Table 9 of U.S. Provisional Application No. 61/754,159 filed Jan. 18, 2013, and in Table 7 of U.S. Provisional Application No. 61/758,921 filed Jan. 31, 2013, each of which are herein incorporated by reference in their entireties.
Lastly, through an understanding of the expression patterns of microRNA in different cosmetic cell types, cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA can be engineered for more targeted expression in specific cell types or only under specific biological conditions. Through introduction of tissue-specific microRNA binding sites, cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA could be designed that would be optimal for cosmetic protein expression in a tissue or in the context of a biological condition. Examples of use of microRNA to drive tissue or disease-specific gene expression are listed (Getner and Naldini, Tissue Antigens. 2012, 80:393-403; herein incorporated by reference in its entirety). In addition, microRNA seed sites can be incorporated into mRNA to decrease expression in certain cells which results in a biological improvement. An example of this is incorporation of miR-142 sites into a UGT1A1-expressing lentiviral vector. The presence of miR-142 seed sites reduced expression in hematopoietic cells, and as a consequence reduced expression in antigen-presenting cells, leading to the absence of an immune response against the virally expressed UGT1A1 (Schmitt et al., Gastroenterology 2010; 139:999-1007; Gonzalez-Asequinolaza et al. Gastroenterology 2010, 139:726-729; both herein incorporated by reference in its entirety). Incorporation of miR-142 sites into modified mRNA could not only reduce expression of the encoded protein in hematopoietic cells, but could also reduce or abolish immune responses to the mRNA-encoded protein. Incorporation of miR-142 seed sites (one or multiple) into mRNA would be important in the case of treatment of patients with complete protein deficiencies (UGT1A1 type I, LDLR-deficient patients, CRIM-negative Pompe patients, etc.).
Transfection experiments can be conducted in relevant cell lines, using engineered cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different microRNA binding site-engineering cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, 72 hour and 7 days post-transfection. In vivo experiments can also be conducted using microRNA-binding site-engineered molecules to examine changes in tissue-specific expression of formulated cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA.
5′ Capping
The 5′ cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5′ proximal introns removal during mRNA splicing.
Endogenous mRNA molecules may be 5′-end capped generating a 5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residue and the 5′-terminal transcribed sense nucleotide of the mRNA molecule. This 5′-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5′ end of the mRNA may optionally also be 2′-O-methylated. 5′-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.
Modifications to the cosmetic polynucleotides, cosmetic primary constructs, and cosmetic mmRNA of the present invention may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap. Additional modified guanosine nucleotides may be used such as α-methyl-phosphonate and seleno-phosphate nucleotides.
Additional modifications include, but are not limited to, 2′-O-methylation of the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of the mRNA (as mentioned above) on the 2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structures can be used to generate the 5′-cap of a nucleic acid molecule, such as an mRNA molecule.
Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5′-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and/or linked to a nucleic acid molecule.
For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5′-5′-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3′-O-methyl group (i.e., N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m7G-3′mppp-G; which may equivalently be designated 3′ O-Me-m7G(5′)ppp(5′)G). The 3′-O atom of the other, unmodified, guanine becomes linked to the 5′-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA). The N7- and 3′-O-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA or mmRNA).
Another exemplary cap is mCAP, which is similar to ARCA but has a 2′-O-methyl group on guanosine (i.e., N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m7Gm-ppp-G).
While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5′-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
Cosmetic polynucleotides, cosmetic primary constructs and cosmetic mmRNA of the invention may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5′-cap structures. As used herein, the phrase “more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a “more authentic” feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5′cap structures of the present invention are those which, among other things, have enhanced binding of cap binding proteins, increased half life, reduced susceptibility to 5′ endonucleases and/or reduced 5′decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural or physiological 5′cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme can create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5′-terminal nucleotide of the mRNA contains a 2′-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5′cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp (cap 1), and 7mG(5′)-ppp(5′)NlmpN2mp (cap 2).
Because the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA may be capped post-transcriptionally, and because this process is more efficient, nearly 100% of the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA may be capped. This is in contrast to  ̃80% when a cap analog is linked to an mRNA in the course of an in vitro transcription reaction.
According to the present invention, 5′ terminal caps may include endogenous caps or cap analogs. According to the present invention, a 5′ terminal cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
Viral Sequences
Additional viral sequences such as, but not limited to, the translation enhancer sequence of the barley yellow dwarf virus (BYDV-PAV), the Jaagsiekte sheep retrovirus (JSRV) and/or the Enzootic nasal tumor virus (See e.g., International Pub. No. WO2012129648; herein incorporated by reference in its entirety) can be engineered and inserted in the 3′ UTR of the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention and can stimulate the translation of the construct in vitro and in vivo. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7 post-transfection.
IRES Sequences
Further, provided are cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA which may contain an internal ribosome entry site (IRES). First identified as a feature Picorna virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5′ cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. Cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA containing more than one functional ribosome binding site may encode several cosmetic peptides or cosmetic polypeptides that are translated independently by the ribosomes (“multicistronic nucleic acid molecules”). When cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).
Poly-A Tails
During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a cosmetic polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3′ end of the transcript may be cleaved to free a 3′ hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 100 and 250 residues long.
It has been discovered that unique poly-A tail lengths provide certain advantages to the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the present invention.
Generally, the length of a poly-A tail of the present invention is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).
In one embodiment, the poly-A tail is designed relative to the length of the overall cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA. This design may be based on the length of the coding region, the length of a particular feature or region (such as the first or flanking regions), or based on the length of the ultimate product expressed from the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA.
In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA or feature thereof. The poly-A tail may also be designed as a fraction of cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA to which it belongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA for Poly-A binding protein may enhance expression.
Additionally, multiple distinct cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA may be linked together to the PABP (Poly-A binding protein) through the 3′-end using modified nucleotides at the 3′-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines and cosmetic protein production can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7 post-transfection.
In one embodiment, the cosmetic polynucleotide and cosmetic primary constructs of the present invention are designed to include a polyA-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. The resultant cosmetic mmRNA construct is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone.
Quantification
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the present invention may be quantified in exosomes derived from one or more bodily fluid. As used herein “bodily fluids” include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
In the quantification method, a sample of not more than 2 mL is obtained from the subject and the exosomes isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof. In the analysis, the level or concentration of a cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA may be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker. The assay may be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
These methods afford the investigator the ability to monitor, in real time, the level of cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA remaining or delivered. This is possible because the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the present invention differ from the endogenous forms due to the structural and/or chemical modifications.
II. Design and Synthesis of mmRNA
Cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA for use in accordance with the invention may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription (IVT) or enzymatic or chemical cleavage of a longer precursor, etc. Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference).
The process of design and synthesis of the cosmetic primary constructs of the invention generally includes the steps of gene construction, mRNA production (either with or without modifications) and purification. In the enzymatic synthesis method, a target cosmetic polynucleotide sequence encoding the cosmetic polypeptide of interest is first selected for incorporation into a vector which will be amplified to produce a cDNA template. Optionally, the target cosmetic polynucleotide sequence and/or any flanking sequences may be codon optimized. The cDNA template is then used to produce mRNA through in vitro transcription (IVT). After production, the mRNA may undergo purification and clean-up processes. The steps of which are provided in more detail below.
Gene Construction
The step of gene construction may include, but is not limited to gene synthesis, vector amplification, plasmid purification, plasmid linearization and clean-up, and cDNA template synthesis and clean-up.
Gene Synthesis
Once a cosmetic polypeptide of interest, or target, is selected for production, a cosmetic primary construct is designed. Within the cosmetic primary construct, a first region of linked nucleosides encoding the polypeptide of interest may be constructed using an open reading frame (ORF) of a selected nucleic acid (DNA or RNA) transcript. The ORF may comprise the wild type ORF, an isoform, variant or a fragment thereof. As used herein, an “open reading frame” or “ORF” is meant to refer to a nucleic acid sequence (DNA or RNA) which is capable of encoding a cosmetic polypeptide of interest. ORFs often begin with the start codon, ATG and end with a nonsense or termination codon or signal.
Further, the nucleotide sequence of the first region may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the mRNA. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. In one embodiment, the ORF sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table 1.







TABLE 1







Codon Options










Single 




Letter 



Amino Acid
Code
Codon Options





Isoleucine
I
ATT, ATC, ATA


 


Leucine
L
CTT, CTC, CTA, CTG, TTA, TTG


 


Valine
V
GTT, GTC, GTA, GTG


 


Phenylalanine
F
TTT, TTC


 


Methionine
M
ATG


 


Cysteine
C
TGT, TGC


 


Alanine
A
GCT, GCC, GCA, GCG


 


Glycine
G
GGT, GGC, GGA, GGG


 


Proline
P
CCT, CCC, CCA, CCG


 


Threonine
T
ACT, ACC, ACA, ACG


 


Serine
S
TCT, TCC, TCA, TCG, AGT, AGC


 


Tyrosine
Y
TAT, TAC


 


Tryptophan
W
TGG


 


Glutamine
Q
CAA, CAG


 


Asparagine
N
AAT, AAC


 


Histidine
H
CAT, CAC


 


Glutamic acid
E
GAA, GAG


 


Aspartic acid
D
GAT, GAC


 


Lysine
K
AAA, AAG


 


Arginine
R
CGT, CGC, CGA, CGG, AGA, AGG


 


Selenocysteine
Sec
UGA in mRNA in presence 




of Selenocystein




insertion element (SECIS)


 


Stop codons
Stop
TAA, TAG, TGA









Features, which may be considered beneficial in some embodiments of the present invention, may be encoded by the cosmetic primary construct and may flank the ORF as a first or second flanking region. The flanking regions may be incorporated into the cosmetic primary construct before and/or after optimization of the ORF. It is not required that a cosmetic primary construct contain both a 5′ and 3′ flanking region. Examples of such features include, but are not limited to, untranslated regions (UTRs), Kozak sequences, an oligo(dT) sequence, and detectable tags and may include multiple cloning sites which may have XbaI recognition.
In some embodiments, a 5′ UTR and/or a 3′ UTR may be provided as flanking regions. Multiple 5′ or 3′ UTRs may be included in the flanking regions and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical modifications, before and/or after codon optimization. Combinations of features may be included in the first and second flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5′ UTR which may contain a strong Kozak translational initiation signal and/or a 3′ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5′UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5′UTRs described in US Patent Application Publication No. 20100293625, herein incorporated by reference in its entirety.
Tables 2 and 3 provide a listing of exemplary UTRs which may be utilized in the cosmetic primary construct of the present invention as flanking regions. Shown in Table 2 is a listing of a 5′-untranslated region of the invention. Variants of 5′ UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.







TABLE 2







5′-Untranslated Regions













SEQ


5′UTR
Name/

ID


Identifier
Description
Sequence
NO.





5UTR-001
Upstream 
GGGAAATAAGAGAGAAAAGAAGAG
1



UTR
TAAGAAGAAATATAAGAGCCACC



 


5UTR-002
Upstream
GGGAGATCAGAGAGAAAAGAAG
2



UTR
AGTAAGAAGAAATATAAGAGCCACC



 


5UTR-003
Upstream 
GGAATAAAAGTCTCAACACAACA
3



UTR
TATACAAAACAAACGAATCTCAA





GCAATCAAGCATTCTACTTCTAT





TGCAGCAATTTAAATCATTTCTT





TTAAAGCAAAAGCAATTTTCTGA





AAATTTTCACCATTTACGAACGA





TAGCAAC



 


5UTR-004
Upstream
GGGAGACAAGCUUGGCAUUCC
4



UTR
GGUACUGUUGGUAAAGCCACC









Shown in Table 3 is a representative listing of 3′-untranslated regions of the invention. Variants of 3′ UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.







TABLE 3







3′-Untranslated Regions













SEQ


3′UTR
Name/

ID


Identifier
Description
Sequence
NO.













3UTR-001
Creatine
GCGCCTGCCCACCTGCCACCGACTGCTGG
5



Kinase
AACCCAGCCAGTGGGAGGGCCTGGCCCA





CCAGAGTCCTGCTCCCTCACTCCTCGCCC





CGCCCCCTGTCCCAGAGTCCCACCTGGGG





GCTCTCTCCACCCTTCTCAGAGTTCCAGT





TTCAACCAGAGTTCCAACCAATGGGCTCC





ATCCTCTGGATTCTGGCCAATGAAATATC





TCCCTGGCAGGGTCCTCTTCTTTTCCCAG





AGCTCCACCCCAACCAGGAGCTCTAGTTA





ATGGAGAGCTCCCAGCACACTCGGAGCT





TGTGCTTTGTCTCCACGCAAAGCGATAAA





TAAAAGCATTGGTGGCCTTTGGTCTTTGA





ATAAAGCCTGAGTAGGAAGTCTAGA



 


3UTR-002
Myoglobin
GCCCCTGCCGCTCCCACCCCCACCCATCT
6




GGGCCCCGGGTTCAAGAGAGAGCGGGGT





CTGATCTCGTGTAGCCATATAGAGTTTGC





TTCTGAGTGTCTGCTTTGTTTAGTAGAGG





TGGGCAGGAGGAGCTGAGGGGCTGGGGC





TGGGGTGTTGAAGTTGGCTTTGCATGCCC





AGCGATGCGCCTCCCTGTGGGATGTCATC





ACCCTGGGAACCGGGAGTGGCCCTTGGC





TCACTGTGTTCTGCATGGTTTGGATCTGA





ATTAATTGTCCTTTCTTCTAAATCCCAACC





GAACTTCTTCCAACCTCCAAACTGGCTGT





AACCCCAAATCCAAGCCATTAACTACACC





TGACAGTAGCAATTGTCTGATTAATCACT





GGCCCCTTGAAGACAGCAGAATGTCCCTT





TGCAATGAGGAGGAGATCTGGGCTGGGC





GGGCCAGCTGGGGAAGCATTTGACTATCT





GGAACTTGTGTGTGCCTCCTCAGGTATGG





CAGTGACTCACCTGGTTTTAATAAAACAA





CCTGCAACATCTCATGGTCTTTGAATAAA





GCCTGAGTAGGAAGTCTAGA



 


3UTR-003
α-actin
ACACACTCCACCTCCAGCACGCGACTTCT
7




CAGGACGACGAATCTTCTCAATGGGGGG





GCGGCTGAGCTCCAGCCACCCCGCAGTC





ACTTTCTTTGTAACAACTTCCGTTGCTGCC





ATCGTAAACTGACACAGTGTTTATAACGT





GTACATACATTAACTTATTACCTCATTTT





GTTATTTTTCGAAACAAAGCCCTGTGGAA





GAAAATGGAAAACTTGAAGAAGCATTAA





AGTCATTCTGTTAAGCTGCGTAAATGGTC





TTTGAATAAAGCCTGAGTAGGAAGTCTA





GA



 


3UTR-004
Albumin
CATCACATTTAAAAGCATCTCAGCCTACC
8




ATGAGAATAAGAGAAAGAAAATGAAGAT





CAAAAGCTTATTCATCTGTTTTTCTTTTTC





GTTGGTGTAAAGCCAACACCCTGTCTAAA





AAACATAAATTTCTTTAATCATTTTGCCT





CTTTTCTCTGTGCTTCAATTAATAAAAAA





TGGAAAGAATCTAATAGAGTGGTACAGC





ACTGTTATTTTTCAAAGATGTGTTGCTAT





CCTGAAAATTCTGTAGGTTCTGTGGAAGT





TCCAGTGTTCTCTCTTATTCCACTTCGGTA





GAGGATTTCTAGTTTCTTGTGGGCTAATT





AAATAAATCATTAATACTCTTCTAATGGT





CTTTGAATAAAGCCTGAGTAGGAAGTCTA





GA



 


3UTR-005
α-globin
GCTGCCTTCTGCGGGGCTTGCCTTCTGGC
9




CATGCCCTTCTTCTCTCCCTTGCACCTGTA





CCTCTTGGTCTTTGAATAAAGCCTGAGTA





GGAAGGCGGCCGCTCGAGCATGCATCTA





GA



 


3UTR-006
G-CSF
GCCAAGCCCTCCCCATCCCATGTATTTAT
10




CTCTATTTAATATTTATGTCTATTTAAGCC





TCATATTTAAAGACAGGGAAGAGCAGAA





CGGAGCCCCAGGCCTCTGTGTCCTTCCCT





GCATTTCTGAGTTTCATTCTCCTGCCTGTA





GCAGTGAGAAAAAGCTCCTGTCCTCCCAT





CCCCTGGACTGGGAGGTAGATAGGTAAA





TACCAAGTATTTATTACTATGACTGCTCC





CCAGCCCTGGCTCTGCAATGGGCACTGGG





ATGAGCCGCTGTGAGCCCCTGGTCCTGAG





GGTCCCCACCTGGGACCCTTGAGAGTATC





AGGTCTCCCACGTGGGAGACAAGAAATC





CCTGTTTAATATTTAAACAGCAGTGTTCC





CCATCTGGGTCCTTGCACCCCTCACTCTG





GCCTCAGCCGACTGCACAGCGGCCCCTGC





ATCCCCTTGGCTGTGAGGCCCCTGGACAA





GCAGAGGTGGCCAGAGCTGGGAGGCATG





GCCCTGGGGTCCCACGAATTTGCTGGGGA





ATCTCGTTTTTCTTCTTAAGACTTTTGGGA





CATGGTTTGACTCCCGAACATCACCGACG





CGTCTCCTGTTTTTCTGGGTGGCCTCGGG





ACACCTGCCCTGCCCCCACGAGGGTCAG





GACTGTGACTCTTTTTAGGGCCAGGCAGG





TGCCTGGACATTTGCCTTGCTGGACGGGG





ACTGGGGATGTGGGAGGGAGCAGACAGG





AGGAATCATGTCAGGCCTGTGTGTGAAA





GGAAGCTCCACTGTCACCCTCCACCTCTT





CACCCCCCACTCACCAGTGTCCCCTCCAC





TGTCACATTGTAACTGAACTTCAGGATAA





TAAAGTGTTTGCCTCCATGGTCTTTGAAT





AAAGCCTGAGTAGGAAGGCGGCCGCTCG





AGCATGCATCTAGA



 


3UTR-007
Colla2; collagen,
ACTCAATCTAAATTAAAAAAGAAAGAAA
11



type I, alpha 2
TTTGAAAAAACTTTCTCTTTGCCATTTCTT




 
CTTCTTCTTTTTTAACTGAAAGCTGAATCC





TTCCATTTCTTCTGCACATCTACTTGCTTA





AATTGTGGGCAAAAGAGAAAAAGAAGGA





TTGATCAGAGCATTGTGCAATACAGTTTC





ATTAACTCCTTCCCCCGCTCCCCCAAAAA





TTTGAATTTTTTTTTCAACACTCTTACACC





TGTTATGGAAAATGTCAACCTTTGTAAGA





AAACCAAAATAAAAATTGAAAAATAAAA





ACCATAAACATTTGCACCACTTGTGGCTT





TTGAATATCTTCCACAGAGGGAAGTTTAA





AACCCAAACTTCCAAAGGTTTAAACTACC





TCAAAACACTTTCCCATGAGTGTGATCCA





CATTGTTAGGTGCTGACCTAGACAGAGAT





GAACTGAGGTCCTTGTTTTGTTTTGTTCAT





AATACAAAGGTGCTAATTAATAGTATTTC





AGATACTTGAAGAATGTTGATGGTGCTAG





AAGAATTTGAGAAGAAATACTCCTGTATT





GAGTTGTATCGTGTGGTGTATTTTTTAAA





AAATTTGATTTAGCATTCATATTTTCCATC





TTATTCCCAATTAAAAGTATGCAGATTAT





TTGCCCAAATCTTCTTCAGATTCAGCATT





TGTTCTTTGCCAGTCTCATTTTCATCTTCT





TCCATGGTTCCACAGAAGCTTTGTTTCTT





GGGCAAGCAGAAAAATTAAATTGTACCT





ATTTTGTATATGTGAGATGTTTAAATAAA





TTGTGAAAAAAATGAAATAAAGCATGTT





TGGTTTTCCAAAAGAACATAT



 


3UTR-008
Col6a2;
CGCCGCCGCCCGGGCCCCGCAGTCGAGG
12



collagen, type
GTCGTGAGCCCACCCCGTCCATGGTGCTA




VI, alpha 2
AGCGGGCCCGGGTCCCACACGGCCAGCA





CCGCTGCTCACTCGGACGACGCCCTGGGC





CTGCACCTCTCCAGCTCCTCCCACGGGGT




 
CCCCGTAGCCCCGGCCCCCGCCCAGCCCC



 

AGGTCTCCCCAGGCCCTCCGCAGGCTGCC





CGGCCTCCCTCCCCCTGCAGCCATCCCAA





GGCTCCTGACCTACCTGGCCCCTGAGCTC





TGGAGCAAGCCCTGACCCAATAAAGGCT





TTGAACCCAT



 


3UTR 009
RPN1;
GGGGCTAGAGCCCTCTCCGCACAGCGTG
13



ribophorin I
GAGACGGGGCAAGGAGGGGGGTTATTAG





GATTGGTGGTTTTGTTTTGCTTTGTTTAAA





GCCGTGGGAAAATGGCACAACTTTACCTC





TGTGGGAGATGCAACACTGAGAGCCAAG





GGGTGGGAGTTGGGATAATTTTTATATAA





AAGAAGTTTTTCCACTTTGAATTGCTAAA





AGTGGCATTTTTCCTATGTGCAGTCACTC





CTCTCATTTCTAAAATAGGGACGTGGCCA





GGCACGGTGGCTCATGCCTGTAATCCCAG





CACTTTGGGAGGCCGAGGCAGGCGGCTC





ACGAGGTCAGGAGATCGAGACTATCCTG





GCTAACACGGTAAAACCCTGTCTCTACTA





AAAGTACAAAAAATTAGCTGGGCGTGGT





GGTGGGCACCTGTAGTCCCAGCTACTCGG





GAGGCTGAGGCAGGAGAAAGGCATGAAT





CCAAGAGGCAGAGCTTGCAGTGAGCTGA





GATCACGCCATTGCACTCCAGCCTGGGCA





ACAGTGTTAAGACTCTGTCTCAAATATAA





ATAAATAAATAAATAAATAAATAAATAA





ATAAAAATAAAGCGAGATGTTGCCCTCA





AA



 


3UTR-010
LRP1; low density
GGCCCTGCCCCGTCGGACTGCCCCCAGAA
14



lipoprotein
AGCCTCCTGCCCCCTGCCAGTGAAGTCCT




receptor-related
TCAGTGAGCCCCTCCCCAGCCAGCCCTTC




protein 1
CCTGGCCCCGCCGGATGTATAAATGTAAA





AATGAAGGAATTACATTTTATATGTGAGC





GAGCAAGCCGGCAAGCGAGCACAGTATT





ATTTCTCCATCCCCTCCCTGCCTGCTCCTT





GGCACCCCCATGCTGCCTTCAGGGAGAC





AGGCAGGGAGGGCTTGGGGCTGCACCTC





CTACCCTCCCACCAGAACGCACCCCACTG





GGAGAGCTGGTGGTGCAGCCTTCCCCTCC





CTGTATAAGACACTTTGCCAAGGCTCTCC





CCTCTCGCCCCATCCCTGCTTGCCCGCTC





CCACAGCTTCCTGAGGGCTAATTCTGGGA





AGGGAGAGTTCTTTGCTGCCCCTGTCTGG





AAGACGTGGCTCTGGGTGAGGTAGGCGG





GAAAGGATGGAGTGTTTTAGTTCTTGGGG





GAGGCCACCCCAAACCCCAGCCCCAACT





CCAGGGGCACCTATGAGATGGCCATGCT





CAACCCCCCTCCCAGACAGGCCCTCCCTG





TCTCCAGGGCCCCCACCGAGGTTCCCAGG





GCTGGAGACTTCCTCTGGTAAACATTCCT





CCAGCCTCCCCTCCCCTGGGGACGCCAAG





GAGGTGGGCCACACCCAGGAAGGGAAAG





CGGGCAGCCCCGTTTTGGGGACGTGAAC





GTTTTAATAATTTTTGCTGAATTCCTTTAC





AACTAAATAACACAGATATTGTTATAAAT





AAAATTGT



 


3UTR-011
Nnt1;
ATATTAAGGATCAAGCTGTTAGCTAATAA
15



cardiotrophm-
TGCCACCTCTGCAGTTTTGGGAACAGGCA




like cytokine
AATAAAGTATCAGTATACATGGTGATGTA




factor 1
CATCTGTAGCAAAGCTCTTGGAGAAAAT





GAAGACTGAAGAAAGCAAAGCAAAAACT





GTATAGAGAGATTTTTCAAAAGCAGTAAT





CCCTCAATTTTAAAAAAGGATTGAAAATT





CTAAATGTCTTTCTGTGCATATTTTTTGTG





TTAGGAATCAAAAGTATTTTATAAAAGG





AGAAAGAACAGCCTCATTTTAGATGTAGT





CCTGTTGGATTTTTTATGCCTCCTCAGTAA





CCAGAAATGTTTTAAAAAACTAAGTGTTT





AGGATTTCAAGACAACATTATACATGGCT





CTGAAATATCTGACACAATGTAAACATTG





CAGGCACCTGCATTTTATGTTTTTTTTTTC





AACAAATGTGACTAATTTGAAACTTTTAT





GAACTTCTGAGCTGTCCCCTTGCAATTCA





ACCGCAGTTTGAATTAATCATATCAAATC





AGTTTTAATTTTTTAAATTGTACTTCAGA





GTCTATATTTCAAGGGCACATTTTCTCAC





TACTATTTTAATACATTAAAGGACTAAAT





AATCTTTCAGAGATGCTGGAAACAAATC





ATTTGCTTTATATGTTTCATTAGAATACC





AATGAAACATACAACTTGAAAATTAGTA





ATAGTATTTTTGAAGATCCCATTTCTAAT





TGGAGATCTCTTTAATTTCGATCAACTTA





TAATGTGTAGTACTATATTAAGTGCACTT





GAGTGGAATTCAACATTTGACTAATAAA





ATGAGTTCATCATGTTGGCAAGTGATGTG





GCAATTATCTCTGGTGACAAAAGAGTAA





AATCAAATATTTCTGCCTGTTACAAATAT





CAAGGAAGACCTGCTACTATGAAATAGA





TGACATTAATCTGTCTTCACTGTTTATAAT





ACGGATGGATTTTTTTTCAAATCAGTGTG





TGTTTTGAGGTCTTATGTAATTGATGACA





TTTGAGAGAAATGGTGGCTTTTTTTAGCT





ACCTCTTTGTTCATTTAAGCACCAGTAAA





GATCATGTCTTTTTATAGAAGTGTAGATT





TTCTTTGTGACTTTGCTATCGTGCCTAAA





GCTCTAAATATAGGTGAATGTGTGATGAA





TACTCAGATTATTTGTCTCTCTATATAATT





AGTTTGGTACTAAGTTTCTCAAAAAATTA





TTAACACATGAAAGACAATCTCTAAACC





AGAAAAAGAAGTAGTACAAATTTTGTTA





CTGTAATGCTCGCGTTTAGTGAGTTTAAA





ACACACAGTATCTTTTGGTTTTATAATCA





GTTTCTATTTTGCTGTGCCTGAGATTAAG





ATCTGTGTATGTGTGTGTGTGTGTGTGTG





CGTTTGTGTGTTAAAGCAGAAAAGACTTT





TTTAAAAGTTTTAAGTGATAAATGCAATT





TGTTAATTGATCTTAGATCACTAGTAAAC





TCAGGGCTGAATTATACCATGTATATTCT





ATTAGAAGAAAGTAAACACCATCTTTATT





CCTGCCCTTTTTCTTCTCTCAAAGTAGTTG





TAGTTATATCTAGAAAGAAGCAATTTTGA





TTTCTTGAAAAGGTAGTTCCTGCACTCAG





TTTAAACTAAAAATAATCATACTTGGATT





TTATTTATTTTTGTCATAGTAAAAATTTTA





ATTTATATATATTTTTATTTAGTATTATCT





TATTCTTTGCTATTTGCCAATCCTTTGTCA





TCAATTGTGTTAAATGAATTGAAAATTCA





TGCCCTGTTCATTTTATTTTACTTTATTGG





TTAGGATATTTAAAGGATTTTTGTATATA





TAATTTCTTAAATTAATATTCCAAAAGGT





TAGTGGACTTAGATTATAAATTATGGCAA





AAATCTAAAAACAACAAAAATGATTTTT 





ATACATTCTATTTCATTATTCCTCTTTTTC





CAATAAGTCATACAATTGGTAGATATGAC





TTATTTTATTTTTGTATTATTCACTATATC





TTTATGATATTTAAGTATAAATAATTAAA





AAAATTTATTGTACCTTATAGTCTGTCAC





CAAAAAAAAAAAATTATCTGTAGGTAGT





GAAATGCTAATGTTGATTTGTCTTTAAGG





GCTTGTTAACTATCCTTTATTTTCTCATTT





GTCTTAAATTAGGAGTTTGTGTTTAAATT





ACTCATCTAAGCAAAAAATGTATATAAAT





CCCATTACTGGGTATATACCCAAAGGATT





ATAAATCATGCTGCTATAAAGACACATGC





ACACGTATGTTTATTGCAGCACTATTCAC





AATAGCAAAGACTTGGAACCAACCCAAA





TGTCCATCAATGATAGACTTGATTAAGAA





AATGTGCACATATACACCATGGAATACTA





TGCAGCCATAAAAAAGGATGAGTTCATG





TCCTTTGTAGGGACATGGATAAAGCTGGA





AACCATCATTCTGAGCAAACTATTGCAAG





GACAGAAAACCAAACACTGCATGTTCTC





ACTCATAGGTGGGAATTGAACAATGAGA





ACACTTGGACACAAGGTGGGGAACACCA





CACACCAGGGCCTGTCATGGGGTGGGGG





GAGTGGGGAGGGATAGCATTAGGAGATA





TACCTAATGTAAATGATGAGTTAATGGGT





GCAGCACACCAACATGGCACATGTATAC





ATATGTAGCAAACCTGCACGTTGTGCACA





TGTACCCTAGAACTTAAAGTATAATTAAA





AAAAAAAAGAAAACAGAAGCTATTTATA





AAGAAGTTATTTGCTGAAATAAATGTGAT





CTTTCCCATTAAAAAAATAAAGAAATTTT





GGGGTAAAAAAACACAATATATTGTATT





CTTGAAAAATTCTAAGAGAGTGGATGTG





AAGTGTTCTCACCACAAAAGTGATAACTA





ATTGAGGTAATGCACATATTAATTAGAAA





GATTTTGTCATTCCACAATGTATATATAC





TTAAAAATATGTTATACACAATAAATACA





TACATTAAAAAATAAGTAAATGTA



 


3UTR-012
Col6a1;
CCCACCCTGCACGCCGGCACCAAACCCTG
16



collagen, type VI,
TCCTCCCACCCCTCCCCACTCATCACTAA




alpha 1
ACAGAGTAAAATGTGATGCGAATTTTCCC





GACCAACCTGATTCGCTAGATTTTTTTTA





AGGAAAAGCTTGGAAAGCCAGGACACAA





CGCTGCTGCCTGCTTTGTGCAGGGTCCTC





CGGGGCTCAGCCCTGAGTTGGCATCACCT



 

GCGCAGGGCCCTCTGGGGCTCAGCCCTG





AGCTAGTGTCACCTGCACAGGGCCCTCTG





AGGCTCAGCCCTGAGCTGGCGTCACCTGT





GCAGGGCCCTCTGGGGCTCAGCCCTGAG





CTGGCCTCACCTGGGTTCCCCACCCCGGG





CTCTCCTGCCCTGCCCTCCTGCCCGCCCTC





CCTCCTGCCTGCGCAGCTCCTTCCCTAGG





CACCTCTGTGCTGCATCCCACCAGCCTGA





GCAAGACGCCCTCTCGGGGCCTGTGCCGC





ACTAGCCTCCCTCTCCTCTGTCCCCATAG





CTGGTTTTTCCCACCAATCCTCACCTAAC





AGTTACTTTACAATTAAACTCAAAGCAAG





CTCTTCTCCTCAGCTTGGGGCAGCCATTG





GCCTCTGTCTCGTTTTGGGAAACCAAGGT





CAGGAGGCCGTTGCAGACATAAATCTCG





GCGACTCGGCCCCGTCTCCTGAGGGTCCT





GCTGGTGACCGGCCTGGACCTTGGCCCTA





CAGCCCTGGAGGCCGCTGCTGACCAGCA





CTGACCCCGACCTCAGAGAGTACTCGCA





GGGGCGCTGGCTGCACTCAAGACCCTCG





AGATTAACGGTGCTAACCCCGTCTGCTCC





TCCCTCCCGCAGAGACTGGGGCCTGGACT





GGACATGAGAGCCCCTTGGTGCCACAGA





GGGCTGTGTCTTACTAGAAACAACGCAA





ACCTCTCCTTCCTCAGAATAGTGATGTGT





TCGACGTTTTATCAAAGGCCCCCTTTCTA





TGTTCATGTTAGTTTTGCTCCTTCTGTGTT





TTTTTCTGAACCATATCCATGTTGCTGACT





TTTCCAAATAAAGGTTTTCACTCCTCTC



 


3UTR-013
Calr;
AGAGGCCTGCCTCCAGGGCTGGACTGAG
17



calreticulin
GCCTGAGCGCTCCTGCCGCAGAGCTGGCC





GCGCCAAATAATGTCTCTGTGAGACTCGA





GAACTTTCATTTTTTTCCAGGCTGGTTCG





GATTTGGGGTGGATTTTGGTTTTGTTCCC





CTCCTCCACTCTCCCCCACCCCCTCCCCG





CCCTTTTTTTTTTTTTTTTTTAAACTGGTAT





TTTATCTTTGATTCTCCTTCAGCCCTCACC





CCTGGTTCTCATCTTTCTTGATCAACATCT





TTTCTTGCCTCTGTCCCCTTCTCTCATCTC





TTAGCTCCCCTCCAACCTGGGGGGCAGTG





GTGTGGAGAAGCCACAGGCCTGAGATTT





CATCTGCTCTCCTTCCTGGAGCCCAGAGG





AGGGCAGCAGAAGGGGGTGGTGTCTCCA





ACCCCCCAGCACTGAGGAAGAACGGGGC





TCTTCTCATTTCACCCCTCCCTTTCTCCCC





TGCCCCCAGGACTGGGCCACTTCTGGGTG





GGGCAGTGGGTCCCAGATTGGCTCACACT





GAGAATGTAAGAACTACAAACAAAATTT





CTATTAAATTAAATTTTGTGTCTCC



 


3UTR-014
Colla1;
CTCCCTCCATCCCAACCTGGCTCCCTCCC
18



collagen type I
ACCCAACCAACTTTCCCCCCAACCCGGAA




alpha 1
ACAGACAAGCAACCCAAACTGAACCCCC





TCAAAAGCCAAAAAATGGGAGACAATTT





CACATGGACTTTGGAAAATATTTTTTTCC



 

TTTGCATTCATCTCTCAAACTTAGTTTTTA





TCTTTGACCAACCGAACATGACCAAAAA





CCAAAAGTGCATTCAACCTTACCAAAAA





AAAAAAAAAAAAAAGAATAAATAAATA





ACTTTTTAAAAAAGGAAGCTTGGTCCACT





TGCTTGAAGACCCATGCGGGGGTAAGTC





CCTTTCTGCCCGTTGGGCTTATGAAACCC





CAATGCTGCCCTTTCTGCTCCTTTCTCCAC





ACCCCCCTTGGGGCCTCCCCTCCACTCCT





TCCCAAATCTGTCTCCCCAGAAGACACAG





GAAACAATGTATTGTCTGCCCAGCAATCA





AAGGCAATGCTCAAACACCCAAGTGGCC





CCCACCCTCAGCCCGCTCCTGCCCGCCCA





GCACCCCCAGGCCCTGGGGGACCTGGGG





TTCTCAGACTGCCAAAGAAGCCTTGCCAT





CTGGCGCTCCCATGGCTCTTGCAACATCT





CCCCTTCGTTTTTGAGGGGGTCATGCCGG





GGGAGCCACCAGCCCCTCACTGGGTTCG





GAGGAGAGTCAGGAAGGGCCACGACAAA





GCAGAAACATCGGATTTGGGGAACGCGT





GTCAATCCCTTGTGCCGCAGGGCTGGGCG





GGAGAGACTGTTCTGTTCCTTGTGTAACT





GTGTTGCTGAAAGACTACCTCGTTCTTGT





CTTGATGTGTCACCGGGGCAACTGCCTGG





GGGCGGGGATGGGGGCAGGGTGGAAGCG





GCTCCCCATTTTATACCAAAGGTGCTACA





TCTATGTGATGGGTGGGGTGGGGAGGGA





ATCACTGGTGCTATAGAAATTGAGATGCC





CCCCCAGGCCAGCAAATGTTCCTTTTTGT





TCAAAGTCTATTTTTATTCCTTGATATTTT





TCTTTTTTTTTTTTTTTTTTTGTGGATGGG





GACTTGTGAATTTTTCTAAAGGTGCTATT





TAACATGGGAGGAGAGCGTGTGCGGCTC





CAGCCCAGCCCGCTGCTCACTTTCCACCC





TCTCTCCACCTGCCTCTGGCTTCTCAGGC





CTCTGCTCTCCGACCTCTCTCCTCTGAAA





CCCTCCTCCACAGCTGCAGCCCATCCTCC





CGGCTCCCTCCTAGTCTGTCCTGCGTCCT





CTGTCCCCGGGTTTCAGAGACAACTTCCC





AAAGCACAAAGCAGTTTTTCCCCCTAGGG





GTGGGAGGAAGCAAAAGACTCTGTACCT





ATTTTGTATGTGTATAATAATTTGAGATG





TTTTTAATTATTTTGATTGCTGGAATAAA





GCATGTGGAAATGACCCAAACATAATCC





GCAGTGGCCTCCTAATTTCCTTCTTTGGA





GTTGGGGGAGGGGTAGACATGGGGAAGG





GGCTTTGGGGTGATGGGCTTGCCTTCCAT





TCCTGCCCTTTCCCTCCCCACTATTCTCTT





CTAGATCCCTCCATAACCCCACTCCCCTT





TCTCTCACCCTTCTTATACCGCAAACCTTT





CTACTTCCTCTTTCATTTTCTATTCTTGCA





ATTTCCTTGCACCTTTTCCAAATCCTCTTC





TCCCCTGCAATACCATACAGGCAATCCAC





GTGCACAACACACACACACACTCTTCACA





TCTGGGGTTGTCCAAACCTCATACCCACT





CCCCTTCAAGCCCATCCACTCTCCACCCC





CTGGATGCCCTGCACTTGGTGGCGGTGGG





ATGCTCATGGATACTGGGAGGGTGAGGG





GAGTGGAACCCGTGAGGAGGACCTGGGG





GCCTCTCCTTGAACTGACATGAAGGGTCA





TCTGGCCTCTGCTCCCTTCTCACCCACGCT





GACCTCCTGCCGAAGGAGCAACGCAACA





GGAGAGGGGTCTGCTGAGCCTGGCGAGG





GTCTGGGAGGGACCAGGAGGAAGGCGTG





CTCCCTGCTCGCTGTCCTGGCCCTGGGGG





AGTGAGGGAGACAGACACCTGGGAGAGC





TGTGGGGAAGGCACTCGCACCGTGCTCTT





GGGAAGGAAGGAGACCTGGCCCTGCTCA





CCACGGACTGGGTGCCTCGACCTCCTGAA





TCCCCAGAACACAACCCCCCTGGGCTGG





GGTGGTCTGGGGAACCATCGTGCCCCCGC





CTCCCGCCTACTCCTTTTTAAGCTT



 


3UTR-015
Plod1;
TTGGCCAGGCCTGACCCTCTTGGACCTTT
19



procollagen-
CTTCTTTGCCGACAACCACTGCCCAGCAG




lysine, 2-
CCTCTGGGACCTCGGGGTCCCAGGGAAC




oxoglutarate 5-
CCAGTCCAGCCTCCTGGCTGTTGACTTCC




dioxygenase 1
CATTGCTCTTGGAGCCACCAATCAAAGAG





ATTCAAAGAGATTCCTGCAGGCCAGAGG





CGGAACACACCTTTATGGCTGGGGCTCTC





CGTGGTGTTCTGGACCCAGCCCCTGGAGA





CACCATTCACTTTTACTGCTTTGTAGTGA





CTCGTGCTCTCCAACCTGTCTTCCTGAAA





AACCAAGGCCCCCTTCCCCCACCTCTTCC





ATGGGGTGAGACTTGAGCAGAACAGGGG





CTTCCCCAAGTTGCCCAGAAAGACTGTCT





GGGTGAGAAGCCATGGCCAGAGCTTCTC





CCAGGCACAGGTGTTGCACCAGGGACTT





CTGCTTCAAGTTTTGGGGTAAAGACACCT





GGATCAGACTCCAAGGGCTGCCCTGAGT





CTGGGACTTCTGCCTCCATGGCTGGTCAT





GAGAGCAAACCGTAGTCCCCTGGAGACA





GCGACTCCAGAGAACCTCTTGGGAGACA





GAAGAGGCATCTGTGCACAGCTCGATCTT





CTACTTGCCTGTGGGGAGGGGAGTGACA





GGTCCACACACCACACTGGGTCACCCTGT





CCTGGATGCCTCTGAAGAGAGGGACAGA





CCGTCAGAAACTGGAGAGTTTCTATTAAA





GGTCATTTAAACCA



 


3UTR-016
Nucb1;
TCCTCCGGGACCCCAGCCCTCAGGATTCC
20



nucleobindin 1
TGATGCTCCAAGGCGACTGATGGGCGCT





GGATGAAGTGGCACAGTCAGCTTCCCTG





GGGGCTGGTGTCATGTTGGGCTCCTGGGG





CGGGGGCACGGCCTGGCATTTCACGCATT





GCTGCCACCCCAGGTCCACCTGTCTCCAC





TTTCACAGCCTCCAAGTCTGTGGCTCTTC





CCTTCTGTCCTCCGAGGGGCTTGCCTTCT





CTCGTGTCCAGTGAGGTGCTCAGTGATCG





GCTTAACTTAGAGAAGCCCGCCCCCTCCC





CTTCTCCGTCTGTCCCAAGAGGGTCTGCT





CTGAGCCTGCGTTCCTAGGTGGCTCGGCC





TCAGCTGCCTGGGTTGTGGCCGCCCTAGC





ATCCTGTATGCCCACAGCTACTGGAATCC





CCGCTGCTGCTCCGGGCCAAGCTTCTGGT





TGATTAATGAGGGCATGGGGTGGTCCCTC





AAGACCTTCCCCTACCTTTTGTGGAACCA





GTGATGCCTCAAAGACAGTGTCCCCTCCA





CAGCTGGGTGCCAGGGGCAGGGGATCCT





CAGTATAGCCGGTGAACCCTGATACCAG





GAGCCTGGGCCTCCCTGAACCCCTGGCTT





CCAGCCATCTCATCGCCAGCCTCCTCCTG





GACCTCTTGGCCCCCAGCCCCTTCCCCAC





ACAGCCCCAGAAGGGTCCCAGAGCTGAC





CCCACTCCAGGACCTAGGCCCAGCCCCTC





AGCCTCATCTGGAGCCCCTGAAGACCAGT





CCCACCCACCTTTCTGGCCTCATCTGACA





CTGCTCCGCATCCTGCTGTGTGTCCTGTTC





CATGTTCCGGTTCCATCCAAATACACTTT





CTGGAACAAA



 


3UTR-017
α-globin
GCTGGAGCCTCGGTGGCCATGCTTCTTGC
21




CCCTTGGGCCTCCCCCCAGCCCCTCCTCC





CCTTCCTGCACCCGTACCCCCGTGGTCTT





TGAATAAAGTCTGAGTGGGCGGC









It should be understood that those listed in the previous tables are examples and that any UTR from any gene may be incorporated into the respective first or second flanking region of the primary construct. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide artificial UTRs which are not variants of wild type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made chimeric with one or more other 5′ UTRs or 3′ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3′ or 5′ UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR.
In one embodiment, a double, triple or quadruple UTR such as a 5′ or 3′ UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3′ UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety.
It is also within the scope of the present invention to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.
In one embodiment, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property. For example, cosmetic polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new chimeric primary transcript. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more cosmetic polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
After optimization (if desired), the cosmetic primary construct components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes. For example, the optimized construct may be reconstituted and transformed into chemically competent E. coli, yeast, neurospora, maize, drosophila, etc. where high copy plasmid-like or chromosome structures occur by methods described herein.
The untranslated region may also include translation enhancer elements (TEE). As a non-limiting example, the TEE may include those described in US Application No. 20090226470, herein incorporated by reference in its entirety, and those known in the art.
Stop Codons
In one embodiment, the cosmetic primary constructs of the present invention may include at least two stop codons before the 3′ untranslated region (UTR). The stop codon may be selected from TGA, TAA and TAG. In one embodiment, the cosmetic primary constructs of the present invention include the stop codon TGA and one additional stop codon. In a further embodiment the addition stop codon may be TAA. In another embodiment, the primary constructs of the present invention include three stop codons.
Vector Amplification
The vector containing the cosmetic primary construct is then amplified and the plasmid isolated and purified using methods known in the art such as, but not limited to, a maxi prep using the Invitrogen PURELINKTM HiPure Maxiprep Kit (Carlsbad, Calif.).
Plasmid Linearization
The plasmid may then be linearized using methods known in the art such as, but not limited to, the use of restriction enzymes and buffers. The linearization reaction may be purified using methods including, for example Invitrogen's PURELINKTM PCR Micro Kit (Carlsbad, Calif.), and HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen's standard PURELINKTM PCR Kit (Carlsbad, Calif.). The purification method may be modified depending on the size of the linearization reaction which was conducted. The linearized plasmid is then used to generate cDNA for in vitro transcription (IVT) reactions.
cDNA Template Synthesis
A cDNA template may be synthesized by having a linearized plasmid undergo polymerase chain reaction (PCR). Table 4 is a listing of primers and probes that may be useful in the PCR reactions of the present invention. It should be understood that the listing is not exhaustive and that primer-probe design for any amplification is within the skill of those in the art. Probes may also contain chemically modified bases to increase base-pairing fidelity to the target molecule and base-pairing strength. Such modifications may include 5-methyl-Cytidine, 2,6-di-amino-purine, 2′-fluoro, phosphoro-thioate, or locked nucleic acids.







TABLE 4







Primers and Probes










Primer/


SEQ


Probe

Hybridization
ID


Identifier
Sequence (5′-3′)
target
NO.





UFP
TTGGACCCTCGTACA
cDNA Template
22



GAAGCTAATACG




 


URP
Tx160CTTCCTACTCAGGC
cDNA Template
23



TTTATTCAAAGACCA




 


GBA1
CCTTGACCTTCTGGAACTTC
Acid
24




glucocere-





brosidase



 


GBA2
CCAAGCACTGAAACGGATAT
Acid
25




glucocere-





brosidase



 


LUC1
GATGAAAAGTGCTCCAAGGA
Luciferase
26


 


LUC2
AACCGTGATGAAAAGGTACC
Luciferase
27


 


LUC3
TCATGCAGATTGGAAAGGTC
Luciferase
28


 


G-CSF1
CTTCTTGGACTGTCCAGAGG
G-CSF
29


 


G-CSF2
GCAGTCCCTGATACAAGAAC
G-CSF
30


 


G-CSF3
GATTGAAGGTGGCTCGCTAC
G-CSF
31





*UFP is universal forward primer; URP is universal reverse primer.






In one embodiment, the cDNA may be submitted for sequencing analysis before undergoing transcription.
mRNA Production
The process of mRNA or mmRNA production may include, but is not limited to, in vitro transcription, cDNA template removal and RNA clean-up, and mRNA capping and/or tailing reactions.
In Vitro Transcription
The cDNA produced in the previous step may be transcribed using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to be incorporated into modified nucleic acids.
RNA Polymerases
Any number of RNA polymerases or variants may be used in the design of the cosmetic primary constructs of the present invention.
RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence. As a non-limiting example, the RNA polymerase may be modified to exhibit an increased ability to incorporate a 2′-modified nucleotide triphosphate compared to an unmodified RNA polymerase (see International Publication WO2008078180 and U.S. Pat. No. 8,101,385; herein incorporated by reference in their entireties).
Variants may be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and/or nucleic acid sequence and/or by using other methods known in the art. As a non-limiting example, T7 RNA polymerase variants may be evolved using the continuous directed evolution system set out by Esvelt et al. (Nature (2011) 472(7344):499-503; herein incorporated by reference in its entirety) where clones of T7 RNA polymerase may encode at least one mutation such as, but not limited to, lysine at position 93 substituted for threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A, H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E, N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limiting example, T7 RNA polymerase variants may encode at least mutation as described in U.S. Pub. Nos. 20100120024 and 20070117112; herein incorporated by reference in their entireties. Variants of RNA polymerase may also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, deletional variants and/or covalent derivatives.
In one embodiment, the cosmetic primary construct may be designed to be recognized by the wild type or variant RNA polymerases. In doing so, the cosmetic primary construct may be modified to contain sites or regions of sequence changes from the wild type or parent primary construct.
In one embodiment, the cosmetic primary construct may be designed to include at least one substitution and/or insertion upstream of an RNA polymerase binding or recognition site, downstream of the RNA polymerase binding or recognition site, upstream of the TATA box sequence, downstream of the TATA box sequence of the cosmetic primary construct but upstream of the coding region of the cosmetic primary construct, within the 5′UTR, before the 5′UTR and/or after the 5′UTR.
In one embodiment, the 5′UTR of the cosmetic primary construct may be replaced by the insertion of at least one region and/or string of nucleotides of the same base. The region and/or string of nucleotides may include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 nucleotides and the nucleotides may be natural and/or unnatural. As a non-limiting example, the group of nucleotides may include 5-8 adenine, cytosine, thymine, a string of any of the other nucleotides disclosed herein and/or combinations thereof.
In one embodiment, the 5′UTR of the cosmetic primary construct may be replaced by the insertion of at least two regions and/or strings of nucleotides of two different bases such as, but not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein and/or combinations thereof. For example, the 5′UTR may be replaced by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine bases. In another example, the 5′UTR may be replaced by inserting 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.
In one embodiment, the cosmetic primary construct may include at least one substitution and/or insertion downstream of the transcription start site which may be recognized by an RNA polymerase. As a non-limiting example, at least one substitution and/or insertion may occur downstream the transcription start site by substituting at least one nucleic acid in the region just downstream of the transcription start site (such as, but not limited to, +1 to +6). Changes to region of nucleotides just downstream of the transcription start site may affect initiation rates, increase apparent nucleotide triphosphate (NTP) reaction constant values, and increase the dissociation of short transcripts from the transcription complex curing initial transcription (Brieba et al, Biochemistry (2002) 41: 5144-5149; herein incorporated by reference in its entirety). The modification, substitution and/or insertion of at least one nucleic acid may cause a silent mutation of the nucleic acid sequence or may cause a mutation in the amino acid sequence.
In one embodiment, the cosmetic primary construct may include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or at least 13 guanine bases downstream of the transcription start site.
In one embodiment, the cosmetic primary construct may include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 guanine bases in the region just downstream of the transcription start site. As a non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 adenine nucleotides. In another non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 cytosine bases. In another non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 thymine, and/or any of the nucleotides described herein.
In one embodiment, the cosmetic primary construct may include at least one substitution and/or insertion upstream of the start codon. For the purpose of clarity, one of skill in the art would appreciate that the start codon is the first codon of the protein coding region whereas the transcription start site is the site where transcription begins. The cosmetic primary construct may include, but is not limited to, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 substitutions and/or insertions of nucleotide bases. The nucleotide bases may be inserted or substituted at 1, at least 1, at least 2, at least 3, at least 4 or at least 5 locations upstream of the start codon. The nucleotides inserted and/or substituted may be the same base (e.g., all A or all C or all T or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T or A, C and T) or at least four different bases. As a non-limiting example, the guanine base upstream of the coding region in the cosmetic primary construct may be substituted with adenine, cytosine, thymine, or any of the nucleotides described herein. In another non-limiting example the substitution of guanine bases in the cosmetic primary construct may be designed so as to leave one guanine base in the region downstream of the transcription start site and before the start codon (see Esvelt et al. Nature (2011) 472(7344):499-503; herein incorporated by reference in its entirety). As a non-limiting example, at least 5 nucleotides may be inserted at 1 location downstream of the transcription start site but upstream of the start codon and the at least 5 nucleotides may be the same base type.
cDNA Template Removal and Clean-Up
The cDNA template may be removed using methods known in the art such as, but not limited to, treatment with Deoxyribonuclease I (DNase I). RNA clean-up may also include a purification method such as, but not limited to, AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Mass.), HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
Capping and/or Tailing Reactions
The cosmetic primary construct or cosmetic mmRNA may also undergo capping and/or tailing reactions. A capping reaction may be performed by methods known in the art to add a 5′ cap to the 5′ end of the cosmetic primary construct. Methods for capping include, but are not limited to, using a Vaccinia Capping enzyme (New England Biolabs, Ipswich, Mass.).
A poly-A tailing reaction may be performed by methods known in the art, such as, but not limited to, 2′ O-methyltransferase and by methods as described herein. If the cosmetic primary construct generated from cDNA does not include a poly-T, it may be beneficial to perform the poly-A-tailing reaction before the cosmetic primary construct is cleaned.
mRNA Purification
Primary construct or mmRNA purification may include, but is not limited to, mRNA or mmRNA clean-up, quality assurance and quality control. mRNA or mmRNA clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in relation to a polynucleotide such as a “purified mRNA or mmRNA” refers to one that is separated from at least one contaminant. As used herein, a “contaminant” is any substance which makes another unfit, impure or inferior. Thus, a purified cosmetic polynucleotide (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.
A quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
In another embodiment, the cosmetic mRNA or cosmetic mmRNA may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.
In one embodiment, the cosmetic mRNA or cosmetic mmRNA may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, Mass.). The quantified cosmetic mRNA or cosmetic mmRNA may be analyzed in order to determine if the cosmetic mRNA or cosmetic mmRNA may be of proper size, check that no degradation of the cosmetic mRNA or cosmetic mmRNA has occurred. Degradation of the cosmetic mRNA and/or cosmetic mmRNA may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
Signal Sequences
The cosmetic primary constructs or cosmetic mmRNA may also encode additional features which facilitate trafficking of the cosmetic polypeptides to therapeutically relevant sites. One such feature which aids in protein trafficking is the signal sequence. As used herein, a “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-60 amino acids) in length which is incorporated at the 5′ (or N-terminus) of the coding region or polypeptide encoded, respectively. Addition of these sequences result in trafficking of the encoded cosmetic polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported.
Table 5 is a representative listing of protein signal sequences which may be incorporated for encoding by the cosmetic polynucleotides, cosmetic primary constructs or cosmetic mmRNA of the invention.







TABLE 5







Signal Sequences















SEQ

SEQ




NUCLEOTIDE SEQUENCE
ID
ENCODED
ID


ID
Description
(5'-3')
NO.
PEPTIDE
NO.















SS-001
α-1-
ATGATGCCATCCTCAGTCTCAT
32
MMPSSVSW
94



antitrypsin
GGGGTATTTTGCTCTTGGCGGG

GILLAGLCC





TCTGTGCTGTCTCGTGCCGGTGT

LVPVSLA





CGCTCGCA





 


SS-002
G-CSF
ATGGCCGGACCGGCGACTCAGT
33
MAGPATQS
95




CGCCCATGAAACTCATGGCCCT

PMKLMALQ





GCAGTTGTTGCTTTGGCACTCA

LLLWHSAL





GCCCTCTGGACCGTCCAAGAGG

WTVQEA





CG





 


SS-003
Factor IX
ATGCAGAGAGTGAACATGATTA
34
MQRVNMIM
96




TGGCCGAGTCCCCATCGCTCAT

AESPSLITIC





CACAATCTGCCTGCTTGGTACC

LLGYLLSAE





TGCTTTCCGCCGAATGCACTGT

CTVFLDHE





CTTTCTGGATCACGAGAATGCG

NANKILNRP





AATAAGATCTTGAACCGACCCA

KR





AACGG





 


SS-004 
Prolactin
ATGAAAGGATCATTGCTGTTGC
35
MKGSLLLL
97




TCCTCGTGTCGAACCTTCTGCTT

LVSNLLLCQ





TGCCAGTCCGTAGCCCCC

SVAP



 


SS-005
Albumin
ATGAAATGGGTGACGTTCATCT
36
MKWVTFIS
98




CACTGTTGTTTTTGTTCTCGTCC

LLFLFSSAY





GCCTACTCCAGGGGAGTATTCC

SRG VFRR





GCCGA





 


SS-006
HMMSP38
ATGTGGTGGCGGCTCTGGTGGC
37
MWWRLW
99




TGCTCCTGTTGCTCCTCTTGCTG

WLLLLLLLL





TGGCCCATGGTGTGGGCA

PMWA



 


MLS-001
ornithine
TGCTCTTTAACCTCCGCATCCTG
38
MLFNLRILL
100



carbamoyl-
TTGAATAACGCTGCGTTCCGAA

NNAAFRNG




transferase
ATGGGCATAACTTCATGGTACG

HNFMVRNF





CAACTTCAGATGCGGCCAGCCA

RCGQPLQ





CTCCAG





 


MLS-002
Cytochrome 
ATGTCCGTCTTGACACCCCTGC
39
MSVLTPLLL
101



C Oxidase
TCTTGAGAGGGCTGACGGGGTC

RGLTGSAR




subunit 8A
CGCTAGACGCCTGCCGGTACCG

RLPVPRAKI





CGAGCGAAGATCCACTCCCTG

HSL



 


MLS-003
Cytochrome 
ATGAGCGTGCTCACTCCGTTGC
40
MSVLTPLLL
102



C Oxidase
TTCTTCGAGGGCTTACGGGATC

RGLTGSAR




subunit 8A
GGCTCGGAGGTTGCCCGTCCCG

RLPVPRAKI





AGAGCGAAGATCCATTCGTTG

HSL



 


SS-007
Type III,
TGACAAAAATAACTTTATCTCC
41
MVTKITLSP
103



bacterial
CCAGAATTTTAGAATCCAAAAA

QNFRIQKQE





CAGGAAACCACACTACTAAAA

TTLLKEKST





GAAAAATCAACCGAGAAAAAT

EKNSLAKSI





TCTTTAGCAAAAAGTATTCTCG

LAVKNHFIE





CAGTAAAAATCACTTCATCGAA

LRSKLSERF





TTAAGGTCAAAATTATCGGAAC

ISHKNT





GTTTTATTTCGCATAAGAACAC







T





 


SS-008
Viral
ATGCTGAGCTTTGTGGATACCC
42
MLSFVDTR
104




GCACCCTGCTGCTGCTGGCGGT

TLLLLAVTS





GACCAGCTGCCTGGCGACCTGC

CLATCQ





CAG





 


SS-009
viral
ATGGGCAGCAGCCAGGCGCCG
43
MGSSQAPR
105




CGCATGGGCAGCGTGGGCGGCC

MGSVGGHG





ATGGCCTGATGGCGCTGCTGAT

LMALLMAG





GGCGGGCCTGATTCTGCCGGGC

LILPGILA





ATTCTGGCG





 


SS-010
Viral
ATGGCGGGCATTTTTTATTTTCT
44
MAGIFYFLF
106




GTTTAGCTTTCTGTTTGGCATTT

SFLFGICD





GCGAT





 


SS-011
Viral
ATGGAAAACCGCCTGCTGCGCG
45
MENRLLRV
107




TGTTTCTGGTGTGGGCGGCGCT

FLVWAALT





GACCATGGATGGCGCGAGCGC

MDGASA





G





 


SS-012
Viral
ATGGCGCGCCAGGGCTGCTTTG
46
MARQGCFG
108




GCAGCTATCAGGTGATTAGCCT

SYQVISLFT





GTTTACCTTTGCGATTGGCGTG

FAIGVNLCL





AACCTGTGCCTGGGC

G



 


SS-013
Bacillus
ATGAGCCGCCTGCCGGTGCTGC
47
MSRLPVLLL
109




TGCTGCTGCAGCTGCTGGTGCG

LQLLVRPGL





CCCGGGCCTGCAG

Q



 


SS-014
Bacillus
ATGAAACAGCAGAAACGCCTGT
48
MKQQKRLY
110




ATGCGCGCCTGCTGACCCTGCT

ARLLTLLFA





GTTTGCGCTGATTTTTCTGCTGC

LIFLLPHSSA





CGCATAGCAGCGCGAGCGCG

SA



 


SS-015 
Secretion
ATGGCGACGCCGCTGCCTCCGC
49
MATPLPPPS 
111



signal
CCTCCCCGCGGCACCTGCGGCT

PRHLRLLRL





GCTGCGGCTGCTGCTCTCCGCC

LLSG





CTCGTCCTCGGC





 


SS-016 
Secretion
ATGAAGGCTCCGGGTCGGCTCG
50
MKAPGRLV
112



signal
TGCTCATCATCCTGTGCTCCGTG

LIILCSVVFS





GTCTTCTCT





 


SS-017 
Secretion
ATGCTTCAGCTTTGGAAACTTG
51
MLQLWKLL
113



signal
TTCTCCTGTGCGGCGTGCTCACT

CGVLT



 


SS-018 
Secretion
ATGCTTTATCTCCAGGGTTGGA
52
MLYLQGWS
114



signal
GCATGCCTGCTGTGGCA

MPAVA



 


SS-019 
Secretion
ATGGATAACGTGCAGCCGAAA
53
MDNVQPKI
115



signal
ATAAAACATCGCCCCTTCTGCT

KHRPFCFSV





TCAGTGTGAAAGGCCACGTGAA

KGHVKMLR





GATGCTGCGGCTGGATATTATC

LDIINSLVTT





AACTCACTGGTAACAACAGTAT

VFMLIVSVL





TCATGCTCATCGTATCTGTGTTG

ALIP





GCACTGATACCA





 


SS-020 
Secretion
ATGCCCTGCCTAGACCAACAGC
54
MPCLDQQL
116



signal
TCACTGTTCATGCCCTACCCTGC

TVHALPCP





CCTGCCCAGCCCTCCTCTCTGG

AQPSSLAFC





CCTTCTGCCAAGTGGGGTTCTT

QVGFLTA





AACAGCA





 


SS-021 
Secretion
ATGAAAACCTTGTTCAATCCAG
55
MKTLFNPA
117



signal
CCCCTGCCATTGCTGACCTGGA

PAIADLDPQ





TCCCCAGTTCTACACCCTCTCA

FYTLSDVFC





GATGTGTTCTGCTGCAATGAAA

CNESEAEIL





GTGAGGCTGAGATTTTAACTGG

TGLTVGSA





CCTCACGGTGGGCAGCGCTGCA

ADA





GATGCT





 


SS-022 
Secretion
ATGAAGCCTCTCCTTGTTGTG 
56
MKPLLVVF
118



signal
TTTGTCTTTCTTTTCCTTTG

VFLFLWDP





GGATCCAGTGCTGGCA

VLA



 


SS-023 
Secretion
ATGTCCTGTTCCCTAAAGTT 
57
MSCSLKFTL 
119



signal
TACTTTGATTGTAATTTTTT

IVIFFTCTLS





TTTACTGTTGGCTTTCATCC

SS





AGC





 


SS-024 
Secretion
ATGGTTCTTACTAAACCTCTT 
58
MVLTKPLQ
120



signal
CAAAGAAATGGCAGCATGATG

RNGSMMSF





AGCTTTGAAAATGTGAAAGAA

ENVKEKSR





AAGAGCAGAGAAGGAGGGCCC

EGGPHAHT





CATGCACACACACCCGAAGAA

PEEELCFVV





GAATTGTGTTTCGTGGTAACA

THTPQVQT





CACTACCCTCAGGTTCAGACC

TLNLFFHIF





ACACTCAACCTGTTTTTCCAT

KVLTQPLSL





ATATTCAAGGTTCTTACTCAA

LWG





CCACTTTCCCTTCTGTGGGGT





 


SS-025 
Secretion
ATGGCCACCCCGCCATTCCGGC
59
MATPPFRLI 
121



signal
TGATAAGGAAGATGTTTTCCTT

RKMFSFKV





CAAGGTGAGCAGATGGATGGG

SRWMGLAC





GCTTGCCTGCTTCCGGTCCCTG

FRSLAAS





GCGGCATCC





 


SS-026  
Secretion
ATGAGCTTTTTCCAACTCCTGAT
60
MSFFQLLM
122



signal
GAAAAGGAAGGAACTCATTCCC

KRKELIPLV





TTGGTGGTGTTCATGACTGTGG

VFMTVAAG





CGGCGGGTGGAGCCTCATCT

GASS



 


SS-027 
Secretion
ATGGTCTCAGCTCTGCGGGGAG
61
MVSALRGA
123



signal
CACCCCTGATCAGGGTGCACTC

PLIRVHSSP





AAGCCCTGTTTCTTCTCCTTCTG

VSSPSVSGP





TGAGTGGACCACGGAGGCTGGT

AALVSCLSS





GAGCTGCCTGTCATCCCAAAGC

QSSALS





TCAGCTCTGAGC





 


SS-028 
Secretion
ATGATGGGGTCCCCAGTGAGTC
62
MMGSPVSH
124



signal
ATCTGCTGGCCGGCTTCTGTGT

LLAGFCVW





GTGGGTCGTCTTGGGC

VVLG



 


SS-029 
Secretion
ATGGCAAGCATGGCTGCCGTGC
63
MASMAAVL
125



signal
TCACCTGGGCTCTGGCTCTTCTT

TWALALLS





TCAGCGTTTTCGGCCACCCAGG

AFSATQA





CA





 


SS-030 
Secretion
ATGGTGCTCATGTGGACCAGTG
64
MVLMWTS
126



signal
GTGACGCCTTCAAGACGGCCTA

GDAFKTAY





CTTCCTGCTGAAGGGTGCCCCT

FLLKGAPLQ





CTGCAGTTCTCCGTGTGCGGCC

FSVCGLLQ





TGCTGCAGGTGCTGGTGGACCT

VLVDLAILG





GGCCATCCTGGGGCAGGCCTAC

QATA





GCC





 


SS-031 
Secretion
ATGGATTTTGTCGCTGGAGCCA
65
MDFVAGAI
127



signal
TCGGAGGCGTCTGCGGTGTTGC

GGVCGVAV





TGTGGGCTACCCCCTGGACACG

GYPLDTVK





GTGAAGGTCAGGATCCAGACG

VRIQTEPLY





GAGCCAAAGTACACAGGCATCT

TGIWHCVR





GGCACTGCGTCCGGGATACGTA

DTYHRERV





TCACCGAGAGCGCGTGTGGG

WGFYRGLS





GCTTCTACCGGGGCCTCTCGCT

LPVCTVSLV





GCCCGTGTGCACGGTGTCCCTG

SS





GTATCTTCC





 


SS-032 
Secretion
ATGGAGAAGCCCCTCTTCCCAT
66
MEKPLFPLV 
128



signal
TAGTGCCTTTGCATTGGTTTGGC

PLHWFGFG





TTTGGCTACACAGCACTGGTTG

YTALVVSG





TTTCTGGTGGGATCGTTGGCTA

GIVGYVKT





TGTAAAAACAGGCAGCGTGCCG

GSVPSLAA





TCCCTGGCTGCAGGGCTGCTCT

GLLFGSLA





TCGGCAGTCTAGCC





 


SS-033 
Secretion
ATGGGTCTGCTCCTTCCCCTGG
67
MGLLLPLA
129



signal
CACTCTGCATCCTAGTCCTGTG

LCILVLC





C





 


SS-034 
Secretion
ATGGGGATCCAGACGAGCCCCG
68
MGIQTSPVL 
130



signal
TCCTGCTGGCCTCCCTGGGGGT

LASLGVGL





GGGGCTGGTCACTCTGCTCGGC

VTLLGLAV





CTGGCTGTGGGC

G



 


SS-035
Secretion
ATGTCGGACCTGCTACTACTGG
69
MSDLLLLG
131



signal
GCCTGATTGGGGGCCTGACTCT

LIGGLTLLL





CTTACTGCTGCTGACGCTGCTA

LLTLLAFA





GCCTTTGCC





 


SS-036
Secretion
ATGGAGACTGTGGTGATTGTTG
70
METVVIVAI
132



signal
CCATAGGTGTGCTGGCCACCAT

GVLATIFLA





GTTTCTGGCTTCGTTTGCAGCCT

SFAALVLV





TGGTGCTGGTTTGCAGGCAG

CRQ



 


SS-037
Secretion
ATGCGCGGCTCTGTGGAGTGCA
71
MAGSVECT
133



signal
CCTGGGGTTGGGGGCACTGTGC

WGWGHCA





CCCCAGCCCCCTGCTCCTTTGG

PSPLLLWTL





ACTCTACTTCTGTTTGCAGCCCC

LLFAAPFGL





ATTTGGCCTGCTGGGG

LG



 


SS-038
Secretion
ATGATGCCGTCCCGTACCAACC
72
MMPSRTNL
134



signal
TGGCTACTGGAATCCCCAGTAG

ATGIPSSKV





TAAAGTGAAATATTCAAGGCTC

KYSRLSSTD





TCCAGCACAGACGATGGCTACA

DGYIDLQFK





TTGACCTTCAGTTTAAGAAAAC

KTPPKIPYK





CCCTCCTAAGATCCCTTATAAG

AIALATVLF





GCCATCGCACTTGCCACTGTGC

LIGA





TGTTTTTGATTGGCGCC





 


SS-039
Secretion
ATGGCCCTGCCCCAGATGTGTG
73
MALPQMCD
135



signal
ACGGGAGCCACTTGGCCTCCAC

GSHLASTLR





CCTCCGCTATTGCATGACAGTC

YCMTVSGT





AGCGGCACAGTGGTTCTGGTGG

VVLVAGTL





CCGGGACGCTCTGCTTCGCT

CFA



 


SS-041
Vrg-6
TGAAAAAGTGGTTCGTTGCTGC
74
MKKWFVA
136




CGGCATCGGCGCTGCCGGACTC

AGIGAGLL





ATGCTCTCCAGCGCCGCCA

MLSSAA



 


SS-042
PhoA
ATGAAACAGAGCACCATTGCGC
75
MKQSTIAL
137




TGGCGCTGCTGCCGCTGCTGTT

ALLPLLFTP





TACCCCGGTGACCAAAGCG

VTKA



 


SS-043
OmpA
ATGAAAAAAACCGCGATTGCG
76
MKKTAIAIA
138




ATTGCGGTGGCGCTGGCGGGCT

VALAGFAT





TTGCGACCGTGGCGCAGGCG

VAQA



 


SS-044
STI
ATGAAAAAACTGATGCTGGCGA
77
MKKLMLAI
139




TTTTTTTTAGCGTGCTGAGCTTT

FFSVLSFPSF





CCGAGCTTTAGCCAGAGC

SQS



 


SS-045
STII
ATGAAAAAAAACATTGCGTTTC
78
MKKNIAFL
140




TGCTGGCGAGCATGTTTGTGTT

LASMFVFSI





TAGCATTGCGACCAACGCGTAT

ATNAYA





GCG





SS-046
Amylase
ATGTTTGCGAAACGCTTTAAAA
79
MFAKRFKT
141




CCAGCCTGCTGCCGCTGTTTGC

SLLPLFAGF





GGGCTTTCTGCTGCTGTTTCATC

LLLFHLVLA





TGGTGCTGGCGGGCCCGGCGGC

GPAAAS





GGCGAGC





 


SS-047
Alpha 
ATGCGCTTTCCGAGCATTTTTAC
80
MRFPSIFTA
142



Factor
CGCGGTGCTGTTTGCGGCGAGC

VLFAASSAL





AGCGCGCTGGCG

A



 


SS-048
Alpha  
ATGCGCTTTCCGAGCATTTTTAC
81
MRFPSIFTT
143



Factor
CACCGTGCTGTTTGCGGCGAGC

VLFAASSAL





AGCGCGCTGGCG

A



 


SS-049
Alpha  
ATGCGCTTTCCGAGCATTTTTAC
82
MRFPSIFTS
144



Factor
CAGCGTGCTGTTTGCGGCGAGC

VLFAASSAL





AGCGCGCTGGCG

A



 


SS-050
Alpha  
ATGCGCTTTCCGAGCATTTTTAC
83
MRFPSIFTH
145



Factor
CCATGTGCTGTTTGCGGCGAGC

VLFAASSAL





AGCGCGCTGGCG

A



 


SS-051
Alpha  
ATGCGCTTTCCGAGCATTTTTAC
84
MRFPSIFTIV
146



Factor
CATTGTGCTGTTTGCGGCGAGC

LFAASSALA





AGCGCGCTGGCG





 


SS-052
Alpha  
ATGCGCTTTCCGAGCATTTTTAC
85
MRFPSIFTF
147



Factor
CTTTGTGCTGTTTGCGGCGAGC

VLFAASSAL





AGCGCGCTGGCG

A



 


SS-053
Alpha  
ATGCGCTTTCCGAGCATTTTTAC
86
MRFPSIFTE
148



Factor
CGAAGTGCTGTTTGCGGCGAGC

VLFAASSAL





AGCGCGCTGGCG

A



 


SS-054
Alpha  
ATGCGCTTTCCGAGCATTTTTAC
87
MRFPSIFTG
149



Factor
CGGCGTGCTGTTTGCGGCGAGC

VLFAASSAL





AGCGCGCTGGCG

A



 


SS-055
Endoglu- 
ATGCGTTCCTCCCCCCTCCTCCG
88
MRSSPLLRS
150



canas eV
CTCCGCCGTTGTGGCCGCCCTG

AVVAALPV





CCGGTGTTGGCCCTTGCC

LALA



 


SS-056
Secretion
ATGGGCGCGGCGGCCGTGCGCT
89
MGAAAVR
151



signal
GGCACTTGTGCGTGCTGCTGGC

WHLCVLLA





CCTGGGCACACGCGGGCGGCTG

LGTRGRL



 


SS-057
Fungal
ATGAGGAGCTCCCTTGTGCTGT
90
MRSSLVLFF
152




TCTTTGTCTCTGCGTGGACGGC

VSAWTALA





CTTGGCCAG





 


SS-058
Fibronectin
ATGCTCAGGGGTCCGGGACCCG
91
MLRGPGPG
153




GGCGGCTGCTGCTGCTAGCAGT

RLLLLAVLC





CCTGTGCCTGGGGACATCGGTG

LGTSVRCTE





CGCTGCACCGAAACCGGGAAG

TGKSKR





AGCAAGAGG





 


SS-059
Fibronectin
ATGCTTAGGGGTCCGGGGCCCG
92
MLRGPGPG
154




GGCTGCTGCTGCTGGCCGTCCA

LLLLAVQC





GCTGGGGACAGCGGTGCCCTCC

LGTAVPSTG





ACG

A



 


SS-060
Fibronectin
ATGCGCCGGGGGGCCCTGACCG
93
MRRGALTG
155




GGCTGCTCCTGGTCCTGTGCCT

LLLVLCLSV





GAGTGTTGTGCTACGTGCAGCC

VLRAAPSA





CCCTCTGCAACAAGCAAGAAGC

TSKKRR





GCAGG









In the table, SS is secretion signal and MLS is mitochondrial leader signal. The cosmetic primary constructs or cosmetic mmRNA of the present invention may be designed to encode any of the signal sequences of SEQ ID NOs 94-155, or fragments or variants thereof. These sequences may be included at the beginning of the cosmetic polypeptide coding region, in the middle or at the terminus or alternatively into a flanking region. Further, any of the cosmetic polynucleotide primary constructs of the present invention may also comprise one or more of the sequences defined by SEQ ID NOs 32-93. These may be in the first region or either flanking region.
Additional signal sequences which may be utilized in the present invention include those taught in, for example, databases such as those found at www.signalpeptide.de/ or proline.bic.nus.edu.sg/spdb/. Those described in U.S. Pat. Nos. 8,124,379; 7,413,875 and 7,385,034 are also within the scope of the invention and the contents of each are incorporated herein by reference in their entirety.
Target Selection
According to the present invention, the cosmetic primary constructs comprise at least a first region of linked nucleosides encoding at least one cosmetic polypeptide of interest. The cosmetic polypeptides of interest or “targets” or cosmetic proteins and cosmetic peptides of the present invention are listed in Tables 6 and 7. Shown in Table 6, in addition to the target number (Target No), name and description of the gene encoding the cosmetic polypeptide of interest (Target Description) are the ENSEMBL Transcript ID (ENST) and SEQ ID NO (Trans SEQ ID NO), the ENSEMBL Protein ID (ENSP) and SEQ ID NO (Peptide SEQ ID NO) and when available the optimized ORF identifier (Optimized ORF SEQ ID). For any particular gene there may exist one or more variants or isoforms. Where these exist, they are shown in the tables as well. It will be appreciated by those of skill in the art that disclosed in the Tables are potential flanking regions. These are encoded in each ENST transcript or NCBI nucleotide sequence either to the 5′ (upstream) or 3′ (downstream) of the ORF or coding region. The coding region is definitively and specifically disclosed by teaching the ENSP protein sequence. Consequently, the sequences taught flanking that encoding the protein are considered flanking regions. It is also possible to further characterize the 5′ and 3′ flanking regions by utilizing one or more available databases or algorithms. Databases have annotated the features contained in the flanking regions of the ENST transcripts and these are available in the art.







TABLE 6







Cosmetic Targets
















Trans

Peptide



Target


SEQ

SEQ
Optimized


No.
Target Description
ENST
ID NO
ENSP
ID NO
ORF SEQ ID
















1
actin gamma 1
573283
156
458435
884
1612, 2337,








3062, 3787,








4512


2
actin, alpha 2, smooth muscle,
224784
157
224784
885
1613, 2338,



aorta




3063, 3788,








4513


3
actin, alpha 2, smooth muscle,
415557
158
396730
886
1614, 2339,



aorta




3064, 3789,








4514


4
actin, alpha 2, smooth muscle,
458159
159
398239
887
1615, 2340,



aorta




3065, 3790,








4515


5
actin, alpha 2, smooth muscle,
458208
160
402373
888
1616, 2341,



aorta




3066, 3791,








4516


6
actin, alpha 2, smooth muscle,
544901
161
439477
889
1617, 2342,



aorta




3067, 3792,








4517


7
actin, alpha, cardiac muscle 1
290378
162
290378
890
1618, 2343,








3068, 3793,








4518


8
actin, alpha, cardiac muscle 1
544062
163
445797
891
1619, 2344,








3069, 3794,








4519


9
actin, beta
320713
164
440549
892
1620, 2345,








3070, 3795,








4520


10
actin, beta
331789
165
349960
893
1621, 2346,








3071, 3796,








4521


11
actin, beta
400179
166
438481
894
1622, 2347,








3072, 3797,








4522


12
actin, beta
414620
167
401032
895
1623, 2348,








3073, 3798,








4523


13
actin, beta
417101
168
399487
896
1624, 2349,








3074, 3799,








4524


14
actin, beta
432588
169
407473
897
1625, 2350,








3075, 3800,








4525


15
actin, beta
443528
170
393951
898
1626, 2351,








3076, 3801,








4526


16
actin, beta
445914
171
414839
899
1627, 2352,








3077, 3802,








4527


17
actin, beta-like 2
423391
172
416706
900
1628, 2353,








3078, 3803,








4528


18
actin, gamma 1
331925
173
331514
901
1629, 2354,








3079, 3804,








4529


19
actin, gamma 1
447294
174
438755
902
1630, 2355,








3080, 3805,








4530


20
actin, gamma 2, smooth
345517
175
295137
903
1631, 2356,



muscle, enteric




3081, 3806,








4531


21
actin, gamma 2, smooth
409624
176
386857
904
1632, 2357,



muscle, enteric




3082, 3807,








4532


22
actin-like 6A
392662
177
376430
905
1633, 2358,








3083, 3808,








4533


23
actin-like 6A
429709
178
397552
906
1634, 2359,








3084, 3809,








4534


24
actin-like 6A
450518
179
394014
907
1635, 2360,








3085, 3810,








4535


25
actin-like 6A
490364
180
420153
908
1636, 2361,








3086, 3811,








4536


26
actin-like 6B
160382
181
160382
909
1637, 2362,








3087, 3812,








4537, 5237,








5352


27
actin-like 7A
333999
182
334300
910
1638, 2363,








3088, 3813,








4538


28
actin-like 7B
374667
183
363799
911
1639, 2364,








3089, 3814,








4539


29
actin-like 8
375406
184
364555
912
1640, 2365,








3090, 3815,








4540


30
actin-like 9
324436
185
316674
913
1641, 2366,








3091, 3816,








4541


31
androgen receptor
374690
186
363822
914
1642, 2367,








3092, 3817,








4542


32
androgen receptor
396043
187
379358
915
1643, 2368,








3093, 3818,








4543


33
androgen receptor
396044
188
379359
916
1644, 2369,








3094, 3819,








4544


34
androgen receptor
538891
189
445174
917
1645, 2370,








3095, 3820,








4545


35
androgen receptor
544984
190
438166
918
1646, 2371,








3096, 3821,








4546


36
attractin
262919
191
262919
919
1647, 2372,








3097, 3822,








4547


37
attractin
340500
192
344367
920
1648, 2373,








3098, 3823,








4548


38
attractin
446916
193
416587
921
1649, 2374,








3099, 3824,








4549


39
brain-derived neurotrophic
314915
194
320002
922
1650, 2375,



factor




3100, 3825,








4550


40
brain-derived neurotrophic
356660
195
349084
923
1651, 2376,



factor




3101, 3826,








4551


41
brain-derived neurotrophic
395978
196
379302
924
1652, 2377,



factor




3102, 3827,








4552


42
brain-derived neurotrophic
395980
197
379304
925
1653, 2378,



factor




3103, 3828,








4553


43
brain-derived neurotrophic
395981
198
379305
926
1654, 2379,



factor




3104, 3829,








4554


44
brain-derived neurotrophic
395983
199
379307
927
1655, 2380,



factor




3105, 3830,








4555


45
brain-derived neurotrophic
395986
200
379309
928
1656, 2381,



factor




3106, 3831,








4556


46
brain-derived neurotrophic
418212
201
400502
929
1657, 2382,



factor




3107, 3832,








4557


47
brain-derived neurotrophic
420794
202
389564
930
1658, 2383,



factor




3108, 3833,








4558


48
brain-derived neurotrophic
438929
203
414303
931
1659, 2384,



factor




3109, 3834,








4559


49
brain-derived neurotrophic
439476
204
389345
932
1660, 2385,



factor




3110, 3835,








4560


50
brain-derived neurotrophic
525528
205
437138
933
1661, 2386,



factor




3111, 3836,








4561


51
brain-derived neurotrophic
525950
206
432035
934
1662, 2387,



factor




3112, 3837,








4562


52
brain-derived neurotrophic
530861
207
435564
935
1663, 2388,



factor




3113, 3838,








4563


53
brain-derived neurotrophic
532997
208
435805
936
1664, 2389,



factor




3114, 3839,








4564


54
brain-derived neurotrophic
533131
209
432727
937
1665, 2390,



factor




3115, 3840,








4565


55
brain-derived neurotrophic
533246
210
432376
938
1666, 2391,



factor




3116, 3841,








4566


56
brain-derived neurotrophic
530786
211
433003
939
1667, 2392,



factor




3117, 3842,








4567


57
calponin 1, basic, smooth
252456
212
252456
940
1668, 2393,



muscle




3118, 3843,








4568


58
calponin 1, basic, smooth
535659
213
442031
941
1669, 2394,



muscle




3119, 3844,








4569


59
calponin 1, basic, smooth
544952
214
437470
942
1670, 2395,



muscle




3120, 3845,








4570


60
calponin 2
263097
215
263097
943
1671, 2396,








3121, 3846,








4571


61
calponin 2
348419
216
340129
944
1672, 2397,








3122, 3847,








4572


62
calponin 2
442531
217
411272
945
1673, 2398,








3123, 3848,








4573


63
calponin 3, acidic
370206
218
359225
946
1674, 2399,








3124, 3849,








4574


64
calponin 3, acidic
394202
219
377752
947
1675, 2400,








3125, 3850,








4575


65
calponin 3, acidic
415017
220
401452
948
1676, 2401,








3126, 3851,








4576


66
calponin 3, acidic
538964
221
437665
949
1677, 2402,








3127, 3852,








4577


67
calponin 3, acidic
545882
222
440081
950
1678, 2403,








3128, 3853,








4578


68
caspase recruitment domain
570421
223
461806
951
1679, 2404,



family member 14




3129, 3854,








4579


69
caspase recruitment domain
573882
224
458715
952
1680, 2405,



family member 14




3130, 3855,








4580


70
caspase recruitment domain
309710
225
308507
953
1681, 2406,



family, member 14




3131, 3856,








4581


71
caspase recruitment domain
344227
226
344549
954
1682, 2407,



family, member 14




3132, 3857,








4582


72
caspase recruitment domain
392434
227
376229
955
1683, 2408,



family, member 14




3133, 3858,








4583


73
coiled-coil alpha-helical rod
376266
228
365442
956
1684, 2409,



protein 1




3134, 3859,








4584


74
coiled-coil alpha-helical rod
383341
229
372832
957
1685, 2410,



protein 1




3135, 3860,








4585


75
coiled-coil alpha-helical rod
383527
230
373019
958
1686, 2411,



protein 1




3136, 3861,








4586


76
coiled-coil alpha-helical rod
396263
231
379561
959
1687, 2412,



protein 1




3137, 3862,








4587


77
coiled-coil alpha-helical rod
396268
232
379566
960
1688, 2413,



protein 1




3138, 3863,








4588


78
coiled-coil alpha-helical rod
400351
233
383204
961
1689, 2414,



protein 1




3139, 3864,








4589


79
coiled-coil alpha-helical rod
400352
234
383205
962
1690, 2415,



protein 1




3140, 3865,








4590


80
coiled-coil alpha-helical rod
400406
235
383257
963
1691, 2416,



protein 1




3141, 3866,








4591


81
coiled-coil alpha-helical rod
400412
236
383263
964
1692, 2417,



protein 1




3142, 3867,








4592


82
coiled-coil alpha-helical rod
412245
237
413000
965
1693, 2418,



protein 1




3143, 3868,








4593


83
coiled-coil alpha-helical rod
412577
238
399555
966
1694, 2419,



protein 1




3144, 3869,








4594


84
coiled-coil alpha-helical rod
414614
239
398934
967
1695, 2420,



protein 1




3145, 3870,








4595


85
coiled-coil alpha-helical rod
416163
240
408012
968
1696, 2421,



protein 1




3146, 3871,








4596


86
coiled-coil alpha-helical rod
416392
241
403628
969
1697, 2422,



protein 1




3147, 3872,








4597


87
coiled-coil alpha-helical rod
416552
242
396696
970
1698, 2423,



protein 1




3148, 3873,








4598


88
coiled-coil alpha-helical rod
416850
243
410052
971
1699, 2424,



protein 1




3149, 3874,








4599


89
coiled-coil alpha-helical rod
417501
244
394964
972
1700, 2425,



protein 1




3150, 3875,








4600


90
coiled-coil alpha-helical rod
417776
245
414578
973
1701, 2426,



protein 1




3151, 3876,








4601


91
coiled-coil alpha-helical rod
419528
246
391181
974
1702, 2427,



protein 1




3152, 3877,








4602


92
coiled-coil alpha-helical rod
419592
247
403227
975
1703, 2428,



protein 1




3153, 3878,








4603


93
coiled-coil alpha-helical rod
420262
248
390479
976
1704, 2429,



protein 1




3154, 3879,








4604


94
coiled-coil alpha-helical rod
423825
249
408031
977
1705, 2430,



protein 1




3155, 3880,








4605


95
coiled-coil alpha-helical rod
425351
250
410018
978
1706, 2431,



protein 1




3156, 3881,








4606


96
coiled-coil alpha-helical rod
425500
251
400469
979
1707, 2432,



protein 1




3157, 3882,








4607


97
coiled-coil alpha-helical rod
425620
252
393042
980
1708, 2433,



protein 1




3158, 3883,








4608


98
coiled-coil alpha-helical rod
426967
253
402432
981
1709, 2434,



protein 1




3159, 3884,








4609


99
coiled-coil alpha-helical rod
427654
254
405010
982
1710, 2435,



protein 1




3160, 3885,








4610


100
coiled-coil alpha-helical rod
428174
255
389303
983
1711, 2436,



protein 1




3161, 3886,








4611


101
coiled-coil alpha-helical rod
428677
256
416029
984
1712, 2437,



protein 1




3162, 3887,








4612


102
coiled-coil alpha-helical rod
429943
257
404757
985
1713, 2438,



protein 1




3163, 3888,








4613


103
coiled-coil alpha-helical rod
431539
258
390347
986
1714, 2439,



protein 1




3164, 3889,








4614


104
coiled-coil alpha-helical rod
432736
259
388264
987
1715, 2440,



protein 1




3165, 3890,








4615


105
coiled-coil alpha-helical rod
433804
260
414411
988
1716, 2441,



protein 1




3166, 3891,








4616


106
coiled-coil alpha-helical rod
437027
261
409907
989
1717, 2442,



protein 1




3167, 3892,








4617


107
coiled-coil alpha-helical rod
437918
262
412157
990
1718, 2443,



protein 1




3168, 3893,








4618


108
coiled-coil alpha-helical rod
438424
263
393508
991
1719, 2444,



protein 1




3169, 3894,








4619


109
coiled-coil alpha-helical rod
438739
264
404715
992
1720, 2445,



protein 1




3170, 3895,








4620


110
coiled-coil alpha-helical rod
438812
265
396200
993
1721, 2446,



protein 1




3171, 3896,








4621


111
coiled-coil alpha-helical rod
439536
266
391018
994
1722, 2447,



protein 1




3172, 3897,








4622


112
coiled-coil alpha-helical rod
439820
267
388748
995
1723, 2448,



protein 1




3173, 3898,








4623


113
coiled-coil alpha-helical rod
440185
268
397125
996
1724, 2449,



protein 1




3174, 3899,








4624


114
coiled-coil alpha-helical rod
444737
269
409157
997
1725, 2450,



protein 1




3175, 3900,








4625


115
coiled-coil alpha-helical rod
444855
270
397856
998
1726, 2451,



protein 1




3176, 3901,








4626


116
coiled-coil alpha-helical rod
447252
271
405118
999
1727, 2452,



protein 1




3177, 3902,








4627


117
coiled-coil alpha-helical rod
447874
272
412354
1000
1728, 2453,



protein 1




3178, 3903,








4628


118
coiled-coil alpha-helical rod
448141
273
414323
1001
1729, 2454,



protein 1




3179, 3904,








4629


119
coiled-coil alpha-helical rod
448162
274
390027
1002
1730, 2455,



protein 1




3180, 3905,








4630


120
coiled-coil alpha-helical rod
449364
275
408519
1003
1731, 2456,



protein 1




3181, 3906,








4631


121
coiled-coil alpha-helical rod
450631
276
399391
1004
1732, 2457,



protein 1




3182, 3907,








4632


122
coiled-coil alpha-helical rod
451337
277
396155
1005
1733, 2458,



protein 1




3183, 3908,








4633


123
coiled-coil alpha-helical rod
451470
278
390313
1006
1734, 2459,



protein 1




3184, 3909,








4634


124
coiled-coil alpha-helical rod
451521
279
401039
1007
1735, 2460,



protein 1




3185, 3910,








4635


125
coiled-coil alpha-helical rod
452333
280
404917
1008
1736, 2461,



protein 1




3186, 3911,








4636


126
coiled-coil alpha-helical rod
452419
281
389758
1009
1737, 2462,



protein 1




3187, 3912,








4637


127
coiled-coil alpha-helical rod
453360
282
397446
1010
1738, 2463,



protein 1




3188, 3913,








4638


128
coiled-coil alpha-helical rod
454570
283
401869
1011
1739, 2464,



protein 1




3189, 3914,








4639


129
coiled-coil alpha-helical rod
455279
284
398715
1012
1740, 2465,



protein 1




3190, 3915,








4640


130
coiled-coil alpha-helical rod
455669
285
408590
1013
1741, 2466,



protein 1




3191, 3916,








4641


131
coiled-coil alpha-helical rod
456365
286
395434
1014
1742, 2467,



protein 1




3192, 3917,








4642


132
coiled-coil alpha-helical rod
456712
287
391707
1015
1743, 2468,



protein 1




3193, 3918,








4643


133
coiled-coil alpha-helical rod
457929
288
389131
1016
1744, 2469,



protein 1




3194, 3919,








4644


134
coiled-coil alpha-helical rod
502557
289
425377
1017
1745, 2470,



protein 1




3195, 3920,








4645


135
coiled-coil alpha-helical rod
503934
290
425595
1018
1746, 2471,



protein 1




3196, 3921,








4646


136
coiled-coil alpha-helical rod
506831
291
425435
1019
1747, 2472,



protein 1




3197, 3922,








4647


137
coiled-coil alpha-helical rod
507226
292
424335
1020
1748, 2473,



protein 1




3198, 3923,








4648


138
coiled-coil alpha-helical rod
507751
293
420941
1021
1749, 2474,



protein 1




3199, 3924,








4649


139
coiled-coil alpha-helical rod
507829
294
420911
1022
1750, 2475,



protein 1




3200, 3925,








4650


140
coiled-coil alpha-helical rod
507892
295
424164
1023
1751, 2476,



protein 1




3201, 3926,








4651


141
coiled-coil alpha-helical rod
508683
296
421393
1024
1752, 2477,



protein 1




3202, 3927,








4652


142
coiled-coil alpha-helical rod
513222
297
425682
1025
1753, 2478,



protein 1




3203, 3928,








4653


143
coiled-coil alpha-helical rod
515274
298
422975
1026
1754, 2479,



protein 1




3204, 3929,








4654


144
collagen type VI alpha 3
392004
299
375861
1027
1755, 2480,








3205, 3930,








4655


145
collagen type VI alpha 3
472056
300
418285
1028
1756, 2481,








3206, 3931,








4656


146
collagen type XXV alpha 1
399127
301
382078
1029
1757, 2482,








3207, 3932,








4657


147
collagen, type I, alpha 1
225964
302
225964
1030
1758, 2483,








3208, 3933,








4658


148
collagen, type II, alpha 1
337299
303
338213
1031
1759, 2484,








3209, 3934,








4659


149
collagen, type II, alpha 1
380518
304
369889
1032
1760, 2485,








3210, 3935,








4660


150
collagen, type II, alpha 1
395281
305
378696
1033
1761, 2486,








3211, 3936,








4661


151
collagen, type IV, alpha 1
375815
306
364973
1034
1762, 2487,








3212, 3937,








4662, 5238,








5353


152
collagen, type IV, alpha 1
375820
307
364979
1035
1763, 2488,








3213, 3938,








4663, 5239,








5354


153
collagen, type IV, alpha 1
397198
308
380382
1036
1764, 2489,








3214, 3939,








4664, 5240,








5355


154
collagen, type IV, alpha 1
543140
309
443348
1037
1765, 2490,








3215, 3940,








4665, 5241,








5356


155
collagen, type IV, alpha 3
261415
310
261415
1038
1766, 2491,



(Goodpasture antigen)




3216, 3941,



binding protein




4666


156
collagen, type IV, alpha 3
357457
311
350046
1039
1767, 2492,



(Goodpasture antigen)




3217, 3942,



binding protein




4667


157
collagen, type IV, alpha 3
380494
312
369862
1040
1768, 2493,



(Goodpasture antigen)




3218, 3943,



binding protein




4668


158
collagen, type IV, alpha 3
405807
313
383996
1041
1769, 2494,



(Goodpasture antigen)




3219, 3944,



binding protein




4669


159
collagen, type IV, alpha 3
508809
314
424244
1042
1770, 2495,



(Goodpasture antigen)




3220, 3945,



binding protein




4670


160
collagen, type IV, alpha 4
329662
315
328553
1043
1771, 2496,








3221, 3946,








4671


161
collagen, type IV, alpha 4
396625
316
379866
1044
1772, 2497,








3222, 3947,








4672


162
collagen, type IV, alpha 5
328300
317
331902
1045
1773, 2498,








3223, 3948,








4673


163
collagen, type IV, alpha 5
361603
318
354505
1046
1774, 2499,








3224, 3949,








4674


164
collagen, type IV, alpha 5
508186
319
425614
1047
1775, 2500,








3225, 3950,








4675


165
collagen, type IV, alpha 6
334504
320
334733
1048
1776, 2501,








3226, 3951,








4676


166
collagen, type IV, alpha 6
372216
321
361290
1049
1777, 2502,








3227, 3952,








4677


167
collagen, type IV, alpha 6
394872
322
378340
1050
1778, 2503,








3228, 3953,








4678


168
collagen, type IV, alpha 6
418180
323
406002
1051
1779, 2504,








3229, 3954,








4679


169
collagen, type IV, alpha 6
538570
324
445236
1052
1780, 2505,








3230, 3955,








4680


170
collagen, type IV, alpha 6
545689
325
443707
1053
1781, 2506,








3231, 3956,








4681


171
collagen, type IX, alpha 1
320755
326
315252
1054
1782, 2507,








3232, 3957,








4682


172
collagen, type IX, alpha 1
357250
327
349790
1055
1783, 2508,








3233, 3958,








4683


173
collagen, type IX, alpha 1
370496
328
359527
1056
1784, 2509,








3234, 3959,








4684


174
collagen, type IX, alpha 1
370499
329
359530
1057
1785, 2510,








3235, 3960,








4685


175
collagen, type IX, alpha 2
372736
330
361821
1058
1786, 2511,








3236, 3961,








4686, 5242,








5357


176
collagen, type IX, alpha 2
372748
331
361834
1059
1787, 2512,








3237, 3962,








4687, 5243,








5358


177
collagen, type IX, alpha 3
343916
332
341640
1060
1788, 2513,








3238, 3963,








4688, 5244,








5359


178
collagen, type IX, alpha 3
452372
333
394280
1061
1789, 2514,








3239, 3964,








4689, 5245,








5360


179
collagen, type IX, alpha 3
537652
334
438537
1062
1790, 2515,








3240, 3965,








4690, 5246,








5361


180
collagen, type V, alpha 1
355306
335
347458
1063
1791, 2516,








3241, 3966,








4691, 5247,








5362


181
collagen, type V, alpha 1
371817
336
360882
1064
1792, 2517,








3242, 3967,








4692, 5248,








5363


182
collagen, type V, alpha 1
371820
337
360885
1065
1793, 2518,








3243, 3968,








4693, 5249,








5364


183
collagen, type V, alpha 3
264828
338
264828
1066
1794, 2519,








3244, 3969,








4694


184
collagen, type VI, alpha 3
295550
339
295550
1067
1795, 2520,








3245, 3970,








4695, 5250,








5365


185
collagen, type VI, alpha 3
346358
340
295546
1068
1796, 2521,








3246, 3971,








4696, 5251,








5366


186
collagen, type VI, alpha 3
347401
341
315609
1069
1797, 2522,








3247, 3972,








4697, 5252,








5367


187
collagen, type VI, alpha 3
353578
342
315873
1070
1798, 2523,








3248, 3973,








4698, 5253,








5368


188
collagen, type VI, alpha 3
392003
343
375860
1071
1799, 2524,








3249, 3974,








4699, 5254,








5369


189
collagen, type VI, alpha 3
409809
344
386844
1072
1800, 2525,








3250, 3975,








4700, 5255,








5370


190
collagen, type VI, alpha 5
265379
345
265379
1073
1801, 2526,








3251, 3976,








4701, 5256,








5371


191
collagen, type VI, alpha 5
312481
346
309762
1074
1802, 2527,








3252, 3977,








4702, 5257,








5372


192
collagen, type VI, alpha 5
373157
347
362250
1075
1803, 2528,








3253, 3978,








4703, 5258,








5373


193
collagen, type VI, alpha 5
432398
348
390895
1076
1804, 2529,








3254, 3979,








4704, 5259,








5374


194
collagen, type VI, alpha 5
512482
349
424968
1077
1805, 2530,








3255, 3980,








4705, 5260,








5375


195
collagen, type VI, alpha 6
358511
350
351310
1078
1806, 2531,








3256, 3981,








4706, 5261,








5376


196
collagen, type VI, alpha 6
453409
351
399236
1079
1807, 2532,








3257, 3982,








4707, 5262,








5377


197
collagen, type VIII, alpha 2
303143
352
305913
1080
1808, 2533,








3258, 3983,








4708


198
collagen, type VIII, alpha 2
373172
353
362267
1081
1809, 2534,








3259, 3984,








4709


199
collagen, type VIII, alpha 2
397799
354
380901
1082
1810, 2535,








3260, 3985,








4710


200
collagen, type VIII, alpha 2
481785
355
436433
1083
1811, 2536,








3261, 3986,








4711


201
collagen, type X, alpha 1
243222
356
243222
1084
1812, 2537,








3262, 3987,








4712


202
collagen, type X, alpha 1
327673
357
327368
1085
1813, 2538,








3263, 3988,








4713


203
collagen, type X, alpha 1
418500
358
392712
1086
1814, 2539,








3264, 3989,








4714


204
collagen, type X, alpha 1
452729
359
411285
1087
1815, 2540,








3265, 3990,








4715


205
collagen, type XI, alpha 2
341947
360
339915
1088
1816, 2541,








3266, 3991,








4716, 5263,








5378


206
collagen, type XI, alpha 2
357486
361
350079
1089
1817, 2542,








3267, 3992,








4717, 5264,








5379


207
collagen, type XI, alpha 2
361917
362
355123
1090
1818, 2543,








3268, 3993,








4718, 5265,








5380


208
collagen, type XI, alpha 2
374708
363
363840
1091
1819, 2544,








3269, 3994,








4719, 5266,








5381


209
collagen, type XI, alpha 2
374712
364
363844
1092
1820, 2545,








3270, 3995,








4720, 5267,








5382


210
collagen, type XI, alpha 2
374713
365
363845
1093
1821, 2546,








3271, 3996,








4721, 5268,








5383


211
collagen, type XI, alpha 2
374714
366
363846
1094
1822, 2547,








3272, 3997,








4722, 5269,








5384


212
collagen, type XI, alpha 2
383087
367
372565
1095
1823, 2548,








3273, 3998,








4723


213
collagen, type XI, alpha 2
383088
368
372566
1096
1824, 2549,








3274, 3999,








4724


214
collagen, type XI, alpha 2
383219
369
372706
1097
1825, 2550,








3275, 4000,








4725


215
collagen, type XI, alpha 2
395194
370
378620
1098
1826, 2551,








3276, 4001,








4726, 5270,








5385


216
collagen, type XI, alpha 2
395196
371
378622
1099
1827, 2552,








3277, 4002,








4727, 5271,








5386


217
collagen, type XI, alpha 2
395197
372
378623
1100
1828, 2553,








3278, 4003,








4728, 5272,








5387


218
collagen, type XI, alpha 2
420405
373
394196
1101
1829, 2554,








3279, 4004,








4729


219
collagen, type XI, alpha 2
425729
374
408123
1102
1830, 2555,








3280, 4005,








4730


220
collagen, type XI, alpha 2
433947
375
388108
1103
1831, 2556,








3281, 4006,








4731


221
collagen, type XI, alpha 2
434780
376
408523
1104
1832, 2557,








3282, 4007,








4732


222
collagen, type XI, alpha 2
435763
377
396587
1105
1833, 2558,








3283, 4008,








4733


223
collagen, type XI, alpha 2
438711
378
398256
1106
1834, 2559,








3284, 4009,








4734


224
collagen, type XI, alpha 2
439039
379
410284
1107
1835, 2560,








3285, 4010,








4735


225
collagen, type XI, alpha 2
443125
380
402987
1108
1836, 2561,








3286, 4011,








4736


226
collagen, type XI, alpha 2
443138
381
404513
1109
1837, 2562,








3287, 4012,








4737


227
collagen, type XI, alpha 2
447349
382
388309
1110
1838, 2563,








3288, 4013,








4738, 5273,








5388


228
collagen, type XI, alpha 2
447741
383
400813
1111
1839, 2564,








3289, 4014,








4739, 5274,








5389


229
collagen, type XI, alpha 2
447855
384
415296
1112
1840, 2565,








3290, 4015,








4740


230
collagen, type XI, alpha 2
448717
385
408627
1113
1841, 2566,








3291, 4016,








4741


231
collagen, type XI, alpha 2
451040
386
398170
1114
1842, 2567,








3292, 4017,








4742


232
collagen, type XI, alpha 2
452044
387
416619
1115
1843, 2568,








3293, 4018,








4743


233
collagen, type XI, alpha 2
452730
388
388775
1116
1844, 2569,








3294, 4019,








4744, 5275,








5390


234
collagen, type XI, alpha 2
452937
389
406347
1117
1845, 2570,








3295, 4020,








4745


235
collagen, type XI, alpha 2
457788
390
405520
1118
1846, 2571,








3296, 4021,








4746, 5276,








5391


236
collagen, type XI, alpha 2
546402
391
448494
1119
1847, 2572,








3297, 4022,








4747


237
collagen, type XI, alpha 2
546998
392
446968
1120
1848, 2573,








3298, 4023,








4748


238
collagen, type XI, alpha 2
547261
393
450150
1121
1849, 2574,








3299, 4024,








4749


239
collagen, type XI, alpha 2
547561
394
448817
1122
1850, 2575,








3300, 4025,








4750


240
collagen, type XI, alpha 2
547597
395
449605
1123
1851, 2576,








3301, 4026,








4751


241
collagen, type XI, alpha 2
547846
396
447511
1124
1852, 2577,








3302, 4027,








4752, 5277,








5392


242
collagen, type XI, alpha 2
547883
397
448628
1125
1853, 2578,








3303, 4028,








4753


243
collagen, type XI, alpha 2
547999
398
446903
1126
1854, 2579,








3304, 4029,








4754


244
collagen, type XI, alpha 2
548137
399
448090
1127
1855, 2580,








3305, 4030,








4755


245
collagen, type XI, alpha 2
548637
400
447422
1128
1856, 2581,








3306, 4031,








4756


246
collagen, type XI, alpha 2
548739
401
448990
1129
1857, 2582,








3307, 4032,








4757


247
collagen, type XI, alpha 2
549088
402
449993
1130
1858, 2583,








3308, 4033,








4758, 5278,








5393


248
collagen, type XI, alpha 2
549289
403
448643
1131
1859, 2584,








3309, 4034,








4759


249
collagen, type XI, alpha 2
549290
404
446543
1132
1860, 2585,








3310, 4035,








4760


250
collagen, type XI, alpha 2
549381
405
448464
1133
1861, 2586,








3311, 4036,








4761


251
collagen, type XI, alpha 2
549491
406
450084
1134
1862, 2587,








3312, 4037,








4762


252
collagen, type XI, alpha 2
549811
407
449275
1135
1863, 2588,








3313, 4038,








4763


253
collagen, type XI, alpha 2
549836
408
448701
1136
1864, 2589,








3314, 4039,








4764


254
collagen, type XI, alpha 2
549885
409
448931
1137
1865, 2590,








3315, 4040,








4765, 5279,








5394


255
collagen, type XI, alpha 2
550101
410
446840
1138
1866, 2591,








3316, 4041,








4766


256
collagen, type XI, alpha 2
550517
411
448206
1139
1867, 2592,








3317, 4042,








4767


257
collagen, type XI, alpha 2
550561
412
449393
1140
1868, 2593,








3318, 4043,








4768


258
collagen, type XI, alpha 2
550955
413
447413
1141
1869, 2594,








3319, 4044,








4769, 5280,








5395


259
collagen, type XI, alpha 2
550998
414
450046
1142
1870, 2595,








3320, 4045,








4770


260
collagen, type XI, alpha 2
551234
415
446501
1143
1871, 2596,








3321, 4046,








4771


261
collagen, type XI, alpha 2
551262
416
450208
1144
1872, 2597,








3322, 4047,








4772


262
collagen, type XI, alpha 2
551413
417
448715
1145
1873, 2598,








3323, 4048,








4773


263
collagen, type XI, alpha 2
551536
418
447371
1146
1874, 2599,








3324, 4049,








4774


264
collagen, type XI, alpha 2
551542
419
447864
1147
1875, 2600,








3325, 4050,








4775


265
collagen, type XI, alpha 2
551559
420
449887
1148
1876, 2601,








3326, 4051,








4776


266
collagen, type XI, alpha 2
551572
421
448571
1149
1877, 2602,








3327, 4052,








4777


267
collagen, type XI, alpha 2
551637
422
447869
1150
1878, 2603,








3328, 4053,








4778, 5281,








5396


268
collagen, type XI, alpha 2
551758
423
447062
1151
1879, 2604,








3329, 4054,








4779


269
collagen, type XI, alpha 2
551785
424
448880
1152
1880, 2605,








3330, 4055,








4780


270
collagen, type XI, alpha 2
551786
425
446509
1153
1881, 2606,








3331, 4056,








4781


271
collagen, type XI, alpha 2
552134
426
449320
1154
1882, 2607,








3332, 4057,








4782


272
collagen, type XI, alpha 2
552473
427
446870
1155
1883, 2608,








3333, 4058,








4783, 5282,








5397


273
collagen, type XI, alpha 2
552556
428
448922
1156
1884, 2609,








3334, 4059,








4784


274
collagen, type XI, alpha 2
552765
429
448124
1157
1885, 2610,








3335, 4060,








4785, 5283,








5398


275
collagen, type XI, alpha 2
552796
430
448204
1158
1886, 2611,








3336, 4061,








4786


276
collagen, type XI, alpha 2
552971
431
450055
1159
1887, 2612,








3337, 4062,








4787


277
collagen, type XI, alpha 2
553210
432
447529
1160
1888, 2613,








3338, 4063,








4788, 5284,








5399


278
collagen, type XI, alpha 2
553240
433
448622
1161
1889, 2614,








3339, 4064,








4789


279
collagen, type XII, alpha 1
322507
434
325146
1162
1890, 2615,








3340, 4065,








4790, 5285,








5400


280
collagen, type XII, alpha 1
345356
435
305147
1163
1891, 2616,








3341, 4066,








4791, 5286,








5401


281
collagen, type XII, alpha 1
432784
436
406049
1164
1892, 2617,








3342, 4067,








4792, 5287,








5402


282
collagen, type XIII, alpha 1
354547
437
346553
1165
1893, 2618,








3343, 4068,








4793


283
collagen, type XIII, alpha 1
356340
438
348695
1166
1894, 2619,








3344, 4069,








4794


284
collagen, type XIII, alpha 1
357811
439
350463
1167
1895, 2620,








3345, 4070,








4795


285
collagen, type XIII, alpha 1
398964
440
381936
1168
1896, 2621,








3346, 4071,








4796


286
collagen, type XIII, alpha 1
398966
441
381938
1169
1897, 2622,








3347, 4072,








4797


287
collagen, type XIII, alpha 1
398968
442
381940
1170
1898, 2623,








3348, 4073,








4798


288
collagen, type XIII, alpha 1
398969
443
381941
1171
1899, 2624,








3349, 4074,








4799


289
collagen, type XIII, alpha 1
398971
444
381943
1172
1900, 2625,








3350, 4075,








4800


290
collagen, type XIII, alpha 1
398972
445
381944
1173
1901, 2626,








3351, 4076,








4801


291
collagen, type XIII, alpha 1
398973
446
381945
1174
1902, 2627,








3352, 4077,








4802


292
collagen, type XIII, alpha 1
398974
447
381946
1175
1903, 2628,








3353, 4078,








4803


293
collagen, type XIII, alpha 1
398978
448
381949
1176
1904, 2629,








3354, 4079,








4804


294
collagen, type XIII, alpha 1
517713
449
430061
1177
1905, 2630,








3355, 4080,








4805


295
collagen, type XIII, alpha 1
520133
450
430173
1178
1906, 2631,








3356, 4081,








4806


296
collagen, type XIII, alpha 1
520267
451
428057
1179
1907, 2632,








3357, 4082,








4807


297
collagen, type XIV, alpha 1
247781
452
247781
1180
1908, 2633,








3358, 4083,








4808


298
collagen, type XIV, alpha 1
297848
453
297848
1181
1909, 2634,








3359, 4084,








4809


299
collagen, type XIV, alpha 1
309791
454
311809
1182
1910, 2635,








3360, 4085,








4810


300
collagen, type XIV, alpha 1
537875
455
443974
1183
1911, 2636,








3361, 4086,








4811


301
collagen, type XIX, alpha 1
322773
456
316030
1184
1912, 2637,








3362, 4087,








4812


302
collagen, type XIX, alpha 1
393333
457
377006
1185
1913, 2638,








3363, 4088,








4813


303
collagen, type XV, alpha 1
375001
458
364140
1186
1914, 2639,








3364, 4089,








4814


304
collagen, type XV, alpha 1
536083
459
445833
1187
1915, 2640,








3365, 4090,








4815


305
collagen, type XVI, alpha 1
271069
460
271069
1188
1916, 2641,








3366, 4091,








4816


306
collagen, type XVI, alpha 1
373672
461
362776
1189
1917, 2642,








3367, 4092,








4817


307
collagen, type XVII, alpha 1
353479
462
340937
1190
1918, 2643,








3368, 4093,








4818, 5288,








5403


308
collagen, type XVII, alpha 1
393211
463
376905
1191
1919, 2644,








3369, 4094,








4819, 5289,








5404


309
collagen, type XVII, alpha 1
433822
464
388832
1192
1920, 2645,








3370, 4095,








4820, 5290,








5405


310
collagen, type XVII, alpha 1
541872
465
438701
1193
1921, 2646,








3371, 4096,








4821, 5291,








5406


311
collagen, type XX, alpha 1
326996
466
323077
1194
1922, 2647,








3372, 4097,








4822, 5292,








5407


312
collagen, type XX, alpha 1
358894
467
351767
1195
1923, 2648,








3373, 4098,








4823, 5293,








5408


313
collagen, type XX, alpha 1
422202
468
414753
1196
1924, 2649,








3374, 4099,








4824, 5294,








5409


314
collagen, type XX, alpha 1
435874
469
408690
1197
1925, 2650,








3375, 4100,








4825, 5295,








5410


315
collagen, type XXI, alpha 1
244728
470
244728
1198
1926, 2651,








3376, 4101,








4826


316
collagen, type XXI, alpha 1
370808
471
359844
1199
1927, 2652,








3377, 4102,








4827


317
collagen, type XXI, alpha 1
370811
472
359847
1200
1928, 2653,








3378, 4103,








4828


318
collagen, type XXI, alpha 1
370817
473
359853
1201
1929, 2654,








3379, 4104,








4829


319
collagen, type XXI, alpha 1
370819
474
359855
1202
1930, 2655,








3380, 4105,








4830


320
collagen, type XXI, alpha 1
535941
475
444384
1203
1931, 2656,








3381, 4106,








4831


321
collagen, type XXII, alpha 1
303045
476
303153
1204
1932, 2657,








3382, 4107,








4832


322
collagen, type XXII, alpha 1
435777
477
387655
1205
1933, 2658,








3383, 4108,








4833


323
collagen, type XXII, alpha 1
545577
478
443522
1206
1934, 2659,








3384, 4109,








4834


324
collagen, type XXIV, alpha 1
370571
479
359603
1207
1935, 2660,








3385, 4110,








4835


325
collagen, type XXIV, alpha 1
436319
480
392531
1208
1936, 2661,








3386, 4111,








4836


326
collagen, type XXIV, alpha 1
496682
481
434740
1209
1937, 2662,








3387, 4112,








4837


327
collagen, type XXV, alpha 1
333642
482
329626
1210
1938, 2663,








3388, 4113,








4838


328
collagen, type XXV, alpha 1
399126
483
382077
1211
1939, 2664,








3389, 4114,








4839


329
collagen, type XXV, alpha 1
399132
484
382083
1212
1940, 2665,








3390, 4115,








4840


330
collagen, type XXV, alpha 1
401873
485
385796
1213
1941, 2666,








3391, 4116,








4841


331
collagen, type XXV, alpha 1
443653
486
388334
1214
1942, 2667,








3392, 4117,








4842


332
collagen, type XXV, alpha 1
505591
487
422266
1215
1943, 2668,








3393, 4118,








4843


333
collagen, type XXV, alpha 1
512961
488
426841
1216
1944, 2669,








3394, 4119,








4844


334
collagen, type XXVII, alpha 1
356083
489
348385
1217
1945, 2670,








3395, 4120,








4845


335
collagen, type XXVII, alpha 1
357257
490
349802
1218
1946, 2671,








3396, 4121,








4846


336
collagen, type XXVII, alpha 1
374106
491
363219
1219
1947, 2672,








3397, 4122,








4847


337
collagen, type XXVIII, alpha
399419
492
382347
1220
1948, 2673,



1




3398, 4123,








4848


338
collagen, type XXVIII, alpha
399429
493
382356
1221
1949, 2674,



1




3399, 4124,








4849


339
collagen, type XXVIII, alpha
448652
494
399021
1222
1950, 2675,



1




3400, 4125,








4850


340
collagen, type XXVIII, alpha
453441
495
391380
1223
1951, 2676,



1




3401, 4126,








4851


341
corneodesmosin
259726
496
259726
1224
1952, 2677,








3402, 4127,








4852


342
corneodesmosin
376288
497
365465
1225
1953, 2678,








3403, 4128,








4853


343
corneodesmosin
383531
498
373023
1226
1954, 2679,








3404, 4129,








4854


344
corneodesmosin
418599
499
392863
1227
1955, 2680,








3405, 4130,








4855


345
corneodesmosin
445893
500
388386
1228
1956, 2681,








3406, 4131,








4856


346
corneodesmosin
457875
501
399604
1229
1957, 2682,








3407, 4132,








4857


347
dopachrome tautomerase
377021
502
366220
1230
1958, 2683,



(dopachrome delta-isomerase,




3408, 4133,



tyrosine-related protein 2)




4858


348
dopachrome tautomerase
377028
503
366227
1231
1959, 2684,



(dopachrome delta-isomerase,




3409, 4134,



tyrosine-related protein 2)




4859


349
dopachrome tautomerase
446125
504
392762
1232
1960, 2685,



(dopachrome delta-isomerase,




3410, 4135,



tyrosine-related protein 2)




4860


350
ectodysplasin A receptor
258443
505
258443
1233
1961, 2686,








3411, 4136,








4861


351
ectodysplasin A receptor
376651
506
365839
1234
1962, 2687,








3412, 4137,








4862


352
ectodysplasin A receptor
409271
507
386371
1235
1963, 2688,








3413, 4138,








4863


353
ectodysplasin A2 receptor
253392
508
253392
1236
1964, 2689,








3414, 4139,








4864


354
ectodysplasin A2 receptor
374719
509
363851
1237
1965, 2690,








3415, 4140,








4865


355
ectodysplasin A2 receptor
396050
510
379365
1238
1966, 2691,








3416, 4141,








4866


356
ectodysplasin A2 receptor
450752
511
402929
1239
1967, 2692,








3417, 4142,








4867


357
ectodysplasin A2 receptor
451436
512
415242
1240
1968, 2693,








3418, 4143,








4868


358
ectodysplasin A2 receptor
456230
513
393935
1241
1969, 2694,








3419, 4144,








4869


359
EDAR-associated death
334232
514
335076
1242
1970, 2695,



domain




3420, 4145,








4870


360
EDAR-associated death
359362
515
352320
1243
1971, 2696,



domain




3421, 4146,








4871


361
EDAR-associated death
439430
516
405815
1244
1972, 2697,



domain




3422, 4147,








4872


362
elastin
252034
517
252034
1245
1973, 2698,








3423, 4148,








4873, 5296,








5411


363
elastin
320399
518
313565
1246
1974, 2699,








3424, 4149,








4874, 5297,








5412


364
elastin
357036
519
349540
1247
1975, 2700,








3425, 4150,








4875, 5298,








5413


365
elastin
358929
520
351807
1248
1976, 2701,








3426, 4151,








4876, 5299,








5414


366
elastin
380575
521
369949
1249
1977, 2702,








3427, 4152,








4877, 5300,








5415


367
elastin
380576
522
369950
1250
1978, 2703,








3428, 4153,








4878, 5301,








5416


368
elastin
417091
523
411092
1251
1979, 2704,








3429, 4154,








4879, 5302,








5417


369
elastin
428787
524
399499
1252
1980, 2705,








3430, 4155,








4880, 5303,








5418


370
elastin
429192
525
391129
1253
1981, 2706,








3431, 4156,








4881, 5304,








5419


371
elastin
438880
526
389206
1254
1982, 2707,








3432, 4157,








4882, 5305,








5420


372
elastin
438906
527
406949
1255
1983, 2708,








3433, 4158,








4883, 5306,








5421


373
elastin
442310
528
403961
1256
1984, 2709,








3434, 4159,








4884, 5307,








5422


374
elastin
442462
529
403940
1257
1985, 2710,








3435, 4160,








4885, 5308,








5423


375
elastin
445912
530
389857
1258
1986, 2711,








3436, 4161,








4886, 5309,








5424


376
elastin
458204
531
403162
1259
1987, 2712,








3437, 4162,








4887, 5310,








5425


377
fibroblast growth factor 11
293829
532
293829
1260
1988, 2713,








3438, 4163,








4888


378
fibroblast growth factor 12
264730
533
264730
1261
1989, 2714,








3439, 4164,








4889


379
fibroblast growth factor 12
392454
534
376248
1262
1990, 2715,








3440, 4165,








4890


380
fibroblast growth factor 12
418610
535
395517
1263
1991, 2716,








3441, 4166,








4891


381
fibroblast growth factor 12
445105
536
393686
1264
1992, 2717,








3442, 4167,








4892


382
fibroblast growth factor 12
450716
537
397635
1265
1993, 2718,








3443, 4168,








4893


383
fibroblast growth factor 12
454309
538
413496
1266
1994, 2719,








3444, 4169,








4894


384
fibroblast growth factor 13
305414
539
303391
1267
1995, 2720,








3445, 4170,








4895


385
fibroblast growth factor 13
315930
540
322390
1268
1996, 2721,








3446, 4171,








4896


386
fibroblast growth factor 13
370603
541
359635
1269
1997, 2722,








3447, 4172,








4897


387
fibroblast growth factor 13
421460
542
388688
1270
1998, 2723,








3448, 4173,








4898


388
fibroblast growth factor 13
436198
543
396198
1271
1999, 2724,








3449, 4174,








4899


389
fibroblast growth factor 13
441825
544
409276
1272
2000, 2725,








3450, 4175,








4900


390
fibroblast growth factor 13
448673
545
411999
1273
2001, 2726,








3451, 4176,








4901


391
fibroblast growth factor 13
455663
546
406916
1274
2002, 2727,








3452, 4177,








4902


392
fibroblast growth factor 13
541469
547
437903
1275
2003, 2728,








3453, 4178,








4903


393
fibroblast growth factor 14
376131
548
365301
1276
2004, 2729,








3454, 4179,








4904


394
fibroblast growth factor 14
376143
549
365313
1277
2005, 2730,








3455, 4180,








4905


395
fibroblast growth factor 16
439435
550
399324
1278
2006, 2731,








3456, 4181,








4906


396
fibroblast growth factor 17
359441
551
352414
1279
2007, 2732,








3457, 4182,








4907, 5311,








5426


397
fibroblast growth factor 17
518533
552
431041
1280
2008, 2733,








3458, 4183,








4908, 5312,








5427


398
fibroblast growth factor 18
274625
553
274625
1281
2009, 2734,








3459, 4184,








4909, 5467-








5553


399
fibroblast growth factor 19
294312
554
294312
1282
2010, 2735,








3460, 4185,








4910


400
fibroblast growth factor 20
180166
555
180166
1283
2011, 2736,








3461, 4186,








4911


401
fibroblast growth factor 20
381981
556
371411
1284
2012, 2737,








3462, 4187,








4912


402
fibroblast growth factor 22
166133
557
166133
1285
2013, 2738,








3463, 4188,








4913, 5313,








5428


403
fibroblast growth factor 22
215530
558
215530
1286
2014, 2739,








3464, 4189,








4914


404
fibroblast growth factor 23
237837
559
237837
1287
2015, 2740,








3465, 4190,








4915, 5554-








5640


405
fibroblast growth factor 3
334134
560
334122
1288
2016, 2741,








3466, 4191,








4916


406
fibroblast growth factor 4
168712
561
168712
1289
2017, 2742,








3467, 4192,








4917, 5314,








5429


407
fibroblast growth factor 5
312465
562
311697
1290
2018, 2743,








3468, 4193,








4918


408
fibroblast growth factor 5
456523
563
398353
1291
2019, 2744,








3469, 4194,








4919


409
fibroblast growth factor 6
228837
564
228837
1292
2020, 2745,








3470, 4195,








4920


410
fibroblast growth factor 8
320185
565
321797
1293
2021, 2746,



(androgen-induced)




3471, 4196,








4921


411
fibroblast growth factor 8
344255
566
340039
1294
2022, 2747,



(androgen-induced)




3472, 4197,








4922


412
fibroblast growth factor 8
346714
567
344306
1295
2023, 2748,



(androgen-induced)




3473, 4198,








4923


413
fibroblast growth factor 8
347978
568
321945
1296
2024, 2749,



(androgen-induced)




3474, 4199,








4924


414
fibroblast growth factor 9
382353
569
371790
1297
2025, 2750,



(glia-activating factor)




3475, 4200,








4925


415
filaggrin family member 2
388718
570
373370
1298
2026, 2751,








3476, 4201,








4926


416
heparin-binding EGF-like
230990
571
230990
1299
2027, 2752,



growth factor




3477, 4202,








4927


417
heparin-binding EGF-like
507104
572
425696
1300
2028, 2753,



growth factor




3478, 4203,








4928


418
insulin-like growth factor
233809
573
233809
1301
2029, 2754,



binding protein 2, 36 kDa




3479, 4204,








4929


419
insulin-like growth factor
301464
574
301464
1302
2030, 2755,



binding protein 6




3480, 4205,








4930, 5315,








5430


420
insulin-like growth factor
548176
575
449344
1303
2031, 2756,



binding protein 6




3481, 4206,








4931, 5316,








5431


421
insulin-like growth factor
548547
576
448953
1304
2032, 2757,



binding protein 6




3482, 4207,








4932, 5317,








5432


422
insulin-like growth factor
377694
577
366923
1305
2033, 2758,



binding protein-like 1




3483, 4208,








4933, 5318,








5433


423
interleukin 1 family, member
337569
578
338418
1306
2034, 2759,



10 (theta)




3484, 4209,








4934, 5319,








5434


424
interleukin 1 family, member
341010
579
341794
1307
2035, 2760,



10 (theta)




3485, 4210,








4935, 5320,








5435


425
interleukin 1 family, member
393197
580
376893
1308
2036, 2761,



10 (theta)




3486, 4211,








4936, 5321,








5436


426
interleukin 1, alpha
263339
581
263339
1309
2037, 2762,








3487, 4212,








4937


427
interleukin 11
264563
582
264563
1310
2038, 2763,








3488, 4213,








4938, 5322,








5437


428
interleukin 12B (natural killer
231228
583
231228
1311
2039, 2764,



cell stimulatory factor 2,




3489, 4214,



cytotoxic lymphocyte




4939



maturation factor 2, p40)


429
interleukin 13
304506
584
304915
1312
2040, 2765,








3490, 4215,








4940


430
interleukin 16
302987
585
302935
1313
2041, 2766,








3491, 4216,








4941


431
interleukin 16
329842
586
329317
1314
2042, 2767,








3492, 4217,








4942


432
interleukin 16
355368
587
347528
1315
2043, 2768,








3493, 4218,








4943


433
interleukin 16
394652
588
378147
1316
2044, 2769,








3494, 4219,








4944


434
interleukin 16
394653
589
378148
1317
2045, 2770,








3495, 4220,








4945


435
interleukin 16
394655
590
378150
1318
2046, 2771,








3496, 4221,








4946


436
interleukin 16
394656
591
378151
1319
2047, 2772,








3497, 4222,








4947


437
interleukin 16
394660
592
378155
1320
2048, 2773,








3498, 4223,








4948


438
interleukin 16
559383
593
453250
1321
2049, 2774,








3499, 4224,








4949


439
interleukin 17A
340057
594
344192
1322
2050, 2775,








3500, 4225,








4950


440
interleukin 17B
261796
595
261796
1323
2051, 2776,








3501, 4226,








4951


441
interleukin 17C
244241
596
244241
1324
2052, 2777,








3502, 4227,








4952


442
interleukin 17D
304920
597
302924
1325
2053, 2778,








3503, 4228,








4953


443
interleukin 17F
336123
598
337432
1326
2054, 2779,








3504, 4229,








4954


444
interleukin 20
367096
599
356063
1327
2055, 2780,








3505, 4230,








4955


445
interleukin 20
367098
600
356065
1328
2056, 2781,








3506, 4231,








4956


446
interleukin 20
391930
601
375796
1329
2057, 2782,








3507, 4232,








4957


447
interleukin 22
328087
602
329384
1330
2058, 2783,








3508, 4233,








4958


448
interleukin 22
538666
603
442424
1331
2059, 2784,








3509, 4234,








4959


449
interleukin 23 receptor
347310
604
321345
1332
2060, 2785,








3510, 4235,








4960


450
interleukin 23 receptor
371002
605
360041
1333
2061, 2786,








3511, 4236,








4961


451
interleukin 23 receptor
395227
606
378652
1334
2062, 2787,








3512, 4237,








4962


452
interleukin 23 receptor
416525
607
388654
1335
2063, 2788,








3513, 4238,








4963


453
interleukin 23 receptor
425614
608
387640
1336
2064, 2789,








3514, 4239,








4964


454
interleukin 23 receptor
431791
609
395010
1337
2065, 2790,








3515, 4240,








4965


455
interleukin 23 receptor
441823
610
399293
1338
2066, 2791,








3516, 4241,








4966


456
interleukin 23 receptor
540775
611
444746
1339
2067, 2792,








3517, 4242,








4967


457
interleukin 23 receptor
540911
612
445271
1340
2068, 2793,








3518, 4243,








4968


458
interleukin 23 receptor
543799
613
443793
1341
2069, 2794,








3519, 4244,








4969


459
interleukin 23, alpha subunit
228534
614
228534
1342
2070, 2795,



p19




3520, 4245,








4970


460
interleukin 24
294984
615
294984
1343
2071, 2796,








3521, 4246,








4971, 5323,








5438


461
interleukin 24
367093
616
356060
1344
2072, 2797,








3522, 4247,








4972, 5324,








5439


462
interleukin 24
391929
617
375795
1345
2073, 2798,








3523, 4248,








4973, 5325,








5440


463
interleukin 25
329715
618
328111
1346
2074, 2799,








3524, 4249,








4974


464
interleukin 25
397242
619
380417
1347
2075, 2800,








3525, 4250,








4975


465
interleukin 26
229134
620
229134
1348
2076, 2801,








3526, 4251,








4976


466
interleukin 27
356897
621
349365
1349
2077, 2802,








3527, 4252,








4977


467
interleukin 28A (interferon,
331982
622
333639
1350
2078, 2803,



lambda 2)




3528, 4253,








4978


468
interleukin 28B (interferon,
413851
623
409000
1351
2079, 2804,



lambda 3)




3529, 4254,








4979


469
interleukin 29 (interferon,
333625
624
329991
1352
2080, 2805,



lambda 1)




3530, 4255,








4980


470
interleukin 31
377035
625
366234
1353
2081, 2806,








3531, 4256,








4981, 5326,








5441


471
interleukin 32
8180
626
8180
1354
2082, 2807,








3532, 4257,








4982, 5327,








5442


472
interleukin 32
325568
627
324742
1355
2083, 2808,








3533, 4258,








4983


473
interleukin 32
382213
628
371648
1356
2084, 2809,








3534, 4259,








4984


474
interleukin 32
396887
629
380096
1357
2085, 2810,








3535, 4260,








4985


475
interleukin 32
396890
630
380099
1358
2086, 2811,








3536, 4261,








4986


476
interleukin 32
440815
631
405063
1359
2087, 2812,








3537, 4262,








4987


477
interleukin 32
444393
632
411958
1360
2088, 2813,








3538, 4263,








4988


478
interleukin 32
525643
633
432218
1361
2089, 2814,








3539, 4264,








4989


479
interleukin 32
526464
634
450364
1362
2090, 2815,








3540, 4265,








4990


480
interleukin 32
528163
635
432850
1363
2091, 2816,








3541, 4266,








4991


481
interleukin 32
529550
636
437020
1364
2092, 2817,








3542, 4267,








4992


482
interleukin 32
530538
637
436929
1365
2093, 2818,








3543, 4268,








4993


483
interleukin 32
530890
638
433747
1366
2094, 2819,








3544, 4269,








4994


484
interleukin 32
531965
639
433177
1367
2095, 2820,








3545, 4270,








4995


485
interleukin 32
533097
640
432917
1368
2096, 2821,








3546, 4271,








4996


486
interleukin 32
534507
641
431775
1369
2097, 2822,








3547, 4272,








4997


487
interleukin 32
548476
642
449483
1370
2098, 2823,








3548, 4273,








4998


488
interleukin 32
548652
643
446624
1371
2099, 2824,








3549, 4274,








4999


489
interleukin 32
548807
644
448354
1372
2100, 2825,








3550, 4275,








5000


490
interleukin 32
549213
645
447812
1373
2101, 2826,








3551, 4276,








5001


491
interleukin 32
551122
646
447496
1374
2102, 2827,








3552, 4277,








5002


492
interleukin 32
552356
647
446978
1375
2103, 2828,








3553, 4278,








5003


493
interleukin 32
552664
648
448683
1376
2104, 2829,








3554, 4279,








5004


494
interleukin 33
381434
649
370842
1377
2105, 2830,








3555, 4280,








5005


495
interleukin 33
417746
650
394039
1378
2106, 2831,








3556, 4281,








5006


496
interleukin 33
456383
651
414238
1379
2107, 2832,








3557, 4282,








5007


497
interleukin 34
288098
652
288098
1380
2108, 2833,








3558, 4283,








5008


498
interleukin 34
429149
653
397863
1381
2109, 2834,








3559, 4284,








5009


499
interleukin 36, alpha
259211
654
259211
1382
2110, 2835,








3560, 4285,








5010


500
interleukin 36, alpha
397653
655
380773
1383
2111, 2836,








3561, 4286,








5011


501
interleukin 36, beta
259213
656
259213
1384
2112, 2837,








3562, 4287,








5012


502
interleukin 36, beta
327407
657
328420
1385
2113, 2838,








3563, 4288,








5013


503
interleukin 36, gamma
259205
658
259205
1386
2114, 2839,








3564, 4289,








5014


504
interleukin 36, gamma
447128
659
411639
1387
2115, 2840,








3565, 4290,








5015


505
interleukin 37
263326
660
263326
1388
2116, 2841,








3566, 4291,








5016


506
interleukin 37
311328
661
309883
1389
2117, 2842,








3567, 4292,








5017


507
interleukin 37
349806
662
263328
1390
2118, 2843,








3568, 4293,








5018


508
interleukin 37
352179
663
263327
1391
2119, 2844,








3569, 4294,








5019


509
interleukin 37
353225
664
309208
1392
2120, 2845,








3570, 4295,








5020


510
interleukin 4 induced 1
341114
665
342557
1393
2121, 2846,








3571, 4296,








5021


511
interleukin 4 induced 1
391826
666
375702
1394
2122, 2847,








3572, 4297,








5022


512
interleukin 4 induced 1
595948
667
472474
1395
2123, 2848,








3573, 4298,








5023


513
interleukin 5 (colony-
231454
668
231454
1396
2124, 2849,



stimulating factor, eosinophil)




3574, 4299,








5024


514
interleukin 9
274520
669
274520
1397
2125, 2850,








3575, 4300,








5025


515
interleukin enhancer binding
589998
670
465219
1398
2126, 2851,



factor 3 90 kDa




3576, 4301,








5026


516
interleukin enhancer binding
250241
671
250241
1399
2127, 2852,



factor 3, 90 kDa




3577, 4302,








5027


517
interleukin enhancer binding
318511
672
315205
1400
2128, 2853,



factor 3, 90 kDa




3578, 4303,








5028


518
interleukin enhancer binding
336059
673
337305
1401
2129, 2854,



factor 3, 90 kDa




3579, 4304,








5029


519
interleukin enhancer binding
407004
674
384660
1402
2130, 2855,



factor 3, 90 kDa




3580, 4305,








5030


520
interleukin enhancer binding
420083
675
405436
1403
2131, 2856,



factor 3, 90 kDa




3581, 4306,








5031


521
interleukin enhancer binding
449870
676
404121
1404
2132, 2857,



factor 3, 90 kDa




3582, 4307,








5032


522
kallikrein-related peptidase 5
336334
677
337733
1405
2133, 2858,








3583, 4308,








5033


523
kallikrein-related peptidase 5
391809
678
375685
1406
2134, 2859,








3584, 4309,








5034


524
kallikrein-related peptidase 5
593428
679
471966
1407
2135, 2860,








3585, 4310,








5035


525
kinesin family member 3A
378735
680
368009
1408
2136, 2861,








3586, 4311,








5036


526
kinesin family member 3A
378746
681
368020
1409
2137, 2862,








3587, 4312,








5037


527
kinesin family member 3A
450914
682
407601
1410
2138, 2863,








3588, 4313,








5038


528
kinesin family member 3A
541316
683
445791
1411
2139, 2864,








3589, 4314,








5039


529
lysophosphatidic acid receptor
345941
684
344353
1412
2140, 2865,



6




3590, 4315,








5040


530
lysophosphatidic acid receptor
378434
685
367691
1413
2141, 2866,



6




3591, 4316,








5041


531
macrophage stimulating 1
383728
686
373234
1414
2142, 2867,



(hepatocyte growth factor-




3592, 4317,



like)




5042


532
macrophage stimulating 1
449682
687
414287
1415
2143, 2868,



(hepatocyte growth factor-




3593, 4318,



like)




5043


533
macrophage stimulating 1
545762
688
437535
1416
2144, 2869,



(hepatocyte growth factor-




3594, 4319,



like)




5044


534
macrophage stimulating 1
334998
689
439273
1417
2145, 2870,



(hepatocyte growth factor-




3595, 4320,



like) pseudogene 9




5045, 5328,








5443


535
macrophage stimulating 1
389184
690
445850
1418
2146, 2871,



(hepatocyte growth factor-




3596, 4321,



like) pseudogene 9




5046


536
macrophage stimulating 1
442552
691
438833
1419
2147, 2872,



(hepatocyte growth factor-




3597, 4322,



like) pseudogene 9




5047, 5329,








5444


537
major histocompatibility
376228
692
365402
1420
2148, 2873,



complex, class I, C




3598, 4323,








5048


538
major histocompatibility
376237
693
365412
1421
2149, 2874,



complex, class I, C




3599, 4324,








5049


539
major histocompatibility
383323
694
372813
1422
2150, 2875,



complex, class I, C




3600, 4325,








5050


540
major histocompatibility
383329
695
372819
1423
2151, 2876,



complex, class I, C




3601, 4326,








5051


541
major histocompatibility
383483
696
372975
1424
2152, 2877,



complex, class I, C




3602, 4327,








5052


542
major histocompatibility
383487
697
372979
1425
2153, 2878,



complex, class I, C




3603, 4328,








5053


543
major histocompatibility
396254
698
379553
1426
2154, 2879,



complex, class I, C




3604, 4329,








5054


544
major histocompatibility
400341
699
383195
1427
2155, 2880,



complex, class I, C




3605, 4330,








5055


545
major histocompatibility
400394
700
383244
1428
2156, 2881,



complex, class I, C




3606, 4331,








5056


546
major histocompatibility
400395
701
383245
1429
2157, 2882,



complex, class I, C




3607, 4332,








5057


547
major histocompatibility
414249
702
390282
1430
2158, 2883,



complex, class I, C




3608, 4333,








5058


548
major histocompatibility
415537
703
400410
1431
2159, 2884,



complex, class I, C




3609, 4334,








5059


549
major histocompatibility
419135
704
407431
1432
2160, 2885,



complex, class I, C




3610, 4335,








5060


550
major histocompatibility
419590
705
390860
1433
2161, 2886,



complex, class I, C




3611, 4336,








5061


551
major histocompatibility
420206
706
410214
1434
2162, 2887,



complex, class I, C




3612, 4337,








5062


552
major histocompatibility
422726
707
389248
1435
2163, 2888,



complex, class I, C




3613, 4338,








5063


553
major histocompatibility
422921
708
397867
1436
2164, 2889,



complex, class I, C




3614, 4339,








5064


554
major histocompatibility
423509
709
403624
1437
2165, 2890,



complex, class I, C




3615, 4340,








5065


555
major histocompatibility
424832
710
414063
1438
2166, 2891,



complex, class I, C




3616, 4341,








5066


556
major histocompatibility
429840
711
413189
1439
2167, 2892,



complex, class I, C




3617, 4342,








5067


557
major histocompatibility
430940
712
413992
1440
2168, 2893,



complex, class I, C




3618, 4343,








5068


558
major histocompatibility
433153
713
415025
1441
2169, 2894,



complex, class I, C




3619, 4344,








5069


559
major histocompatibility
434884
714
398169
1442
2170, 2895,



complex, class I, C




3620, 4345,








5070


560
major histocompatibility
437368
715
407494
1443
2171, 2896,



complex, class I, C




3621, 4346,








5071


561
major histocompatibility
438171
716
393374
1444
2172, 2897,



complex, class I, C




3622, 4347,








5072


562
major histocompatibility
445075
717
412426
1445
2173, 2898,



complex, class I, C




3623, 4348,








5073


563
major histocompatibility
448362
718
410661
1446
2174, 2899,



complex, class I, C




3624, 4349,








5074


564
major histocompatibility
449884
719
411867
1447
2175, 2900,



complex, class I, C




3625, 4350,








5075


565
major histocompatibility
453809
720
388708
1448
2176, 2901,



complex, class I, C




3626, 4351,








5076


566
major histocompatibility
454033
721
391250
1449
2177, 2902,



complex, class I, C




3627, 4352,








5077


567
major histocompatibility
456487
722
393458
1450
2178, 2903,



complex, class I, C




3628, 4353,








5078


568
major histocompatibility
457806
723
407051
1451
2179, 2904,



complex, class I, C




3629, 4354,








5079


569
major histocompatibility
457903
724
390851
1452
2180, 2905,



complex, class I, C




3630, 4355,








5080


570
major histocompatibility
458192
725
404526
1453
2181, 2906,



complex, class I, C




3631, 4356,








5081


571
major histocompatibility
458668
726
403541
1454
2182, 2907,



complex, class I, C




3632, 4357,








5082


572
major histocompatibility
458701
727
410579
1455
2183, 2908,



complex, class I, C




3633, 4358,








5083


573
major histocompatibility
461937
728
436842
1456
2184, 2909,



complex, class I, C




3634, 4359,








5084


574
major histocompatibility
464914
729
433897
1457
2185, 2910,



complex, class I, C




3635, 4360,








5085


575
major histocompatibility
469544
730
436915
1458
2186, 2911,



complex, class I, C




3636, 4361,








5086


576
major histocompatibility
476248
731
435957
1459
2187, 2912,



complex, class I, C




3637, 4362,








5087


577
major histocompatibility
479595
732
433088
1460
2188, 2913,



complex, class I, C




3638, 4363,








5088


578
major histocompatibility
485321
733
431141
1461
2189, 2914,



complex, class I, C




3639, 4364,








5089


579
major histocompatibility
488273
734
431347
1462
2190, 2915,



complex, class I, C




3640, 4365,








5090


580
major histocompatibility
491984
735
433409
1463
2191, 2916,



complex, class I, C




3641, 4366,








5091


581
major histocompatibility
539307
736
440406
1464
2192, 2917,



complex, class I, C




3642, 4367,








5092


582
major histocompatibility
549351
737
447743
1465
2193, 2918,



complex, class I, C




3643, 4368,








5093


583
major histocompatibility
552865
738
448156
1466
2194, 2919,



complex, class I, C




3644, 4369,








5094


584
matrix metallopeptidase 15
219271
739
219271
1467
2195, 2920,



(membrane-inserted)




3645, 4370,








5095


585
matrix metallopeptidase 16
286614
740
286614
1468
2196, 2921,



(membrane-inserted)




3646, 4371,








5096


586
matrix metallopeptidase 1 7
360564
741
353767
1469
2197, 2922,



(membrane-inserted)




3647, 4372,








5097


587
matrix metallopeptidase 17
535291
742
441106
1470
2198, 2923,



(membrane-inserted)




3648, 4373,








5098


588
matrix metallopeptidase 17
545790
743
441710
1471
2199, 2924,



(membrane-inserted)




3649, 4374,








5099


589
matrix metallopeptidase 19
322569
744
313437
1472
2200, 2925,








3650, 4375,








5100, 5330,








5445


590
matrix metallopeptidase 19
394182
745
377736
1473
2201, 2926,








3651, 4376,








5101, 5331,








5446


591
matrix metallopeptidase 19
409200
746
386625
1474
2202, 2927,








3652, 4377,








5102, 5332,








5447


592
matrix metallopeptidase 20
260228
747
260228
1475
2203, 2928,








3653, 4378,








5103


593
matrix metallopeptidase 21
368808
748
357798
1476
2204, 2929,








3654, 4379,








5104


594
matrix metallopeptidase 23B
356026
749
348308
1477
2205, 2930,








3655, 4380,








5105, 5333,








5448


595
matrix metallopeptidase 23B
378675
750
367945
1478
2206, 2931,








3656, 4381,








5106, 5334,








5449


596
matrix metallopeptidase 23B
412415
751
411590
1479
2207, 2932,








3657, 4382,








5107, 5335,








5450


597
matrix metallopeptidase 23B
512731
752
423780
1480
2208, 2933,








3658, 4383,








5108, 5336,








5451


598
matrix metallopeptidase 24
246186
753
246186
1481
2209, 2934,



(membrane-inserted)




3659, 4384,








5109, 5337,








5452


599
matrix metallopeptidase 24
540655
754
441902
1482
2210, 2935,



(membrane-inserted)




3660, 4385,








5110


600
matrix metallopeptidase 25
325800
755
324953
1483
2211, 2936,








3661, 4386,








5111


601
matrix metallopeptidase 25
336577
756
337816
1484
2212, 2937,








3662, 4387,








5112


602
matrix metallopeptidase 26
300762
757
300762
1485
2213, 2938,








3663, 4388,








5113


603
matrix metallopeptidase 26
380390
758
369753
1486
2214, 2939,








3664, 4389,








5114


604
matrix metallopeptidase 27
260229
759
260229
1487
2215, 2940,








3665, 4390,








5115, 5338,








5453


605
matrix metallopeptidase 28
250144
760
250144
1488
2216, 2941,








3666, 4391,








5116, 5339,








5454


606
matrix metallopeptidase 28
338839
761
340652
1489
2217, 2942,








3667, 4392,








5117, 5340,








5455


607
matrix metallopeptidase 28
538544
762
437605
1490
2218, 2943,








3668, 4393,








5118, 5341,








5456


608
matrix metallopeptidase 28
589103
763
468709
1491
2219, 2944,








3669, 4394,








5119, 5342,








5457


609
matrix metallopeptidase 8
236826
764
236826
1492
2220, 2945,



(neutrophil collagenase)




3670, 4395,








5120


610
matrix metallopeptidase 8
534942
765
440388
1493
2221, 2946,



(neutrophil collagenase)




3671, 4396,








5121


611
matrix metallopeptidase 8
544383
766
446018
1494
2222, 2947,



(neutrophil collagenase)




3672, 4397,








5122


612
melanocortin 1 receptor
555147
767
451605
1495
2223, 2948,



(alpha melanocyte stimulating




3673, 4398,



hormone receptor)




5123


613
melanocortin 2 receptor
327606
768
333821
1496
2224, 2949,



(adrenocorticotropic




3674, 4399,



hormone)




5124


614
melanocortin 2 receptor
399821
769
382718
1497
2225, 2950,



(adrenocorticotropic




3675, 4400,



hormone)




5125


615
melanocortin 3 receptor
243911
770
243911
1498
2226, 2951,








3676, 4401,








5126


616
melanocortin 4 receptor
299766
771
299766
1499
2227, 2952,








3677, 4402,








5127


617
melanocortin 5 receptor
324750
772
318077
1500
2228, 2953,








3678, 4403,








5128


618
microphthalmia-associated
314557
773
324246
1501
2229, 2954,



transcription factor




3679, 4404,








5129


619
microphthalmia-associated
314589
774
324443
1502
2230, 2955,



transcription factor




3680, 4405,








5130


620
microphthalmia-associated
328528
775
327867
1503
2231, 2956,



transcription factor




3681, 4406,








5131


621
microphthalmia-associated
352241
776
295600
1504
2232, 2957,



transcription factor




3682, 4407,








5132


622
microphthalmia-associated
394351
777
377880
1505
2233, 2958,



transcription factor




3683, 4408,








5133


623
microphthalmia-associated
394355
778
377884
1506
2234, 2959,



transcription factor




3684, 4409,








5134


624
microphthalmia-associated
448226
779
391803
1507
2235, 2960,



transcription factor




3685, 4410,








5135


625
microphthalmia-associated
451708
780
398639
1508
2236, 2961,



transcription factor




3686, 4411,








5136


626
microphthalmia-associated
457080
781
391276
1509
2237, 2962,



transcription factor




3687, 4412,








5137


627
microphthalmia-associated
531774
782
435909
1510
2238, 2963,



transcription factor




3688, 4413,








5138


628
microphthalmia-associated
472437
783
418845
1511
2239, 2964,



transcription factor




3689, 4414,








5139


629
NLR family pyrin domain
572272
784
460475
1512
2240, 2965,



containing 1




3690, 4415,








5140


630
NLR family pyrin domain
577119
785
460216
1513
2241, 2966,



containing 1




3691, 4416,








5141


631
NLR family, pyrin domain
262467
786
262467
1514
2242, 2967,



containing 1




3692, 4417,








5142


632
NLR family, pyrin domain
269280
787
269280
1515
2243, 2968,



containing 1




3693, 4418,








5143


633
NLR family, pyrin domain
345221
788
324366
1516
2244, 2969,



containing 1




3694, 4419,








5144


634
NLR family, pyrin domain
354411
789
346390
1517
2245, 2970,



containing 1




3695, 4420,








5145


635
NLR family, pyrin domain
537069
790
438391
1518
2246, 2971,



containing 1




3696, 4421,








5146


636
NLR family, pyrin domain
544378
791
442029
1519
2247, 2972,



containing 1




3697, 4422,








5147


637
nuclear factor of kappa light
189444
792
189444
1520
2248, 2973,



polypeptide gene enhancer in




3698, 4423,



B-cells 2 (p49/p100)




5148


638
nuclear factor of kappa light
336486
793
337001
1521
2249, 2974,



polypeptide gene enhancer in




3699, 4424,



B-cells 2 (p49/p100)




5149


639
nuclear factor of kappa light
369966
794
358983
1522
2250, 2975,



polypeptide gene enhancer in




3700, 4425,



B-cells 2 (p49/p100)




5150


640
nuclear factor of kappa light
428099
795
410256
1523
2251, 2976,



polypeptide gene enhancer in




3701, 4426,



B-cells 2 (p49/p100)




5151


641
ovo-like 1 (Drosophila)
335987
796
337862
1524
2252, 2977,








3702, 4427,








5152


642
phospholipase C, gamma 2
563193
797
455533
1525



(phosphatidylinositol-



specific)


643
phospholipase C, gamma 2
563375
798
454536
1526



(phosphatidylinositol-



specific)


644
phospholipase C, gamma 2
565054
799
455956
1527



(phosphatidylinositol-



specific)


645
platelet derived growth factor
302251
800
302193
1528
2253, 2978,



D




3703, 4428,








5153, 5343,








5458


646
platelet derived growth factor
393158
801
376865
1529
2254, 2979,



D




3704, 4429,








5154, 5344,








5459


647
platelet-derived growth factor
331163
802
330382
1530
2255, 2980,



beta polypeptide




3705, 4430,








5155, 5345,








5460


648
platelet-derived growth factor
381551
803
370963
1531
2256, 2981,



beta polypeptide




3706, 4431,








5156, 5346,








5461


649
platelet-derived growth factor
440375
804
405780
1532
2257, 2982,



beta polypeptide




3707, 4432,








5157, 5347,








5462


650
platelet-derived growth factor
455790
805
402306
1533
2258, 2983,



beta polypeptide




3708, 4433,








5158, 5348,








5463


651
proopiomelanocortin
264708
806
264708
1534
2259, 2984,








3709, 4434,








5159


652
proopiomelanocortin
380794
807
370171
1535
2260, 2985,








3710, 4435,








5160


653
proopiomelanocortin
395826
808
379170
1536
2261, 2986,








3711, 4436,








5161


654
proopiomelanocortin
405623
809
384092
1537
2262, 2987,








3712, 4437,








5162


655
proopiomelanocortin
449220
810
387993
1538
2263, 2988,








3713, 4438,








5163


656
psoriasis susceptibility 1
259881
811
259881
1539
2264, 2989,



candidate 1




3714, 4439,








5164


657
psoriasis susceptibility 1
376289
812
365466
1540
2265, 2990,



candidate 1




3715, 4440,








5165


658
psoriasis susceptibility 1
420214
813
396568
1541
2266, 2991,



candidate 1




3716, 4441,








5166


659
psoriasis susceptibility 1
433334
814
391443
1542
2267, 2992,



candidate 1




3717, 4442,








5167


660
psoriasis susceptibility 1
441092
815
398278
1543
2268, 2993,



candidate 1




3718, 4443,








5168


661
psoriasis susceptibility 1
448455
816
389875
1544
2269, 2994,



candidate 1




3719, 4444,








5169


662
psoriasis susceptibility 1
549149
817
446589
1545
2270, 2995,



candidate 1




3720, 4445,








5170


663
psoriasis susceptibility 1
259845
818
259845
1546
2271, 2996,



candidate 2




3721, 4446,








5171, 5349,








5464


664
psoriasis susceptibility 1
383530
819
373022
1547
2272, 2997,



candidate 2




3722, 4447,








5172


665
psoriasis susceptibility 1
413924
820
398734
1548
2273, 2998,



candidate 2




3723, 4448,








5173


666
psoriasis susceptibility 1
416027
821
390931
1549
2274, 2999,



candidate 2




3724, 4449,








5174


667
psoriasis susceptibility 1
422316
822
403456
1550
2275, 3000,



candidate 2




3725, 4450,








5175


668
psoriasis susceptibility 1
458589
823
414952
1551
2276, 3001,



candidate 2




3726, 4451,








5176


669
Putative caspase-14-like
428155
824
400592
1552
2277, 3002,



protein




3727, 4452,








5177


670
ribosomal protein S6 kinase,
366959
825
355926
1553
2278, 3003,



52 kDa, polypeptide 1




3728, 4453,








5178


671
ribosomal protein S6 kinase,
366960
826
355927
1554
2279, 3004,



52 kDa, polypeptide 1




3729, 4454,








5179


672
ribosomal protein S6 kinase,
543354
827
439282
1555
2280, 3005,



52 kDa, polypeptide 1




3730, 4455,








5180


673
ribosomal protein S6 kinase,
543470
828
442306
1556
2281, 3006,



52 kDa, polypeptide 1




3731, 4456,








5181


674
ribosomal protein S6 kinase,
312629
829
308413
1557
2282, 3007,



70 kDa, polypeptide 2




3732, 4457,








5182


675
ribosomal protein S6 kinase,
528964
830
432847
1558
2283, 3008,



70 kDa, polypeptide 2




3733, 4458,








5183


676
ribosomal protein S6 kinase,
539188
831
442949
1559
2284, 3009,



70 kDa, polypeptide 2




3734, 4459,








5184


677
ribosomal protein S6 kinase,
379548
832
368865
1560
2285, 3010,



90 kDa, polypeptide 3




3735, 4460,








5185


678
ribosomal protein S6 kinase,
379565
833
368884
1561
2286, 3011,



90 kDa, polypeptide 3




3736, 4461,








5186


679
ribosomal protein S6 kinase,
438357
834
388512
1562
2287, 3012,



90 kDa, polypeptide 3




3737, 4462,








5187


680
ribosomal protein S6 kinase,
457145
835
407655
1563
2288, 3013,



90 kDa, polypeptide 3




3738, 4463,








5188


681
ribosomal protein S6 kinase,
540702
836
444837
1564
2289, 3014,



90 kDa, polypeptide 3




3739, 4464,








5189


682
ribosomal protein S6 kinase,
544447
837
440220
1565
2290, 3015,



90 kDa, polypeptide 3




3740, 4465,








5190


683
ribosomal protein S6 kinase,
294261
838
294261
1566
2291, 3016,



90 kDa, polypeptide 4




3741, 4466,








5191


684
ribosomal protein S6 kinase,
334205
839
333896
1567
2292, 3017,



90 kDa, polypeptide 4




3742, 4467,








5192


685
ribosomal protein S6 kinase,
528057
840
435580
1568
2293, 3018,



90 kDa, polypeptide 4




3743, 4468,








5193


686
ribosomal protein S6 kinase,
530504
841
432945
1569
2294, 3019,



90 kDa, polypeptide 4




3744, 4469,








5194


687
ribosomal protein S6 kinase,
261991
842
261991
1570
2295, 3020,



90 kDa, polypeptide 5




3745, 4470,








5195


688
ribosomal protein S6 kinase,
418736
843
402787
1571
2296, 3021,



90 kDa, polypeptide 5




3746, 4471,








5196


689
ribosomal protein S6 kinase,
536315
844
442803
1572
2297, 3022,



90 kDa, polypeptide 5




3747, 4472,








5197


690
ribosomal protein S6 kinase,
262752
845
262752
1573
2298, 3023,



90 kDa, polypeptide 6




3748, 4473,








5198


691
ribosomal protein S6 kinase,
543399
846
440830
1574
2299, 3024,



90 kDa, polypeptide 6




3749, 4474,








5199


692
ribosomal protein S6 kinase-
354625
847
346644
1575
2300, 3025,



like 1




3750, 4475,








5200


693
ribosomal protein S6 kinase-
358328
848
351086
1576
2301, 3026,



like 1




3751, 4476,








5201


694
ribosomal protein S6 kinase-
555647
849
452027
1577
2302, 3027,



like 1




3752, 4477,








5202


695
ribosomal protein S6 kinase-
556776
850
451338
1578
2303, 3028,



like 1




3753, 4478,








5203


696
ribosomal protein S6 kinase-
557413
851
450567
1579
2304, 3029,



like 1




3754, 4479,








5204


697
solute carrier family 24
298877
852
298877
1580
2305, 3030,



(sodium/potassium/calcium




3755, 4480,



exchanger), member 4




5205


698
solute carrier family 24
318079
853
316957
1581
2306, 3031,



(sodium/potassium/calcium




3756, 4481,



exchanger), member 4




5206


699
solute carrier family 24
351924
854
337789
1582
2307, 3032,



(sodium/potassium/calcium




3757, 4482,



exchanger), member 4




5207


700
solute carrier family 24
393265
855
376948
1583
2308, 3033,



(sodium/potassium/calcium




3758, 4483,



exchanger), member 4




5208


701
solute carrier family 24
531433
856
433302
1584
2309, 3034,



(sodium/potassium/calcium




3759, 4484,



exchanger), member 4




5209


702
solute carrier family 24
532405
857
431840
1585
2310, 3035,



(sodium/potassium/calcium




3760, 4485,



exchanger), member 4




5210


703
solute carrier family 24,
341459
858
341550
1586
2311, 3036,



member 5




3761, 4486,








5211


704
solute carrier family 24,
449382
859
389966
1587
2312, 3037,



member 5




3762, 4487,








5212


705
superoxide dismutase 2,
337404
860
337127
1588
2313, 3038,



mitochondrial




3763, 4488,








5213


706
superoxide dismutase 2,
367054
861
356021
1589
2314, 3039,



mitochondrial




3764, 4489,








5214


707
superoxide dismutase 2,
367055
862
356022
1590
2315, 3040,



mitochondrial




3765, 4490,








5215


708
superoxide dismutase 2,
538183
863
446252
1591
2316, 3041,



mitochondrial




3766, 4491,








5216


709
superoxide dismutase 2,
546087
864
442920
1592
2317, 3042,



mitochondrial




3767, 4492,








5217


710
superoxide dismutase 3,
382120
865
371554
1593
2318, 3043,



extracellular




3768, 4493,








5218


711
tachykinin, precursor 1
319273
866
321106
1594
2319, 3044,








3769, 4494,








5219


712
tachykinin, precursor 1
346867
867
289574
1595
2320, 3045,








3770, 4495,








5220


713
tachykinin, precursor 1
350485
868
289576
1596
2321, 3046,








3771, 4496,








5221


714
thyroid stimulating hormone,
256592
869
256592
1597
2322, 3047,



beta




3772, 4497,








5222, 5350,








5465


715
thyroid stimulating hormone,
369517
870
358530
1598
2323, 3048,



beta




3773, 4498,








5223, 5351,








5466


716
transmembrane channel-like 6
590602
871
465261
1599
2324, 3049,








3774, 4499,








5224


717
transmembrane channel-like 8
301627
872
301627
1600
2325, 3050,








3775, 4500,








5225


718
transmembrane channel-like 8
318430
873
325561
1601
2326, 3051,








3776, 4501,








5226


719
tumor necrosis factor
376122
874
365290
1602
2327, 3052,








3777, 4502,








5227


720
tumor necrosis factor
383496
875
372988
1603
2328, 3053,








3778, 4503,








5228


721
tumor necrosis factor
412275
876
392858
1604
2329, 3054,








3779, 4504,








5229


722
tumor necrosis factor
420425
877
410668
1605
2330, 3055,








3780, 4505,








5230


723
tumor necrosis factor
443707
878
389492
1606
2331, 3056,








3781, 4506,








5231


724
tumor necrosis factor
448781
879
389490
1607
2332, 3057,








3782, 4507,








5232


725
tumor necrosis factor
449264
880
398698
1608
2333, 3058,








3783, 4508,








5233


726
tyrosinase-related protein 1
381137
881
370529
1609
2334, 3059,








3784, 4509,








5234


727
tyrosinase-related protein 1
388918
882
373570
1610
2335, 3060,








3785, 4510,








5235


728
tyrosinase-related protein 1
473763
883
419006
1611
2336, 3061,








3786, 4511,








5236









Protein Cleavage Signals and Sites

In one embodiment, the cosmetic polypeptides of the present invention may include at least one protein cleavage signal containing at least one protein cleavage site. The protein cleavage site may be located at the N-terminus, the C-terminus, at any space between the N- and the C-termini such as, but not limited to, half-way between the N- and C-termini, between the N-terminus and the half way point, between the half way point and the C-terminus, and combinations thereof.
The cosmetic polypeptides of the present invention may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin or Factor Xa protein cleavage signal. Proprotein convertases are a family of nine proteinases, comprising seven basic amino acid-specific subtilisin-like serine proteinases related to yeast kexin, known as prohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basic amino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilases that cleave at non-basic residues, called subtilisin kexin isozyme 1 (SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9). Non-limiting examples of protein cleavage signal amino acid sequences are listing in Table 7. In Table 7, “X” refers to any amino acid, “n” may be 0, 2, 4 or 6 amino acids and “*” refers to the protein cleavage site. In Table 7, SEQ ID NO: 5643 refers to when n=4 and SEQ ID NO: 5644 refers to when n=6.







TABLE 7







Protein Cleavage Site Sequences









Protein Cleavage Signal
Amino Acid Cleavage Sequence
SEQ ID NO





Proprotein convertase
R-X-X-R*
5641



R-X-K/R-R*
5642



K/R-Xn-K/R*
5643 




or 5644


 


Thrombin
L-V-P-R*-G-S
5645



L-V-P-R*
5646



A/F/G/I/L/T/V/M-A/F/G/I/L/T/V/
5647



W-P-R*



 


Factor Xa
I-E-G-R*
5648



I-D-G-R*
5649



A-E-G-R*
5650



A/F/G/I/L/T/V/M-D/E-G-R*
5651









In one embodiment, the cosmetic primary constructs and the cosmetic mmRNA of the present invention may be engineered such that the cosmetic primary construct or cosmetic mmRNA contains at least one encoded protein cleavage signal. The encoded protein cleavage signal may be located before the start codon, after the start codon, before the coding region, within the coding region such as, but not limited to, half way in the coding region, between the start codon and the half way point, between the half way point and the stop codon, after the coding region, before the stop codon, between two stop codons, after the stop codon and combinations thereof.
In one embodiment, the cosmetic primary constructs or cosmetic mmRNA of the present invention may include at least one encoded protein cleavage signal containing at least one protein cleavage site. The encoded protein cleavage signal may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin and/or Factor Xa protein cleavage signal. One of skill in the art may use Table 1 above or other known methods to determine the appropriate encoded protein cleavage signal to include in the cosmetic primary constructs or mmRNA of the present invention. For example, starting with the signal of Table 7 and considering the codons of Table 1 one can design a signal for the cosmetic primary construct which can produce a protein signal in the resulting cosmetic polypeptide.
In one embodiment, the cosmetic polypeptides of the present invention include at least one protein cleavage signal and/or site.
As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No. 20090227660, herein incorporated by reference in their entireties, use a furin cleavage site to cleave the N-terminal methionine of GLP-1 in the expression product from the Golgi apparatus of the cells. In one embodiment, the polypeptides of the present invention include at least one protein cleavage signal and/or site with the proviso that the polypeptide is not GLP-1.
In one embodiment, the cosmetic primary constructs or cosmetic mmRNA of the present invention includes at least one encoded protein cleavage signal and/or site.
In one embodiment, the cosmetic primary constructs or cosmetic mmRNA of the present invention includes at least one encoded protein cleavage signal and/or site with the proviso that the cosmetic primary construct or cosmetic mmRNA does not encode GLP-1.
In one embodiment, the cosmetic primary constructs or cosmetic mmRNA of the present invention may include more than one coding region. Where multiple coding regions are present in the cosmetic primary construct or cosmetic mmRNA of the present invention, the multiple coding regions may be separated by encoded protein cleavage sites. As a non-limiting example, the cosmetic primary construct or cosmetic mmRNA may be signed in an ordered pattern. On such pattern follows AXBY form where A and B are coding regions which may be the same or different coding regions and/or may encode the same or different cosmetic polypeptides, and X and Y are encoded protein cleavage signals which may encode the same or different protein cleavage signals. A second such pattern follows the form AXYBZ where A and B are coding regions which may be the same or different coding regions and/or may encode the same or different cosmetic polypeptides, and X, Y and Z are encoded protein cleavage signals which may encode the same or different protein cleavage signals. A third pattern follows the form ABXCY where A, B and C are coding regions which may be the same or different coding regions and/or may encode the same or different cosmetic polypeptides, and X and Y are encoded protein cleavage signals which may encode the same or different protein cleavage signals.
In one embodiment, the cosmetic polypeptides, cosmetic primary constructs and cosmetic mmRNA can also contain sequences that encode protein cleavage sites so that the cosmetic polypeptides, cosmetic primary constructs and cosmetic mmRNA can be released from a carrier region or a fusion partner by treatment with a specific protease for said protein cleavage site.
In one embodiment, the cosmetic polypeptides, primary constructs and mmRNA of the present invention may include a sequence encoding the 2A peptide. In one embodiment, this sequence may be used to separate the coding region of two or more polypeptides of interest. As a non-limiting example, the sequence encoding the 2A peptide may be between coding region A and coding region B (A-2Apep-B). The presence of the 2A peptide would result in the cleavage of one long protein into protein A, protein B and the 2A peptide. Protein A and protein B may be the same or different polypeptides of interest. In another embodiment, the 2A peptide may be used in the cosmetic polynucleotides, primary constructs and/or mmRNA of the present invention to produce two, three, four, five, six, seven, eight, nine, ten or more proteins.
Incorporating Post Transcriptional Control Modulators
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA of the present invention may include at least one post transcriptional control modulator. These post transcriptional control modulators may be, but are not limited to, small molecules, compounds and regulatory sequences. As a non-limiting example, post transcriptional control may be achieved using small molecules identified by PTC Therapeutics Inc. (South Plainfield, N.J.) using their GEMSTM (Gene Expression Modulation by Small-Moleclues) screening technology.
The post transcriptional control modulator may be a gene expression modulator which is screened by the method detailed in or a gene expression modulator described in International Publication No. WO2006022712, herein incorporated by reference in its entirety. Methods identifying RNA regulatory sequences involved in translational control are described in International Publication No. WO2004067728, herein incorporated by reference in its entirety; methods identifying compounds that modulate untranslated region dependent expression of a gene are described in International Publication No. WO2004065561, herein incorporated by reference in its entirety.
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA of the present invention may include at least one post transcriptional control modulator is located in the 5′ and/or the 3′ untranslated region of the cosmetic polynucleotides, primary constructs and/or mmRNA of the present invention
In another embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA of the present invention may include at least one post transcription control modulator to modulate premature translation termination. The post transcription control modulators may be compounds described in or a compound found by methods outlined in International Publication Nos. WO2004010106, WO2006044456, WO2006044682, WO2006044503 and WO2006044505, each of which is herein incorporated by reference in its entirety. As a non-limiting example, the compound may bind to a region of the 28S ribosomal RNA in order to modulate premature translation termination (See e.g., WO2004010106, herein incorporated by reference in its entirety).
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA of the present invention may include at least one post transcription control modulator to alter protein expression. As a non-limiting example, the expression of VEGF may be regulated using the compounds described in or a compound found by the methods described in International Publication Nos. WO2005118857, WO2006065480, WO2006065479 and WO2006058088, each of which is herein incorporated by reference in its entirety.
The cosmetic polynucleotides, primary constructs and/or mmRNA of the present invention may include at least one post transcription control modulator to control translation. In one embodiment, the post transcription control modulator may be a RNA regulatory sequence. As a non-limiting example, the RNA regulatory sequence may be identified by the methods described in International Publication No. WO2006071903, herein incorporated by reference in its entirety.
III. Modifications
Herein, in a cosmetic polynucleotide (such as a cosmetic primary construct or a cosmetic mRNA molecule), the terms “modification” or, as appropriate, “modified” refer to modification with respect to A, G, U or C ribonucleotides. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties. In a polypeptide, the term “modification” refers to a modification as compared to the canonical set of 20 amino acids, moiety)
The modifications may be various distinct modifications. In some embodiments, the coding region, the flanking regions and/or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA introduced to a cell may exhibit reduced degradation in the cell, as compared to an unmodified cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA.
The cosmetic polynucleotides, cosmetic primary constructs, and cosmetic mmRNA can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications according to the present invention may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.
As described herein, the cosmetic polynucleotides, cosmetic primary constructs, and cosmetic mmRNA of the invention do not substantially induce an innate immune response of a cell into which the mRNA is introduced. Features of an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc, and/or 3) termination or reduction in protein translation.
In certain embodiments, it may desirable to intracellularly degrade a modified nucleic acid molecule introduced into the cell. For example, degradation of a modified cosmetic nucleic acid molecule may be preferable if precise timing of protein production is desired. Thus, in some embodiments, the invention provides a modified cosmetic nucleic acid molecule containing a degradation domain, which is capable of being acted on in a directed manner within a cell. In another aspect, the present disclosure provides cosmetic polynucleotides comprising a nucleoside or nucleotide that can disrupt the binding of a major groove interacting, e.g. binding, partner with the polynucleotide (e.g., where the modified nucleotide has decreased binding affinity to major groove interacting partner, as compared to an unmodified nucleotide).
The cosmetic polynucleotides, cosmetic primary constructs, and cosmetic mmRNA can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.). In some embodiments, the cosmetic polynucleotides, cosmetic primary constructs, or cosmetic mmRNA may include one or more messenger RNAs (mRNAs) and one or more modified nucleoside or nucleotides (e.g., mmRNA molecules). Details for these cosmetic polynucleotides, cosmetic primary constructs, and cosmetic mmRNA follow.
Cosmetic Polynucleotides and Cosmetic Primary Constructs
The cosmetic polynucleotides, primary constructs, and mmRNA of the invention includes a first region of linked nucleosides encoding a polypeptide of interest, a first flanking region located at the 5′ terminus of the first region, and a second flanking region located at the 3′ terminus of the first region.
In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (Ia) or Formula (Ia-1):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
U is O, S, N(RU)nu, or C(RU)nu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl;
- - - is a single bond or absent;
each of R1′, R2′, R1′′, R2′′, R1, R2, R3, R4, and R5 is independently, if present, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent; wherein the combination of R3 with one or more of R1′, R1′′, R2′, R2′′, or R5 (e.g., the combination of R1′ and R3, the combination of R1′′ and R3, the combination of R2′ and R3, the combination of R2′′ and R3, or the combination of R5 and R3) can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); wherein the combination of R5 with one or more of R1′, R1′′, R2′, or R2′′ (e.g., the combination of R1′ and R5, the combination of R1′′ and R5, the combination of R2′ and R5, or the combination of R2′′ and R5) can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); and wherein the combination of R4 and one or more of R1′, R1′′, R2′, R2′′, R3, or R5 can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); each of m′ and m′′ is, independently, an integer from 0 to 3 (e.g., from 0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);
each of Y1, Y2, and Y3, is, independently, O, S, Se, —NRN1—, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent;
each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
each Y5 is, independently, O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;
n is an integer from 1 to 100,000; and
B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof), wherein the combination of B and R1′′, the combination of B and R2′, the combination of B and R1′′, or the combination of B and R2′′ can, taken together with the carbons to which they are attached, optionally form a bicyclic group (e.g., a bicyclic heterocyclyl) or wherein the combination of B, R1′′, and R3 or the combination of B, R2′′, and R3 can optionally form a tricyclic or tetracyclic group (e.g., a tricyclic or tetracyclic heterocyclyl, such as in Formula (IIo)-(IIp) herein), In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA includes a modified ribose. In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (Ia-2)-(Ia-5) or a pharmaceutically acceptable salt or stereoisomer thereof.




In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (Ib) or Formula (Ib-1):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
U is O, S, N(RU)nu, or C(RU)nu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl;
- - - is a single bond or absent;
each of R1, R3′, R3′′, and R4 is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent; and wherein the combination of R1 and R3′ or the combination of R1 and R3′′ can be taken together to form optionally substituted alkylene or optionally substituted heteroalkylene (e.g., to produce a locked nucleic acid);
each R5 is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, or absent;
each of Y1, Y2, and Y3 is, independently, O, S, Se, —NRN1—, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;
each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
n is an integer from 1 to 100,000; and
B is a nucleobase.
In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (Ic):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
U is O, S, N(RU)nu, or C(RU)nu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl;
- - - is a single bond or absent;
each of B1, B2, and B3 is, independently, a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof, as described herein), H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl, wherein one and only one of B1, B2, and B3 is a nucleobase;
each of Rb1, Rb2, Rb3, R3, and R5 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl or optionally substituted aminoalkynyl;
each of Y1, Y2, and Y3, is, independently, O, S, Se, —NRN1—, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;
each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
each Y5 is, independently, O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;
n is an integer from 1 to 100,000; and
wherein the ring including U can include one or more double bonds.
In particular embodiments, the ring including U does not have a double bond between U-CB3Rb3 or between CB3Rb3—CB2Rb2.
In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (Id):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
U is O, S, N(RU)nu, or C(RU)nu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl;
each R3 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl;
each of Y1, Y2, and Y3, is, independently, O, S, Se, —NRN1—, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;
each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
each Y5 is, independently, O, S, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;
n is an integer from 1 to 100,000; and
B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof).
In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (Ie):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each of U′ and U′′ is, independently, O, S, N(RU)nu, or C(RU)nu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl;
each R6 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl;
each Y5′ is, independently, O, S, optionally substituted alkylene (e.g., methylene or ethylene), or optionally substituted heteroalkylene;
n is an integer from 1 to 100,000; and
B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof).
In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (If) or (If-1):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each of U′ and U′′ is, independently, O, S, N, N(RU)nu, or C(RU)nu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl (e.g., U′ is O and U′′ is N);
- - - is a single bond or absent;
each of R1′, R2′, R1′′, R2′′, R3, and R4 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent; and wherein the combination of R1′ and R3, the combination of R1′′ and R3, the combination of R2′ and R3, or the combination of R2′′ and R3 can be taken together to form optionally substituted alkylene or optionally substituted heteroalkylene (e.g., to produce a locked nucleic acid); each of m′ and m′′ is, independently, an integer from 0 to 3 (e.g., from 0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);
each of Y1, Y2, and Y3, is, independently, O, S, Se, —NRN1—, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent;
each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
each Y5 is, independently, O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;
n is an integer from 1 to 100,000; and
B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof).
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If), and (IIa)-(IIp)), the ring including U has one or two double bonds.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), each of R1, R1′, and R1′′, if present, is H. In further embodiments, each of R2, R2′, and R2′′, if present, is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl). In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′ is C1-6 alkyl.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), each of R2, R2′, and R2′′, if present, is H. In further embodiments, each of R1, R1′, and R1′′, if present, is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl). In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′ is C1-6 alkyl.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), each of R3, R4, and R5 is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In particular embodiments, R3 is H, R4 is H, R5 is H, or R3, R4, and R5 are all H. In particular embodiments, R3 is C1-6 alkyl, R4 is C1-6 alkyl, R5 is C1-6 alkyl, or R3, R4, and R5 are all C1-6 alkyl. In particular embodiments, R3 and R4 are both H, and R5 is C1-6 alkyl.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), R3 and R5 join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl, such as trans-3′,4′ analogs, wherein R3 and R5 join together to form heteroalkylene (e.g., —(CH2)b1O(CH2)b2O(CH2)b3—, wherein each of b1, b2, and b3 are, independently, an integer from 0 to 3).
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), R3 and one or more of R1′, R1′′, R2′, R2′′, or R5 join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl, R3 and one or more of R1′, R1′′, R2′, R2′′, or R5 join together to form heteroalkylene (e.g., —(CH2)b1O(CH2)b2O(CH2)b3—, wherein each of b1, b2, and b3 are, independently, an integer from 0 to 3).
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), R5 and one or more of R1′, R1′′, R2′, or R2′′ join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl, R5 and one or more of R1′, R1′′, R2′, or R2′′ join together to form heteroalkylene (e.g., —(CH2)b1O(CH2)b2O(CH2)b3—, wherein each of b1, b2, and b3 are, independently, an integer from 0 to 3).
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), each Y2 is, independently, O, S, or —NRN1—, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl. In particular embodiments, Y2 is NRN1—, wherein RN1 is H or optionally substituted alkyl (e.g., C1-6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl).
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), each Y3 is, independently, O or S.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), R1 is H; each R2 is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g., —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′ is C1-6 alkyl); each Y2 is, independently, O or —NRN1—, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted alkyl (e.g., C1-6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y3 is, independently, O or S (e.g., S). In further embodiments, R3 is H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In yet further embodiments, each Y1 is, independently, O or —NRN1—, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted alkyl (e.g., C1-6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), each R1 is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g., —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′ is C1-6 alkyl); R2 is H; each Y2 is, independently, O or —NRN1—, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted alkyl (e.g., C1-6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y3 is, independently, O or S (e.g., S). In further embodiments, R3 is H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In yet further embodiments, each Y1 is, independently, O or —NRN1—, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted alkyl (e.g., C1-6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), the ring including U is in the β-D (e.g., β-D-ribo) configuration.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), the ring including U is in the α-L (e.g., α-L-ribo) configuration.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), one or more B is not pseudouridine (ψ) or 5-methyl-cytidine (m5C). In some embodiments, about 10% to about 100% of n number of B nucleobases is not ψ or m5C (e.g., from 10% to 20%, from 10% to 35%, from 10% to 50%, from 10% to 60%, from 10% to 75%, from 10% to 90%, from 10% to 95%, from 10% to 98%, from 10% to 99%, from 20% to 35%, from 20% to 50%, from 20% to 60%, from 20% to 75%, from 20% to 90%, from 20% to 95%, from 20% to 98%, from 20% to 99%, from 20% to 100%, from 50% to 60%, from 50% to 75%, from 50% to 90%, from 50% to 95%, from 50% to 98%, from 50% to 99%, from 50% to 100%, from 75% to 90%, from 75% to 95%, from 75% to 98%, from 75% to 99%, and from 75% to 100% of n number of B is not ψ or m5C). In some embodiments, B is not ψ or m5C.
In some embodiments of the cosmetic polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Y1, Y2, or Y3 is not O.
In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA includes a modified ribose. In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (IIa)-(IIc):




or a pharmaceutically acceptable salt or stereoisomer thereof. In particular embodiments, U is O or C(RU)nu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl (e.g., U is —CH2— or —CH—). In other embodiments, each of R1, R2, R3, R4, and R5 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent (e.g., each R1 and R2 is, independently, H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy; each R3 and R4 is, independently, H or optionally substituted alkyl; and R5 is H or hydroxy), and  is a single bond or double bond. In particular embodiments, the polynucleotidesor mmRNA includes n number of linked nucleosides having Formula (IIb-1)-(IIb-2):





or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, U is O or C(RU)nu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl (e.g., U is —CH2— or —CH—). In other embodiments, each of R1 and R2 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent (e.g., each R1 and R2 is, independently, H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, e.g., H, halo, hydroxy, alkyl, or alkoxy). In particular embodiments, R2 is hydroxy or optionally substituted alkoxy (e.g., methoxy, ethoxy, or any described herein).

In particular embodiments, the cosmetic polynucleotide, primary construct, or mmRNA includes n number of linked nucleosides having Formula (IIc-1)-(IIc-4):




or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, U is O or C(RU)nu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl (e.g., U is —CH2— or —CH—). In some embodiments, each of R1, R2, and R3 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent (e.g., each R1 and R2 is, independently, H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, e.g., H, halo, hydroxy, alkyl, or alkoxy; and each R3 is, independently, H or optionally substituted alkyl)). In particular embodiments, R2 is optionally substituted alkoxy (e.g., methoxy or ethoxy, or any described herein). In particular embodiments, R1 is optionally substituted alkyl, and R2 is hydroxy. In other embodiments, R1 is hydroxy, and R2 is optionally substituted alkyl. In further embodiments, R3 is optionally substituted alkyl.

In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA includes an acyclic modified ribose. In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (IId)-(IIf):




or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA includes an acyclic modified hexitol. In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides Formula (IIg)-(IIj):




or a pharmaceutically acceptable salt or stereoisomer thereof

In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA includes a sugar moiety having a contracted or an expanded ribose ring. In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula
(IIk)-(IIm):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each of R1′, R1′′, R2′, and R2′′ is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, or absent; and wherein the combination of R2′ and R3 or the combination of R2′′ and R3 can be taken together to form optionally substituted alkylene or optionally substituted heteroalkylene.

In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA includes a locked modified ribose. In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (IIn):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R3′ is O, S, or —NRN1—, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl and R3′′ is optionally substituted alkylene (e.g., —CH2—, —CH2CH2—, or —CH2CH2CH2—) or optionally substituted heteroalkylene (e.g., —CH2NH—, —CH2CH2NH—, —CH2OCH2—, or —CH2CH2OCH2—)(e.g., R3′ is O and R3′′ is optionally substituted alkylene (e.g., —CH2—, —CH2CH2—, or —CH2CH2CH2—)).

In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA includes n number of linked nucleosides having Formula (IIn-1)-(II-n2):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R3′ is O, S, or —NRN1—, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl and R3′′ is optionally substituted alkylene (e.g., —CH2—, —CH2CH2—, or —CH2CH2CH2—) or optionally substituted heteroalkylene (e.g., —CH2NH—, —CH2CH2NH—, —CH2OCH2—, or —CH2CH2OCH2—) (e.g., R3′ is O and R3′′ is optionally substituted alkylene (e.g., —CH2—, —CH2CH2—, or —CH2CH2CH2—)).

In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA includes a locked modified ribose that forms a tetracyclic heterocyclyl. In some embodiments, the cosmetic polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (IIo):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R12a, R12c, T1′, T1′′, T2′, T2′′, V1, and V3 are as described herein.

Any of the formulas for the cosmetic polynucleotides, primary constructs, or mmRNA can include one or more nucleobases described herein (e.g., Formulas (b1)-(b43)).
In one embodiment, the present invention provides methods of preparing a polynucleotide, primary construct, or mmRNA, wherein the polynucleotide comprises n number of nucleosides having Formula (Ia), as defined herein:




the method comprising reacting a compound of Formula (IIIa), as defined herein:




with an RNA polymerase, and a cDNA template.
In a further embodiment, the present invention provides methods of amplifying a polynucleotide, primary construct, or mmRNA comprising at least one nucleotide (e.g., mmRNA molecule), the method comprising:
reacting a compound of Formula (IIIa), as defined herein, with a primer, a cDNA template, and an RNA polymerase.
In one embodiment, the present invention provides methods of preparing a polynucleotide, primary construct, or mmRNA comprising at least one nucleotide (e.g., mmRNA molecule), wherein the polynucleotide comprises n number of nucleosides having Formula (Ia), as defined herein:




the method comprising reacting a compound of Formula (IIIa-1), as defined herein:




with an RNA polymerase, and a cDNA template.
In a further embodiment, the present invention provides methods of amplifying a polynucleotide, primary construct, or mmRNA comprising at least one nucleotide (e.g., mmRNA molecule), the method comprising:

    
    
        reacting a compound of Formula (IIIa-1), as defined herein, with a primer, a cDNA template, and an RNA polymerase.
    
    


In one embodiment, the present invention provides methods of preparing a modified mRNA comprising at least one nucleotide (e.g., mmRNA molecule), wherein the polynucleotide comprises n number of nucleosides having Formula (Ia-2), as defined herein:




the method comprising reacting a compound of Formula (IIIa-2), as defined herein:




with an RNA polymerase, and a cDNA template.
In a further embodiment, the present invention provides methods of amplifying a modified mRNA comprising at least one nucleotide (e.g., mmRNA molecule), the method comprising:
reacting a compound of Formula (IIIa-2), as defined herein, with a primer, a cDNA template, and an RNA polymerase.
In some embodiments, the reaction may be repeated from 1 to about 7,000 times. In any of the embodiments herein, B may be a nucleobase of Formula (b1)-(b43).
The cosmetic polynucleotides, primary constructs, and mmRNA can optionally include 5′ and/or 3′ flanking regions, which are described herein.
Modified RNA (mmRNA) Molecules
The present invention also includes building blocks, e.g., modified ribonucleosides, modified ribonucleotides, of modified RNA (mmRNA) molecules. For example, these building blocks can be useful for preparing the cosmetic polynucleotides, primary constructs, or mmRNA of the invention.
In some embodiments, the building block molecule has Formula (IIIa) or (IIIa-1):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the substituents are as described herein (e.g., for Formula (Ia) and (Ia-1)), and wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Y1, Y2, or Y3 is not O.

In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IVa)-(IVb):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, Formula (IVa) or (IVb) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). In particular embodiments, Formula (IVa) or (IVb) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, Formula (IVa) or (IVb) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, Formula (IVa) or (IVb) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IVc)-(IVk):







or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IVc)-(IVk) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IVc)-(IVk) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, one of Formulas (IVc)-(IVk) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IVc)-(IVk) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).

In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (Va) or (Vb):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)).

In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IXa)-(IXd):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IXa)-(IXd) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IXa)-(IXd) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, one of Formulas (IXa)-(IXd) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IXa)-(IXd) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).

In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IXe)-(IXg):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IXe)-(IXg) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IXe)-(IXg) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, one of Formulas (IXe)-(IXg) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IXe)-(IXg) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).

In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IXh)-(IXk):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IXh)-(IXk) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IXh)-(IXk) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, one of Formulas (IXh)-(IXk) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IXh)-(IXk) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).

In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IXl)-(IXr):




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r1 and r2 is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5) and B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IXl)-(IXr) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IXl)-(IXr) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, one of Formulas (IXl)-(IXr) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IXl)-(IXr) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be selected from the group consisting of:










or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be selected from the group consisting of:







or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5) and s1 is as described herein.

In some embodiments, the building block molecule, which may be incorporated into a nucleic acid (e.g., RNA, mRNA, polynucleotide, primary construct, or mmRNA), is a modified uridine (e.g., selected from the group consisting of:
































































or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y1, Y3, Y4, Y6, and r are as described herein (e.g., each r is, independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3, or from 1 to 5)).

In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, is a modified cytidine (e.g., selected from the group consisting of:



















or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y1, Y3, Y4, Y6, and r are as described herein (e.g., each r is, independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3, or from 1 to 5)). For example, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be:





or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, is a modified adenosine (e.g., selected from the group consisting of:






















or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y1, Y3, Y4, Y6, and r are as described herein (e.g., each r is, independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3, or from 1 to 5)).

In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, is a modified guanosine (e.g., selected from the group consisting of:






















or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y1, Y3, Y4, Y6, and r are as described herein (e.g., each r is, independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3, or from 1 to 5)).

In some embodiments, the chemical modification can include replacement of C group at C-5 of the ring (e.g., for a pyrimidine nucleoside, such as cytosine or uracil) with N (e.g., replacement of the >CH group at C-5 with >NRN1 group, wherein RN1 is H or optionally substituted alkyl). For example, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be:




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).

In another embodiment, the chemical modification can include replacement of the hydrogen at C-5 of cytosine with halo (e.g., Br, Cl, F, or I) or optionally substituted alkyl (e.g., methyl). For example, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be:




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).

In yet a further embodiment, the chemical modification can include a fused ring that is formed by the NH2 at the C-4 position and the carbon atom at the C-5 position. For example, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be:




or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).

Modifications on the Sugar

The modified nucleosides and nucleotides (e.g., building block molecules), which may be incorporated into a polynucleotide, primary construct, or mmRNA (e.g., RNA or mRNA, as described herein), can be modified on the sugar of the ribonucleic acid. For example, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl-C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), —O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA) in which the 2′-hydroxyl is connected by a C1-6 alkylene or C1-6 heteroalkylene bridge to the 4′-carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino as defined herein; and amino acid, as defined herein
Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide, primary construct, or mmRNA molecule can include nucleotides containing, e.g., arabinose, as the sugar.
Modifications on the Nucleobase
The present disclosure provides for modified nucleosides and nucleotides. As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group. The modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides).
The modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil.
The modified nucleosides and nucleotides can include a modified nucleobase. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases can be modified or wholly replaced to provide cosmetic polynucleotides, primary constructs, or mmRNA molecules having enhanced properties, e.g., resistance to nucleases through disruption of the binding of a major groove binding partner. Table 8 below identifies the chemical faces of each canonical nucleotide. Circles identify the atoms comprising the respective chemical regions.











TABLE 8









Watson-Crick




Major Groove
Minor Groove
Base-pairing




Face
Face
Face







Pyrim- idines
Cytidine:














 



Uridine:














 


Purines
Adenosine:














 



Guanosine:





















In some embodiments, B is a modified uracil. Exemplary modified uracils include those having Formula (b1)-(b5):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
 is a single or double bond;
each of T1′, T1′′, T2′, and T2′′ is, independently, H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or the combination of T1′ and T1′′ or the combination of T2′ and T2′′ join together (e.g., as in T2) to form O (oxo), S (thio), or Se (seleno);
each of V1 and V2 is, independently, O, S, N(RVb)nv, or C(RVb)nv, wherein nv is an integer from 0 to 2 and each RVb is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl);
R10 is H, halo, optionally substituted amino acid, hydroxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aminoalkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl;
R11 is H or optionally substituted alkyl;
R12a is H, optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl; and
R12c is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl.
Other exemplary modified uracils include those having Formula (b6)-(b9):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
 is a single or double bond;
each of T1′, T1′′, T2′, and T2′′ is, independently, H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or the combination of T1′ and T1′′ join together (e.g., as in T1) or the combination of T2′ and T2′′ join together (e.g., as in T2) to form O (oxo), S (thio), or Se (seleno), or each T1 and T2 is, independently, O (oxo), S (thio), or Se (seleno);
each of W1 and W2 is, independently, N(RWa)nw or C(RWa)nw, wherein nw is an integer from 0 to 2 and each ea is, independently, H, optionally substituted alkyl, or optionally substituted alkoxy;
each V3 is, independently, O, S, N(RVa)nv, or C(RVa)nv, wherein nv is an integer from 0 to 2 and each RVa is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy, optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an O-protecting group), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl), and wherein RVa and R12c taken together with the carbon atoms to which they are attached can form optionally substituted cycloalkyl, optionally substituted aryl, or optionally substituted heterocyclyl (e.g., a 5- or 6-membered ring);
R12a is H, optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an O-protecting group), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, optionally substituted carbamoylalkyl, or absent;
R12b is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkaryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted amino acid, optionally substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an O-protecting group), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl,
wherein the combination of R12b and T1′ or the combination of R12b and R12c can join together to form optionally substituted heterocyclyl; and
R12c is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl.
Further exemplary modified uracils include those having Formula (b28)-(b31):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each of T1 and T2 is, independently, O (oxo), S (thio), or Se (seleno);
each RVb′ and RVb′′ is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an O-protecting group), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl) (e.g., RVb′ is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted aminoalkyl, e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl);
R12a is H, optionally substituted alkyl, optionally substituted carboxyaminoalkyl, optionally substituted aminoalkyl (e.g., e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl; and
R12b is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl (e.g., e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl),
optionally substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl.
In particular embodiments, T1 is O (oxo), and T2 is S (thio) or Se (seleno). In other embodiments, T1 is S (thio), and T2 is O (oxo) or Se (seleno). In some embodiments, RVb′ is H, optionally substituted alkyl, or optionally substituted alkoxy.
In other embodiments, each R12a and R12b is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted hydroxyalkyl. In particular embodiments, R12a is H. In other embodiments, both R12a and R12b are H.
In some embodiments, each RVb′ of R12b is, independently, optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl). In some embodiments, the amino and/or alkyl of the optionally substituted aminoalkyl is substituted with one or more of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted sulfoalkyl, optionally substituted carboxy (e.g., substituted with an O-protecting group), optionally substituted hydroxy (e.g., substituted with an O-protecting group), optionally substituted carboxyalkyl (e.g., substituted with an O-protecting group), optionally substituted alkoxycarbonylalkyl (e.g., substituted with an O-protecting group), or N-protecting group. In some embodiments, optionally substituted aminoalkyl is substituted with an optionally substituted sulfoalkyl or optionally substituted alkenyl. In particular embodiments, R12a and RVb′ are both H. In particular embodiments, T1 is O (oxo), and T2 is S (thio) or Se (seleno).
In some embodiments, RVb′ is optionally substituted alkoxycarbonylalkyl or optionally substituted carbamoylalkyl.
In particular embodiments, the optional substituent for R12a, R12b, R12c, or RVa is a polyethylene glycol group (e.g., —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl); or an amino-polyethylene glycol group (e.g., —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl).
In some embodiments, B is a modified cytosine. Exemplary modified cytosines include compounds of Formula (b10)-(b14):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each of T3′ and T3′′ is, independently, H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or the combination of T3′ and T3′′ join together (e.g., as in T3) to form O (oxo), S (thio), or Se (seleno);
each V4 is, independently, O, S, N(RVc)nv, or C(RVc)nv, wherein nv is an integer from 0 to 2 and each RVc is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl), wherein the combination of R13b and RVc can be taken together to form optionally substituted heterocyclyl;
each V5 is, independently, N(RVd)nv, or C(RVd)nv, wherein nv is an integer from 0 to 2 and each RVd is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl) (e.g., V5 is —CH or N);
each of R13a and R13b is, independently, H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy, wherein the combination of R13b and R14 can be taken together to form optionally substituted heterocyclyl;
each R14 is, independently, H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl (e.g., substituted with an O-protecting group), optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted acyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H, alkyl, aryl, or phosphoryl), azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkyl; and
each of R15 and R16 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.
Further exemplary modified cytosines include those having Formula (b32)-(b35):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each of T1 and T3 is, independently, O (oxo), S (thio), or Se (seleno);
each of R13a and R13b is, independently, H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy, wherein the combination of R13b and R14 can be taken together to form optionally substituted heterocyclyl;
each R14 is, independently, H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl (e.g., substituted with an O-protecting group), optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted acyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H, alkyl, aryl, or phosphoryl), azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted aminoalkyl (e.g., hydroxyalkyl, alkyl, alkenyl, or alkynyl), optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl; and
each of R15 and R16 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl (e.g., R15 is H, and R16 is H or optionally substituted alkyl).
In some embodiments, R15 is H, and R16 is H or optionally substituted alkyl. In particular embodiments, R14 is H, acyl, or hydroxyalkyl. In some embodiments, R14 is halo. In some embodiments, both R14 and R15 are H. In some embodiments, both R15 and R16 are H. In some embodiments, each of R14 and R15 and R16 is H. In further embodiments, each of R13a and R13b is independently, H or optionally substituted alkyl.
Further non-limiting examples of modified cytosines include compounds of Formula (b36):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each R13b is, independently, H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy, wherein the combination of R13b and R14b can be taken together to form optionally substituted heterocyclyl;
each R14a and R14b is, independently, H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl (e.g., substituted with an O-protecting group), optionally substituted hydroxyalkenyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted acyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H, alkyl, aryl, phosphoryl, optionally substituted aminoalkyl, or optionally substituted carboxyaminoalkyl), azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl; and
each of R15 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.
In particular embodiments, R14b is an optionally substituted amino acid (e.g., optionally substituted lysine). In some embodiments, R14a is H.
In some embodiments, B is a modified guanine Exemplary modified guanines include compounds of Formula (b15)-(b17):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each of T4′, T4′′, T5′, T5′′, T6′, and T6′′ is independently, H, optionally substituted alkyl, or optionally substituted alkoxy, and wherein the combination of T4′ and T4′′ (e.g., as in T4) or the combination of T5′ and T5′′ (e.g., as in T5) or the combination of T6′ and T6′′ (e.g., as in T6) join together form O (oxo), S (thio), or Se (seleno);
each of V5 and V6 is, independently, O, S, N(RVd)nv, or C(RVd)nv, wherein nv is an integer from 0 to 2 and each RVd is, independently, H, halo, thiol, optionally substituted amino acid, cyano, amidine, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl), optionally substituted thioalkoxy, or optionally substituted amino; and
each of R17, R18, R19a, R19b, R21, R22, R23, and R24 is, independently, H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted amino acid.
Exemplary modified guanosines include compounds of Formula (b37)-(b40):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each of T4′ is, independently, H, optionally substituted alkyl, or optionally substituted alkoxy, and each T4 is, independently, O (oxo), S (thio), or Se (seleno);
each of R18, R19a, R19b, and R21 is, independently, H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted amino acid.
In some embodiments, R18 is H or optionally substituted alkyl. In further embodiments, T4 is oxo. In some embodiments, each of R19a and R19b is, independently, H or optionally substituted alkyl.
In some embodiments, B is a modified adenine. Exemplary modified adenines include compounds of Formula (b18)-(b20):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each V7 is, independently, O, S, N(RVe)nv, or C(RVe)nv, wherein nv is an integer from 0 to 2 and each RVe is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl);
each R25 is, independently, H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino;
each of R26a and R26b is, independently, H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoylalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, or polyethylene glycol group (e.g., —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl); or an amino-polyethylene glycol group (e.g., —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl);
each R27 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy or optionally substituted amino;
each R28 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and
each R29 is, independently, H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoylalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted alkoxy, or optionally substituted amino.
Exemplary modified adenines include compounds of Formula (b41)-(b43):




or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein
each R25 is, independently, H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino;
each of R26a and R26b is, independently, H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoylalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, or polyethylene glycol group (e.g., —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl); or an amino-polyethylene glycol group (e.g., —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl); and
each R27 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy, or optionally substituted amino.
In some embodiments, R26a is H, and R26b is optionally substituted alkyl. In some embodiments, each of R26a and R26b is, independently, optionally substituted alkyl. In particular embodiments, R27 is optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy. In other embodiments, R25 is optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy.
In particular embodiments, the optional substituent for R26a, R26b, or R29 is a polyethylene glycol group (e.g., —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl); or an amino-polyethylene glycol group (e.g., —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl).
In some embodiments, B may have Formula (b21):




wherein X12 is, independently, O, S, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene, xa is an integer from 0 to 3, and R12a and T2 are as described herein.

In some embodiments, B may have Formula (b22):




wherein R10′ is, independently, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl, and R11, R12a, T1, and T2 are as described herein.

In some embodiments, B may have Formula (b23):




wherein R10 is optionally substituted heterocyclyl (e.g., optionally substituted furyl, optionally substituted thienyl, or optionally substituted pyrrolyl), optionally substituted aryl (e.g., optionally substituted phenyl or optionally substituted naphthyl), or any substituent described herein (e.g., for R10); and wherein R11 (e.g., H or any substituent described herein), R12a (e.g., H or any substituent described herein), T1 (e.g., oxo or any substituent described herein), and T2 (e.g., oxo or any substituent described herein) are as described herein.

In some embodiments, B may have Formula (b24):




wherein R14′ is, independently, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkaryl, optionally substituted alkheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl, and R13a, R13b, R15, and T3 are as described herein.

In some embodiments, B may have Formula (b25):




wherein R14′ is optionally substituted heterocyclyl (e.g., optionally substituted furyl, optionally substituted thienyl, or optionally substituted pyrrolyl), optionally substituted aryl (e.g., optionally substituted phenyl or optionally substituted naphthyl), or any substituent described herein (e.g., for R14 or R14′); and wherein R13a (e.g., H or any substituent described herein), R13b (e.g., H or any substituent described herein), R15 (e.g., H or any substituent described herein), and T3 (e.g., oxo or any substituent described herein) are as described herein.

In some embodiments, B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil. In some embodiments, B may be:




In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methylpseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine (also known as 1-methylpseudouridine (m1ψ)), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um), 3,2′-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine.
In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O-dimethyl-cytidine (m5Cm), N4-acetyl-2′-O-methyl-cytidine (ac4Cm), N4,2′-O-dimethyl-cytidine (m4Cm), 5-formyl-2′-O-methyl-cytidine (f5Cm), N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.
In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m′A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2 m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am), N6,2′-O-dimethyl-adenosine (m6Am), N6,N6,2′-O-trimethyl-adenosine (m62Am), 1,2′-O-dimethyl-adenosine (m1Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.
In some embodiments, the modified nucleobase is a modified guanine Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m22Gm), 1-methyl-2′-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2′-O-methyl-guanosine (m2,7Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m′Im), and 2′-O-ribosylguanosine (phosphate) (Gr(p)).
The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and/or derivatives thereof, e.g., A includes adenine or adenine analogs, e.g., 7-deaza adenine).
Modifications on the Internucleoside Linkage
The modified nucleotides, which may be incorporated into a polynucleotide, primary construct, or mmRNA molecule, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
The α-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked cosmetic polynucleotides, primary constructs, or mmRNA molecules are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseudouridine).
Other internucleoside linkages that may be employed according to the present invention, including internucleoside linkages which do not contain a phosphorous atom, are described herein below.
Combinations of Modified Sugars, Nucleobases, and Internucleoside Linkages
The cosmetic polynucleotides, primary constructs, and mmRNA of the invention can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein. For examples, any of the nucleotides described herein in Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr) can be combined with any of the nucleobases described herein (e.g., in Formulas (b1)-(b43) or any other described herein).
Synthesis of Cosmetic Polypeptides, Primary Constructs, and mmRNA Molecules
The cosmetic polypeptides, cosmetic primary constructs, and cosmetic mmRNA molecules for use in accordance with the invention may be prepared according to any useful technique, as described herein. The modified nucleosides and nucleotides used in the synthesis of cosmetic polynucleotides, cosmetic primary constructs, and cosmetic mmRNA molecules disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditions (e.g., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are provided, a skilled artisan would be able to optimize and develop additional process conditions. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
Preparation of cosmetic polypeptides, cosmetic primary constructs, and cosmetic mmRNA molecules of the present invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
The reactions of the processes described herein can be carried out in suitable solvents, which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.
Resolution of racemic mixtures of modified nucleosides and nucleotides can be carried out by any of numerous methods known in the art. An example method includes fractional recrystallization using a “chiral resolving acid” which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.
Modified nucleosides and nucleotides (e.g., building block molecules) can be prepared according to the synthetic methods described in Ogata et al., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res. 22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568 (1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each of which are incorporated by reference in their entirety.
The cosmetic polypeptides, cosmetic primary constructs, and cosmetic mmRNA of the invention may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may or may not be uniformly modified in a cosmetic polynucleotide of the invention, or in a given predetermined sequence region thereof (e.g. one or more of the sequence regions represented in FIG. 1). In some embodiments, all nucleotides X in a cosmetic polynucleotide of the invention (or in a given sequence region thereof) are modified, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA such that the function of the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA is not substantially decreased. A modification may also be a 5′ or 3′ terminal modification. The cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).
In some embodiments, the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA includes a modified pyrimidine (e.g., a modified uracil/uridine/U or modified cytosine/cytidine/C). In some embodiments, the uracil or uridine (generally: U) in the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA molecule may be replaced with from about 1% to about 100% of a modified uracil or modified uridine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100% of a modified uracil or modified uridine). The modified uracil or uridine can be replaced by a compound having a single unique structure or by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures, as described herein). In some embodiments, the cytosine or cytidine (generally: C) in the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA molecule may be replaced with from about 1% to about 100% of a modified cytosine or modified cytidine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100% of a modified cytosine or modified cytidine). The modified cytosine or cytidine can be replaced by a compound having a single unique structure or by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures, as described herein).
In some embodiments, the present disclosure provides methods of synthesizing a cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA (e.g., the first region, first flanking region, or second flanking region) including n number of linked nucleosides having Formula (Ia-1):




comprising:

a) reacting a nucleotide of Formula (IV-1):




with a phosphoramidite compound of Formula (V-1):




wherein Y9 is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino, thiol, optionally substituted amino acid, or a peptide (e.g., including from 2 to 12 amino acids); and each P1, P2, and P3 is, independently, a suitable protecting group; and  denotes a solid support;
to provide a cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA of Formula (VI-1):




and

b) oxidizing or sulfurizing the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA of Formula (V) to yield a cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA of Formula (VII-1):




and

c) removing the protecting groups to yield the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA of Formula (Ia).
In some embodiments, steps a) and b) are repeated from 1 to about 10,000 times. In some embodiments, the methods further comprise a nucleotide (e.g., mmRNA molecule) selected from the group consisting of A, C, G and U adenosine, cytosine, guanosine, and uracil. In some embodiments, the nucleobase may be a pyrimidine or derivative thereof. In some embodiments, the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA is translatable.
Other components of cosmetic polynucleotides, cosmetic primary constructs, and cosmetic mmRNA are optional, and are beneficial in some embodiments. For example, a 5′ untranslated region (UTR) and/or a 3′UTR are provided, wherein either or both may independently contain one or more different nucleotide modifications. In such embodiments, nucleotide modifications may also be present in the translatable region. Also provided are cosmetic polynucleotides, cosmetic primary constructs, and cosmetic mmRNA containing a Kozak sequence.
Exemplary syntheses of modified nucleotides, which are incorporated into a modified nucleic acid or mmRNA, e.g., RNA or mRNA, are provided below in Scheme 1 through Scheme 11. Scheme 1 provides a general method for phosphorylation of nucleosides, including modified nucleosides.




Various protecting groups may be used to control the reaction. For example, Scheme 2 provides the use of multiple protecting and deprotecting steps to promote phosphorylation at the 5′ position of the sugar, rather than the 2′ and 3′ hydroxyl groups.




Modified nucleotides can be synthesized in any useful manner. Schemes 3, 4, and 7 provide exemplary methods for synthesizing modified nucleotides having a modified purine nucleobase; and Schemes 5 and 6 provide exemplary methods for synthesizing modified nucleotides having a modified pseudouridine or pseudoisocytidine, respectively.




















Schemes 8 and 9 provide exemplary syntheses of modified nucleotides. Scheme 10 provides a non-limiting biocatalytic method for producing nucleotides.












Scheme 11 provides an exemplary synthesis of a modified uracil, where the N1 position is modified with R12b, as provided elsewhere, and the 5′-position of ribose is phosphorylated. T1, T2, R12a, R12b, and r are as provided herein. This synthesis, as well as optimized versions thereof, can be used to modify other pyrimidine nucleobases and purine nucleobases (see e.g., Formulas (b1)-(b43)) and/or to install one or more phosphate groups (e.g., at the 5′ position of the sugar). This alkylating reaction can also be used to include one or more optionally substituted alkyl group at any reactive group (e.g., amino group) in any nucleobase described herein (e.g., the amino groups in the Watson-Crick base-pairing face for cytosine, uracil, adenine, and guanine)




Combinations of Nucleotides in mmRNA

Further examples of modified nucleotides and modified nucleotide combinations are provided below in Table 9. These combinations of modified nucleotides can be used to form the cosmetic polypeptides, cosmetic primary constructs, or cosmetic mmRNA of the invention.
Unless otherwise noted, the modified nucleotides may be completely substituted for the natural nucleotides of the modified nucleic acids or mmRNA of the invention. As a non-limiting example, the natural nucleotide uridine may be substituted with a modified nucleoside described herein. In another non-limiting example, the natural nucleotide uridine may be partially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%) with at least one of the modified nucleoside disclosed herein.








TABLE 9





Modified Nucleotide
Modified Nucleotide Combination







α-thio-cytidine
α-thio-cytidine/5-iodo-uridine



α-thio-cytidine/N1-methyl-pseudouridine



α-thio-cytidine/α-thio-uridine



α-thio-cytidine/5-methyl-uridine



α-thio-cytidine/pseudo-uridine



about 50% of the cytosines are α-thio-cytidine


pseudoisocytidine
pseudoisocytidine/5-iodo-uridine



pseudoisocytidine/N1-methyl-pseudouridine



pseudoisocytidine/α-thio-uridine



pseudoisocytidine/5-methyl-uridine



pseudoisocytidine/pseudouridine



about 25% of cytosines are pseudoisocytidine



pseudoisocytidine/about 50% of uridines are N1-



methyl-pseudouridine and about 50% of uridines are



pseudouridine



pseudoisocytidine/about 25% of uridines are N1-



methyl-pseudouridine and about 25% of uridines are



pseudouridine


pyrrolo-cytidine
pyrrolo-cytidine/5-iodo-uridine



pyrrolo-cytidine/N1-methyl-pseudouridine



pyrrolo-cytidine/α-thio-uridine



pyrrolo-cytidine/5-methyl-uridine



pyrrolo-cytidine/pseudouridine



about 50% of the cytosines are pyrrolo-cytidine


5-methyl-cytidine
5-methyl-cytidine/5-iodo-uridine



5-methyl-cytidine/N1-methyl-pseudouridine



5-methyl-cytidine/α-thio-uridine



5-methyl-cytidine/5-methyl-uridine



5-methyl-cytidine/pseudouridine



about 25% of cytosines are 5-methyl-cytidine



about 50% of cytosines are 5-methyl-cytidine



5-methyl-cytidine/5-methoxy-uridine



5-methyl-cytidine/5-bromo-uridine



5-methyl-cytidine/2-thio-uridine



5-methyl-cytidine/about 50% of uridines are 2-thio-



uridine



about 50% of uridines are 5-methyl-cytidine/about



50% of uridines are 2-thio-uridine


N4-acetyl-cytidine
N4-acetyl-cytidine/5-iodo-uridine



N4-acetyl-cytidine/N1-methyl-pseudouridine



N4-acetyl-cytidine/α-thio-uridine



N4-acetyl-cytidine/5-methyl-uridine



N4-acetyl-cytidine/pseudouridine



about 50% of cytosines are N4-acetyl-cytidine



about 25% of cytosines are N4-acetyl-cytidine



N4-acetyl-cytidine/5-methoxy-uridine



N4-acetyl-cytidine/5-bromo-uridine



N4-acetyl-cytidine/2-thio-uridine



about 50% of cytosines are N4-acetyl-cytidine/about



50% of uridines are 2-thio-uridine









Further examples of modified nucleotide combinations are provided below in Table 10. These combinations of modified nucleotides can be used to form the polypeptides, primary constructs, or mmRNA of the invention.








TABLE 10





Modified Nucleotide
Modified Nucleotide Combination







modified cytidine having
modified cytidine with (b10)/pseudouridine


one or more nucleobases
modified cytidine with (b10)/N1-methyl-


of Formula (b10)
pseudouridine



modified cytidine with (b10)/5-methoxy-uridine



modified cytidine with (b10)/5-methyl-uridine



modified cytidine with (b10)/5-bromo-uridine



modified cytidine with (b10)/2-thio-uridine



about 50% of cytidine substituted with



modified cytidine (b10)/about 50% of



uridines are 2-thio-uridine


modified cytidine having
modified cytidine with (b32)/pseudouridine


one or more nucleobases
modified cytidine with (b32)/N1-methyl-


of Formula (b32)
pseudouridine



modified cytidine with (b32)/5-methoxy-uridine



modified cytidine with (b32)/5-methyl-uridine



modified cytidine with (b32)/5-bromo-uridine



modified cytidine with (b32)/2-thio-uridine



about 50% of cytidine substituted with



modified cytidine (b32)/about 50% of



uridines are 2-thio-uridine


modified uridine having
modified uridine with (b1)/N4-acetyl-cytidine


one or more nucleobases
modified uridine with (b1)/5-methyl-cytidine


of Formula (b1)


modified uridine having
modified uridine with (b8)/N4-acetyl-cytidine


one or more nucleobases
modified uridine with (b8)/5-methyl-cytidine


of Formula (b8)


modified uridine having
modified uridine with (b28)/N4-acetyl-cytidine


one or more nucleobases
modified uridine with (b28)/5-methyl-cytidine


of Formula (b28)


modified uridine having
modified uridine with (b29)/N4-acetyl-cytidine


one or more nucleobases
modified uridine with (b29)/5-methyl-cytidine


of Formula (b29)


modified uridine having
modified uridine with (b30)/N4-acetyl-cytidine


one or more nucleobases
modified uridine with (b30)/5-methyl-cytidine


of Formula (b30)









In some embodiments, at least 25% of the cytosines are replaced by a compound of Formula (b10)-(b14) (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).
In some embodiments, at least 25% of the uracils are replaced by a compound of Formula (b1)-(b9) (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).
In some embodiments, at least 25% of the cytosines are replaced by a compound of Formula (b10)-(b14), and at least 25% of the uracils are replaced by a compound of Formula (b1)-(b9) (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).
IV. Pharmaceutical Compositions
Formulation, Administration, Delivery and Dosing
The present invention provides cosmetic polynucleotides, cosmetic primary constructs and cosmetic mmRNA compositions and complexes in combination with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g. therapeutically and/or prophylactically active substances. General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to cosmetic polynucleotides, cosmetic primary constructs and cosmetic mmRNA to be delivered as described herein.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
A pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
Formulations
The cosmetic polynucleotide, cosmetic primary construct, and cosmetic mmRNA of the invention can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the cosmetic polynucleotide, cosmetic primary construct, or mmRNA); (4) alter the biodistribution (e.g., target the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present invention can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with cosmetic polynucleotide, primary cosmetic construct, or cosmetic mmRNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof. Accordingly, the formulations of the invention can include one or more excipients, each in an amount that together increases the stability of the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA, increases cell transfection by the cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA, increases the expression of cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA encoded protein, and/or alters the release profile of cosmetic polynucleotide, cosmetic primary construct, or cosmetic mmRNA encoded proteins. Further, the cosmetic primary construct and cosmetic mmRNA of the present invention may be formulated using self-assembled nucleic acid nanoparticles.
Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient may generally be equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage including, but not limited to, one-half or one-third of such a dosage.
Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.
In some embodiments, the formulations described herein may contain at least one cosmetic mmRNA. As a non-limiting example, the formulations may contain 1, 2, 3, 4 or 5 cosmetic mmRNA. In one embodiment the formulation may contain modified mRNA encoding proteins selected from categories such as, cosmetic proteins. In one embodiment, the formulation contains at least three cosmetic modified mRNA encoding cosmetic proteins. In one embodiment, the formulation contains at least five cosmetic modified mRNA encoding cosmetic proteins. Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
In some embodiments, the particle size of the lipid nanoparticle may be increased and/or decreased. The change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the cosmetic modified mRNA delivered to mammals.
Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, surface active agents and/or emulsifiers, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in the pharmaceutical formulations of the invention.
Lipidoids
The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of cosmetic polynucleotides, primary constructs or mmRNA (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all of which are incorporated herein in their entireties).
While these lipidoids have been used to effectively deliver double stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; all of which is incorporated herein in their entirety), the present disclosure describes their formulation and use in delivering single stranded cosmetic polynucleotides, primary constructs, or mmRNA. Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in an effective delivery of the cosmetic polynucleotide, primary construct, or mmRNA, as judged by the production of an encoded protein, following the injection of a lipidoid formulation via localized and/or systemic routes of administration. Lipidoid complexes of cosmetic polynucleotides, primary constructs, or mmRNA can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.
In vivo delivery of nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety). As an example, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy. Formulations with the different lipidoids, including, but not limited to penta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401:61 (2010); herein incorporated by reference in its entirety), C12-200 (including derivatives and variants), and MD1, can be tested for in vivo activity.
The lipidoid referred to herein as “98N12-5” is disclosed by Akinc et al., Mol Ther. 2009 17:872-879 and is incorporated by reference in its entirety. (See FIG. 2)
The lipidoid referred to herein as “C12-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869 (see FIG. 2) and Liu and Huang, Molecular Therapy. 2010 669-670 (see FIG. 2); both of which are herein incorporated by reference in their entirety. The lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to cosmetic polynucleotide, primary construct, or mmRNA. As an example, formulations with certain lipidoids, include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (C14 alkyl chain length). As another example, formulations with certain lipidoids, include, but are not limited to, C12-200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.
In one embodiment, a cosmetic polynucleotide, primary construct, or mmRNA formulated with a lipidoid for systemic intravenous administration can target the liver. For example, a final optimized intravenous formulation using a cosmetic polynucleotide, primary construct, or mmRNA, and comprising a lipid molar composition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipid with a final weight ratio of about 7.5 to 1 total lipid to cosmetic polynucleotide, primary construct, or mmRNA, and a C14 alkyl chain length on the PEG lipid, with a mean particle size of roughly 50-60 nm, can result in the distribution of the formulation to be greater than 90% to the liver. (see, Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety). In another example, an intravenous formulation using a C12-200 (see U.S. provisional application 61/175,770 and published international application WO2010129709, each of which is herein incorporated by reference in their entirety) lipidoid may have a molar ratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG, with a weight ratio of 7 to 1 total lipid to cosmetic polynucleotide, primary construct, or mmRNA, and a mean particle size of 80 nm may be effective to deliver cosmetic polynucleotide, primary construct, or mmRNA to hepatocytes (see, Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869 herein incorporated by reference in its entirety). In another embodiment, an MD1 lipidoid-containing formulation may be used to effectively deliver cosmetic polynucleotide, primary construct, or mmRNA to hepatocytes in vivo. The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879 herein incorporated by reference in its entirety), use of a lipidoid-formulated cosmetic polynucleotide, primary construct, or mmRNA to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited. Use of lipidoid formulations to deliver siRNA in vivo to other non-hepatocyte cells such as myeloid cells and endothelium has been reported (see Akinc et al., Nat Biotechnol. 2008 26:561-569; Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; Cho et al. Adv. Funct. Mater. 2009 19:3112-3118; 8th International Judah Folkman Conference, Cambridge, Mass. Oct. 8-9, 2010; each of which is herein incorporated by reference in its entirety). Effective delivery to myeloid cells, such as monocytes, lipidoid formulations may have a similar component molar ratio. Different ratios of lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formulation of the cosmetic polynucleotide, primary construct, or mmRNA for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc. For example, the component molar ratio may include, but is not limited to, 50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG (see Leuschner et al., Nat Biotechnol 2011 29:1005-1010; herein incorporated by reference in its entirety). The use of lipidoid formulations for the localized delivery of nucleic acids to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, may not require all of the formulation components desired for systemic delivery, and as such may comprise only the lipidoid and the cosmetic polynucleotide, primary construct, or mmRNA.
Combinations of different lipidoids may be used to improve the efficacy of cosmetic polynucleotide, primary construct, or mmRNA directed protein production as the lipidoids may be able to increase cell transfection by the cosmetic polynucleotide, primary construct, or mmRNA; and/or increase the translation of encoded protein (see Whitehead et al., Mol. Ther. 2011, 19:1688-1694, herein incorporated by reference in its entirety).
Liposomes, Lipoplexes, and Lipid Nanoparticles
The cosmetic polynucleotide, primary construct, and mmRNA of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceutical compositions of polynucleotide, primary construct, or mmRNA include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).
In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; all of which are incorporated herein in their entireties). The original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. The liposome formulations are composed of 3 to 4 lipid components in addition to the cosmetic polynucleotide, primary construct, or mmRNA. As an example a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.
In one embodiment, pharmaceutical compositions may include liposomes which may be formed to deliver mmRNA which may encode at least one immunogen. The mmRNA may be encapsulated by the liposome and/or it may be contained in an aqueous core which may then be encapsulated by the liposome (see International Pub. Nos. WO2012031046, WO2012031043, WO2012030901 and WO2012006378; each of which is herein incorporated by reference in their entirety). In another embodiment, the mmRNA which may encode an immunogen may be formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid which can interact with the mmRNA anchoring the molecule to the emulsion particle (see International Pub. No. WO2012006380; herein incorporated by reference in its entirety). In yet another embodiment, the lipid formulation may include at least cationic lipid, a lipid which may enhance transfection and a least one lipid which contains a hydrophilic head group linked to a lipid moiety (International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; each of which is herein incorporated by reference in their entirety). In another embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA encoding an immunogen may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers (see U.S. Pub. No. 20120177724, herein incorporated by reference in its entirety).
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA may be formulated in a liposome comprising a cationic lipid. The liposome may have a molar ratio of nitrogen atoms in the cationic lipid to the phophates in the RNA (N:P ratio) of between 1:1 and 20:1 as described in International Publication No. WO2013006825, herein incorporated by reference in its entirety. In another embodiment, the liposome may have a N:P ratio of greater than 20:1 or less than 1:1.
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid-polycation complex. The formation of the lipid-polycation complex may be accomplished by methods known in the art and/or as described in U.S. Pub. No. 20120178702, herein incorporated by reference in its entirety. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in International Pub. No. WO2012013326; herein incorporated by reference in its entirety. In another embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid-polycation complex which may further include a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
The liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. In one example by Semple et al. (Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated by reference in its entirety), the liposome formulation was composed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA. As another example, changing the composition of the cationic lipid could more effectively deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated by reference in its entirety).
In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations. As a non-limiting example, LNP formulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol. In another embodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.
In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA may be formulated in a lipid nanoparticle such as those described in International Publication No. WO2012170930, herein incorporated by reference in its entirety.
In one embodiment, the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865 and WO2008103276, U.S. Pat. Nos. 7,893,302, 7,404,969 and 8,283,333 and US Patent Publication No. US20100036115 and US20120202871; each of which is herein incorporated by reference in their entirety. In another embodiment, the cationic lipid may be selected from, but not limited to, formula A described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365 and WO2012044638; each of which is herein incorporated by reference in their entirety. In yet another embodiment, the cationic lipid may be selected from, but not limited to, formula CLI-CLXXIX of International Publication No. WO2008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969 and formula I-VI of US Patent Publication No. US20100036115; each of which is herein incorporated by reference in their entirety. As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)—N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21 Z)—N,N-dimethylheptacosa-18,21-dien-8-amine, (17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)—N,N-dimetylheptacos-18-en-10-amine, (17Z)—N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine, (17Z)—N,N-dimethylnonacos-17-en-10-amine, (24Z)—N,N-dimethyltritriacont-24-en-10-amine, (20Z)—N,N-dimethylnonacos-20-en-10-amine, (22Z)—N,N-dimethylhentriacont-22-en-10-amine, (16Z)—N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcycIopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecyIcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof
In one embodiment, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety.
In one embodiment, the cationic lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 and WO201021865; each of which is herein incorporated by reference in their entirety.
In one embodiment, the LNP formulations of the cosmetic polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG at 3% lipid molar ratio. In another embodiment, the LNP formulations polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG at 1.5% lipid molar ratio.
In one embodiment, the pharmaceutical compositions of the cosmetic polynucleotides, primary constructs and/or mmRNA may include at least one of the PEGylated lipids described in International Publication No. 2012099755, herein incorporated by reference.
In one embodiment, the LNP formulation may contain PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-2000). In one embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component. In another embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol. As a non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012; PMID: 22908294; herein incorporated by reference in its entirety). As another non-limiting example, modified RNA described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U.S. Pub. No. 20120207845; herein incorporated by reference in its entirety.
In one embodiment, the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or WO2008103276, each of which is herein incorporated by reference in their entirety. As a non-limiting example, modified RNA described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or WO2008103276; each of which is herein incorporated by reference in their entirety.
In one embodiment, LNP formulations described herein may comprise a polycationic composition. As a non-limiting example, the polycationic composition may be selected from formula 1-60 of US Patent Publication No. US20050222064; herein incorporated by reference in its entirety. In another embodiment, the LNP formulations comprising a polycationic composition may be used for the delivery of the modified RNA described herein in vivo and/or in vitro.
In one embodiment, the LNP formulations described herein may additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US Patent Publication No. US20050222064; herein incorporated by reference in its entirety.
In one embodiment, the pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
The nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a modified nucleic acid molecule (e.g., mmRNA). As a non-limiting example, the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. WO2012109121; herein incorporated by reference in its entirety).
Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity. The rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation. The ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.
In one embodiment, the internal ester linkage may be located on either side of the saturated carbon. Non-limiting examples of reLNPs include,




In one embodiment, an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen. (U.S. Publication No. 20120189700 and International Publication No. WO2012099805; each of which is herein incorporated by reference in their entirety). The polymer may encapsulate the nanospecies or partially encapsulate the nanospecies. The immunogen may be a recombinant protein, a modified RNA and/or a primary construct described herein. In one embodiment, the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen.
Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier. Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes). Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosla tissue within seconds or within a few hours. Large polymeric nanoparticles (200 nm-500 nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; each of which is herein incorporated by reference in their entirety). The transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT). As a non-limiting example, compositions which can penetrate a mucosal barrier may be made as described in U.S. Pat. No. 8,241,670, herein incorporated by reference in its entirety.
The lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and/or biocompatible. The polymeric material may additionally be irradiated. As a non-limiting example, the polymeric material may be gamma irradiated (See e.g., International App. No. WO201282165, herein incorporated by reference in its entirety). Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), and trimethylene carbonate, polyvinylpyrrolidone. The lipid nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer (such as a branched polyether-polyamide block copolymer described in International Publication No. WO2013012476, herein incorporated by reference in its entirety), and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., US Publication 20120121718 and US Publication 20100003337 and U.S. Pat. No. 8,263,665; each of which is herein incorporated by reference in their entirety). The co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created. For example, the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; herein incorporated by reference in its entirety).
The vitamin of the polymer-vitamin conjugate may be vitamin E. The vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).
The lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, mmRNA, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase. The surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle. (see e.g., US Publication 20100215580 and US Publication 20080166414; each of which is herein incorporated by reference in their entirety).
The mucus penetrating lipid nanoparticles may comprise at least one mmRNA described herein. The mmRNA may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the particle. The mmRNA may be covalently coupled to the lipid nanoparticle. Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.
In one embodiment, the cosmetic polynucleotide, primary construct, or mmRNA is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293 Weide et al. J Immunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6; 104:4095-4100; deFougerolles Hum Gene Ther. 2008 19:125-132; all of which are incorporated herein by reference in its entirety).
In one embodiment such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all of which are incorporated herein by reference in its entirety). One example of passive targeting of formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticle formulations which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein incorporated by reference in its entirety). Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all of which are incorporated herein by reference in its entirety).
In one embodiment, the cosmetic polynucleotide, primary construct, or mmRNA is formulated as a solid lipid nanoparticle. A solid lipid nanoparticle (SLN) may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers. In a further embodiment, the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; herein incorporated by reference in its entirety).
Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of polynucleotide, primary construct, or mmRNA directed protein production as these formulations may be able to increase cell transfection by the cosmetic polynucleotide, primary construct, or mmRNA; and/or increase the translation of encoded protein. One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; herein incorporated by reference in its entirety). The liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the cosmetic polynucleotide, primary construct, or mmRNA.
In one embodiment, the polynucleotides, primary constructs, and/or the mmRNA of the present invention can be formulated for controlled release and/or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In one embodiment, the polynucleotides, primary constructs or the mmRNA may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term “encapsulate” means to enclose, surround or encase. As it relates to the formulation of the compounds of the invention, encapsulation may be substantial, complete or partial. The term “substitantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulation” means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the invention using fluorescence and/or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the delivery agent.
In one embodiment, the controlled release formulation may include, but is not limited to, tri-block co-polymers. As a non-limiting example, the formulation may include two different types of tri-block co-polymers (International Pub. No. WO2012131104 and WO2012131106; each of which is herein incorporated by reference in its entirety).
In another embodiment, the polynucleotides, primary constructs, or the mmRNA may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art. As a non-limiting example, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).
In another embodiment, the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject. As another non-limiting example, the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
In one embodiment, the cosmetic polynucleotide, primary construct, or mmRNA formulation for controlled release and/or targeted delivery may also include at least one controlled release coating. Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).
In one embodiment, the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
In one embodiment, the polynucleotides, primary constructs, and/or the mmRNA of the present invention may be encapsulated in a therapeutic nanoparticle. Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286 and US20120288541 and U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and 8,318,211 each of which is herein incorporated by reference in their entirety. In another embodiment, therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, herein incorporated by reference in its entirety.
In one embodiment, the therapeutic nanoparticle may be formulated for sustained release. As used herein, “sustained release” refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the polynucleotides, primary constructs, and mmRNA of the present invention (see International Pub No. 2010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, each of which is herein incorporated by reference in their entirety).
In one embodiment, the therapeutic nanoparticles may be formulated to be target specific. As a non-limiting example, the therapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO2011084518; herein incorporated by reference in its entirety). In one embodiment, the therapeutic nanoparticles may be formulated to be cancer specific. As a non-limiting example, the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in their entirety.
In one embodiment, the nanoparticles of the present invention may comprise a polymeric matrix. As a non-limiting example, the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
In one embodiment, the therapeutic nanoparticle comprises a diblock copolymer. In one embodiment, the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof
As a non-limiting example the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat. No. 8,236,330, each of which is herein incorporated by reference in their entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968, and International Publication No. WO2012166923, each of which is herein incorporated by reference in its entirety).
In one embodiment, the therapeutic nanoparticle may comprise a multiblock copolymer (See e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910; each of which is herein incorporated by reference in its entirety).
In one embodiment, the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer. (See e.g., U.S. Pub. No. 20120076836; herein incorporated by reference in its entirety).
In one embodiment, the therapeutic nanoparticle may comprise at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof
In one embodiment, the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.
In one embodiment, the therapeutic nanoparticles may comprise at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No. 8,287,849; herein incorporated by reference in its entirety) and combinations thereof.
In one embodiment, the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
In another embodiment, the therapeutic nanoparticle may include a conjugation of at least one targeting ligand. The targeting ligand may be any ligand known in the art such as, but not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; herein incorporated by reference in its entirety).
In one embodiment, the therapeutic nanoparticle may be formulated in an aqueous solution which may be used to target cancer (see International Pub No. WO2011084513 and US Pub No. US20110294717, each of which is herein incorporated by reference in their entirety).
In one embodiment, the polynucleotides, primary constructs, or mmRNA may be encapsulated in, linked to and/or associated with synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in International Pub. Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149411 WO2012149454 and WO2013019669, and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US20120244222, each of which is herein incorporated by reference in their entirety. The synthetic nanocarriers may be formulated using methods known in the art and/or described herein. As a non-limiting example, the synthetic nanocarriers may be formulated by the methods described in International Pub Nos. WO2010005740, WO2010030763 and WO201213501 and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US2012024422, each of which is herein incorporated by reference in their entirety. In another embodiment, the synthetic nanocarrier formulations may be lyophilized by methods described in International Pub. No. WO2011072218 and U.S. Pat. No. 8,211,473; each of which is herein incorporated by reference in their entirety.
In one embodiment, the synthetic nanocarriers may contain reactive groups to release the cosmetic polynucleotides, primary constructs and/or mmRNA described herein (see International Pub. No. WO20120952552 and US Pub No. US20120171229, each of which is herein incorporated by reference in their entirety).
In one embodiment, the synthetic nanocarriers may contain an immunostimulatory agent to enhance the immune response from delivery of the synthetic nanocarrier. As a non-limiting example, the synthetic nanocarrier may comprise a Th1 immunostimulatory agent which may enhance a Th1-based response of the immune system (see International Pub No. WO2010123569 and US Pub. No. US20110223201, each of which is herein incorporated by reference in its entirety).
In one embodiment, the synthetic nanocarriers may be formulated for targeted release. In one embodiment, the synthetic nanocarrier is formulated to release the cosmetic polynucleotides, primary constructs and/or mmRNA at a specified pH and/or after a desired time interval. As a non-limiting example, the synthetic nanoparticle may be formulated to release the cosmetic polynucleotides, primary constructs and/or mmRNA after 24 hours and/or at a pH of 4.5 (see International Pub. Nos. WO2010138193 and WO2010138194 and US Pub Nos. US20110020388 and US20110027217, each of which is herein incorporated by reference in their entireties).
In one embodiment, the synthetic nanocarriers may be formulated for controlled and/or sustained release of the cosmetic polynucleotides, primary constructs and/or mmRNA described herein. As a non-limiting example, the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Pub No. WO2010138192 and US Pub No. 20100303850, each of which is herein incorporated by reference in their entirety.
In one embodiment, the synthetic nanocarrier may be formulated for use as a vaccine. In one embodiment, the synthetic nanocarrier may encapsulate at least one polynucleotide, primary construct and/or mmRNA which encode at least one antigen. As a non-limiting example, the synthetic nanocarrier may include at least one antigen and an excipient for a vaccine dosage form (see International Pub No. WO2011150264 and US Pub No. US20110293723, each of which is herein incorporated by reference in their entirety). As another non-limiting example, a vaccine dosage form may include at least two synthetic nanocarriers with the same or different antigens and an excipient (see International Pub No. WO2011150249 and US Pub No. US20110293701, each of which is herein incorporated by reference in their entirety). The vaccine dosage form may be selected by methods described herein, known in the art and/or described in International Pub No. WO2011150258 and US Pub No. US20120027806, each of which is herein incorporated by reference in their entirety).
In one embodiment, the synthetic nanocarrier may comprise at least one polynucleotide, primary construct and/or mmRNA which encodes at least one adjuvant. As non-limiting example, the adjuvant may comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (See e.g, U.S. Pat. No. 8,241,610; herein incorporated by reference in its entirety). In another embodiment, the synthetic nanocarrier may comprise at least one polynucleotide, primary construct and/or mmRNA and an adjuvant. As a non-limiting example, the synthetic nanocarrier comprising and adjuvant may be formulated by the methods described in International Pub No. WO2011150240 and US Pub No. US20110293700, each of which is herein incorporated by reference in its entirety.
In one embodiment, the synthetic nanocarrier may encapsulate at least one polynucleotide, primary construct and/or mmRNA which encodes a peptide, fragment or region from a virus. As a non-limiting example, the synthetic nanocarrier may include, but is not limited to, the nanocarriers described in International Pub No. WO2012024621, WO201202629, WO2012024632 and US Pub No. US20120064110, US20120058153 and US20120058154, each of which is herein incorporated by reference in their entirety.
In one embodiment, the synthetic nanocarrier may be coupled to a cosmetic polynucleotide, primary construct or mmRNA which may be able to trigger a humoral and/or cytotoxic T lymphocyte (CTL) response (See e.g., International Publication No. WO2013019669, herein incorporated by reference in its entirety).
In one embodiment, the nanoparticle may be optimized for oral administration. The nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof. As a non-limiting example, the nanoparticle may be formulated by the methods described in U.S. Pub. No. 20120282343; herein incorporated by reference in its entirety.
Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles
The cosmetic polynucleotide, primary construct, and mmRNA of the invention can be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers which may be used for delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Reasearch Corp., Pasadena, Calif.) formulations from MIRUS® Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PHASERX® polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM (Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers. RONDELTM (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, Calif.) and pH responsive co-block polymers such as, but not limited to, PHASERX® (Seattle, Wash.).
A non-limiting example of chitosan formulation includes a core of positively charged chitosan and an outer portion of negatively charged substrate (U.S. Pub. No. 20120258176; herein incorporated by reference in its entirety). Chitosan includes, but is not limited to N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof
In one embodiment, the polymers used in the present invention have undergone processing to reduce and/or inhibit the attachment of unwanted substances such as, but not limited to, bacteria, to the surface of the polymer. The polymer may be processed by methods known and/or described in the art and/or described in International Pub. No. WO2012150467, herein incorporated by reference in its entirety.
A non-limiting example of PLGA formulations include, but are not limited to, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
Many of these polymer approaches have demonstrated efficacy in delivering oligonucleotides in vivo into the cell cytoplasm (reviewed in deFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated by reference in its entirety). Two polymer approaches that have yielded robust in vivo delivery of nucleic acids, in this case with small interfering RNA (siRNA), are dynamic polyconjugates and cyclodextrin-based nanoparticles. The first of these delivery approaches uses dynamic polyconjugates and has been shown in vivo in mice to effectively deliver siRNA and silence endogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887; herein incorporated by reference in its entirety). This particular approach is a multicomponent polymer system whose key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and N-acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887; herein incorporated by reference in its entirety). On binding to the hepatocyte and entry into the endosome, the polymer complex disassembles in the low-pH environment, with the polymer exposing its positive charge, leading to endosomal escape and cytoplasmic release of the siRNA from the polymer. Through replacement of the N-acetylgalactosamine group with a mannose group, it was shown one could alter targeting from asialoglycoprotein receptor-expressing hepatocytes to sinusoidal endothelium and Kupffer cells. Another polymer approach involves using transferrin-targeted cyclodextrin-containing polycation nanoparticles. These nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene product in transferrin receptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et al., Cancer Res. 2005 65: 8984-8982; herein incorporated by reference in its entirety) and siRNA formulated in these nanoparticles was well tolerated in non-human primates (Heidel et al., Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated by reference in its entirety). Both of these delivery strategies incorporate rational approaches using both targeted delivery and endosomal escape mechanisms.
The polymer formulation can permit the sustained or delayed release of polynucleotide, primary construct, or mmRNA (e.g., following intramuscular or subcutaneous injection). The altered release profile for the cosmetic polynucleotide, primary construct, or mmRNA can result in, for example, translation of an encoded protein over an extended period of time. The polymer formulation may also be used to increase the stability of the cosmetic polynucleotide, primary construct, or mmRNA. Biodegradable polymers have been previously used to protect nucleic acids other than mmRNA from degradation and been shown to result in sustained release of payloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 2010 7:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct. 1; Chu et al., Acc Chem Res. 2012 Jan. 13; Manganiello et al., Biomaterials. 2012 33:2301-2309; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Singha et al., Nucleic Acid Ther. 2011 2:133-147; deFougerolles Hum Gene Ther. 2008 19:125-132; Schaffert and Wagner, Gene Ther. 2008 16:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 2011 8:1455-1468; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010 464:1067-1070; each of which is herein incorporated by reference in its entirety).
In one embodiment, the pharmaceutical compositions may be sustained release formulations. In a further embodiment, the sustained release formulations may be for subcutaneous delivery. Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).
As a non-limiting example modified mRNA may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the modified mRNA in the PLGA microspheres while maintaining the integrity of the modified mRNA during the encapsulation process. EVAc are non-biodegradeable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications (e.g., extended release products Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a sustained release progesterone intrauterine device; transdermal delivery systems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosity at temperatures less than 5° C. and forms a solid gel at temperatures greater than 15° C. PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interaction to provide a stabilizing effect.
Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by, but not limited by, folate, transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010 464:1067-1070; each of which is herein incorporated by reference in its entirety).
The modified nucleic acid, and mmRNA of the invention may be formulated with or in a polymeric compound. The polymer may include at least one polymer such as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic multi-block copolymers, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containing polymers, dextran polymers, dextran polymer derivatives or combinations thereof.
As a non-limiting example, the modified nucleic acid or mmRNA of the invention may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No. 6,177,274; herein incorporated by reference in its entirety. The formulation may be used for transfecting cells in vitro or for in vivo delivery of the modified nucleic acid and mmRNA. In another example, the modified nucleic acid and mmRNA may be suspended in a solution or medium with a cationic polymer, in a dry pharmaceutical composition or in a solution that is capable of being dried as described in U.S. Pub. Nos. 20090042829 and 20090042825; each of which are herein incorporated by reference in their entireties.
As another non-limiting example the cosmetic polynucleotides, primary constructs or mmRNA of the invention may be formulated with a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat. No. 8,236,330, herein incorporated by reference in their entireties) or PLGA-PEG-PLGA block copolymers (See U.S. Pat. No. 6,004,573, herein incorporated by reference in its entirety). As a non-limiting example, the cosmetic polynucleotides, primary constructs or mmRNA of the invention may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968, herein incorporated by reference in its entirety).
A polyamine derivative may be used to deliver nucleic acids or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No. 20100260817 herein incorporated by reference in its entirety). As a non-limiting example, a pharmaceutical composition may include the modified nucleic acids and mmRNA and the polyamine derivative described in U.S. Pub. No. 20100260817 (the contents of which are incorporated herein by reference in its entirety. As a non-limiting example the cosmetic polynucleotides, primary constructs and mmRNA of the present invention may be delivered using a polyaminde polymer such as, but not limited to, a polymer comprising a 1,3-dipolar addition polymer prepared by combining a carbohydrate diazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280; herein incorporated by reference in its entirety).
In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA of the present invention may be formulated with at least one polymer and/or derivatives thereof described in International Publication Nos. WO2011115862, WO2012082574 and WO2012068187 and U.S. Pub. No. 20120283427, each of which are herein incorporated by reference in their entireties. In another embodiment, the modified nucleic acid or mmRNA of the present invention may be formulated with a polymer of formula Z as described in WO2011115862, herein incorporated by reference in its entirety. In yet another embodiment, the modified nucleic acid or mmRNA may be formulated with a polymer of formula Z, Z′ or Z′′ as described in International Pub. Nos. WO2012082574 or WO2012068187 and U.S. Pub. No. 2012028342, each of which are herein incorporated by reference in their entireties. The polymers formulated with the modified RNA of the present invention may be synthesized by the methods described in International Pub. Nos. WO2012082574 or WO2012068187, each of which are herein incorporated by reference in their entireties.
The cosmetic polynucleotides, primary constructs or mmRNA of the invention may be formulated with at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof
Formulations of cosmetic polynucleotides, primary constructs or mmRNA of the invention may include at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers or combinations thereof
For example, the modified nucleic acid or mmRNA of the invention may be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof. The biodegradable cationic lipopolymer may be made by methods known in the art and/or described in U.S. Pat. No. 6,696,038, U.S. App. Nos. 20030073619 and 20040142474 each of which is herein incorporated by reference in their entireties. The poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Pub. No. 20100004315, herein incorporated by reference in its entirety. The biodegradabale polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Pat. Nos. 6,517,869 and 6,267,987, the contents of which are each incorporated herein by reference in their entirety. The linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,652,886. The PAGA polymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,217,912 herein incorporated by reference in its entirety. The PAGA polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines, polylactides and poly(lactide-co-glycolides). The biodegradable cross-linked cationic multi-block copolymers may be made my methods known in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 each of which are herein incorporated by reference in their entireties. For example, the multi-block copolymers may be synthesized using linear polyethyleneimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines. Further, the composition or pharmaceutical composition may be made by the methods known in the art, described herein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 each of which are herein incorporated by reference in their entireties.
The polynucleotides, primary constructs, and mmRNA of the invention may be formulated with at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
The polynucleotides, primary construct, mmRNA of the invention may be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include those known in the art and described in US Pub. No. 20120269761, herein incorporated by reference in its entirety.
In one embodiment, the polymers described herein may be conjugated to a lipid-terminating PEG. As a non-limiting example, PLGA may be conjugated to a lipid-terminating PEG forming PLGA-DSPE-PEG. As another non-limiting example, PEG conjugates for use with the present invention are described in International Publication No. WO2008103276, herein incorporated by reference in its entirety. The polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. Pat. No. 8,273,363, herein incorporated by reference in its entirety.
In one embodiment, the modified RNA described herein may be conjugated with another compound. Non-limiting examples of conjugates are described in U.S. Pat. Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties. In another embodiment, modified RNA of the present invention may be conjugated with conjugates of formula 1-122 as described in U.S. Pat. Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties. The cosmetic polynucleotides, primary constructs and/or mmRNA described herein may be conjugated with a metal such as, but not limited to, gold. (See e.g., Giljohann et al. Journ. Amer. Chem. Soc. 2009 131(6): 2072-2073; herein incorporated by reference in its entirety). In another embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA described herein may be conjugated and/or encapsulated in gold-nanoparticles. (Interantional Pub. No. WO201216269 and U.S. Pub. No. 20120302940; each of which is herein incorporated by reference in its entirety).
As described in U.S. Pub. No. 20100004313, herein incorporated by reference in its entirety, a gene delivery composition may include a nucleotide sequence and a poloxamer. For example, the modified nucleic acid and mmRNA of the present invention may be used in a gene delivery composition with the poloxamer described in U.S. Pub. No. 20100004313.
In one embodiment, the polymer formulation of the present invention may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups. The polymer formulation may be contacted with a cationic lipopolymer using the methods described in U.S. Pub. No. 20090042829 herein incorporated by reference in its entirety. The cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B—[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) and combinations thereof.
The cosmetic polynucleotides, primary constructs and/or mmRNA of the invention may be formulated in a polyplex of one or more polymers (U.S. Pub. No. 20120237565 and 20120270927; each of which is herein incorporated by reference in its entirety). In one embodiment, the polyplex comprises two or more cationic polymers. The catioinic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI.
The cosmetic polynucleotide, primary construct, and mmRNA of the invention can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate. Components may be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-tuning of the nanoparticle so to delivery of the polynucleotide, primary construct and mmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller et al., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug Deliv Rev. 2011 63:748-761; Endres et al., Biomaterials. 2011 32:7721-7731; Su et al., Mol Pharm. 2011 Jun. 6; 8(3):774-87; herein incorporated by reference in its entirety). As a non-limiting example, the nanoparticle may comprise a plurality of polymers such as, but not limited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (International Pub. No. WO20120225129; herein incorporated by reference in its entirety).
Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver polynucleotides, primary constructs and mmRNA in vivo. In one embodiment, a lipid coated calcium phosphate nanoparticle, which may also contain a targeting ligand such as anisamide, may be used to deliver the polynucleotide, primary construct and mmRNA of the present invention. For example, to effectively deliver siRNA in a mouse metastatic lung model a lipid coated calcium phosphate nanoparticle was used (Li et al., J Contr Rel. 2010 142: 416-421; Li et al., J Contr Rel. 2012 158:108-114; Yang et al., Mol Ther. 2012 20:609-615; herein incorporated by reference in its entirety). This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the siRNA.
In one embodiment, calcium phosphate with a PEG-polyanion block copolymer may be used to delivery polynucleotides, primary constructs and mmRNA (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel. 2006 111:368-370; herein incorporated by reference in its entirety).
In one embodiment, a PEG-charge-conversional polymer (Pitella et al., Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle to deliver the cosmetic polynucleotides, primary constructs and mmRNA of the present invention. The PEG-charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a polycation at acidic pH, thus enhancing endosomal escape.
The use of core-shell nanoparticles has additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-13001). The complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle. For example, the core-shell nanoparticles may efficiently deliver siRNA to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.
In one embodiment, a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG may be used to delivery of the polynucleotide, primary construct and mmRNA of the present invention. As a non-limiting example, in mice bearing a luciferease-expressing tumor, it was determined that the lipid-polymer-lipid hybrid nanoparticle significantly suppressed luciferase expression, as compared to a conventional lipoplex (Shi et al, Angew Chem Int Ed. 2011 50:7027-7031; herein incorporated by reference in its entirety).
In one embodiment, the lipid nanoparticles may comprise a core of the modified nucleic acid molecules disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the modified nucleic acids in the core.
Core-shell nanoparticles for use with the modified nucleic acid molecules of the present invention are described and may be formed by the methods described in U.S. Pat. No. 8,313,777 herein incorporated by reference in its entirety.
In one embodiment, the core-shell nanoparticles may comprise a core of the modified nucleic acid molecules disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the modified nucleic acid molecules in the core. As a non-limiting example, the core-shell nanoparticle may be used to treat an eye disease or disorder (See e.g. US Publication No. 20120321719, herein incorporated by reference in its entirety).
In one embodiment, the polymer used with the formulations described herein may be a modified polymer (such as, but not limited to, a modified polyacetal) as described in International Publication No. WO2011120053, herein incorporated by reference in its entirety.
Peptides and Proteins
The cosmetic polynucleotide, primary construct, and mmRNA of the invention can be formulated with peptides and/or proteins in order to increase transfection of cells by the cosmetic polynucleotide, primary construct, or mmRNA. In one embodiment, peptides such as, but not limited to, cell penetrating peptides and proteins and peptides that enable intracellular delivery may be used to deliver pharmaceutical formulations. A non-limiting example of a cell penetrating peptide which may be used with the pharmaceutical formulations of the present invention includes a cell-penetrating peptide sequence attached to polycations that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cell-penetrating peptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton Fla., 2002); El-Andaloussi et al., Curr. Pharm. Des. 11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci. 62(16):1839-49 (2005), all of which are incorporated herein by reference in their entirety). The compositions can also be formulated to include a cell penetrating agent, e.g., liposomes, which enhance delivery of the compositions to the intracellular space. Polynucleotides, primary constructs, and mmRNA of the invention may be complexed to peptides and/or proteins such as, but not limited to, peptides and/or proteins from Aileron Therapeutics (Cambridge, Mass.) and Permeon Biologics (Cambridge, Mass.) in order to enable intracellular delivery (Cronican et al., ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl. Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 2009 73:3-6; Verdine and Hilinski, Methods Enzymol. 2012; 503:3-33; all of which are herein incorporated by reference in its entirety).
In one embodiment, the cell-penetrating polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein, “protein-binding partner” includes, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. The cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner. The cell-penetrating polypeptide may be capable of being secreted from a cell where the cosmetic polynucleotide, primary construct, or mmRNA may be introduced.
Formulations of the including peptides or proteins may be used to increase cell transfection by the cosmetic polynucleotide, primary construct, or mmRNA, alter the biodistribution of the cosmetic polynucleotide, primary construct, or mmRNA (e.g., by targeting specific tissues or cell types), and/or increase the translation of encoded protein. (See e.g., International Pub. No. WO2012110636; herein incorporated by reference in its entirety).
Cells
The cosmetic polynucleotide, primary construct, and mmRNA of the invention can be transfected ex vivo into cells, which are subsequently transplanted into a subject. As non-limiting examples, the pharmaceutical compositions may include red blood cells to deliver modified RNA to liver and myeloid cells, virosomes to deliver modified RNA in virus-like particles (VLPs), and electroporated cells such as, but not limited to, from MAXCYTE® (Gaithersburg, Md.) and from ERYTECH® (Lyon, France) to deliver modified RNA. Examples of use of red blood cells, viral particles and electroporated cells to deliver payloads other than mmRNA have been documented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133; Fang et al., Expert Opin Biol Ther. 2012 12:385-389; Hu et al., Proc Natl Acad Sci USA. 2011 108:10980-10985; Lund et al., Pharm Res. 2010 27:400-420; Huckriede et al., J Liposome Res. 2007; 17:39-47; Cusi, Hum Vaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all of which are herein incorporated by reference in its entirety).
The cosmetic polynucleotides, primary constructs and mmRNA may be delivered in synthetic VLPs synthesized by the methods described in International Pub No. WO2011085231 and US Pub No. 20110171248, each of which are herein incorporated by reference in their entireties.
Cell-based formulations of the cosmetic polynucleotide, primary construct, and mmRNA of the invention may be used to ensure cell transfection (e.g., in the cellular carrier), alter the biodistribution of the cosmetic polynucleotide, primary construct, or mmRNA (e.g., by targeting the cell carrier to specific tissues or cell types), and/or increase the translation of encoded protein.
A variety of methods are known in the art and suitable for introduction of nucleic acid into a cell, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microproj ectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.
The technique of sonoporation, or cellular sonication, is the use of sound (e.g., ultrasonic frequencies) for modifying the permeability of the cell plasma membrane. Sonoporation methods are known to those in the art and are used to deliver nucleic acids in vivo (Yoon and Park, Expert Opin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr Pharm Biotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 2007 14:465-475; all herein incorporated by reference in their entirety). Sonoporation methods are known in the art and are also taught for example as it relates to bacteria in US Patent Publication 20100196983 and as it relates to other cell types in, for example, US Patent Publication 20100009424, each of which are incorporated herein by reference in their entirety.
Electroporation techniques are also well known in the art and are used to deliver nucleic acids in vivo and clinically (Andre et al., Curr Gene Ther. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 2010 10:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all herein incorporated by reference in their entirety). In one embodiment, polynucleotides, primary constructs or mmRNA may be delivered by electroporation as described in Example 8.
Hyaluronidase
The intramuscular or subcutaneous localized injection of polynucleotide, primary construct, or mmRNA of the invention can include hyaluronidase, which catalyzes the hydrolysis of hyaluronan. By catalyzing the hydrolysis of hyaluronan, a constituent of the interstitial barrier, hyaluronidase lowers the viscosity of hyaluronan, thereby increasing tissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440; herein incorporated by reference in its entirety). It is useful to speed their dispersion and systemic distribution of encoded proteins produced by transfected cells. Alternatively, the hyaluronidase can be used to increase the number of cells exposed to a cosmetic polynucleotide, primary construct, or mmRNA of the invention administered intramuscularly or subcutaneously.
Nanoparticle Mimics
The polynucleotide, primary construct or mmRNA of the invention may be encapsulated within and/or absorbed to a nanoparticle mimic. A nanoparticle mimic can mimic the delivery function organisms or particles such as, but not limited to, pathogens, viruses, bacteria, fungus, parasites, prions and cells. As a non-limiting example the polynucleotide, primary construct or mmRNA of the invention may be encapsulated in a non-viron particle which can mimic the delivery function of a virus (see International Pub. No. WO2012006376 herein incorporated by reference in its entirety).
Nanotubes
The cosmetic polynucleotides, primary constructs or mmRNA of the invention can be attached or otherwise bound to at least one nanotube such as, but not limited to, rosette nanotubes, rosette nanotubes having twin bases with a linker, carbon nanotubes and/or single-walled carbon nanotubes, The cosmetic polynucleotides, primary constructs or mmRNA may be bound to the nanotubes through forces such as, but not limited to, steric, ionic, covalent and/or other forces.
In one embodiment, the nanotube can release one or more polynucleotides, primary constructs or mmRNA into cells. The size and/or the surface structure of at least one nanotube may be altered so as to govern the interaction of the nanotubes within the body and/or to attach or bind to the cosmetic polynucleotides, primary constructs or mmRNA disclosed herein. In one embodiment, the building block and/or the functional groups attached to the building block of the at least one nanotube may be altered to adjust the dimensions and/or properties of the nanotube. As a non-limiting example, the length of the nanotubes may be altered to hinder the nanotubes from passing through the holes in the walls of normal blood vessels but still small enough to pass through the larger holes in the blood vessels of tumor tissue.
In one embodiment, at least one nanotube may also be coated with delivery enhancing compounds including polymers, such as, but not limited to, polyethylene glycol. In another embodiment, at least one nanotube and/or the cosmetic polynucleotides, primary constructs or mmRNA may be mixed with pharmaceutically acceptable excipients and/or delivery vehicles.
In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA are attached and/or otherwise bound to at least one rosette nanotube. The rosette nanotubes may be formed by a process known in the art and/or by the process described in International Publication No. WO2012094304, herein incorporated by reference in its entirety. At least one polynucleotide, primary construct and/or mmRNA may be attached and/or otherwise bound to at least one rosette nanotube by a process as described in International Publication No. WO2012094304, herein incorporated by reference in its entirety, where rosette nanotubes or modules forming rosette nanotubes are mixed in aqueous media with at least one polynucleotide, primary construct and/or mmRNA under conditions which may cause at least one polynucleotide, primary construct or mmRNA to attach or otherwise bind to the rosette nanotubes.
In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA may be attached to and/or otherwise bound to at least one carbon nanotube. As a non-limiting example, the cosmetic polynucleotides, primary constructs or mmRNA may be bound to a linking agent and the linked agent may be bound to the carbon nanotube (See e.g., U.S. Pat. No. 8,246,995; herein incorporated by reference in its entirety). The carbon nanotube may be a single-walled nanotube (See e.g., U.S. Pat. No. 8,246,995; herein incorporated by reference in its entirety).
Conjugates
The polynucleotides, primary constructs, and mmRNA of the invention include conjugates, such as a cosmetic polynucleotide, primary construct, or mmRNA covalently linked to a carrier or targeting group, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting group and therapeutic protein or peptide).
The conjugates of the invention include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
Representative U.S. patents that teach the preparation of polynucleotide conjugates, particularly to RNA, include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which is herein incorporated by reference in their entireties.
In one embodiment, the conjugate of the present invention may function as a carrier for the modified nucleic acids and mmRNA of the present invention. The conjugate may comprise a cationic polymer such as, but not limited to, polyamine, polylysine, polyalkylenimine, and polyethylenimine which may be grafted to with poly(ethylene glycol). As a non-limiting example, the conjugate may be similar to the polymeric conjugate and the method of synthesizing the polymeric conjugate described in U.S. Pat. No. 6,586,524 herein incorporated by reference in its entirety.
The conjugates can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer.
Targeting groups can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Targeting groups may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, or an activator of p38 MAP kinase.
The targeting group can be any ligand that is capable of targeting a specific receptor. Examples include, without limitation, folate, GalNAc, galactose, mannose, mannose-6P, apatamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In particular embodiments, the targeting group is an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein.
In one embodiment, pharmaceutical compositions of the present invention may include chemical modifications such as, but not limited to, modifications similar to locked nucleic acids.
Representative U.S. patents that teach the preparation of locked nucleic acid (LNA) such as those from Santaris, include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.
Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include polynucleotides, primary constructs or mmRNA with phosphorothioate backbones and oligonucleosides with other modified backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P(O)2—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the polynucletotides featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modifications at the 2′ position may also aid in delivery. Preferably, modifications at the 2′ position are not located in a polypeptide-coding sequence, i.e., not in a translatable region. Modifications at the 2′ position may be located in a 5′UTR, a 3′UTR and/or a tailing region. Modifications at the 2′ position can include one of the following at the 2′ position: H (i.e., 2′-deoxy); F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C1 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, the cosmetic polynucleotides, primary constructs or mmRNA include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties, or a group for improving the pharmacodynamic properties, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below. Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. Polynucleotides of the invention may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920 and each of which is herein incorporated by reference.
In still other embodiments, the cosmetic polynucleotide, primary construct, or mmRNA is covalently conjugated to a cell penetrating polypeptide. The cell-penetrating peptide may also include a signal sequence. The conjugates of the invention can be designed to have increased stability; increased cell transfection; and/or altered the biodistribution (e.g., targeted to specific tissues or cell types).
In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA may be conjugated to an agent to enhance delivery. As a non-limiting example, the agent may be a monomer or polymer such as a targeting monomer or a polymer having targeting blocks as described in International Publication No. WO2011062965, herein incorporated by reference in its entirety. In another non-limiting example, the agent may be a transport agent covalently coupled to the cosmetic polynucleotides, primary constructs or mmRNA of the present invention (See e.g., U.S. Pat. Nos. 6,835,393 and 7,374,778, each of which is herein incorporated by reference in its entirety). In yet another non-limiting example, the agent may be a membrane barrier transport enhancing agent such as those described in U.S. Pat. Nos. 7,737,108 and 8,003,129, each of which is herein incorporated by reference in its entirety.
In another embodiment, the cosmetic polynucleotides, primary constructs or mmRNA may be conjugated to SMARTT POLYMER TECHNOLOGY® (PHASERX®, Inc. Seattle, Wash.).
Self-Assembled Nanoparticles
Nucleic Acid Self-Assembled Nanoparticles
Self-assembled nanoparticles have a well-defined size which may be precisely controlled as the nucleic acid strands may be easily reprogrammable. For example, the optimal particle size for a cancer-targeting nanodelivery carrier is 20-100 nm as a diameter greater than 20 nm avoids renal clearance and enhances delivery to certain tumors through enhanced permeability and retention effect. Using self-assembled nucleic acid nanoparticles a single uniform population in size and shape having a precisely controlled spatial orientation and density of cancer-targeting ligands for enhanced delivery. As a non-limiting example, oligonucleotide nanoparticles were prepared using programmable self-assembly of short DNA fragments and therapeutic siRNAs. These nanoparticles are molecularly identical with controllable particle size and target ligand location and density. The DNA fragments and siRNAs self-assembled into a one-step reaction to generate DNA/siRNA tetrahedral nanoparticles for targeted in vivo delivery. (Lee et al., Nature Nanotechnology 2012 7:389-393; herein incorporated by reference in its entirety).
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein may be formulated as self-assembled nanoparticles. As a non-limiting example, nucleic acids may be used to make nanoparticles which may be used in a delivery system for the cosmetic polynucleotides, primary constructs and/or mmRNA of the present invention (See e.g., International Pub. No. WO2012125987; herein incorporated by reference in its entirety).
In one embodiment, the nucleic acid self-assembled nanoparticles may comprise a core of the cosmetic polynucleotides, primary constructs or mmRNA disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the polynucleotides, primary constructs and mmRNA in the core.
Polymer-Based Self-Assembled Nanoparticles
Polymers may be used to form sheets which self-assembled into nanoparticles. These nanoparticles may be used to deliver the cosmetic polynucleotides, primary constructs and mmRNA of the present invention. In one embodiment, these self-assembled nanoparticles may be microsponges formed of long polymers of RNA hairpins which form into crystalline ‘pleated’ sheets before self-assembling into microsponges. These microsponges are densely-packed sponge like microparticles which may function as an efficient carrier and may be able to deliver cargo to a cell. The microsponges may be from 1 um to 300 nm in diameter. The microsponges may be complexed with other agents known in the art to form larger microsponges. As a non-limiting example, the microsponge may be complexed with an agent to form an outer layer to promote cellular uptake such as polycation polyethyleneime (PEI). This complex can form a 250-nm diameter particle that can remain stable at high temperatures (150° C.) (Grabow and Jaegar, Nature Materials 2012, 11:269-269; herein incorporated by reference in its entirety). Additionally these microsponges may be able to exhibit an extraordinary degree of protection from degradation by ribonucleases.
In another embodiment, the polymer-based self-assembled nanoparticles such as, but not limited to, microsponges, may be fully programmable nanoparticles. The geometry, size and stoichiometry of the nanoparticle may be precisely controlled to create the optimal nanoparticle for delivery of cargo such as, but not limited to, polynucleotides, primary constructs and/or mmRNA.
In one embodiment, the polymer based nanoparticles may comprise a core of the cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the polynucleotides, primary construct and/or mmRNA in the core.
In yet another embodiment, the polymer based nanoparticle may comprise a non-nucleic acid polymer comprising a plurality of heterogenous monomers such as those described in Interantional Publication No. WO2013009736, herein incorporated by reference in its entirety.
Inorganic Nanoparticles
The cosmetic polynucleotides, primary constructs and/or mmRNAs of the present invention may be formulated in inorganic nanoparticles (U.S. Pat. No. 8,257,745, herein incorporated by reference in its entirety). The inorganic nanoparticles may include, but are not limited to, clay substances that are water swellable. As a non-limiting example, the inorganic nanoparticle may include synthetic smectite clays which are made from simple silicates (See e.g., U.S. Pat. Nos. 5,585,108 and 8,257,745 each of which are herein incorporated by reference in their entirety).
In one embodiment, the inorganic nanoparticles may comprise a core of the modified nucleic acids disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the modified nucleic acids in the core.
Semi-Conductive and Metallic Nanoparticles
The cosmetic polynucleotides, primary constructs and/or mmRNAs of the present invention may be formulated in water-dispersible nanoparticle comprising a semiconductive or metallic material (U.S. Pub. No. 20120228565; herein incorporated by reference in its entirety) or formed in a magnetic nanoparticle (U.S. Pub. No. 20120265001 and 20120283503; each of which is herein incorporated by reference in its entirety). The water-dispersible nanoparticles may be hydrophobic nanoparticles or hydrophilic nanoparticles.
In one embodiment, the semi-conductive and/or metallic nanoparticles may comprise a core of the cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the cosmetic polynucleotides, primary constructs and/or mmRNA in the core.
Gels and Hydrogels
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein may be encapsulated into any hydrogel known in the art which may form a gel when injected into a subject. Hydrogels are a network of polymer chains that are hydrophilic, and are sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (they can contain over 99% water) natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. The hydrogel described herein may used to encapsulate lipid nanoparticles which are biocompatible, biodegradable and/or porous.
As a non-limiting example, the hydrogel may be an aptamer-functionalized hydrogel. The aptamer-functionalized hydrogel may be programmed to release one or more polynucleotides, primary constructs and/or mmRNA using nucleic acid hybridization. (Battig et al., J. Am. Chem. Society. 2012 134:12410-12413; herein incorporated by reference in its entirety).
As another non-limiting example, the hydrogel may be a shaped as an inverted opal. The opal hydrogels exhibit higher swelling ratios and the swelling kinetics is an order of magnitude faster as well. Methods of producing opal hydrogels and description of opal hydrogels are described in International Pub. No. WO2012148684, herein incorporated by reference in its entirety.
In yet another non-limiting example, the hydrogel may be an antibacterial hydrogel. The antibacterial hydrogel may comprise a pharmaceutical acceptable salt or organic material such as, but not limited to pharmaceutical grade and/or medical grade silver salt and aloe vera gel or extract. (International Pub. No. WO2012151438, herein incorporated by reference in its entirety).
In one embodiment, the modified mRNA may be encapsulated in a lipid nanoparticle and then the lipid nanoparticle may be encapsulated into a hyrdogel.
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein may be encapsulated into any gel known in the art. As a non-limiting example the gel may be a fluorouracil injectable gel or a fluorouracil injectable gel containing a chemical compound and/or drug known in the art. As another example, the cosmetic polynucleotides, primary constructs and/or mmRNA may be encapsulated in a fluorouracil gel containing epinephrine (See e.g., Smith et al. Cancer Chemotherapty and Pharmacology, 1999 44(4):267-274; herein incorporated by reference in its entirety).
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein may be encapsulated into a fibrin gel, fibrin hydrogel or fibrin glue. In another embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid nanoparticle or a rapidly eliminated lipid nanoparticle prior to being encapsulated into a fibrin gel, fibrin hydrogel or a fibrin glue. In yet another embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA may be formulated as a lipoplex prior to being encapsulated into a fibrin gel, hydrogel or a fibrin glue. Fibrin gels, hydrogels and glues comprise two components, a fibrinogen solution and a thrombin solution which is rich in calcium (See e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148: 49-55; Kidd et al. Journal of Controlled Release 2012. 157:80-85; each of which is herein incorporated by reference in its entirety). The concentration of the components of the fibrin gel, hydrogel and/or glue can be altered to change the characteristics, the network mesh size, and/or the degradation characteristics of the gel, hydrogel and/or glue such as, but not limited to changing the release characteristics of the fibrin gel, hydrogel and/or glue. (See e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148: 49-55; Kidd et al. Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128; each of which is herein incorporated by reference in its entirety). This feature may be advantageous when used to deliver the modified mRNA disclosed herein. (See e.g., Kidd et al. Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128; each of which is herein incorporated by reference in its entirety).
Cations and Anions
Formulations of polynucleotides, primary constructs and/or mmRNA disclosed herein may include cations or anions. In one embodiment, the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ and combinations thereof. As a non-limiting example, formulations may include polymers and a polynucleotides, primary constructs and/or mmRNA complexed with a metal cation (See e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, each of which is herein incorporated by reference in its entirety).
Molded Nanoparticles and Microparticles
The cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein may be formulated in nanoparticles and/or microparticles. These nanoparticles and/or microparticles may be molded into any size shape and chemistry. As an example, the nanoparticles and/or microparticles may be made using the PRINT® technology by LIQUIDA TECHNOLOGIES® (Morrisville, N.C.) (See e.g., International Pub. No. WO2007024323; herein incorporated by reference in its entirety).
In one embodiment, the molded nanoparticles may comprise a core of the cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the polynucleotides, primary construct and/or mmRNA in the core.
NanoJackets and NanoLiposomes
The cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein may be formulated in NanoJackets and NanoLiposomes by Keystone Nano (State College, Pa.). NanoJackets are made of compounds that are naturally found in the body including calcium, phosphate and may also include a small amount of silicates. Nanojackets may range in size from 5 to 50 nm and may be used to deliver hydrophilic and hydrophobic compounds such as, but not limited to, polynucleotides, primary constructs and/or mmRNA.
NanoLiposomes are made of lipids such as, but not limited to, lipids which naturally occur in the body. NanoLiposomes may range in size from 60-80 nm and may be used to deliver hydrophilic and hydrophobic compounds such as, but not limited to, polynucleotides, primary constructs and/or mmRNA. In one aspect, the cosmetic polynucleotides, primary constructs and/or mmRNA disclosed herein are formulated in a NanoLiposome such as, but not limited to, Ceramide NanoLiposomes.
Excipients
Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.
In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEENn®60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof
Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONETM, KATHONTM, and/or EUXYL®.
Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or combinations thereof
Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof
Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
Delivery
The present disclosure encompasses the delivery of cosmetic polynucleotides, primary constructs or mmRNA for any of therapeutic, pharmaceutical, diagnostic or imaging by any appropriate route taking into consideration likely advances in the sciences of drug delivery. Delivery may be naked or formulated.
Naked Delivery
The cosmetic polynucleotides, primary constructs or mmRNA of the present invention may be delivered to a cell naked. As used herein in, “naked” refers to delivering polynucleotides, primary constructs or mmRNA free from agents which promote transfection. For example, the cosmetic polynucleotides, primary constructs or mmRNA delivered to the cell may contain no modifications. The naked polynucleotides, primary constructs or mmRNA may be delivered to the cell using routes of administration known in the art and described herein.
Formulated Delivery
The cosmetic polynucleotides, primary constructs or mmRNA of the present invention may be formulated, using the methods described herein. The formulations may contain polynucleotides, primary constructs or mmRNA which may be modified and/or unmodified. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot. The formulated polynucleotides, primary constructs or mmRNA may be delivered to the cell using routes of administration known in the art and described herein.
The compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.
Administration
The cosmetic polynucleotides, primary constructs or mmRNA of the present invention may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), or in ear drops. In specific embodiments, compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier. Non-limiting routes of administration for the cosmetic polynucleotides, primary constructs or mmRNA of the present invention are described below.
Parenteral and Injectable Administration
Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.
Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
Rectal and Vaginal Administration
Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
Oral Administration
Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g. agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g. paraffin), absorption accelerators (e.g. quaternary ammonium compounds), wetting agents (e.g. cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin and bentonite clay), and lubricants (e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.
Topical or Transdermal Administration
As described herein, compositions containing the cosmetic polynucleotides, primary constructs or mmRNA of the invention may be formulated for administration topically. The skin may be an ideal target site for delivery as it is readily accessible. Gene expression may be restricted not only to the skin, potentially avoiding nonspecific toxicity, but also to specific layers and cell types within the skin.
The site of cutaneous expression of the delivered compositions will depend on the route of nucleic acid delivery. Three routes are commonly considered to deliver polynucleotides, primary constructs or mmRNA to the skin: (i) topical application (e.g. for local/regional treatment and/or cosmetic applications); (ii) intradermal injection (e.g. for local/regional treatment and/or cosmetic applications); and (iii) systemic delivery (e.g. for treatment of dermatologic diseases that affect both cutaneous and extracutaneous regions). Polynucleotides, primary constructs or mmRNA can be delivered to the skin by several different approaches known in the art. Most topical delivery approaches have been shown to work for delivery of DNA, such as but not limited to, topical application of non-cationic liposome-DNA complex, cationic liposome-DNA complex, particle-mediated (gene gun), puncture-mediated gene transfections, and viral delivery approaches. After delivery of the nucleic acid, gene products have been detected in a number of different skin cell types, including, but not limited to, basal keratinocytes, sebaceous gland cells, dermal fibroblasts and dermal macrophages.
In one embodiment, the invention provides for a variety of dressings (e.g., wound dressings) or bandages (e.g., adhesive bandages) for conveniently and/or effectively carrying out methods of the present invention. Typically dressing or bandages may comprise sufficient amounts of pharmaceutical compositions and/or polynucleotides, primary constructs or mmRNA described herein to allow a user to perform multiple treatments of a subject(s).
In one embodiment, the invention provides for the cosmetic polynucleotides, primary constructs or mmRNA compositions to be delivered in more than one injection.
In one embodiment, before topical and/or transdermal administration at least one area of tissue, such as skin, may be subjected to a device and/or solution which may increase permeability. In one embodiment, the tissue may be subjected to an abrasion device to increase the permeability of the skin (see U.S. Patent Publication No. 20080275468, herein incorporated by reference in its entirety). In another embodiment, the tissue may be subjected to an ultrasound enhancement device. An ultrasound enhancement device may include, but is not limited to, the devices described in U.S. Publication No. 20040236268 and U.S. Pat. Nos. 6,491,657 and 6,234,990; each of which are herein incorporated by reference in their entireties. Methods of enhancing the permeability of tissue are described in U.S. Publication Nos. 20040171980 and 20040236268 and U.S. Pat. No. 6,190,315; each of which are herein incorporated by reference in their entireties.
In one embodiment, a device may be used to increase permeability of tissue before delivering formulations of modified mRNA described herein. The permeability of skin may be measured by methods known in the art and/or described in U.S. Pat. No. 6,190,315, herein incorporated by reference in its entirety. As a non-limiting example, a modified mRNA formulation may be delivered by the drug delivery methods described in U.S. Pat. No. 6,190,315, herein incorporated by reference in its entirety.
In another non-limiting example tissue may be treated with a eutectic mixture of local anesthetics (EMLA) cream before, during and/or after the tissue may be subjected to a device which may increase permeability. Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated by reference in its entirety) showed that using the EMLA cream in combination with a low energy, an onset of superficial cutaneous analgesia was seen as fast as 5 minutes after a pretreatment with a low energy ultrasound.
In one embodiment, enhancers may be applied to the tissue before, during, and/or after the tissue has been treated to increase permeability. Enhancers include, but are not limited to, transport enhancers, physical enhancers, and cavitation enhancers. Non-limiting examples of enhancers are described in U.S. Pat. No. 6,190,315, herein incorporated by reference in its entirety.
In one embodiment, a device may be used to increase permeability of tissue before delivering formulations of modified mRNA described herein, which may further contain a substance that invokes an immune response. In another non-limiting example, a formulation containing a substance to invoke an immune response may be delivered by the methods described in U.S. Publication Nos. 20040171980 and 20040236268; each of which are herein incorporated by reference in their entireties.
Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required. Additionally, the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium. Alternatively or additionally, rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.
Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Topically-administrable formulations may, for example, comprise from about 0.1% to about 10% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
Depot Administration
As described herein, in some embodiments, the composition is formulated in depots for extended release. Generally, a specific organ or tissue (a “target tissue”) is targeted for administration.
In some aspects of the invention, the cosmetic polynucleotides, primary constructs or mmRNA are spatially retained within or proximal to a target tissue. Provided are method of providing a composition to a target tissue of a mammalian subject by contacting the target tissue (which contains one or more target cells) with the composition under conditions such that the composition, in particular the nucleic acid component(s) of the composition, is substantially retained in the target tissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the composition is retained in the target tissue. Advantageously, retention is determined by measuring the amount of the nucleic acid present in the composition that enters one or more target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the nucleic acids administered to the subject are present intracellularly at a period of time following administration. For example, intramuscular injection to a mammalian subject is performed using an aqueous composition containing a ribonucleic acid and a transfection reagent, and retention of the composition is determined by measuring the amount of the ribonucleic acid present in the muscle cells.
Aspects of the invention are directed to methods of providing a composition to a target tissue of a mammalian subject, by contacting the target tissue (containing one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue. The composition contains an effective amount of a polynucleotides, primary constructs or mmRNA such that the polypeptide of interest is produced in at least one target cell. The compositions generally contain a cell penetration agent, although “naked” nucleic acid (such as nucleic acids without a cell penetration agent or other agent) is also contemplated, and a pharmaceutically acceptable carrier.
In some circumstances, the amount of a protein produced by cells in a tissue is desirably increased. Preferably, this increase in protein production is spatially restricted to cells within the target tissue. Thus, provided are methods of increasing production of a protein of interest in a tissue of a mammalian subject. A composition is provided that contains polynucleotides, primary constructs or mmRNA characterized in that a unit quantity of composition has been determined to produce the polypeptide of interest in a substantial percentage of cells contained within a predetermined volume of the target tissue.
In some embodiments, the composition includes a plurality of different polynucleotides, primary constructs or mmRNA, where one or more than one of the cosmetic polynucleotides, primary constructs or mmRNA encodes a polypeptide of interest. Optionally, the composition also contains a cell penetration agent to assist in the intracellular delivery of the composition. A determination is made of the dose of the composition required to produce the polypeptide of interest in a substantial percentage of cells contained within the predetermined volume of the target tissue (generally, without inducing significant production of the polypeptide of interest in tissue adjacent to the predetermined volume, or distally to the target tissue). Subsequent to this determination, the determined dose is introduced directly into the tissue of the mammalian subject.
In one embodiment, the invention provides for the cosmetic polynucleotides, primary constructs or mmRNA to be delivered in more than one injection or by split dose injections.
In one embodiment, the invention may be retained near target tissue using a small disposable drug reservoir, patch pump or osmotic pump. Non-limiting examples of patch pumps include those manufactured and/or sold by BD® (Franklin Lakes, N.J.), Insulet Corporation (Bedford, Mass.), SteadyMed Therapeutics (San Francisco, Calif.), Medtronic (Minneapolis, Minn.) (e.g., MiniMed), UniLife (York, Pa.), Valeritas (Bridgewater, N.J.), and SpringLeaf Therapeutics (Boston, Mass.). A non-limiting example of an osmotic pump include those manufactured by DURECT® (Cupertino, Calif.) (e.g., DUROS® and ALZET®).
Pulmonary Administration
A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. Such compositions are suitably in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50% to 99.9% (w/w) of the composition, and active ingredient may constitute 0.1% to 20% (w/w) of the composition. A propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
As a non-limiting example, the cosmetic polynucleotides, primary constructs and/or mmRNA described herein may be formulated for pulmonary delivery by the methods described in U.S. Pat. No. 8,257,685; herein incorporated by reference in its entirety.
Pharmaceutical compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.
Intranasal, Nasal and Buccal Administration
Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 μm to 500 μm. Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, 0.1% to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
Ophthalmic Administration
A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein. Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this invention. A multilayer thin film device may be prepared to contain a pharmaceutical composition for delivery to the eye and/or surrounding tissue.
Payload Administration: Detectable Agents and Therapeutic Agents
The cosmetic polynucleotides, primary constructs or mmRNA described herein can be used in a number of different scenarios in which delivery of a substance (the “payload”) to a biological target is desired, for example delivery of detectable substances for detection of the target, or delivery of a therapeutic agent. Detection methods can include, but are not limited to, both imaging in vitro and in vivo imaging methods, e.g., immunohistochemistry, bioluminescence imaging (BLI), Magnetic Resonance Imaging (MRI), positron emission tomography (PET), electron microscopy, X-ray computed tomography, Raman imaging, optical coherence tomography, absorption imaging, thermal imaging, fluorescence reflectance imaging, fluorescence microscopy, fluorescence molecular tomographic imaging, nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging, photoacoustic imaging, lab assays, or in any situation where tagging/staining/imaging is required.
The cosmetic polynucleotides, primary constructs or mmRNA can be designed to include both a linker and a payload in any useful orientation. For example, a linker having two ends is used to attach one end to the payload and the other end to the nucleobase, such as at the C-7 or C-8 positions of the deaza-adenosine or deaza-guanosine or to the N-3 or C-5 positions of cytosine or uracil. The polynucleotide of the invention can include more than one payload (e.g., a label and a transcription inhibitor), as well as a cleavable linker. In one embodiment, the modified nucleotide is a modified 7-deaza-adenosine triphosphate, where one end of a cleavable linker is attached to the C7 position of 7-deaza-adenine, the other end of the linker is attached to an inhibitor (e.g., to the C5 position of the nucleobase on a cytidine), and a label (e.g., Cy5) is attached to the center of the linker (see, e.g., compound 1 of A*pCp C5 Parg Capless in FIG. 5 and columns 9 and 10 of U.S. Pat. No. 7,994,304, incorporated herein by reference). Upon incorporation of the modified 7-deaza-adenosine triphosphate to an encoding region, the resulting polynucleotide having a cleavable linker attached to a label and an inhibitor (e.g., a polymerase inhibitor). Upon cleavage of the linker (e.g., with reductive conditions to reduce a linker having a cleavable disulfide moiety), the label and inhibitor are released. Additional linkers and payloads (e.g., therapeutic agents, detectable labels, and cell penetrating payloads) are described herein.
Scheme 12 below depicts an exemplary modified nucleotide wherein the nucleobase, adenine, is attached to a linker at the C-7 carbon of 7-deaza adenine. In addition, Scheme 12 depicts the modified nucleotide with the linker and payload, e.g., a detectable agent, incorporated onto the 3′ end of the mRNA. Disulfide cleavage and 1,2-addition of the thiol group onto the propargyl ester releases the detectable agent. The remaining structure (depicted, for example, as pApC5Parg in Scheme 12) is the inhibitor. The rationale for the structure of the modified nucleotides is that the tethered inhibitor sterically interferes with the ability of the polymerase to incorporate a second base. Thus, it is critical that the tether be long enough to affect this function and that the inhibitor be in a stereochemical orientation that inhibits or prohibits second and follow on nucleotides into the growing polynucleotide strand.




For example, the cosmetic polynucleotides, primary constructs or mmRNA described herein can be used in reprogramming induced pluripotent stem cells (iPS cells), which can directly track cells that are transfected compared to total cells in the cluster. In another example, a drug that may be attached to the cosmetic polynucleotides, primary constructs or mmRNA via a linker and may be fluorescently labeled can be used to track the drug in vivo, e.g. intracellularly. Other examples include, but are not limited to, the use of a polynucleotides, primary constructs or mmRNA in reversible drug delivery into cells.
The cosmetic polynucleotides, primary constructs or mmRNA described herein can be used in intracellular targeting of a payload, e.g., detectable or therapeutic agent, to specific organelle. Exemplary intracellular targets can include, but are not limited to, the nuclear localization for advanced mRNA processing, or a nuclear localization sequence (NLS) linked to the mRNA containing an inhibitor.
In addition, the cosmetic polynucleotides, primary constructs or mmRNA described herein can be used to deliver therapeutic agents to cells or tissues, e.g., in living animals. For example, the cosmetic polynucleotides, primary constructs or mmRNA described herein can be used to deliver highly polar chemotherapeutics agents to kill cancer cells. The cosmetic polynucleotides, primary constructs or mmRNA attached to the therapeutic agent through a linker can facilitate member permeation allowing the therapeutic agent to travel into a cell to reach an intracellular target.
In one example, the linker is attached at the 2′-position of the ribose ring and/or at the 3′ and/or 5′ position of the polynucleotides, primary constructs mmRNA (See e.g., International Pub. No. WO2012030683, herein incorporated by reference in its entirety). The linker may be any linker disclosed herein, known in the art and/or disclosed in International Pub. No. WO2012030683, herein incorporated by reference in its entirety.
In another example, the cosmetic polynucleotides, primary constructs or mmRNA can be attached to the cosmetic polynucleotides, primary constructs or mmRNA a viral inhibitory peptide (VIP) through a cleavable linker. The cleavable linker can release the VIP and dye into the cell. In another example, the cosmetic polynucleotides, primary constructs or mmRNA can be attached through the linker to an ADP-ribosylate, which is responsible for the actions of some bacterial toxins, such as cholera toxin, diphtheria toxin, and pertussis toxin. These toxin proteins are ADP-ribosyltransferases that modify target proteins in human cells. For example, cholera toxin ADP-ribosylates G proteins modifies human cells by causing massive fluid secretion from the lining of the small intestine, which results in life-threatening diarrhea.
In some embodiments, the payload may be a therapeutic agent such as a cytotoxin, radioactive ion, chemotherapeutic, or other therapeutic agent. A cytotoxin or cytotoxic agent includes any agent that may be detrimental to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein in its entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092, 5,585,499, and 5,846,545, all of which are incorporated herein by reference), and analogs or homologs thereof. Radioactive ions include, but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids).
In some embodiments, the payload may be a detectable agent, such as various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials (e.g., 18F, 67Ga, 81mKr, 82Rb, 111In, 123I, 133Xe, 201Tl, 125I, 35S, 14C, 3H, or 99mTc (e.g., as pertechnetate (technetate(VII), TcO4−)), and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexol), microbubbles, or perfluorocarbons). Such optically-detectable labels include for example, without limitation, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives (e.g., acridine and acridine isothiocyanate); 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives (e.g., coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), and 7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′ 5′′-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives (e.g., eosin and eosin isothiocyanate); erythrosin and derivatives (e.g., erythrosin B and erythrosin isothiocyanate); ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein, fluorescein isothiocyanate, X-rhodamine-5-(and -6)-isothiocyanate (QFITC or XRITC), and fluorescamine); 2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indolium hydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144); 5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethyl benzothiazolium perchlorate (IR140); Malachite Green isothiocyanate; 4-methylumbelliferone orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl 1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRONTM Brilliant Red 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolic acid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5); cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.
In some embodiments, the detectable agent may be a non-detectable pre-cursor that becomes detectable upon activation (e.g., fluorogenic tetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). In vitro assays in which the enzyme labeled compositions can be used include, but are not limited to, enzyme linked immunosorbent assays (ELISAs), immunoprecipitation assays, immunofluorescence, enzyme immunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.
Combinations
The cosmetic polynucleotides, primary constructs or mmRNA may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body. As a non-limiting example, the nucleic acids or mmRNA may be used in combination with a pharmaceutical agent for the treatment of cancer or to control hyperproliferative cells. In U.S. Pat. No. 7,964,571, herein incorporated by reference in its entirety, a combination therapy for the treatment of solid primary or metastasized tumor is described using a pharmaceutical composition including a DNA plasmid encoding for interleukin-12 with a lipopolymer and also administering at least one anticancer agent or chemotherapeutic. Further, the nucleic acids and mmRNA of the present invention that encodes anti-proliferative molecules may be in a pharmaceutical composition with a lipopolymer (see e.g., U.S. Pub. No. 20110218231, herein incorporated by reference in its entirety, claiming a pharmaceutical composition comprising a DNA plasmid encoding an anti-proliferative molecule and a lipopolymer) which may be administered with at least one chemotherapeutic or anticancer agent.
It will further be appreciated that therapeutically, prophylactically, diagnostically, or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. In one embodiment, the combinations, each or together may be administered according to the split dosing regimens described herein.
Dosing
The present invention provides methods comprising administering modified mRNAs and their encoded proteins or complexes in accordance with the invention to a subject in need thereof. Nucleic acids, proteins or complexes, or pharmaceutical, imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and/or condition (e.g., a disease, disorder, and/or condition relating to working memory deficits). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
In certain embodiments, compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used.
According to the present invention, it has been discovered that administration of mmRNA in split-dose regimens produce higher levels of proteins in mammalian subjects. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administer in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose. In one embodiment, the mmRNA of the present invention are administer to a subject in split doses. The mmRNA may be formulated in buffer only or in a formulation described herein.
Dosage Forms
A pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).
Liquid Dosage Forms
Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, compositions may be mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
Injectable
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.
Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
In order to prolong the effect of an active ingredient, it may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the polynucleotide, primary construct or mmRNA then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered polynucleotide, primary construct or mmRNA may be accomplished by dissolving or suspending the polynucleotide, primary construct or mmRNA in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the polynucleotide, primary construct or mmRNA in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of polynucleotide, primary construct or mmRNA to polymer and the nature of the particular polymer employed, the rate of polynucleotide, primary construct or mmRNA release can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the polynucleotide, primary construct or mmRNA in liposomes or microemulsions which are compatible with body tissues.
Pulmonary
Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 μm to 500 μm. Such a formulation may be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
Coatings or Shells
Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
Properties of Pharmaceutical Compositions
The pharmaceutical compositions described herein can be characterized by one or more of bioavailability, therapeutic window and/or volume of distribution.
Bioavailability
The cosmetic polynucleotides, primary constructs or mmRNA, when formulated into a composition with a delivery agent as described herein, can exhibit an increase in bioavailability as compared to a composition lacking a delivery agent as described herein. As used herein, the term “bioavailability” refers to the systemic availability of a given amount of cosmetic polynucleotides, primary constructs or mmRNA administered to a mammal. Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration (Cmax) of the unchanged form of a compound following administration of the compound to a mammal. AUC is a determination of the area under the curve plotting the serum or plasma concentration of a compound along the ordinate (Y-axis) against time along the abscissa (X-axis). Generally, the AUC for a particular compound can be calculated using methods known to those of ordinary skill in the art and as described in G. S. Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, New York, Inc., 1996, herein incorporated by reference in its entirety.
The Cmax value is the maximum concentration of the compound achieved in the serum or plasma of a mammal following administration of the compound to the mammal. The Cmax value of a particular compound can be measured using methods known to those of ordinary skill in the art. The phrases “increasing bioavailability” or “improving the pharmacokinetics,” as used herein mean that the systemic availability of a first polynucleotide, primary construct or mmRNA, measured as AUC, Cmax, or Cmin in a mammal is greater, when co-administered with a delivery agent as described herein, than when such co-administration does not take place. In some embodiments, the bioavailability of the polynucleotide, primary construct or mmRNA can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
Therapeutic Window
The cosmetic polynucleotides, primary constructs or mmRNA, when formulated into a composition with a delivery agent as described herein, can exhibit an increase in the therapeutic window of the administered polynucleotide, primary construct or mmRNA composition as compared to the therapeutic window of the administered polynucleotide, primary construct or mmRNA composition lacking a delivery agent as described herein. As used herein “therapeutic window” refers to the range of plasma concentrations, or the range of levels of therapeutically active substance at the site of action, with a high probability of eliciting a therapeutic effect. In some embodiments, the therapeutic window of the polynucleotide, primary construct or mmRNA when co-administered with a delivery agent as described herein can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
Volume of Distribution
The cosmetic polynucleotides, primary constructs or mmRNA, when formulated into a composition with a delivery agent as described herein, can exhibit an improved volume of distribution (Vdist), e.g., reduced or targeted, relative to a composition lacking a delivery agent as described herein. The volume of distribution (Vdist) relates the amount of the drug in the body to the concentration of the drug in the blood or plasma. As used herein, the term “volume of distribution” refers to the fluid volume that would be required to contain the total amount of the drug in the body at the same concentration as in the blood or plasma: Vdist equals the amount of drug in the body/concentration of drug in blood or plasma. For example, for a 10 mg dose and a plasma concentration of 10 mg/L, the volume of distribution would be 1 liter. The volume of distribution reflects the extent to which the drug is present in the extravascular tissue. A large volume of distribution reflects the tendency of a compound to bind to the tissue components compared with plasma protein binding. In a clinical setting, Vdist can be used to determine a loading dose to achieve a steady state concentration. In some embodiments, the volume of distribution of the polynucleotide, primary construct or mmRNA when co-administered with a delivery agent as described herein can decrease at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%.
Biological Effect
In one embodiment, the biological effect of the modified mRNA delivered to the animals may be categorized by analyzing the protein expression in the animals. The protein expression may be determined from analyzing a biological sample collected from a mammal administered the modified mRNA of the present invention. In one embodiment, the expression protein encoded by the modified mRNA administered to the mammal of at least 50 pg/ml may be preferred. For example, a protein expression of 50-200 pg/ml for the protein encoded by the modified mRNA delivered to the mammal may be seen as a therapeutically effective amount of protein in the mammal.
Detection of Modified Nucleic Acids by Mass Spectrometry
Mass spectrometry (MS) is an analytical technique that can provide structural and molecular mass/concentration information on molecules after their conversion to ions. The molecules are first ionized to acquire positive or negative charges and then they travel through the mass analyzer to arrive at different areas of the detector according to their mass/charge (m/z) ratio.
Mass spectrometry is performed using a mass spectrometer which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. For example ionization of the sample may be performed by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. The skilled artisan will understand that the choice of ionization method can be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.
After the sample has been ionized, the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio (i.e., m/z). Suitable analyzers for determining mass-to-charge ratios include quadropole analyzers, ion traps analyzers, and time-of-flight analyzers. The ions may be detected using several detection modes. For example, selected ions may be detected (i.e., using a selective ion monitoring mode (SIM)), or alternatively, ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).
Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupled with stable isotope labeled dilution of peptide standards has been shown to be an effective method for protein verification (e.g., Keshishian et al., Mol Cell Proteomics 2009 8: 2339-2349; Kuhn et al., Clin Chem 2009 55:1108-1117; Lopez et al., Clin Chem 2010 56:281-290; each of which are herein incorporated by reference in its entirety). Unlike untargeted mass spectrometry frequently used in biomarker discovery studies, targeted MS methods are peptide sequence-based modes of MS that focus the full analytical capacity of the instrument on tens to hundreds of selected peptides in a complex mixture. By restricting detection and fragmentation to only those peptides derived from proteins of interest, sensitivity and reproducibility are improved dramatically compared to discovery-mode MS methods. This method of mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples.
In one embodiment, a biological sample which may contain at least one protein encoded by at least one modified mRNA of the present invention may be analyzed by the method of MRM-MS. The quantification of the biological sample may further include, but is not limited to, isotopically labeled peptides or proteins as internal standards.
According to the present invention, the biological sample, once obtained from the subject, may be subjected to enzyme digestion. As used herein, the term “digest” means to break apart into shorter peptides. As used herein, the phrase “treating a sample to digest proteins” means manipulating a sample in such a way as to break down proteins in a sample. These enzymes include, but are not limited to, trypsin, endoproteinase Glu-C and chymotrypsin. In one embodiment, a biological sample which may contain at least one protein encoded by at least one modified mRNA of the present invention may be digested using enzymes.
In one embodiment, a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed for protein using electrospray ionization. Electrospray ionization (ESI) mass spectrometry (ESIMS) uses electrical energy to aid in the transfer of ions from the solution to the gaseous phase before they are analyzed by mass spectrometry. Samples may be analyzed using methods known in the art (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12; herein incorporated by reference in its entirety). The ionic species contained in solution may be transferred into the gas phase by dispersing a fine spray of charge droplets, evaporating the solvent and ejecting the ions from the charged droplets to generate a mist of highly charged droplets. The mist of highly charged droplets may be analyzed using at least 1, at least 2, at least 3 or at least 4 mass analyzers such as, but not limited to, a quadropole mass analyzer. Further, the mass spectrometry method may include a purification step. As a non-limiting example, the first quadrapole may be set to select a single m/z ratio so it may filter out other molecular ions having a different m/z ratio which may eliminate complicated and time-consuming sample purification procedures prior to MS analysis.
In one embodiment, a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed for protein in a tandem ESIMS system (e.g., MS/MS). As non-limiting examples, the droplets may be analyzed using a product scan (or daughter scan) a precursor scan (parent scan) a neutral loss or a multiple reaction monitoring.
In one embodiment, a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MALDIMS). MALDI provides for the nondestructive vaporization and ionization of both large and small molecules, such as proteins. In MALDI analysis, the analyte is first co-crystallized with a large molar excess of a matrix compound, which may also include, but is not limited to, an ultraviolet absorbing weak organic acid. Non-limiting examples of matrices used in MALDI are α-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid. Laser radiation of the analyte-matrix mixture may result in the vaporization of the matrix and the analyte. The laser induced desorption provides high ion yields of the intact analyte and allows for measurement of compounds with high accuracy. Samples may be analyzed using methods known in the art (e.g., Lewis, Wei and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894; herein incorporated by reference in its entirety). As non-limiting examples, mass analyzers used in the MALDI analysis may include a linear time-of-flight (TOF), a TOF reflectron or a Fourier transform mass analyzer.
In one embodiment, the analyte-matrix mixture may be formed using the dried-droplet method. A biologic sample is mixed with a matrix to create a saturated matrix solution where the matrix-to-sample ratio is approximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of the saturated matrix solution is then allowed to dry to form the analyte-matrix mixture.
In one embodiment, the analyte-matrix mixture may be formed using the thin-layer method. A matrix homogeneous film is first formed and then the sample is then applied and may be absorbed by the matrix to form the analyte-matrix mixture.
In one embodiment, the analyte-matrix mixture may be formed using the thick-layer method. A matrix homogeneous film is formed with a nitro-cellulose matrix additive. Once the uniform nitro-cellulose matrix layer is obtained the sample is applied and absorbed into the matrix to form the analyte-matrix mixture.
In one embodiment, the analyte-matrix mixture may be formed using the sandwich method. A thin layer of matrix crystals is prepared as in the thin-layer method followed by the addition of droplets of aqueous trifluoroacetic acid, the sample and matrix. The sample is then absorbed into the matrix to form the analyte-matrix mixture.
V. Uses of Cosmetic Polynucleotides, Primary Constructs and mmRNA of the Invention
The cosmetic polynucleotides, primary constructs and mmRNA of the present invention are designed, in preferred embodiments, to provide for avoidance or evasion of deleterious bio-responses such as the immune response and/or degradation pathways, overcoming the threshold of expression and/or improving protein production capacity, improved expression rates or translation efficiency, improved drug or protein half life and/or protein concentrations, optimized protein localization, to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, secretion efficiency (when applicable), accessibility to circulation, and/or modulation of a cell's status, function and/or activity.
Therapeutics
Therapeutic Agents
The cosmetic polynucleotides, primary constructs or mmRNA of the present invention, such as modified nucleic acids and modified RNAs, and the proteins translated from them described herein can be used as therapeutic or prophylactic agents. They are provided for use in medicine. For example, a polynucleotide, primary construct or mmRNA described herein can be administered to a subject, wherein the polynucleotide, primary construct or mmRNA is translated in vivo to produce a therapeutic or prophylactic polypeptide in the subject. Provided are compositions, methods, kits, and reagents for diagnosis, treatment or prevention of a disease or condition in humans and other mammals. The active therapeutic agents of the invention include polynucleotides, primary constructs or mmRNA, cells containing polynucleotides, primary constructs or mmRNA or polypeptides translated from the cosmetic polynucleotides, primary constructs or mmRNA.
In certain embodiments, provided herein are combination therapeutics containing one or more polynucleotide, primary construct or mmRNA containing translatable regions that encode for a protein or proteins that boost a mammalian subject's immunity along with a protein that induces antibody-dependent cellular toxicity. For example, provided herein are therapeutics containing one or more nucleic acids that encode trastuzumab and granulocyte-colony stimulating factor (G-CSF). In particular, such combination therapeutics are useful in Her2+ breast cancer patients who develop induced resistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy. 2(6):795-8 (2010)).
Provided herein are methods of inducing translation of a recombinant polypeptide in a cell population using the polynucleotide, primary construct or mmRNA described herein. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell population is contacted with an effective amount of a composition containing a nucleic acid that has at least one nucleoside modification, and a translatable region encoding the recombinant polypeptide. The population is contacted under conditions such that the nucleic acid is localized into one or more cells of the cell population and the recombinant polypeptide is translated in the cell from the nucleic acid.
An “effective amount” of the composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the nucleic acid (e.g., size, and extent of modified nucleosides), and other determinants. In general, an effective amount of the composition provides efficient protein production in the cell, preferably more efficient than a composition containing a corresponding unmodified nucleic acid. Increased efficiency may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the nucleic acid), increased protein translation from the nucleic acid, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified nucleic acid), or reduced innate immune response of the host cell.
Aspects of the invention are directed to methods of inducing in vivo translation of a recombinant polypeptide in a mammalian subject in need thereof. Therein, an effective amount of a composition containing a nucleic acid that has at least one structural or chemical modification and a translatable region encoding the recombinant polypeptide is administered to the subject using the delivery methods described herein. The nucleic acid is provided in an amount and under other conditions such that the nucleic acid is localized into a cell of the subject and the recombinant polypeptide is translated in the cell from the nucleic acid. The cell in which the nucleic acid is localized, or the tissue in which the cell is present, may be targeted with one or more than one rounds of nucleic acid administration.
In certain embodiments, the administered polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that provide a functional activity which is substantially absent in the cell, tissue or organism in which the recombinant polypeptide is translated. For example, the missing functional activity may be enzymatic, structural, or gene regulatory in nature. In related embodiments, the administered polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that increases (e.g., synergistically) a functional activity which is present but substantially deficient in the cell in which the recombinant polypeptide is translated.
In other embodiments, the administered polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that replace a polypeptide (or multiple polypeptides) that is substantially absent in the cell in which the recombinant polypeptide is translated. Such absence may be due to genetic mutation of the encoding gene or regulatory pathway thereof. In some embodiments, the recombinant polypeptide increases the level of an endogenous protein in the cell to a desirable level; such an increase may bring the level of the endogenous protein from a subnormal level to a normal level or from a normal level to a super-normal level.
Alternatively, the recombinant polypeptide functions to antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell. Usually, the activity of the endogenous protein is deleterious to the subject; for example, due to mutation of the endogenous protein resulting in altered activity or localization. Additionally, the recombinant polypeptide antagonizes, directly or indirectly, the activity of a biological moiety present in, on the surface of, or secreted from the cell. Examples of antagonized biological moieties include lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), a nucleic acid, a carbohydrate, a protein toxin such as shiga and tetanus toxins, or a small molecule toxin such as botulinum, cholera, and diphtheria toxins. Additionally, the antagonized biological molecule may be an endogenous protein that exhibits an undesirable activity, such as a cytotoxic or cytostatic activity.
The recombinant proteins described herein may be engineered for localization within the cell, potentially within a specific compartment such as the nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
In some embodiments, modified mRNAs and their encoded polypeptides in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to one or more of the following: autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders (e.g. arthritis, pelvic inflammatory disease); infectious diseases (e.g. viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungal infections, sepsis); neurological disorders (e g. Alzheimer's disease, Huntington's disease; autism; Duchenne muscular dystrophy); cardiovascular disorders (e.g. atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders, angiogenic disorders such as macular degeneration); proliferative disorders (e.g. cancer, benign neoplasms); respiratory disorders (e.g. chronic obstructive pulmonary disease); digestive disorders (e.g. inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g. diabetes, osteoporosis); urological disorders (e.g. renal disease); psychological disorders (e.g. depression, schizophrenia); skin disorders (e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia, hemophilia); etc.
Diseases characterized by dysfunctional or aberrant protein activity include cystic fibrosis, sickle cell anemia, epidermolysis bullosa, amyotrophic lateral sclerosis, and glucose-6-phosphate dehydrogenase deficiency. The present invention provides a method for treating such conditions or diseases in a subject by introducing nucleic acid or cell-based therapeutics containing the polynucleotide, primary construct or mmRNA provided herein, wherein the polynucleotide, primary construct or mmRNA encode for a protein that antagonizes or otherwise overcomes the aberrant protein activity present in the cell of the subject. Specific examples of a dysfunctional protein are the missense mutation variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produce a dysfunctional protein variant of CFTR protein, which causes cystic fibrosis.
Diseases characterized by missing (or substantially diminished such that proper (normal or physiological protein function does not occur) protein activity include cystic fibrosis, Niemann-Pick type C, β thalassemia major, Duchenne muscular dystrophy, Hurler Syndrome, Hunter Syndrome, and Hemophilia A. Such proteins may not be present, or are essentially non-functional. The present invention provides a method for treating such conditions or diseases in a subject by introducing nucleic acid or cell-based therapeutics containing the polynucleotide, primary construct or mmRNA provided herein, wherein the polynucleotide, primary construct or mmRNA encode for a protein that replaces the protein activity missing from the target cells of the subject. Specific examples of a dysfunctional protein are the nonsense mutation variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produce a nonfunctional protein variant of CFTR protein, which causes cystic fibrosis.
Thus, provided are methods of treating cystic fibrosis in a mammalian subject by contacting a cell of the subject with a polynucleotide, primary construct or mmRNA having a translatable region that encodes a functional CFTR polypeptide, under conditions such that an effective amount of the CTFR polypeptide is present in the cell. Preferred target cells are epithelial, endothelial and mesothelial cells, such as the lung, and methods of administration are determined in view of the target tissue; i.e., for lung delivery, the RNA molecules are formulated for administration by inhalation.
In another embodiment, the present invention provides a method for treating hyperlipidemia in a subject, by introducing into a cell population of the subject with a modified mRNA molecule encoding Sortilin, a protein recently characterized by genomic studies, thereby ameliorating the hyperlipidemia in a subject. The SORT1 gene encodes a trans-Golgi network (TGN) transmembrane protein called Sortilin. Genetic studies have shown that one of five individuals has a single nucleotide polymorphism, rs12740374, in the 1p13 locus of the SORT1 gene that predisposes them to having low levels of low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL). Each copy of the minor allele, present in about 30% of people, alters LDL cholesterol by 8 mg/dL, while two copies of the minor allele, present in about 5% of the population, lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have also been shown to have a 40% decreased risk of myocardial infarction. Functional in vivo studies in mice describes that overexpression of SORT1 in mouse liver tissue led to significantly lower LDL-cholesterol levels, as much as 80% lower, and that silencing SORT1 increased LDL cholesterol approximately 200% (Musunuru K et al. From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466: 714-721).
In another embodiment, the present invention provides a method for treating hematopoietic disorders, cardiovascular disease, oncology, diabetes, cystic fibrosis, neurological diseases, inborn errors of metabolism, skin and systemic disorders, and blindness. The identity of molecular targets to treat these specific diseases has been described (Templeton ed., Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, 3rd Edition, Bota Raton, Fla.: CRC Press; herein incorporated by reference in its entirety).
Provided herein, are methods to prevent infection and/or sepsis in a subject at risk of developing infection and/or sepsis, the method comprising administering to a subject in need of such prevention a composition comprising a polynucleotide, primary construct or mmRNA precursor encoding an anti-microbial polypeptide (e.g., an anti-bacterial polypeptide), or a partially or fully processed form thereof in an amount sufficient to prevent infection and/or sepsis. In certain embodiments, the subject at risk of developing infection and/or sepsis may be a cancer patient. In certain embodiments, the cancer patient may have undergone a conditioning regimen. In some embodiments, the conditioning regiment may include, but is not limited to, chemotherapy, radiation therapy, or both.
Further provided herein, are methods to treat infection and/or sepsis in a subject, the method comprising administering to a subject in need of such treatment a composition comprising a polynucleotide, primary construct or mmRNA precursor encoding an anti-microbial polypeptide (e.g., an anti-bacterial polypeptide), e.g., an anti-microbial polypeptide described herein, or a partially or fully processed form thereof in an amount sufficient to treat an infection and/or sepsis. In certain embodiments, the subject in need of treatment is a cancer patient. In certain embodiments, the cancer patient has undergone a conditioning regimen. In some embodiments, the conditioning regiment may include, but is not limited to, chemotherapy, radiation therapy, or both.
In certain embodiments, the subject may exhibits acute or chronic microbial infections (e.g., bacterial infections). In certain embodiments, the subject may have received or may be receiving a therapy. In certain embodiments, the therapy may include, but is not limited to, radiotherapy, chemotherapy, steroids, ultraviolet radiation, or a combination thereof. In certain embodiments, the patient may suffer from a microvascular disorder. In some embodiments, the microvascular disorder may be diabetes. In certain embodiments, the patient may have a wound. In some embodiments, the wound may be an ulcer. In a specific embodiment, the wound may be a diabetic foot ulcer. In certain embodiments, the subject may have one or more burn wounds. In certain embodiments, the administration may be local or systemic. In certain embodiments, the administration may be subcutaneous. In certain embodiments, the administration may be intravenous. In certain embodiments, the administration may be oral. In certain embodiments, the administration may be topical. In certain embodiments, the administration may be by inhalation. In certain embodiments, the administration may be rectal. In certain embodiments, the administration may be vaginal.
Other aspects of the present disclosure relate to transplantation of cells containing polynucleotide, primary construct, or mmRNA to a mammalian subject. Administration of cells to mammalian subjects is known to those of ordinary skill in the art, and include, but is not limited to, local implantation (e.g., topical or subcutaneous administration), organ delivery or systemic injection (e.g., intravenous injection or inhalation), and the formulation of cells in pharmaceutically acceptable carrier. Such compositions containing polynucleotide, primary construct, or mmRNA can be formulated for administration intramuscularly, transarterially, intraperitoneally, intravenously, intranasally, subcutaneously, endoscopically, transdermally, or intrathecally. In some embodiments, the composition may be formulated for extended release.
The subject to whom the therapeutic agent may be administered suffers from or may be at risk of developing a disease, disorder, or deleterious condition. Provided are methods of identifying, diagnosing, and classifying subjects on these bases, which may include clinical diagnosis, biomarker levels, genome-wide association studies (GWAS), and other methods known in the art.
Wound Management
The cosmetic polynucleotides, primary constructs or mmRNA of the present invention may be used for wound treatment, e.g. of wounds exhibiting delayed healing. Provided herein are methods comprising the administration of polynucleotide, primary construct or mmRNA in order to manage the treatment of wounds. The methods herein may further comprise steps carried out either prior to, concurrent with or post administration of the polynucleotide, primary construct or mmRNA. For example, the wound bed may need to be cleaned and prepared in order to facilitate wound healing and hopefully obtain closure of the wound. Several strategies may be used in order to promote wound healing and achieve wound closure including, but not limited to: (i) debridement, optionally repeated, sharp debridement (surgical removal of dead or infected tissue from a wound), optionally including chemical debriding agents, such as enzymes, to remove necrotic tissue; (ii) wound dressings to provide the wound with a moist, warm environment and to promote tissue repair and healing.
Examples of materials that are used in formulating wound dressings include, but are not limited to: hydrogels (e.g., AQUASORB®; DUODERM®), hydrocolloids (e.g., AQUACEL®; COMFEEL®), foams (e.g., LYOFOAM®; SPYROSORB®), and alginates (e.g., ALGISITE®; CURASORB®); (iii) additional growth factors to stimulate cell division and proliferation and to promote wound healing e.g. becaplermin (REGRANEX GEL®), a human recombinant platelet-derived growth factor that is approved by the FDA for the treatment of neuropathic foot ulcers; (iv) soft-tissue wound coverage, a skin graft may be necessary to obtain coverage of clean, non-healing wounds. Examples of skin grafts that may be used for soft-tissue coverage include, but are not limited to: autologous skin grafts, cadaveric skin graft, bioengineered skin substitutes (e.g., APLIGRAF®; DERMAGRAFT®).
In certain embodiments, the polynucleotide, primary construct or mmRNA of the present invention may further include hydrogels (e.g., AQUASORB®; DUODERM®), hydrocolloids (e.g., AQUACEL®; COMFEEL®), foams (e.g., LYOFOAM®; SPYROSORB®), and/or alginates (e.g., ALGISITE®; CURASORB®). In certain embodiments, the polynucleotide, primary construct or mmRNA of the present invention may be used with skin grafts including, but not limited to, autologous skin grafts, cadaveric skin graft, or bioengineered skin substitutes (e.g., APLIGRAF®; DERMAGRAFT®). In some embodiments, the polynucleotide, primary construct or mmRNA may be applied with would dressing formulations and/or skin grafts or they may be applied separately but methods such as, but not limited to, soaking or spraying.
In some embodiments, compositions for wound management may comprise a polynucleotide, primary construct or mmRNA encoding for an anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) and/or an anti-viral polypeptide. A precursor or a partially or fully processed form of the anti-microbial polypeptide may be encoded. The composition may be formulated for administration using a bandage (e.g., an adhesive bandage). The anti-microbial polypeptide and/or the anti-viral polypeptide may be intermixed with the dressing compositions or may be applied separately, e.g., by soaking or spraying.
Production of Antibodies
In one embodiment of the invention, the cosmetic polynucleotides, primary constructs or mmRNA may encode antibodies and fragments of such antibodies. These may be produced by any one of the methods described herein. The antibodies may be of any of the different subclasses or isotypes of immunoglobulin such as, but not limited to, IgA, IgG, or IgM, or any of the other subclasses. Exemplary antibody molecules and fragments that may be prepared according to the invention include, but are not limited to, immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that may contain the paratope. Such portion of antibodies that contain the paratope include, but are not limited to Fab, Fab′, F(ab′)2, F(v) and those portions known in the art.
The polynucleotides of the invention may encode variant antibody polypeptides which may have a certain identity with a reference polypeptide sequence, or have a similar or dissimilar binding characteristic with the reference polypeptide sequence.
Antibodies obtained by the methods of the present invention may be chimeric antibodies comprising non-human antibody-derived variable region(s) sequences, derived from the immunized animals, and human antibody-derived constant region(s) sequences. In addition, they can also be humanized antibodies comprising complementary determining regions (CDRs) of non-human antibodies derived from the immunized animals and the framework regions (FRs) and constant regions derived from human antibodies. In another embodiment, the methods provided herein may be useful for enhancing antibody protein product yield in a cell culture process.
Managing Infection
In one embodiment, provided are methods for treating or preventing a microbial infection (e.g., a bacterial infection) and/or a disease, disorder, or condition associated with a microbial or viral infection, or a symptom thereof, in a subject, by administering a polynucleotide, primary construct or mmRNA encoding an anti-microbial polypeptide. Said administration may be in combination with an anti-microbial agent (e.g., an anti-bacterial agent), e.g., an anti-microbial polypeptide or a small molecule anti-microbial compound described herein. The anti-microbial agents include, but are not limited to, anti-bacterial agents, anti-viral agents, anti-fungal agents, anti-protozoal agents, anti-parasitic agents, and anti-prion agents.
The agents can be administered simultaneously, for example in a combined unit dose (e.g., providing simultaneous delivery of both agents). The agents can also be administered at a specified time interval, such as, but not limited to, an interval of minutes, hours, days or weeks. Generally, the agents may be concurrently bioavailable, e.g., detectable, in the subject. In some embodiments, the agents may be administered essentially simultaneously, for example two unit dosages administered at the same time, or a combined unit dosage of the two agents. In other embodiments, the agents may be delivered in separate unit dosages. The agents may be administered in any order, or as one or more preparations that includes two or more agents. In a preferred embodiment, at least one administration of one of the agents, e.g., the first agent, may be made within minutes, one, two, three, or four hours, or even within one or two days of the other agent, e.g., the second agent. In some embodiments, combinations can achieve synergistic results, e.g., greater than additive results, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than additive results.
Conditions Associated with Bacterial Infection
Diseases, disorders, or conditions which may be associated with bacterial infections include, but are not limited to one or more of the following: abscesses, actinomycosis, acute prostatitis, aeromonas hydrophila, annual ryegrass toxicity, anthrax, bacillary peliosis, bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, bacterium-related cutaneous conditions, bartonellosis, BCG-oma, botryomycosis, botulism, Brazilian purpuric fever, Brodie abscess, brucellosis, Buruli ulcer, campylobacteriosis, caries, Carrion's disease, cat scratch disease, cellulitis, chlamydia infection, cholera, chronic bacterial prostatitis, chronic recurrent multifocal osteomyelitis, clostridial necrotizing enteritis, combined periodontic-endodontic lesions, contagious bovine pleuropneumonia, diphtheria, diphtheritic stomatitis, ehrlichiosis, erysipelas, piglottitis, erysipelas, Fitz-Hugh-Curtis syndrome, flea-borne spotted fever, foot rot (infectious pododermatitis), Garre's sclerosing osteomyelitis, Gonorrhea, Granuloma inguinale, human granulocytic anaplasmosis, human monocytotropic ehrlichiosis, hundred days' cough, impetigo, late congenital syphilitic oculopathy, legionellosis, Lemierre's syndrome, leprosy (Hansen's Disease), leptospirosis, listeriosis, Lyme disease, lymphadenitis, melioidosis, meningococcal disease, meningococcal septicaemia, methicillin-resistant Staphylococcus aureus (MRSA) infection, mycobacterium avium-intracellulare (MAI), mycoplasma pneumonia, necrotizing fasciitis, nocardiosis, noma (cancrum oris or gangrenous stomatitis), omphalitis, orbital cellulitis, osteomyelitis, overwhelming post-splenectomy infection (OPSI), ovine brucellosis, pasteurellosis, periorbital cellulitis, pertussis (whooping cough), plague, pneumococcal pneumonia, Pott disease, proctitis, pseudomonas infection, psittacosis, pyaemia, pyomyositis, Q fever, relapsing fever (typhinia), rheumatic fever, Rocky Mountain spotted fever (RMSF), rickettsiosis, salmonellosis, scarlet fever, sepsis, serratia infection, shigellosis, southern tick-associated rash illness, staphylococcal scalded skin syndrome, streptococcal pharyngitis, swimming pool granuloma, swine brucellosis, syphilis, syphilitic aortitis, tetanus, toxic shock syndrome (TSS), trachoma, trench fever, tropical ulcer, tuberculosis, tularemia, typhoid fever, typhus, urogenital tuberculosis, urinary tract infections, vancomycin-resistant Staphylococcus aureus infection, Waterhouse-Friderichsen syndrome, pseudotuberculosis (Yersinia) disease, and yersiniosis. Other diseases, disorders, and/or conditions associated with bacterial infections can include, for example, Alzheimer's disease, anorexia nervosa, asthma, atherosclerosis, attention deficit hyperactivity disorder, autism, autoimmune diseases, bipolar disorder, cancer (e.g., colorectal cancer, gallbladder cancer, lung cancer, pancreatic cancer, and stomach cancer), chronic fatigue syndrome, chronic obstructive pulmonary disease, Crohn's disease, coronary heart disease, dementia, depression, Guillain-Barré syndrome, metabolic syndrome, multiple sclerosis, myocardial infarction, obesity, obsessive-compulsive disorder, panic disorder, psoriasis, rheumatoid arthritis, sarcoidosis, schizophrenia, stroke, thromboangiitis obliterans (Buerger's disease), and Tourette syndrome.
Bacterial Pathogens
The bacterium described herein can be a Gram-positive bacterium or a Gram-negative bacterium. Bacterial pathogens include, but are not limited to, Acinetobacter baumannii, Bacillus anthracis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, coagulase Negative Staphylococcus, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, E. coli O157:H7, Enterobacter sp., Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Moraxella catarralis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Preteus mirabilis, Proteus sps., Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Serratia marcesens, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, and Yersinia pestis. Bacterial pathogens may also include bacteria that cause resistant bacterial infections, for example, clindamycin-resistant Clostridium difficile, fluoroquinolon-resistant Clostridium difficile, methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Enterococcus faecalis, multidrug-resistant Enterococcus faecium, multidrug-resistance Pseudomonas aeruginosa, multidrug-resistant Acinetobacter baumannii, and vancomycin-resistant Staphylococcus aureus (VRSA).
Antibiotic Combinations
In one embodiment, the modified mRNA of the present invention may be administered in conjunction with one or more antibiotics. These include, but are not limited to Aknilox, Ambisome, Amoxycillin, Ampicillin, Augmentin, Avelox, Azithromycin, Bactroban, Betadine, Betnovate, Blephamide, Cefaclor, Cefadroxil, Cefdinir, Cefepime, Cefix, Cefixime, Cefoxitin, Cefpodoxime, Cefprozil, Cefuroxime, Cefzil, Cephalexin, Cephazolin, Ceptaz, Chloramphenicol, Chlorhexidine, Chloromycetin, Chlorsig, Ciprofloxacin, Clarithromycin, Clindagel, Clindamycin, Clindatech, Cloxacillin, Colistin, Co-trimoxazole, Demeclocycline, Diclocil, Dicloxacillin, Doxycycline, Duricef, Erythromycin, Flamazine, Floxin, Framycetin, Fucidin, Furadantin, Fusidic, Gatifloxacin, Gemifloxacin, Gemifloxacin, Ilosone, Iodine, Levaquin, Levofloxacin, Lomefloxacin, Maxaquin, Mefoxin, Meronem, Minocycline, Moxifloxacin, Myambutol, Mycostatin, Neosporin, Netromycin, Nitrofurantoin, Norfloxacin, Norilet, Ofloxacin, Omnicef, Ospamox, Oxytetracycline, Paraxin, Penicillin, Pneumovax, Polyfax, Povidone, Rifadin, Rifampin, Rifaximin, Rifinah, Rimactane, Rocephin, Roxithromycin, Seromycin, Soframycin, Sparfloxacin, Staphlex, Targocid, Tetracycline, Tetradox, Tetralysal, tobramycin, Tobramycin, Trecator, Tygacil, Vancocin, Velosef, Vibramycin, Xifaxan, Zagam, Zitrotek, Zoderm, Zymar, and Zyvox.
Antibacterial Agents
Exemplary anti-bacterial agents include, but are not limited to, aminoglycosides (e.g., amikacin (AMIKIN®), gentamicin (GARAMYCIN®), kanamycin (KANTREX®), neomycin (MYCIFRADIN®), netilmicin (NETROMYCIN®), tobramycin (NEBCIN®), Paromomycin (HUMATIN®)), ansamycins (e.g., geldanamycin, herbimycin), carbacephem (e.g., loracarbef (LORABID®), Carbapenems (e.g., ertapenem (INVANZ®), doripenem (DORIBAX®), imipenem/cilastatin (PRIMAXIN®), meropenem (MERREM®), cephalosporins (first generation) (e.g., cefadroxil (DURICEF®), cefazolin (ANCEF®), cefalotin or cefalothin (KEFLIN®), cefalexin (KEFLEX®), cephalosporins (second generation) (e.g., cefaclor (CECLOR®), cefamandole (MANDOL®), cefoxitin (MEFOXIN®), cefprozil (CEFZIL®), cefuroxime (CEFTIN®, ZINNAT®)), cephalosporins (third generation) (e.g., cefixime (SUPRAX®), cefdinir (OMNICEF®, CEFDIEL®), cefditoren (SPECTRACEF®), cefoperazone (CEFOBID®), cefotaxime (CLAFORAN®), cefpodoxime (VANTIN®), ceftazidime (FORTAZ®), ceftibuten (CEDAX®), ceftizoxime (CEFIZOX®), ceftriaxone (ROCEPHIN®)), cephalosporins (fourth generation) (e.g., cefepime (MAXIPIME®)), cephalosporins (fifth generation) (e.g., ceftobiprole (ZEFTERA®)), glycopeptides (e.g., teicoplanin (TARGOCID®), vancomycin (VANCOCIN®), telavancin (VIBATIV®)), lincosamides (e.g., clindamycin (CLEOCIN®), lincomycin (LINCOCIN®)), lipopeptide (e.g., daptomycin (CUBICIN®)), macrolides (e.g., azithromycin (ZITHROMAX®, SUMAMED®, ZITROCIN®), clarithromycin (BIAXIN®), dirithromycin (DYNABAC®), erythromycin (ERYTHOCIN®, ERYTHROPED®), roxithromycin, troleandomycin (TAO®), telithromycin (KETEK®), spectinomycin (TROBICIN®)), monobactams (e.g., aztreonam (AZACTAM®)), nitrofurans (e.g., furazolidone (FUROXONE®), nitrofurantoin (MACRODANTIN®, MACROBID®)), penicillins (e.g., amoxicillin (NOVAMOX®, AMOXIL®), ampicillin (PRINCIPEN®), azlocillin, carbenicillin (GEOCILLIN®), cloxacillin (TEGOPEN®), dicloxacillin (DYNAPEN®), flucloxacillin (FLOXAPEN®), mezlocillin (MEZLIN®), methicillin (STAPHCILLIN®), nafcillin (UNIPEN®), oxacillin (PROSTAPHLIN®), penicillin G (PENTIDS®), penicillin V (PEN-VEE-K®), piperacillin (PIPRACIL®), temocillin (NEGABAN®), ticarcillin (TICAR®)), penicillin combinations (e.g., amoxicillin/clavulanate (AUGMENTIN®), ampicillin/sulbactam (UNASYN®), piperacillin/tazobactam (ZOSYN®), ticarcillin/clavulanate (TIMENTIN®)), polypeptides (e.g., bacitracin, colistin (COLY-MYCIN-S®), polymyxin B, quinolones (e.g., ciprofloxacin (CIPRO®, CIPROXIN®, CIPROBAY®), enoxacin (PENETREX®), gatifloxacin (TEQUIN®), levofloxacin (LEVAQUIN®), lomefloxacin (MAXAQUIN®), moxifloxacin (AVELOX®), nalidixic acid (NEGGRAM®), norfloxacin (NOROXIN®), ofloxacin (FLOXIN®, OCUFLOX®), trovafloxacin (TROVAN®), grepafloxacin (RAXAR®), sparfloxacin (ZAGAM®), temafloxacin (OMNIFLOX®)), sulfonamides (e.g., mafenide (SULFAMYLON®), sulfonamidochrysoidine (PRONTOSIL®), sulfacetamide (SULAMYD®, BLEPH-100), sulfadiazine (MICRO-SULFON®), silver sulfadiazine (SILVADENE®), sulfamethizole (THIOSULFIL FORTE®), sulfamethoxazole (GANTANOL®), sulfanilimide, sulfasalazine (AZULFIDINE®), sulfisoxazole (GANTRISIN®), trimethoprim (PROLOPRIM®), TRIMPEX®), trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX) (BACTRIM®, SEPTRA®)), tetracyclines (e.g., demeclocycline (DECLOMYCIN®), doxycycline (VIBRAMYCIN®), minocycline (MINOCIN®), oxytetracycline (TERRAMYCIN®), tetracycline (SUMYCIN®, ACHROMYCIN® V, STECLIN®)), drugs against mycobacteria (e.g., clofazimine (LAMPRENE®), dapsone (AVLOSULFON®), capreomycin (CAPASTAT®), cycloserine (SEROMYCIN®), ethambutol (MYAMBUTOL®), ethionamide (TRECATOR®), isoniazid (I.N.H.®), pyrazinamide (ALDINAMIDE®), rifampin (RIFADIN®, RIMACTANE®), rifabutin (MYCOBUTIN®), rifapentine (PRIFTIN®), streptomycin), and others (e.g., arsphenamine (SALVARSAN®), chloramphenicol (CHLOROMYCETIN®), fosfomycin (MONUROL®), fusidic acid (FUCIDIN®), linezolid (ZYVOX®), metronidazole (FLAGYL®), mupirocin (BACTROBAN®), platensimycin, quinupristin/dalfopristin (SYNERCID®), rifaximin (XIFAXAN®), thiamphenicol, tigecycline (TIGACYL®), tinidazole (TINDAMAX®, FASIGYN®)).
Conditions Associated with Viral Infection
In another embodiment, provided are methods for treating or preventing a viral infection and/or a disease, disorder, or condition associated with a viral infection, or a symptom thereof, in a subject, by administering a polynucleotide, primary construct or mmRNA encoding an anti-viral polypeptide, e.g., an anti-viral polypeptide described herein in combination with an anti-viral agent, e.g., an anti-viral polypeptide or a small molecule anti-viral agent described herein.
Diseases, disorders, or conditions associated with viral infections include, but are not limited to, acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, Coxsackie infections, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, hepatic cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (e.g., herpes labialis and cold sores), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, Cytomegalic inclusion disease, Kaposi sarcoma, multicentric Castleman disease, primary effusion lymphoma, AIDS, influenza, Reye syndrome, measles, postinfectious encephalomyelitis, Mumps, hyperplastic epithelial lesions (e.g., common, flat, plantar and anogenital warts, laryngeal papillomas, epidermodysplasia verruciformis), cervical carcinoma, squamous cell carcinomas, croup, pneumonia, bronchiolitis, common cold, Poliomyelitis, Rabies, bronchiolitis, pneumonia, influenza-like syndrome, severe bronchiolitis with pneumonia, German measles, congenital rubella, Varicella, and herpes zoster.
Viral Pathogens
Viral pathogens include, but are not limited to, adenovirus, coxsackievirus, dengue virus, encephalitis virus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human herpesvirus type 8, human immunodeficiency virus, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, varicella-zoster virus, West Nile virus, and yellow fever virus. Viral pathogens may also include viruses that cause resistant viral infections.
Antiviral Agents
Exemplary anti-viral agents include, but are not limited to, abacavir (ZIAGEN®), abacavir/lamivudine/zidovudine (Trizivir®), aciclovir or acyclovir (CYCLOVIR®, HERPEX®, ACIVIR®, ACIVIRAX®, ZOVIRAX®, ZOVIR®), adefovir (Preveon®, Hepsera®), amantadine (SYMMETREL®), amprenavir (AGENERASE®), ampligen, arbidol, atazanavir (REYATAZ®), boceprevir, cidofovir, darunavir (PREZISTA®), delavirdine (RESCRIPTOR®), didanosine (VIDEX®), docosanol (ABREVA®), edoxudine, efavirenz (SUSTIVA®, STOCRIN®), emtricitabine (EMTRIVA®), emtricitabine/tenofovir/efavirenz (ATRIPLA®), enfuvirtide (FUZEON®), entecavir (BARACLUDE®, ENTAVIR®), famciclovir (FAMVIR®), fomivirsen (VITRAVENE®), fosamprenavir (LEXIVA®, TELZIR®), foscarnet (FOSCAVIR®), fosfonet, ganciclovir (CYTOVENE®, CYMEVENE®, VITRASERT®), GS 9137 (ELVITEGRAVIR®), imiquimod (ALDARA®, ZYCLARA®, BESELNA®), indinavir (CRIXIVAN®), inosine, inosine pranobex (IMUNOVIR®), interferon type I, interferon type II, interferon type III, kutapressin (NEXAVIR®), lamivudine (ZEFFIX®, HEPTOVIR®, EPIVIR®), lamivudine/zidovudine (COMBIVIR®), lopinavir, loviride, maraviroc (SELZENTRY®, CELSENTRI®), methisazone, MK-2048, moroxydine, nelfinavir (VIRACEPT®), nevirapine (VIRAMUNE®), oseltamivir (TAMIFLU®), peginterferon alfa-2a (PEGASYS®), penciclovir (DENAVIR®), peramivir, pleconaril, podophyllotoxin (CONDYLOX®), raltegravir (ISENTRESS®), ribavirin (COPEGUs®, REBETOL®, RIBASPHEREO, VILONA® AND VIRAZOLE®), rimantadine (FLUMADINE®), ritonavir (NORVIR®), pyramidine, saquinavir (INVIRASE®, FORTOVASE®), stavudine, tea tree oil (melaleuca oil), tenofovir (VIREAD®), tenofovir/emtricitabine (TRUVADA®), tipranavir (APTIVUS®), trifluridine (VIROPTIC®), tromantadine (VIRU-MERZ®), valaciclovir (VALTREX®), valganciclovir (VALCYTE®), vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir (RELENZA®), and zidovudine (azidothymidine (AZT), RETROVIR®, RETROVIS®).
Conditions Associated with Fungal Infections
Diseases, disorders, or conditions associated with fungal infections include, but are not limited to, aspergilloses, blastomycosis, candidasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycetomas, paracoccidioidomycosis, and tinea pedis. Furthermore, persons with immuno-deficiencies are particularly susceptible to disease by fungal genera such as Aspergillus, Candida, Cryptoccocus, Histoplasma, and Pneumocystis. Other fungi can attack eyes, nails, hair, and especially skin, the so-called dermatophytic fungi and keratinophilic fungi, and cause a variety of conditions, of which ringworms such as athlete's foot are common. Fungal spores are also a major cause of allergies, and a wide range of fungi from different taxonomic groups can evoke allergic reactions in some people.
Fungal Pathogens
Fungal pathogens include, but are not limited to, Ascomycota (e.g., Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis/posadasii, Candida albicans), Basidiomycota (e.g., Filobasidiella neoformans, Trichosporon), Microsporidia (e.g., Encephalitozoon cuniculi, Enterocytozoon bieneusi), and Mucoromycotina (e.g., Mucor circinelloides, Rhizopus oryzae, Lichtheimia corymbifera).
Anti-Fungal Agents
Exemplary anti-fungal agents include, but are not limited to, polyene antifungals (e.g., natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, hamycin), imidazole antifungals (e.g., miconazole (MICATIN®, DAKTARIN®), ketoconazole (NIZORAL®, FUNGORAL®, SEBIZOLE®), clotrimazole (LOTRIMIN®, LOTRIMIN® AF, CANESTEN®), econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole (ERTACZO®), sulconazole, tioconazole), triazole antifungals (e.g., albaconazole fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole), thiazole antifungals (e.g., abafungin), allylamines (e.g., terbinafine (LAMISIL®), naftifine (NAFTIN®), butenafine (LOTRIMIN® Ultra)), echinocandins (e.g., anidulafungin, caspofungin, micafungin), and others (e.g., polygodial, benzoic acid, ciclopirox, tolnaftate (TINACTIN®, DESENEX®, AFTATE®), undecylenic acid, flucytosine or 5-fluorocytosine, griseofulvin, haloprogin, sodium bicarbonate, allicin).
Conditions Associated with Protozoal Infection
Diseases, disorders, or conditions associated with protozoal infections include, but are not limited to, amoebiasis, giardiasis, trichomoniasis, African Sleeping Sickness, American Sleeping Sickness, leishmaniasis (Kala-Azar), balantidiasis, toxoplasmosis, malaria, acanthamoeba keratitis, and babesiosis.
Protozoan Pathogens
Protozoal pathogens include, but are not limited to, Entamoeba histolytica, Giardia lambila, Trichomonas vaginalis, Trypanosoma brucei, T. cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii, Plasmodium spp., and Babesia microti. 
Anti-Protozoan Agents
Exemplary anti-protozoal agents include, but are not limited to, eflornithine, furazolidone (FUROXONE®, DEPENDAL-M®), melarsoprol, metronidazole (FLAGYL®), ornidazole, paromomycin sulfate (HUMATIN®), pentamidine, pyrimethamine (DARAPRIM®), and tinidazole (TINDAMAX®, FASIGYN®).
Conditions Associated with Parasitic Infection
Diseases, disorders, or conditions associated with parasitic infections include, but are not limited to, acanthamoeba keratitis, amoebiasis, ascariasis, babesiosis, balantidiasis, baylisascariasis, chagas disease, clonorchiasis, cochliomyia, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, fasciolopsiasis, filariasis, giardiasis, gnathostomiasis, hymenolepiasis, isosporiasis, katayama fever, leishmaniasis, lyme disease, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.
Parasitic Pathogens
Parasitic pathogens include, but are not limited to, Acanthamoeba, Anisakis, Ascaris lumbricoides, botfly, Balantidium coli, bedbug, Cestoda, chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, hookworm, Leishmania, Linguatula serrata, liver fluke, Loa boa, Paragonimus, pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, mite, tapeworm, Toxoplasma gondii, Trypanosoma, whipworm, Wuchereria bancrofti. 
Anti-Parasitic Agents
Exemplary anti-parasitic agents include, but are not limited to, antinematodes (e.g., mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin), anticestodes (e.g., niclosamide, praziquantel, albendazole), antitrematodes (e.g., praziquantel), antiamoebics (e.g., rifampin, amphotericin B), and antiprotozoals (e.g., melarsoprol, eflornithine, metronidazole, tinidazole).
Conditions Associated with Prion Infection
Diseases, disorders, or conditions associated with prion infections include, but are not limited to Creutzfeldt-Jakob disease (CJD), iatrogenic Creutzfeldt-Jakob disease (iCJD), variant Creutzfeldt-Jakob disease (vCJD), familial Creutzfeldt-Jakob disease (fCJD), sporadic Creutzfeldt-Jakob disease (sCJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), Kuru, Scrapie, bovine spongiform encephalopathy (BSE), mad cow disease, transmissible mink encephalopathy (TME), chronic wasting disease (CWD), feline spongiform encephalopathy (FSE), exotic ungulate encephalopathy (EUE), and spongiform encephalopathy.
Anti-Prion Agents
Exemplary anti-prion agents include, but are not limited to, flupirtine, pentosan polysuphate, quinacrine, and tetracyclic compounds.
Modulation of the Immune Response
Avoidance of the Immune Response
As described herein, a useful feature of the cosmetic polynucleotides, primary constructs or mmRNA of the invention is the capacity to reduce, evade or avoid the innate immune response of a cell. In one aspect, provided herein are polynucleotides, primary constructs or mmRNA encoding a polypeptide of interest which when delivered to cells, results in a reduced immune response from the host as compared to the response triggered by a reference compound, e.g. an unmodified polynucleotide corresponding to a polynucleotide, primary construct or mmRNA of the invention, or a different polynucleotide, primary construct or mmRNA of the invention. As used herein, a “reference compound” is any molecule or substance which when administered to a mammal, results in an innate immune response having a known degree, level or amount of immune stimulation. A reference compound need not be a nucleic acid molecule and it need not be any of the cosmetic polynucleotides, primary constructs or mmRNA of the invention. Hence, the measure of a polynucleotides, primary constructs or mmRNA avoidance, evasion or failure to trigger an immune response can be expressed in terms relative to any compound or substance which is known to trigger such a response.
The term “innate immune response” includes a cellular response to exogenous single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. As used herein, the innate immune response or interferon response operates at the single cell level causing cytokine expression, cytokine release, global inhibition of protein synthesis, global destruction of cellular RNA, upregulation of major histocompatibility molecules, and/or induction of apoptotic death, induction of gene transcription of genes involved in apoptosis, anti-growth, and innate and adaptive immune cell activation. Some of the genes induced by type I IFNs include PKR, ADAR (adenosine deaminase acting on RNA), OAS (2′,5′-oligoadenylate synthetase), RNase L, and Mx proteins. PKR and ADAR lead to inhibition of translation initiation and RNA editing, respectively. OAS is a dsRNA-dependent synthetase that activates the endoribonuclease RNase L to degrade ssRNA.
In some embodiments, the innate immune response comprises expression of a Type I or Type II interferon, and the expression of the Type I or Type II interferon is not increased more than two-fold compared to a reference from a cell which has not been contacted with a polynucleotide, primary construct or mmRNA of the invention.
In some embodiments, the innate immune response comprises expression of one or more IFN signature genes and where the expression of the one of more IFN signature genes is not increased more than three-fold compared to a reference from a cell which has not been contacted with the polynucleotide, primary construct or mmRNA of the invention.
While in some circumstances, it might be advantageous to eliminate the innate immune response in a cell, the invention provides polynucleotides, primary constructs and mmRNA that upon administration result in a substantially reduced (significantly less) the immune response, including interferon signaling, without entirely eliminating such a response.
In some embodiments, the immune response is lower by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% as compared to the immune response induced by a reference compound. The immune response itself may be measured by determining the expression or activity level of Type 1 interferons or the expression of interferon-regulated genes such as the toll-like receptors (e.g., TLR7 and TLR8). Reduction of innate immune response can also be measured by measuring the level of decreased cell death following one or more administrations to a cell population; e.g., cell death is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequency observed with a reference compound. Moreover, cell death may affect fewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than 0.01% of cells contacted with the polynucleotide, primary construct or mmRNA.
In another embodiment, the polynucleotide, primary construct or mmRNA of the present invention is significantly less immunogenic than an unmodified in vitro-synthesized RNA molecule polynucleotide, or primary construct with the same sequence or a reference compound. As used herein, “significantly less immunogenic” refers to a detectable decrease in immunogenicity. In another embodiment, the term refers to a fold decrease in immunogenicity. In another embodiment, the term refers to a decrease such that an effective amount of the polynucleotide, primary construct or mmRNA can be administered without triggering a detectable immune response. In another embodiment, the term refers to a decrease such that the polynucleotide, primary construct or mmRNA can be repeatedly administered without eliciting an immune response sufficient to detectably reduce expression of the recombinant protein. In another embodiment, the decrease is such that the polynucleotide, primary construct or mmRNA can be repeatedly administered without eliciting an immune response sufficient to eliminate detectable expression of the recombinant protein.
In another embodiment, the polynucleotide, primary construct or mmRNA is 2-fold less immunogenic than its unmodified counterpart or reference compound. In another embodiment, immunogenicity is reduced by a 3-fold factor. In another embodiment, immunogenicity is reduced by a 5-fold factor. In another embodiment, immunogenicity is reduced by a 7-fold factor. In another embodiment, immunogenicity is reduced by a 10-fold factor. In another embodiment, immunogenicity is reduced by a 15-fold factor. In another embodiment, immunogenicity is reduced by a fold factor. In another embodiment, immunogenicity is reduced by a 50-fold factor. In another embodiment, immunogenicity is reduced by a 100-fold factor. In another embodiment, immunogenicity is reduced by a 200-fold factor. In another embodiment, immunogenicity is reduced by a 500-fold factor. In another embodiment, immunogenicity is reduced by a 1000-fold factor. In another embodiment, immunogenicity is reduced by a 2000-fold factor. In another embodiment, immunogenicity is reduced by another fold difference.
Methods of determining immunogenicity are well known in the art, and include, e.g. measuring secretion of cytokines (e.g. IL-12, IFNalpha, TNF-alpha, RANTES, MIP-1alpha or beta, IL-6, IFN-beta, or IL-8), measuring expression of DC activation markers (e.g. CD83, HLA-DR, CD80 and CD86), or measuring ability to act as an adjuvant for an adaptive immune response.
The polynucleotide, primary construct or mmRNA of the invention, including the combination of modifications taught herein may have superior properties making them more suitable as therapeutic modalities.
It has been determined that the “all or none” model in the art is sorely insufficient to describe the biological phenomena associated with the therapeutic utility of modified mRNA. The present inventors have determined that to improve protein production, one may consider the nature of the modification, or combination of modifications, the percent modification and survey more than one cytokine or metric to determine the efficacy and risk profile of a particular modified mRNA.
In one aspect of the invention, methods of determining the effectiveness of a modified mRNA as compared to unmodified involves the measure and analysis of one or more cytokines whose expression is triggered by the administration of the exogenous nucleic acid of the invention. These values are compared to administration of an unmodified nucleic acid or to a standard metric such as cytokine response, PolyIC, R-848 or other standard known in the art.
One example of a standard metric developed herein is the measure of the ratio of the level or amount of encoded polypeptide (protein) produced in the cell, tissue or organism to the level or amount of one or more (or a panel) of cytokines whose expression is triggered in the cell, tissue or organism as a result of administration or contact with the modified nucleic acid. Such ratios are referred to herein as the Protein:Cytokine Ratio or “PC” Ratio. The higher the PC ratio, the more efficacious the modified nucleic acid (polynucleotide encoding the protein measured). Preferred PC Ratios, by cytokine, of the present invention may be greater than 1, greater than 10, greater than 100, greater than 1000, greater than 10,000 or more. Modified nucleic acids having higher PC Ratios than a modified nucleic acid of a different or unmodified construct are preferred.
The PC ratio may be further qualified by the percent modification present in the polynucleotide. For example, normalized to a 100% modified nucleic acid, the protein production as a function of cytokine (or risk) or cytokine profile can be determined.
In one embodiment, the present invention provides a method for determining, across chemistries, cytokines or percent modification, the relative efficacy of any particular modified the polynucleotide, primary construct or mmRNA by comparing the PC Ratio of the modified nucleic acid (polynucleotide, primary construct or mmRNA).
mmRNA containing varying levels of nucleobase substitutions could be produced that maintain increased protein production and decreased immunostimulatory potential. The relative percentage of any modified nucleotide to its naturally occurring nucleotide counterpart can be varied during the IVT reaction (for instance, 100, 50, 25, 10, 5, 2.5, 1, 0.1, 0.01% 5 methyl cytidine usage versus cytidine; 100, 50, 25, 10, 5, 2.5, 1, 0.1, 0.01% pseudouridine or N1-methyl-pseudouridine usage versus uridine). mmRNA can also be made that utilize different ratios using 2 or more different nucleotides to the same base (for instance, different ratios of pseudouridine and N1-methyl-pseudouridine). mmRNA can also be made with mixed ratios at more than 1 “base” position, such as ratios of 5 methyl cytidine/cytidine and pseudouridine/N1-methyl-pseudouridine/uridine at the same time. Use of modified mRNA with altered ratios of modified nucleotides can be beneficial in reducing potential exposure to chemically modified nucleotides. Lastly, positional introduction of modified nucleotides into the mmRNA which modulate either protein production or immunostimulatory potential or both is also possible. The ability of such mmRNA to demonstrate these improved properties can be assessed in vitro (using assays such as the PBMC assay described herein), and can also be assessed in vivo through measurement of both mmRNA-encoded protein production and mediators of innate immune recognition such as cytokines
In another embodiment, the relative immunogenicity of the polynucleotide, primary construct or mmRNA and its unmodified counterpart are determined by determining the quantity of the polynucleotide, primary construct or mmRNA required to elicit one of the above responses to the same degree as a given quantity of the unmodified nucleotide or reference compound. For example, if twice as much polynucleotide, primary construct or mmRNA is required to elicit the same response, than the polynucleotide, primary construct or mmRNA is two-fold less immunogenic than the unmodified nucleotide or the reference compound.
In another embodiment, the relative immunogenicity of the polynucleotide, primary construct or mmRNA and its unmodified counterpart are determined by determining the quantity of cytokine (e.g. IL-12, IFNalpha, TNF-alpha, RANTES, MIP-1alpha or beta, IL-6, IFN-beta, or IL-8) secreted in response to administration of the polynucleotide, primary construct or mmRNA, relative to the same quantity of the unmodified nucleotide or reference compound. For example, if one-half as much cytokine is secreted, than the polynucleotide, primary construct or mmRNA is two-fold less immunogenic than the unmodified nucleotide. In another embodiment, background levels of stimulation are subtracted before calculating the immunogenicity in the above methods.
Provided herein are also methods for performing the titration, reduction or elimination of the immune response in a cell or a population of cells. In some embodiments, the cell is contacted with varied doses of the same polynucleotides, primary constructs or mmRNA and dose response is evaluated. In some embodiments, a cell is contacted with a number of different polynucleotides, primary constructs or mmRNA at the same or different doses to determine the optimal composition for producing the desired effect. Regarding the immune response, the desired effect may be to avoid, evade or reduce the immune response of the cell. The desired effect may also be to alter the efficiency of protein production.
The cosmetic polynucleotides, primary constructs and/or mmRNA of the present invention may be used to reduce the immune response using the method described in International Publication No. WO2013003475, herein incorporated by reference in its entirety.
Activation of the Immune Response: Vaccines
Additionally, certain modified nucleosides, or combinations thereof, when introduced into the cosmetic polynucleotides, primary constructs or mmRNA of the invention will activate the innate immune response. Such activating molecules are useful as adjuvants when combined with polypeptides and/or other vaccines. In certain embodiments, the activating molecules contain a translatable region which encodes for a polypeptide sequence useful as a vaccine, thus providing the ability to be a self-adjuvant.
In one embodiment, the cosmetic polynucleotides, primary constructs and/or mmRNA of the invention may encode an immunogen. The delivery of the cosmetic polynucleotides, primary constructs and/or mmRNA encoding an immunogen may activate the immune response. As a non-limiting example, the cosmetic polynucleotides, primary constructs and/or mmRNA encoding an immunogen may be delivered to cells to trigger multiple innate response pathways (see International Pub. No. WO2012006377; herein incorporated by reference in its entirety). As another non-limiting example, the cosmetic polynucleotides, primary constructs and mmRNA of the present invention encoding an immunogen may be delivered to a vertebrate in a dose amount large enough to be immunogenic to the vertebrate (see International Pub. No. WO2012006372 and WO2012006369; each of which is herein incorporated by reference in their entirety).
The cosmetic polynucleotides, primary constructs or mmRNA of invention may encode a polypeptide sequence for a vaccine and may further comprise an inhibitor. The inhibitor may impair antigen presentation and/or inhibit various pathways known in the art. As a non-limiting example, the cosmetic polynucleotides, primary constructs or mmRNA of the invention may be used for a vaccine in combination with an inhibitor which can impair antigen presentation (see International Pub. No. WO2012089225 and WO2012089338; each of which is herein incorporated by reference in their entirety).
In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA of the invention may be self-replicating RNA. Self-replicating RNA molecules can enhance efficiency of RNA delivery and expression of the enclosed gene product. In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA may comprise at least one modification described herein and/or known in the art. In one embodiment, the self-replicating RNA can be designed so that the self-replicating RNA does not induce production of infectious viral particles. As a non-limiting example the self-replicating RNA may be designed by the methods described in US Pub. No. US20110300205 and International Pub. Nos. WO2011005799, WO2013006838 and WO2013006842, each of which is herein incorporated by reference in their entirety.
In one embodiment, the self-replicating polynucleotides, primary constructs or mmRNA of the invention may encode a protein which may raise the immune response. As a non-limiting example, the cosmetic polynucleotides, primary constructs or mmRNA may be self-replicating mRNA may encode at least one antigen (see US Pub. No. US20110300205 and International Pub. No. WO2011005799; each of which is herein incorporated by reference in their entirety).
In one embodiment, the self-replicating polynucleotides, primary constructs or mmRNA of the invention may be formulated using methods described herein or known in the art. As a non-limiting example, the self-replicating RNA may be formulated for delivery by the methods described in Geall et al (Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012; PMID: 22908294).
In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA of the present invention may encode amphipathic and/or immunogenic amphipathic peptides.
In on embodiment, a formulation of the cosmetic polynucleotides, primary constructs or mmRNA of the present invention may further comprise an amphipathic and/or immunogenic amphipathic peptide. As a non-limiting example, the cosmetic polynucleotides, primary constructs or mmRNA comprising an amphipathic and/or immunogenic amphipathic peptide may be formulated as described in US. Pub. No. US20110250237 and International Pub. Nos. WO2010009277 and WO2010009065; each of which is herein incorporated by reference in their entirety.
In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA of the present invention may be immunostimulatory. As a non-limiting example, the cosmetic polynucleotides, primary constructs or mmRNA may encode all or a part of a positive-sense or a negative-sense stranded RNA virus genome (see International Pub No. WO2012092569 and US Pub No. US20120177701, each of which is herein incorporated by reference in their entirety). In another non-limiting example, the immunostimulatory polynucleotides, primary constructs or mmRNA of the present invention may be formulated with an excipient for administration as described herein and/or known in the art (see International Pub No. WO2012068295 and US Pub No. US20120213812, each of which is herein incorporated by reference in their entirety).
In one embodiment, the response of the vaccine formulated by the methods described herein may be enhanced by the addition of various compounds to induce the therapeutic effect. As a non-limiting example, the vaccine formulation may include a MHC II binding peptide or a peptide having a similar sequence to a MHC II binding peptide (see International Pub Nos. WO2012027365, WO2011031298 and US Pub No. US20120070493, US20110110965, each of which is herein incorporated by reference in their entirety). As another example, the vaccine formulations may comprise modified nicotinic compounds which may generate an antibody response to nicotine residue in a subject (see International Pub No. WO2012061717 and US Pub No. US20120114677, each of which is herein incorporated by reference in their entirety).
Naturally Occurring Mutants
In another embodiment, the cosmetic polynucleotides, primary construct and/or mmRNA can be utilized to express variants of naturally occurring proteins that have an improved disease modifying activity, including increased biological activity, improved patient outcomes, or a protective function, etc. Many such modifier genes have been described in mammals (Nadeau, Current Opinion in Genetics & Development 2003 13:290-295; Hamilton and Yu, PLoS Genet. 2012; 8:e1002644; Corder et al., Nature Genetics 1994 7:180-184; all herein incorporated by reference in their entireties). Examples in humans include Apo E2 protein, Apo A-I variant proteins (Apo A-I Milano, Apo A-I Paris), hyperactive Factor IX protein (Factor IX Padua Arg338Lys), transthyretin mutants (TTR Thr119Met). Expression of ApoE2 (cys112, cys158) has been shown to confer protection relative to other ApoE isoforms (ApoE3 (cys112, arg158), and ApoE4 (arg112, arg158)) by reducing susceptibility to Alzheimer's disease and possibly other conditions such as cardiovascular disease (Corder et al., Nature Genetics 1994 7:180-184; Seripa et al., Rejuvenation Res. 2011 14:491-500; Liu et al. Nat Rev Neurol. 2013 9:106-118; all herein incorporated by reference in their entireties). Expression of Apo A-I variants has been associated with reduced cholesterol (deGoma and Rader, 2011 Nature Rev Cardiol 8:266-271; Nissen et al., 2003 JAMA 290:2292-2300; all herein incorporated by reference in its entirety). The amino acid sequence of ApoA-I in certain populations has been changed to cysteine in Apo A-I Milano (Arg 173 changed to Cys) and in Apo A-I Paris (Arg 151 changed to Cys). Factor IX mutation at position R338L (FIX Padua) results in a Factor IX protein that has  ̃10-fold increased activity (Simioni et al., N Engl J Med. 2009 361:1671-1675; Finn et al., Blood. 2012 120:4521-4523; Cantore et al., Blood. 2012 120:4517-20; all herein incorporated by reference in their entireties). Mutation of transthyretin at positions 104 or 119 (Arg104 His, Thr119Met) has been shown to provide protection to patients also harboring the disease causing Va130Met mutations (Saraiva, Hum Mutat. 2001 17:493-503; DATA BASE ON TRANSTHYRETIN MUTATIONS www.ibmc.up.pt/mjsaraiva/ttrmut.html; all herein incorporated by reference in its entirety). Differences in clinical presentation and severity of symptoms among Portuguese and Japanese Met 30 patients carrying respectively the Met 119 and the His104 mutations are observed with a clear protective effect exerted by the non pathogenic mutant (Coelho et al. 1996 Neuromuscular Disorders (Suppl) 6: S20; Terazaki et al. 1999. Biochem Biophys Res Commun 264: 365-370; all herein incorporated by reference in its entirety), which confer more stability to the molecule. A modified mRNA encoding these protective TTR alleles can be expressed in TTR amyloidosis patients, thereby reducing the effect of the pathogenic mutant TTR protein.
Major Groove Interacting Partners
As described herein, the phrase “major groove interacting partner” refers to RNA recognition receptors that detect and respond to RNA ligands through interactions, e.g. binding, with the major groove face of a nucleotide or nucleic acid. As such, RNA ligands comprising modified nucleotides or nucleic acids such as the polynucleotide, primary construct or mmRNA as described herein decrease interactions with major groove binding partners, and therefore decrease an innate immune response.
Example major groove interacting, e.g. binding, partners include, but are not limited to the following nucleases and helicases. Within membranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single- and double-stranded RNAs. Within the cytoplasm, members of the superfamily 2 class of DEX(D/H) helicases and ATPases can sense RNAs to initiate antiviral responses. These helicases include the RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated gene 5). Other examples include laboratory of genetics and physiology 2 (LGP2), HIN-200 domain containing proteins, or Helicase-domain containing proteins.
Targeting of Pathogenic Organisms or Diseased Cells
Provided herein are methods for targeting pathogenic microorganisms, such as bacteria, yeast, protozoa, helminthes and the like, or diseased cells such as cancer cells using polynucleotides, primary constructs or mmRNA that encode cytostatic or cytotoxic polypeptides. Preferably the mRNA introduced contains modified nucleosides or other nucleic acid sequence modifications that are translated exclusively, or preferentially, in the target pathogenic organism, to reduce possible off-target effects of the therapeutic. Such methods are useful for removing pathogenic organisms or killing diseased cells found in any biological material, including blood, semen, eggs, and transplant materials including embryos, tissues, and organs.
Bioprocessing
The methods provided herein may be useful for enhancing protein product yield in a cell culture process. In a cell culture containing a plurality of host cells, introduction of a polynucleotide, primary construct or mmRNA described herein results in increased protein production efficiency relative to a corresponding unmodified nucleic acid. Such increased protein production efficiency can be demonstrated, e.g., by showing increased cell transfection, increased protein translation from the polynucleotide, primary construct or mmRNA, decreased nucleic acid degradation, and/or reduced innate immune response of the host cell. Protein production can be measured by enzyme-linked immunosorbent assay (ELISA), and protein activity can be measured by various functional assays known in the art. The protein production may be generated in a continuous or a batch-fed mammalian process.
Additionally, it is useful to optimize the expression of a specific polypeptide in a cell line or collection of cell lines of potential interest, particularly a polypeptide of interest such as a protein variant of a reference protein having a known activity. In one embodiment, provided is a method of optimizing expression of a polypeptide of interest in a target cell, by providing a plurality of target cell types, and independently contacting with each of the plurality of target cell types a polynucleotide, primary construct or mmRNA encoding a polypeptide of interest. The cells may be transfected with two or more polynucleotide, primary construct or mmRNA simultaneously or sequentially.
In certain embodiments, multiple rounds of the methods described herein may be used to obtain cells with increased expression of one or more nucleic acids or proteins of interest. For example, cells may be transfected with one or more polynucleotide, primary construct or mmRNA that encode a nucleic acid or protein of interest. The cells may be isolated according to methods described herein before being subjected to further rounds of transfections with one or more other nucleic acids which encode a nucleic acid or protein of interest before being isolated again. This method may be useful for generating cells with increased expression of a complex of proteins, nucleic acids or proteins in the same or related biological pathway, nucleic acids or proteins that act upstream or downstream of each other, nucleic acids or proteins that have a modulating, activating or repressing function to each other, nucleic acids or proteins that are dependent on each other for function or activity, or nucleic acids or proteins that share homology.
Additionally, culture conditions may be altered to increase protein production efficiency. Subsequently, the presence and/or level of the polypeptide of interest in the plurality of target cell types is detected and/or quantitated, allowing for the optimization of a polypeptide's expression by selection of an efficient target cell and cell culture conditions relating thereto. Such methods are particularly useful when the polypeptide contains one or more post-translational modifications or has substantial tertiary structure, situations which often complicate efficient protein production.
In one embodiment, the cells used in the methods of the present invention may be cultured. The cells may be cultured in suspension or as adherent cultures. The cells may be cultured in a varied of vessels including, but not limited to, bioreactors, cell bags, wave bags, culture plates, flasks and other vessels well known to those of ordinary skill in the art. Cells may be cultured in IMDM (Invitrogen, Catalog number 12440-53) or any other suitable media including, but not limited to, chemically defined media formulations. The ambient conditions which may be suitable for cell culture, such as temperature and atmospheric composition, are well known to those skilled in the art. The methods of the invention may be used with any cell that is suitable for use in protein production.
The invention provides for the repeated introduction (e.g., transfection) of modified nucleic acids into a target cell population, e.g., in vitro, ex vivo, in situ, or in vivo. For example, contacting the same cell population may be repeated one or more times (such as two, three, four, five or more than five times). In some embodiments, the step of contacting the cell population with the cosmetic polynucleotides, primary constructs or mmRNA is repeated a number of times sufficient such that a predetermined efficiency of protein translation in the cell population is achieved. Given the often reduced cytotoxicity of the target cell population provided by the nucleic acid modifications, repeated transfections are achievable in a diverse array of cell types and within a variety of tissues, as provided herein.
In one embodiment, the bioprocessing methods of the present invention may be used to produce antibodies or functional fragments thereof. The functional fragments may comprise a Fab, Fab′, F(ab′)2, an Fv domain, an scFv, or a diabody. They may be variable in any region including the complement determining region (CDR). In one embodiment, there is complete diversity in the CDR3 region. In another embodiment, the antibody is substantially conserved except in the CDR3 region.
Antibodies may be made which bind or associate with any biomolecule, whether human, pathogenic or non-human in origin. The pathogen may be present in a non-human mammal, a clinical specimen or from a commercial product such as a cosmetic or pharmaceutical material. They may also bind to any specimen or sample including clinical specimens or tissue samples from any organism.
In some embodiments, the contacting step is repeated multiple times at a frequency selected from the group consisting of: 6 hour, 12 hour, 24 hour, 36 hour, 48 hour, 72 hour, 84 hour, 96 hour, and 108 hour and at concentrations of less than 20 nM, less than 50 nM, less than 80 nM or less than 100 nM. Compositions may also be administered at less than 1 mM, less than 5 mM, less than 10 mM, less than 100 mM or less than 500 mM.
In some embodiments, the cosmetic polynucleotides, primary constructs or mmRNA are added at an amount of 50 molecules per cell, 100 molecules/cell, 200 molecules/cell, 300 molecules/cell, 400 molecules/cell, 500 molecules/cell, 600 molecules/cell, 700 molecules/cell, 800 molecules/cell, 900 molecules/cell, 1000 molecules/cell, 2000 molecules/cell, or 5000 molecules/cell.
In other embodiments, the cosmetic polynucleotides, primary constructs or mmRNA are added at a concentration selected from the group consisting of: 0.01 fmol/106 cells, 0.1 fmol/106 cells, 0.5 fmol/106 cells, 0.75 fmol/106 cells, 1 fmol/106 cells, 2 fmol/106 cells, 5 fmol/106 cells, 10 fmol/106 cells, 20 fmol/106 cells, 30 fmol/106 cells, 40 fmol/106 cells, 50 fmol/106 cells, 60 fmol/106 cells, 100 fmol/106 cells, 200 fmol/106 cells, 300 fmol/106 cells, 400 fmol/106 cells, 500 fmol/106 cells, 700 fmol/106 cells, 800 fmol/106 cells, 900 fmol/106 cells, and 1 pmol/106 cells.
In some embodiments, the production of a biological product upon is detected by monitoring one or more measurable bioprocess parameters, such as a parameter selected from the group consisting of: cell density, pH, oxygen levels, glucose levels, lactic acid levels, temperature, and protein production. Protein production can be measured as specific productivity (SP) (the concentration of a product, such as a heterologously expressed polypeptide, in solution) and can be expressed as mg/L or g/L; in the alternative, specific productivity can be expressed as pg/cell/day. An increase in SP can refer to an absolute or relative increase in the concentration of a product produced under two defined set of conditions (e.g., when compared with controls not treated with modified mRNA(s)).
Cells
In one embodiment, the cells are selected from the group consisting of mammalian cells, bacterial cells, plant, microbial, algal and fungal cells. In some embodiments, the cells are mammalian cells, such as, but not limited to, human, mouse, rat, goat, horse, rabbit, hamster or cow cells. In a further embodiment, the cells may be from an established cell line, including, but not limited to, HeLa, NS0, SP2/0, KEK 293T, Vero, Caco, Caco-2, MDCK, COS-1, COS-7, K562, Jurkat, CHO-K1, DG44, CHOK1SV, CHO-S, Huvec, CV-1, Huh-7, NIH3T3, HEK293, 293, A549, HepG2, IMR-90, MCF-7, U-20S, Per.C6, SF9, SF21 or Chinese Hamster Ovary (CHO) cells.
In certain embodiments, the cells are fungal cells, such as, but not limited to, Chrysosporium cells, Aspergillus cells, Trichoderma cells, Dictyostelium cells, Candida cells, Saccharomyces cells, Schizosaccharomyces cells, and Penicillium cells.
In certain embodiments, the cells are bacterial cells such as, but not limited to, E. coli, B. subtilis, or BL21 cells. Primary and secondary cells to be transfected by the methods of the invention can be obtained from a variety of tissues and include, but are not limited to, all cell types which can be maintained in culture. For examples, primary and secondary cells which can be transfected by the methods of the invention include, but are not limited to, fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), endothelial cells, glial cells, neural cells, formed elements of the blood (e.g., lymphocytes, bone marrow cells), muscle cells and precursors of these somatic cell types. Primary cells may also be obtained from a donor of the same species or from another species (e.g., mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).
Purification and Isolation
Those of ordinary skill in the art should be able to make a determination of the methods to use to purify or isolate of a protein of interest from cultured cells. Generally, this is done through a capture method using affinity binding or non-affinity purification. If the protein of interest is not secreted by the cultured cells, then a lysis of the cultured cells should be performed prior to purification or isolation. One may use unclarified cell culture fluid containing the protein of interest along with cell culture media components as well as cell culture additives, such as anti-foam compounds and other nutrients and supplements, cells, cellular debris, host cell proteins, DNA, viruses and the like in the present invention. The process may be conducted in the bioreactor itself. The fluid may either be preconditioned to a desired stimulus such as pH, temperature or other stimulus characteristic or the fluid can be conditioned upon the addition of polymer(s) or the polymer(s) can be added to a carrier liquid that is properly conditioned to the required parameter for the stimulus condition required for that polymer to be solubilized in the fluid. The polymer may be allowed to circulate thoroughly with the fluid and then the stimulus may be applied (change in pH, temperature, salt concentration, etc) and the desired protein and polymer(s) precipitate can out of the solution. The polymer and the desired protein(s) can be separated from the rest of the fluid and optionally washed one or more times to remove any trapped or loosely bound contaminants. The desired protein may then be recovered from the polymer(s) by, for example, elution and the like. Preferably, the elution may be done under a set of conditions such that the polymer remains in its precipitated form and retains any impurities to it during the selected elution of the desired protein. The polymer and protein as well as any impurities may be solubilized in a new fluid such as water or a buffered solution and the protein may be recovered by a means such as affinity, ion exchanged, hydrophobic, or some other type of chromatography that has a preference and selectivity for the protein over that of the polymer or impurities. The eluted protein may then be recovered and may be subjected to additional processing steps, either batch like steps or continuous flow through steps if appropriate.
In another embodiment, it may be useful to optimize the expression of a specific polypeptide in a cell line or collection of cell lines of potential interest, particularly a polypeptide of interest such as a protein variant of a reference protein having a known activity. In one embodiment, provided is a method of optimizing expression of a polypeptide of interest in a target cell, by providing a plurality of target cell types, and independently contacting with each of the plurality of target cell types a modified mRNA encoding a polypeptide. Additionally, culture conditions may be altered to increase protein production efficiency. Subsequently, the presence and/or level of the polypeptide of interest in the plurality of target cell types is detected and/or quantitated, allowing for the optimization of a polypeptide of interest's expression by selection of an efficient target cell and cell culture conditions relating thereto. Such methods may be useful when the polypeptide of interest contains one or more post-translational modifications or has substantial tertiary structure, which often complicate efficient protein production.
Protein Recovery
The protein of interest may be preferably recovered from the culture medium as a secreted polypeptide, or it can be recovered from host cell lysates if expressed without a secretory signal. It may be necessary to purify the protein of interest from other recombinant proteins and host cell proteins in a way that substantially homogenous preparations of the protein of interest are obtained. The cells and/or particulate cell debris may be removed from the culture medium or lysate. The product of interest may then be purified from contaminant soluble proteins, polypeptides and nucleic acids by, for example, fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC (RP-HPLC), SEPHADEX® chromatography, chromatography on silica or on a cation exchange resin such as DEAE. Methods of purifying a protein heterologous expressed by a host cell are well known in the art.
Methods and compositions described herein may be used to produce proteins which are capable of attenuating or blocking the endogenous agonist biological response and/or antagonizing a receptor or signaling molecule in a mammalian subject. For example, IL-12 and IL-23 receptor signaling may be enhanced in chronic autoimmune disorders such as multiple sclerosis and inflammatory diseases such as rheumatoid arthritis, psoriasis, lupus erythematosus, ankylosing spondylitis and Chron's disease (Kikly K, Liu L, Na S, Sedgwich J D (2006) Cur. Opin. Immunol. 18(6): 670-5). In another embodiment, a nucleic acid encodes an antagonist for chemokine receptors. Chemokine receptors CXCR-4 and CCR-5 are required for HIV entry into host cells (Arenzana-Seisdedos F et al, (1996) Nature. October 3; 383 (6599):400).
Gene Silencing
The cosmetic polynucleotides, primary constructs and mmRNA described herein are useful to silence (i.e., prevent or substantially reduce) expression of one or more target genes in a cell population. A polynucleotide, primary construct or mmRNA encoding a polypeptide of interest capable of directing sequence-specific histone H3 methylation is introduced into the cells in the population under conditions such that the polypeptide is translated and reduces gene transcription of a target gene via histone H3 methylation and subsequent heterochromatin formation. In some embodiments, the silencing mechanism is performed on a cell population present in a mammalian subject. By way of non-limiting example, a useful target gene is a mutated Janus Kinase-2 family member, wherein the mammalian subject expresses the mutant target gene suffers from a myeloproliferative disease resulting from aberrant kinase activity.
Co-administration of cosmetic polynucleotides, primary constructs and mmRNA and RNAi agents are also provided herein.
Modulation of Biological Pathways
The rapid translation polynucleotides, primary constructs and mmRNA introduced into cells provides a desirable mechanism of modulating target biological pathways. Such modulation includes antagonism or agonism of a given pathway. In one embodiment, a method is provided for antagonizing a biological pathway in a cell by contacting the cell with an effective amount of a composition comprising a polynucleotide, primary construct or mmRNA encoding a polypeptide of interest, under conditions such that the cosmetic polynucleotides, primary constructs and mmRNA is localized into the cell and the polypeptide is capable of being translated in the cell from the cosmetic polynucleotides, primary constructs and mmRNA, wherein the polypeptide inhibits the activity of a polypeptide functional in the biological pathway. Exemplary biological pathways are those defective in an autoimmune or inflammatory disorder such as multiple sclerosis, rheumatoid arthritis, psoriasis, lupus erythematosus, ankylosing spondylitis colitis, or Crohn's disease; in particular, antagonism of the IL-12 and IL-23 signaling pathways are of particular utility. (See Kikly K, Liu L, Na S, Sedgwick J D (2006) Curr. Opin. Immunol. 18 (6): 670-5).
Further, provided are polynucleotide, primary construct or mmRNA encoding an antagonist for chemokine receptors; chemokine receptors CXCR-4 and CCR-5 are required for, e.g., HIV entry into host cells (Arenzana-Seisdedos F et al, (1996) Nature. October 3; 383(6599):400).
Alternatively, provided are methods of agonizing a biological pathway in a cell by contacting the cell with an effective amount of a polynucleotide, primary construct or mmRNA encoding a recombinant polypeptide under conditions such that the nucleic acid is localized into the cell and the recombinant polypeptide is capable of being translated in the cell from the nucleic acid, and the recombinant polypeptide induces the activity of a polypeptide functional in the biological pathway. Exemplary agonized biological pathways include pathways that modulate cell fate determination. Such agonization is reversible or, alternatively, irreversible.
Expression of Ligand or Receptor on Cell Surface
In some aspects and embodiments of the aspects described herein, the cosmetic polynucleotides, primary constructs or mmRNA described herein can be used to express a ligand or ligand receptor on the surface of a cell (e.g., a homing moiety). A ligand or ligand receptor moiety attached to a cell surface can permit the cell to have a desired biological interaction with a tissue or an agent in vivo. A ligand can be an antibody, an antibody fragment, an aptamer, a peptide, a vitamin, a carbohydrate, a protein or polypeptide, a receptor, e.g., cell-surface receptor, an adhesion molecule, a glycoprotein, a sugar residue, a therapeutic agent, a drug, a glycosaminoglycan, or any combination thereof. For example, a ligand can be an antibody that recognizes a cancer-cell specific antigen, rendering the cell capable of preferentially interacting with tumor cells to permit tumor-specific localization of a modified cell. A ligand can confer the ability of a cell composition to accumulate in a tissue to be treated, since a preferred ligand may be capable of interacting with a target molecule on the external face of a tissue to be treated. Ligands having limited cross-reactivity to other tissues are generally preferred.
In some cases, a ligand can act as a homing moiety which permits the cell to target to a specific tissue or interact with a specific ligand. Such homing moieties can include, but are not limited to, any member of a specific binding pair, antibodies, monoclonal antibodies, or derivatives or analogs thereof, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((SCFV)2 fragments), diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments; and other homing moieties include for example, aptamers, receptors, and fusion proteins.
In some embodiments, the homing moiety may be a surface-bound antibody, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site. In one embodiment, multiple antibodies are expressed on the surface of a cell, and each antibody can have a different specificity for a desired target. Such approaches can increase the avidity and specificity of homing interactions.
A skilled artisan can select any homing moiety based on the desired localization or function of the cell, for example an estrogen receptor ligand, such as tamoxifen, can target cells to estrogen-dependent breast cancer cells that have an increased number of estrogen receptors on the cell surface. Other non-limiting examples of ligand/receptor interactions include CCRI (e.g., for treatment of inflamed joint tissues or brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8 (e.g., targeting to lymph node tissue), CCR6, CCR9, CCR10 (e.g., to target to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin), CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., for treatment of inflammation and inflammatory disorders, bone marrow), Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4NCAM-1 (e.g., targeting to endothelium). In general, any receptor involved in targeting (e.g., cancer metastasis) can be harnessed for use in the methods and compositions described herein.
Modulation of Cell Lineage
Provided are methods of inducing an alteration in cell fate in a target mammalian cell. The target mammalian cell may be a precursor cell and the alteration may involve driving differentiation into a lineage, or blocking such differentiation. Alternatively, the target mammalian cell may be a differentiated cell, and the cell fate alteration includes driving de-differentiation into a pluripotent precursor cell, or blocking such de-differentiation, such as the dedifferentiation of cancer cells into cancer stem cells. In situations where a change in cell fate is desired, effective amounts of mRNAs encoding a cell fate inductive polypeptide is introduced into a target cell under conditions such that an alteration in cell fate is induced. In some embodiments, the modified mRNAs are useful to reprogram a subpopulation of cells from a first phenotype to a second phenotype. Such a reprogramming may be temporary or permanent. Optionally, the reprogramming induces a target cell to adopt an intermediate phenotype.
Additionally, the methods of the present invention are particularly useful to generate induced pluripotent stem cells (iPS cells) because of the high efficiency of transfection, the ability to re-transfect cells, and the tenability of the amount of recombinant polypeptides produced in the target cells. Further, the use of iPS cells generated using the methods described herein is expected to have a reduced incidence of teratoma formation.
Also provided are methods of reducing cellular differentiation in a target cell population. For example, a target cell population containing one or more precursor cell types is contacted with a composition having an effective amount of a polynucleotides, primary constructs and mmRNA encoding a polypeptide, under conditions such that the polypeptide is translated and reduces the differentiation of the precursor cell. In non-limiting embodiments, the target cell population contains injured tissue in a mammalian subject or tissue affected by a surgical procedure. The precursor cell is, e.g., a stromal precursor cell, a neural precursor cell, or a mesenchymal precursor cell.
In a specific embodiment, provided are polynucleotide, primary construct or mmRNA that encode one or more differentiation factors Gata4, Mef2c and Tbx4. These mRNA-generated factors are introduced into fibroblasts and drive the reprogramming into cardiomyocytes. Such a reprogramming can be performed in vivo, by contacting an mRNA-containing patch or other material to damaged cardiac tissue to facilitate cardiac regeneration. Such a process promotes cardiomyocyte genesis as opposed to fibrosis.
Mediation of Cell Death
In one embodiment, polynucleotides, primary constructs or mmRNA compositions can be used to induce apoptosis in a cell (e.g., a cancer cell) by increasing the expression of a death receptor, a death receptor ligand or a combination thereof. This method can be used to induce cell death in any desired cell and has particular usefulness in the treatment of cancer where cells escape natural apoptotic signals.
Apoptosis can be induced by multiple independent signaling pathways that converge upon a final effector mechanism consisting of multiple interactions between several “death receptors” and their ligands, which belong to the tumor necrosis factor (TNF) receptor/ligand superfamily. The best-characterized death receptors are CD95 (“Fas”), TNFRI (p55), death receptor 3 (DR3 or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). The final effector mechanism of apoptosis may be the activation of a series of proteinases designated as caspases. The activation of these caspases results in the cleavage of a series of vital cellular proteins and cell death. The molecular mechanism of death receptors/ligands-induced apoptosis is well known in the art. For example, Fas/FasL-mediated apoptosis is induced by binding of three FasL molecules which induces trimerization of Fas receptor via C-terminus death domains (DDs), which in turn recruits an adapter protein FADD (Fas-associated protein with death domain) and Caspase-8. The oligomerization of this trimolecular complex, Fas/FAIDD/caspase-8, results in proteolytic cleavage of proenzyme caspase-8 into active caspase-8 that, in turn, initiates the apoptosis process by activating other downstream caspases through proteolysis, including caspase-3. Death ligands in general are apoptotic when formed into trimers or higher order of structures. As monomers, they may serve as antiapoptotic agents by competing with the trimers for binding to the death receptors.
In one embodiment, the cosmetic polynucleotides, primary constructs or mmRNA composition encodes for a death receptor (e.g., Fas, TRAIL, TRAMO, TNFR, TLR etc). Cells made to express a death receptor by transfection of cosmetic polynucleotides, primary constructs and mmRNA become susceptible to death induced by the ligand that activates that receptor. Similarly, cells made to express a death ligand, e.g., on their surface, will induce death of cells with the receptor when the transfected cell contacts the target cell. In another embodiment, the cosmetic polynucleotides, primary constructs and mmRNA composition encodes for a death receptor ligand (e.g., FasL, TNF, etc). In another embodiment, the cosmetic polynucleotides, primary constructs and mmRNA composition encodes a caspase (e.g., caspase 3, caspase 8, caspase 9 etc). Where cancer cells often exhibit a failure to properly differentiate to a non-proliferative or controlled proliferative form, in another embodiment, the synthetic, polynucleotides, primary constructs and mmRNA composition encodes for both a death receptor and its appropriate activating ligand. In another embodiment, the synthetic, polynucleotides, primary constructs and mmRNA composition encodes for a differentiation factor that when expressed in the cancer cell, such as a cancer stem cell, will induce the cell to differentiate to a non-pathogenic or nonself-renewing phenotype (e.g., reduced cell growth rate, reduced cell division etc) or to induce the cell to enter a dormant cell phase (e.g., G0 resting phase).
One of skill in the art will appreciate that the use of apoptosis-inducing techniques may require that the cosmetic polynucleotides, primary constructs or mmRNA are appropriately targeted to e.g., tumor cells to prevent unwanted wide-spread cell death. Thus, one can use a delivery mechanism (e.g., attached ligand or antibody, targeted liposome etc) that recognizes a cancer antigen such that the cosmetic polynucleotides, primary constructs or mmRNA are expressed only in cancer cells.
Cosmetic Applications
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of cosmetic diseases, disorders and conditions. Such disease, disorders and conditions include, but are not limited to, acne vulgaris, acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, actinic keratosis, acne vulgaris, acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, acute urticaria, allergic contact dermatitis, alopecia areata, angioedema, athlete's foot, atopic dermatitis, autoeczematization, baby acne, balding, bastomycosis, blackheads, birthmarks and other skin pigmentation problems, boils, bruises, bug bites and stings, burns, cellulitis, chiggers, chloracne, cholinergic or stress uricara, chronic urticara, cold type urticara, confluent and reticulated papillomatosis, corns, cysts, dandruff, dermatitis herpetiformis, dermatographism, dyshidrotic eczema, diaper rash, dry skin, dyshidrosis, ectodermal dysplasia such as, hyprohidrotic ectodermal dysplasia and X-linked hyprohidrotic ectodermal dysplasia, eczema, epidermaodysplasia verruciformis, erythema nodosum, excoriated acne, exercise-induced anaphylasis folliculitis, excess skin oil, folliculitis, freckles, frostbite, fungal nails, hair density, hair growth rate, halogen acne, hair loss, heat rash, hematoma, herpes simplex infections (non-genital), hidradenitis suppurativa, hives, hyperhidrosis, hyperpigmentation, hypohidrotic ectodermal dysplasia, hypopigmentation, impetigo, ingrown hair, heat type urticara, ingrown toenail, infantile acne or neonatal acne, itch, irritant contact dermatitis, jock itch, keloid, keratosis pilaris, lichen planus, lichen sclerosus, lupus miliaris disseminatus faciei, melasma, moles, molluscum contagiosum, nail growth rate, nail health, neurodermatitis, nummular eczema, occupational acne, oil acne, onychomycosis, physical urticara, pilonidal cyst, pityriasis rosea, pityriasis versicolor, poison ivy, pomade acne, pseudofolliculitis barbae or acne keloidalis nuchae, psoriasis, psoriatic arthritis, pressure or delayed pressue urticara, puncture wounds such as cuts and scrapes, rash, rare or water type urticara, rhinoplasty, ringworm, rosacea, rothmund-thomson syndrome, sagging of the skin, scabis, scars, seborrhea, seborrheic dermatitis, shingles, skin cancer, skin tag, solar type urticara, spider bite, stretch marks, sunburn, tar acne, tropical acne, thinning of skin, thrush, tinea versicolor, transient acantholytic dermatosis, tycoon's cap or acne necrotica miliaris, uneven skin tone, varicose veins, venous eczema, vibratory angioedema, vitiligo, warts, Weber-Christian disease, wrinkles, x-linked hypohidrotic ectodermal dysplasia, xerotic eczema, yeast infection and general signs of aging.
Common Diseases, Disorders and Conditions
Dry Skin
Dry skin often shows as patches of rough, red, and/or itchy areas of skin in places of the body including areas are often exposed to the environment such as, but not limited to, arms, hands and lower legs and other areas which tend to be protected from the environment such as, but not limited to, soles of the feet, thighs and the abdomen. Dry skin can lead to cracks and fissures in the skin which may be worsened in the winter because of low humidity. Dry skin may be hereditary (ichthyosis vulgaris), a result of aging when natural oils diminish, a result of medical conditions such as, but not limited to, asthma and thyroid disease, or a result of daily skin care habits.
Ichthyosis vulgaris (also known as autosomal dominant ichthyosis, ichthyosis simplex or common ichthyosis) is the most common form of the heterogeneous family of genetic skin disorders known as, ichthyosis. Ichthyosis vulgaris is mostly an autosomal dominant inherited skin disorder, but there is a rare non-heritable version called acquired ichthyosis, that causes dry, scaly skin affecting around 1 in 250 people. Many people with ichthyosis have associated conditions such as, but not limited to, problems with sweating due to the build up of scales on the skin, cracking of the skin on the fingers or extremities that create bloody cuts, inflamed and/or tight skin that can be painful and other atopic diseases such as allergies, eczema, asthma, and keratosis pilaris.
A mutation to the gene encoding profilaggrin (a protein which is converted to filaggrin) is believed to cause ichthyosis vulgaris. In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of dry skin and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NO: 1298.
Psoriasis
Psoriasis is an autoimmune disease that appears on the skin when the immune system mistakes the skin cells as a pathogen. The body then sends out signals to speed up the growth cycle of skin cells. Associated conditions with psoriasis include, but are not limited to, an increased risk of stroke, high blood lipid levels, psoriatic nail dystrophy causing small indentations, ridges, pits, discoloration or separation from the nail bed, inflammation of the joints known as psoriatic arthritis and psoriasis of the scalp where the scalp may have fine dry scaly skin or a heavily crusted plaque area.
Plague psoriasis, the most common of the five types of psoriasis which affects 1-2% of the people in the United States, is when skin rapidly accumulates causing the skin to be inflamed, red, and covered with silvery scales. Patches of circular to oval shaped red plaques that itch or burn are typical and are can be found anywhere on the body but are common on the arms, legs, trunk or scalp. The occurrence and length of time psorisasis can be affected by environmental factors such as, but not limited to, smoking, injury to the skin, sun exposure, alcohol, certain drugs and medication, and infection such as HIV.
Guttate psoriasis is small, salmon-pink drops on the skin often triggered by a streptococcal infection but can be triggered by other infections such as, but not limited to, chickenpox or colds. Pustular psoriasis is clearly defined raised bumps on the skin that are filled with pus and the skin under and around these bumps can be reddish. Pustular is an uncommon form of psoriasis and can occur alone or in combination with plaque-type psoriasis. Inverse psoriasis is bright red, smooth patches found in the folds of the skin usually in areas such as, but not limited to, under the breast, in the armpits, near the genital, under the buttocks or in abdominal folds. Erythrodermic psoriasis is the least common type of psoriasis but can be serious as a very large area of the body is bright red and inflamed. The body can appear covered in a red, peeling rash that usually itches and/or burns which increases blood flow and can put a strain on the heart.
There are nine locations, called psoriasis susceptibility 1 through 9 (PSORS1-PSORS9), on different chromosomes that are associated with psoriasis. PSORS1 may be the major determinant of psoriasis and probably accounts for 35-50% of its heritability as it controls genes that affect the immune system or encode proteins that are found in the skin in greater amounts in psoriasis. PSORS1 is located in the major histocompatibility comples (MHC) on chromosome 6 which controls important immune functions. In the PSORS1 locus there are three genes that have a strong association with psoriasis vulgaris, major histocompatibility complex, class I, C (HLA-C) variant HLA-Cw6 which encodes a MHC class I protein, coiled-coil alpha-helical rod protein 1 (CCHR1) variant WWC which encodes a coiled protein that may be overexpressed in psoriatic epidermis and corneodesmosin (CDSN) variant allele 5 which may be expressed in the granular and cornified layers of the epidermis and may be upregulated in psoriasis. Studies have also suggested that a rare mutation on the gene encoding the caspase recruitment domain family, member 14 (CARD14; also known as CARD-containing MAGUK protein 2) protein and an environmental trigger was able to cause plaque psoriasis. Currently, IL-12B on chromosome 5q which expresses interleukin-12B and IL-23R on chromosome 1p which expresses the interleukin-23 receptor are under investigation as they may be involved in T cell differentiation and T cells are involved in the inflammatory process that leads to psoriasis. Further, IL-12B and IL-23R are on the pathway that upregulates two genes involved in inflammation, tumor necrosis factor (TNF and TNF-alpha) and nuclear factor of kappa light polypeptide gene enhancer in B-cells.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of the five types of psoriasis. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1224, 1225, 1226, 1227, 1228, 1229, 1311, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1520, 1521, 1522, 1523, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1602, 1603, 1604, 1605, 1606, 1607 and 1608.
Eczema
Eczema, the general term for inflammation of the skin, is a disease that causes itchy, inflamed skin which can affect the insides of the elbows, backs of the knees, the face and can cover most of the body. Several other skin diseases, disorders and conditions that are also under the term eczema include, but are not limited to, atopic dermatitis, nummular eczema, dyshidrotic eczema, seborrheic dermatitis, irritant contact dermatitis, allergic contact dermatitis, dyshidrosis, venous eczema, dermatitis herpetiformis, neurodermatitis, autoeczematization and xerotic eczema. Flare-ups of eczema can be caused by triggers such as, but not limited to, dry skin, irritants, allergens, emotional stress, heat and sweating, and infections.
Inherited eczema and some related disorders are believed to be caused by the gene that produces the protein filaggrin. Further, three genetic variants, ovo-like 1 (OVOL1), actin-like 9 (ACTL9) and kinesin family member 3A (KIF3A) have been associated with eczema. Itching sensations which are a common symptom in eczema have recently been connected to the genes brain-derived neurotrophic factor (BDNF) and tachykinin, precursor 1 (TAC1)
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of eczema and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 913, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 1408, 1409, 1410, 1411, 1524, 1594, 1595 and 1596.
Hives or Urticaria
Hives, or urticaria, is an outbreak of swollen, pale red bumps or plaques on the skin that appear suddenly which may be a result of the body's reaction to allergens, an acute viral infection, friction, pressure, temperature extremes, exercise and sunlight. Hives can cause itching but may also burn or sting and may appear anywhere on the skin such as the face, lips, tongue, throat, or ears. There are several types of hives such as, but not limited to, acute urticaria, chronic urticara and angioedema, physical urticara, pressure or delayed pressue urticara, cholinergic or stress uricara, cold type urticara, heat type urticara, solar type urticara, rare or water type urticara, vibratory angioedema, exercise-induced anaphylasis and dermatographism.
People that have urticaria, such as cold urticaria, can have mutations in the gene phospholipase C, gamma 2 (PLCG-2) which is an enzyme involved in activating immune cells. The mutations (termed PLAID) will cause the PLCG-2 enzyme to function without shutting off causing excessive or deficient immune system reactions. Targeting the PLCG-2 enzyme may be a way to treat diseases, disorder and conditions associated with the PLAID mutation.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of urticaria and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 1525-1527.
Rosacea
Rosacea involves the swelling of the blood vessels just under the skin which cause symptoms such as, but not limited to, redness of the face, blushing or flusing, red nose, acne-like skin sores, a burning or stinging feeling in the face, irritated, bloodshot, or watery eyes or spider-like blood vessels (telangiectasia) of the face. There are four subtypes of rosacea and subjects having rosacea may suffer from more than one subtype. Erthematotelangiectatic rosacea is permanent redness with a tendency to flush and blush easily. Subjects with papulopustular rosacea, often confused with acne, suffer from some permanent redness with red bumps with some bumps being pus filled. Phymatous rosacea causes thickening skin, irregular surface nodularities of the nose, chin, forehead, cheeks, eyelids and ears. Red, dry and irritated eyes and eyelids are referred to as ocular rosacea.
Subjects who have rosacea have elevated levels of cathelicidin antimicrobial peptide (CAMP) and/or kallikrein-related peptidase 5 (also known as stratum corneum tryptic enzyme (SCTE)). In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of rosacea and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 1405-1407.
Acne
Acne is a general term that is used for acneiform eruptions commonly known as a symptom for acne vulgaris but may also refer to acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, baby acne, blackheads, chloracne, excoriated acne, halogen acne, infantile acne or neonatal acne, lupus miliaris disseminatus faciei, occupational acne, oil acne, pomade acne, tar acne, tropical acne, tycoon's cap or acne necrotica miliaris, pseudofolliculitis barbae or acne keloidalis nuchae, and hidradenitis suppurativa. Acne may be caused by hormonal changes, genetic factors, psychological factors such as, but not limited to, stress, infections and diet.
Matrix metalloproteinases (MMP) have a predominant role in the inflammatory matrix remodeling and hyperproliferative skin disorders. Matrix metalloproteinase-1 (MMP-1 or interstitial collagenase), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-13 (MMP-13), tissue inhibitors of MMP-1 (TIMP-1) and tissue inhibitors of MMP-2 (TIMP-2) were found to be highly expressed in skin from an acne patient suggesting that MP and TIMP of epithelial origin controlling these genes may have therapeutic effects on acne.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of acne and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to the cosmetic targets disclosed herein.
Vitiligo
Vitiligo is a skin condition in which there is a loss of brown color or pigment from areas of the skin which results in irregular white patches. 1 out of every 100 people in the United States is affected by this autoimmune problem where the immune system destroys the cells that produce brown pigment (melanocytes). Vitiligo most often affects the face, elbows, knees, hands, feet and genitals. Vitiligo has been associated with other autoimmune diseases such as, but not limited to, Addison's disease, hyperthyroidism and pernicious anemia.
Mutations on the NLR family, pyrin domain containing 1 gene (NALP1) gene, located on chromosome 17 at 17p13, may be a cause of vitiligo. NALP1 is expressed at high levels in T cells and Langerhan cells which are involved in skin autoimmunity. Caspase 1 and caspase 7 are inflammatory products of NALP1 which activate the inflammatory cytokine interleukin 1-beta (IL-1B) which is often expressed at high levels in patients with vitiligo Inhibiting caspase and IL-1B may be useful therapeutics for vitiligo and its associated diseases.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of vitiligo and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 1512-1519.
Hypohidrotic Ectodermal Dysplasia
Subjects with hyprohidrotic ectodermal dysplasia (HED) have a reduced ability to sweat, sparse scalp and body hair and absent or malformed teeth. They may also have distinctive facial features such as, but not limited to, a prominent forehead, thick lips, flattened bridge of the nose, and thin, wrinkled and dark-color skin around the eyes. Common associated conditions with those who have HED include chronic skin problems such as atopic dermatitis and eczema, and a bad-smelling discharge from the nose.
Hyprohidrotic ectodermal dysplasia is caused by mutations in the ectodysplasin A gene (EDA), receptor (EDAR), and receptor associated death domain (EDARADD). Most cases are inherited in mutations on the EDA gene in an X-linked recessive pattern. Less common are mutations in the EDAR which have an autosomal dominant or autosomal recessive pattern of inheritance and mutations in the EDARADD with have an autosomal recessive pattern of inheritance.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of hyprohidrotic ectodermal dysplasia and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 1233-1235 and 1242-1244.
Balding
Balding, or hair thinning, is the partial or complete loss of hair. Male pattern baldness, or androgenetic alopecia (AGA), may depend on a variant of the androgen receptor (AR) in order to develop. Further the ectodysplasin A2 receptor (EDA2R) has also been pointed out as a gene associated with AGA. Lysophosphatidic acid receptor 6 (P2RY5) has been connected to hair loss as it is linked to hair structure. Certain variants of P2RY5 have lead to baldness at birth and another variant caused wholly hair at birth.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of hyprohidrotic ectodermal dysplasia and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 914, 915, 916, 917, 918, 1236, 1237, 1238, 1239, 1240, 1241, 1412 and 1413.
Scarring and Stretch Marks
Scars are areas of fibrous tissue that replace normal skin after injury. Scar tissue is the same protein (collagen) as the tissue that it replaces but the collagen cross-links and forms a pronounced alignment in a single direction instead of a random basketweave formation in normal tissue. The skin where scars tissue exists is less resistant to ultraviolet radiation and sweat glands and hair follicles do not grow back in the scar tissue.
Strecth marks, called striae, are a form of scarring that is caused when the skin is stretched rapidly, such as during pregnancy, weight gain, or growth spirts, or when skin is under tension during the healing process. Other forms of scarring include hypertropic scars when the body over produces collagen causing the scar to be raised above the surrounding skin and atrophic scars which have a sunken recess in the skin caused when the underlying structures supporting the skin are lost.
Studies have suggested that collagen, ribosomal s6 kinase and sectrected phosphoprotein 1 (also known as osteopontin) play important roles in the formation of scar tissue. Transforming growth factor beta 3 may also be used to have scar tissue more closely resemble normal tissue in appearance and structure.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of scarring and stretch marks and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1469, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578 and 1579.
Epidermaodysplasia Verruciformis
Epidermodysplasia verruciformis (also known as Lutz-Lewandowsky epidermodysplasia) is an extremely rare autosomal recessive genetic hereditary skin disorder often associated with a high risk of skin cancer. The skin disorder is caused by a mutation in either transmembrane channel-like 6 (EVER1) or transmembrane channel-like 8 (EVER2) genes which are located adjacent to one another on chromosome 17. EVER1 and EVER2 help to regulate the distribution of zinc in the cell nucleus and thus the activity of these genes may restrict the access of viral proteins to cellular zinc stores which may limit cell growth.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of scarring and stretch marks and its associated diseases, disorders and/or conditions. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 1600-1601.
Saying Skin, Thinning of Skin, Wrinkles
Skin may have an undesirable change such as sagging, thinning or wrinkling as a result of the substantial and steady decline in the expression of cosmetic proteins such as, but not limited to, collagen, elastin, fibroblast growth factor 7, TIMP metallopeptidase inhibitors, matrix metallopeptidases, superoxide dismutase and other extracellular matrix proteins and proteoglycans. Replacing these cosmetic proteins and/or preventing the decline of these cosmetic proteins may be an effective strategy in maintaining and/or improving the appearance and health of the skin.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of sagging skin, thinning skin and/or wrinkles. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1469, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1063, 1064, 1065, 1066, 1576, 1067, 1068, 1069, 1070, 1027, 1028, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1184, 1185, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1029, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1592, 1590, 1589, 1588, 1591, 1254, 1259, 1255, 1251, 1258, 1256, 1252, 1245, 1253, 1246, 1249, 1250, 1247, 1248, 1257, 1401, 1399, 1403, 1404, 1400, 1402, 1494, 1493, 1492, 1467, 1468, 1471, 1470, 1474, 1473, 1472, 1475, 1476, 1478, 1480, 1477, 1479, 1481, 1482, 1484, 1483, 1485, 1486, 1487, 1489, 1490, 1491 and 1488. Repair and Maintenance of Tissue
Skin cells express a variety of proteins and other factors that may be important to the repair and maintenance of the tissue. Proteins which may be important to the repair and maintenance of the tissue include, but are not limited to, transforming growth factor (TGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), heparin-binding EGF-like growth factor, vascular endothelial growth factor (VEGF), c-fos induced growth factor, colony stimulating factor 2, interleukins, fibroblast growth factor (FGF), connective tissue growth factor (CTGF), growth hormone, colony stimulating factor 1, colony stimulating factor 3, calponin, cysteine-rich, angiogenic inducer, thyroid stimulating hormone (TSH), hepatocyte growth factor (HGF), hypoxia inducible factor 1 (HIF-1) and actin.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of cells which are important to the repair and maintenance of tissue. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1414, 1415, 1416, 1417, 1418, 1419, 1528, 1529, 1530, 1531, 1532, 1533, 1588, 1589, 1590, 1591, 1592, 1597 and 1598.
Skin Tanning
Tanning of the skin is a natural shield against skin cancer. In response to ultraviolet (UV) light from the sun, kerotinocytes being pumping out melanocyte-stimulating hormone which binds to melanocyte-stimulating hormone receptors on melanocytes which leads to melanin production and ultimately a tan. Melanocyte-stimulating hormone is derived from pro-opiomelanocortin. As a non-limiting example, by altering the levels of pro-opiomelanocortin in the body the ability of a subject to tan may be altered.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of cells which are important to the tanning of skin. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 919, 920, 921, 1230, 1231, 1232, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1534, 1535, 1536, 1537, 1538, 1586, 1587, 1593, 1609, 1610 and 1611.
Hair Pigmentation
Hair color is determined by the levels of pigmentation in the hair follicles due to melanin, specifically eumelanin and pheomelanin. There are two types of eumelanin, black eumelanin and brown eumelanin. A pink to red pigmentation is given by pheomelanin. As a non-limiting example, red hair has a large amount of pheomelanin in the hair follicles.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of cells which are important to the pigmentation of hair and hair follicles. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 1495, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587 and 1593.
Neurotoxins
In one embodiment, neurotoxins are used in the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA described herein for the treatment, amelioration or prophylaxis or various diseases, disorder and/or conditions. As used herein, “neurotoxin” refers to a substance that is capable of causing damage to nerves or nerve tissue. Categories of neurotoxins include, but are not limited to, sodium channel inhibitors (e.g., tetrodotoxin), potassium channel inhibitors (e.g., tetraethylammonium), chloride channel inhibitors (e.g., chlorotoxin and curare), calcium channel inhibitors (e.g., conotoxin), synaptic vesicle release inhibitors (e.g., botulinum toxin and tetanus toxin) and blood brain barrier inhibitor (e.g., aluminum and mercury).
The neurotoxins described herein may be used as therapeutic agents in cosmetic and/or medical procedures such as, but not limited, treatment of strabismus (“crossed eyes”), blepharospasm (“uncontrollable blinking”), achalasia, sweating, muscle spasms, upper motor neuron syndrome, cervical dystonia, headaches and chronic migraines.
Tetanus Toxin
Tetanus toxin is a neurotoxin produced by Clostridium tetani which has two separate, distinct parts. In one part of the toxin is the cause of the toxic effects and tetanus symptoms. In the other part, called the carboxy-terminal domain, is non-toxic and is able to penetrate and affect the nervous system. The carboxy-terminal domain is able to inhibit serotonin from being transported through the synaptic membranes. This domain may be useful as a protectant against neurodegenerative and other muscle disorders such as Parkinson's disease as it can prevent the death of neurons when the neurons are faced with aggressive situations.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of neurodegenerative and other muscle disorders. In a further embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, the sequences described in Example 116 (See e.g., Table 178).
Botulinum Toxin
Botulinum toxin is a neurotoxin produced by Clostridium botulinum, which has seven immunologically distinct protein neurotoxins designated as serotypes A through G with a C1 and C2 with a molecular weight of 150 kDa. The botulinum toxin has a disulfide bond between the 50 kDa N-terminal light chain and the 100 kDa C-terminal heavy chain to form the 150 kDa neurotoxin.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of cosmetic diseases, disorders and/or conditions and other muscle disorders.
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a cosmetic polypeptide of interest such as, but not limited to, the sequences described in Example 116 (See e.g., Table 178).
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA encode a light chain of a polypeptide of interest such as, but not limited to, the sequences described in Example 116 (See e.g., Table 178).A light chain of the polypeptide of interest may be produced by a method known in the art. As a non-limiting example, light chains of botulinum neurotoxin may be produced using the methods described in U.S. Pub. No. 20100273211 herein incorporated by reference in its entirety.
The botulinum toxins are released as protein aggregates by Clostridia which are designated as progenitor toxins or toxin complexes. Toxin complexes result from the noncovalent association of the neurotoxin with up to seven other proteins which are known as neurotoxin-associated proteins (NAPs). NAPs include, but are not limited to, nontoxic, non-hemagglutinin protein (NTNH), hemagglutination protein (HA) and other proteins possessing hemagglutination properties. Broadly there are three categories of HA proteins, having designations of HA-1, HA-2, HA-3 (HA-3a & HA-3b are also present in some complexes). The 150 kDa toxin is the active neurotoxin, while the bound NTNH and HA proteins that form the aggregate or complex provide protection or facilitate delivery of the active toxin. The aggregate or complex may be of three sizes designated 12S (300 kDa), 16S (500 kDa), and 19S (900 kDa). The type A serotype produces all three size complexes (12S, 16S, and 19S), type B, C, and D strains produce 12S and 16S complexes, strains E and F produce only 12S complexes, while the G strain only produces the 16S complex.
The 12S complex is botulinum toxin bound to the NTNH protein. All the complexes consist of this bound pair. The 16S aggregate is the addition of HA proteins bound to this core complex. The 19S is a dimer of the 16S aggregate. The HA proteins have specific nomenclature based on their molecular weight (HA-17 weights 17 kDa) and are HA-17, HA-17/15, HA-19/20, HA-26/21, HA-33, HA-33/35, HA-52, HA-55, HA-70. With the type A complex the HA proteins have been correlated as follows: HA-15/17 (HA-2), HA-33/35 (HA-1), and HA-70 (HA-3) that fragments into HA-19/20 (HA-3a) and HA-52 (HA-3b). The C serotype complex is similar with the B and D complexes having only an HA-3 (and no HA-3a or -3b). The G complex is composed of the HA-2 and HA-3 proteins. Example 117 (see e.g., Table 179) describes a non-limiting listing of NAPs for the seven botulinum toxin serotypes.
Botulinum toxins may be used to treat various diseases, disorders and conditions such as, but not limited to, headaches, migraine headaches, tension headaches, sinus headaches, cervicogenic headaches, hormone headaches, sweating disorders, axillary hyperhidrosis, palmar hyperhidrosis, plantar hyperhidrosis, genital hyphydrosis, Frey's syndrome, hyperkinetic skin lines, facial wrinkle, glabellar lines, crow's feet, marionette lines, a nasolabial fold, a skin disorder, achalasia, strabismus, chronic anal fissure, blepharospasm, musculoskeletal pain, fibromyalgia, pancreatitis, tachycardia, prostatic enlargement, prostatitis, urinary retention, urinary incontinence, overactive bladder, hemifacial spasm, tremors, myoclonus, gastrointestinal disorders, diabetes, sialorrhea, detrusor-sphincter dyssynergia, post stroke spasticity, wound healing, juvenile cerebral palsy, smooth muscle spasm, restenosis, a focal dystonia, epilepsy, cervical dystonia, thyroid disorder, hypercalcemia, an obsessive compulsive disorder, arthritic pain, Raynaud's syndrome, striae distensae, peritoneal adhesion, vasospasms, rhinorrhea, muscle contracture, an injured muscle, laryngeal dystonia, writer's cramp, carpel tunnel syndrome, movement disorders such as Parkinson′ disease, tics, tourette's syndrome, sialorrhea in Parkinson's disease, bulbar amyotrophic lateral sclerosis, autonomic hypersecretory disorders, dystonia and other movement disorders, idiopathic and neurogenic detrusor overactivity, vaginismus, temporomandibular joint pain disorders, diabetic neuropathy, vocal cord dysfunction (VCD) including spasmodic dysphonia and tremor, reduction of the Masseter muscle for decreasing the apparent size of the lower jaw and benign prostatic hyperplasia. Botulinum toxin has also been used in clinical settings such as, for example, for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles.
Botulinum toxin type A has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of essential blepharospasm, strabismus and hemifacial spasm in patients over the age of twelve, cervical dystonia, glabellar line (facial) wrinkles and for treating hyperhydrosis. The FDA has also approved a botulinum toxin type B for the treatment of cervical dystonia.
Treatment of Migraines and Headaches
Botulinum toxin may be used to treat migraines and headaches such as, but not limited to, chronic migraines, cluster headaches, sinus headaches, tension headaches and cervicogenic headaches. Botulinum toxin may reduce by peractivity along a neuromuscular pathway by preventing release of acetylcholine, thereby decreasing contractility of musculature and relieving migraine and/or headache pain. In addition, reduction of excess acetylcholine found at the neuromuscular junctions during migraines and headaches through botulinum toxin injection may further result in normalization of muscle activity and decreased pain. Botulinum toxin may further reduce pain by entering into the central nervous system and blocking neurotransmitter exocytosis and nociceptor signalling, thereby decreasing the signal pathway of pain during a migraine and/or headache.
Chronic migraines are neurological disorders characterized by moderate to severe headaches and nausea. Botulinum toxin may be injected into the head and neck to treat these chronic headaches. As a non-limiting example, botulinum toxin may be able to improve headache symptoms in subjects who may have pressure perceived from an outside source, who have had chronic migraines for less than 30 years and/or who may have chronic daily headache due to medication overuse.
Sinus headaches are conditions that arise from inflammation of the paranasal sinuses, often due to infection, allergy or autoimmune conditions, which may lead to chronic facial pain. Subcutaneous injection of botulinum toxin may be able to improve sinus related headache pain in subjects who have developed chronic facial pain as a result of a sinus infection. Injection of botulinum toxin may reduce pain syndromes associated with sinus headaches by impairing secretion of neuro-effectors from mast cells, sensory nerve tissue and blood vessel endothelium, showing an anti-inflammatory effect from botulinum toxin treatment.
Tension headaches are conditions that arise from muscle tension around the head and neck and may be the result of stress, sleep deprivation, hunger, eyestrain and bad posture. Tension headaches may occur as an isolated event, constantly, or daily and pain may last for 30 minutes to 7 days. Direct injection of botulinum toxin at the site of pain or trigger points in facial muscles may be used as a preventative medication for tension headaches. Multiple injections may be required to relieve tension headache symptoms and relief may last for about 3 to 4 months.
Cervicogenic headaches are a chronic, hemicranial pain syndrome in which the sensation of pain originates in the cervical spine or soft tissues of the neck and are often precipitated by neck movement or sustained awkward head positioning. Cervicogenic headaches will last on average between 2 weeks to 7 weeks. Injection of botulinum toxin, into pericranial and cervical muscles may effectively treat cervicogenic headaches. Injection of botulinum toxin may relax tense muscles in the head and neck, which cause cervicogenic headaches, and may result in pain relief
Hormone headaches are pain syndromes often associated with changing hormone levels that occur during menstruation, pregnancy, and menopause in women. Chemically induced hormone changes may be induced by medications such as, but not limited to, birth control pills, may also trigger hormone headaches in women. Injection of botulinum toxin into the endocrine system may help reduce effects of pain associated with hormone headaches by blocking nerve signals carrying pain signals. The effects of botulinum toxin may wear off after 2 to 3 months and may require repeat injections to maintain desired effects.
Treatment of Sweating Conditions
Botulinum toxin may be used to treat hyperhidrosis such as, but not limited to, general sweating conditions, palmer hyperhidrosis, plantar hyperhidrosis, axillary hyperhidrosis and gustatory hyperhidrosis and genital hyperhidrosis. Injection of botulinum toxin at a desired site may prevent cellular exocytosis of acetylcholine and exert an inhibitory effect on secretory cells, resulting in a decrease in sweat production in or around an injected area. Treatment with botulinum toxin may further result in reduction in unpleasant body odor associated with hyperhidrosis by reducing moisture and secretions of sweat glands.
General sweating is a condition characterized by abnormally increased perspiration, in excess of that required to maintain a normal body temperature, of any part of the body. Type A botulinum toxin injections may be used as an effective treatment for general sweating by blocking the secretion of acetylcholine, the chemical responsible for turning on sweat glands. Small injections of botulinum toxin to an affected area is a relatively simple procedure that should cause little pain to the subject, and a single treatment for excessive sweating may have a lasting effect from about 4 months to 12 months.
Palmer hyperhidrosis is a sweating condition specific to the hands. Injection of botulinum toxin type A or type B may be a long-lasting method to treat excessive sweating of the palms by blocking acetylcholine secretion in or around the site of injection into the palms. Treatment of botulinum toxin injections into the palms typically take less than a half of hour and a significant symptomatic improvement can be seen that may last up to 12 months.
Plantar hyperhidrosis is a sweating condition specific to the feet, with over 3% of the world's population having this condition. Direct injection of botulinum toxin may effectively be used to treat plantar hyperhidrosis by blocking acetylcholine at the site of injection and blocking sweat gland secretion. In order to increase the efficacy of botulinum toxin treatment for plantar hyperhidrosis repeated and multiple injections may be required as the feet have over 250,000 sweat glands which may be regulated to manage sweat secretion.
Axillary hyperhidrosis is a sweating condition specific to the armpits, causing profuse sweating under the armpits without the ability to control the rate of perspiration. Axillary hyperhidrosis may be caused by the sympathetic nervous system sending signals to the underarm sweat glands to keep producing sweat. Intradermal injection of botulinum toxin under the armpits may be used to treat axillary hyperhidrosis by inhibiting acetylcholine secretion which initiates sweat glands in the armpits. Treatment of axillary hyperhidrosis with botulinum toxin may last for about 3 weeks to about 17 weeks, thereafter repeated injections may be needed to continue to manage sweat secretion.
Gustatory hyperhidrosis or Frey's syndrome is a sweating condition specific to the forehead, upper lip, perioral region or sternum, often occurring a few moments after eating spicy foods, tomato sauce, chocolate, coffee, tea or hot soups. Frey's Syndrome may be congenital or may be acquired after parotid gland surgery or an injury to the auricotemporal nerve (which carries sympathetic fibers to the sweat glands of the scalp and parasympathetic fibers to the parotid gland). Symptoms of Frey's syndrome include, but are not limited to, sweating on the cheek area adjacent to the ear. Injection of botulinum toxin type A may be an effective treatment for Frey's syndrome. In addition to inhibiting acetylcholine at the nerve endplates, thereby blocking signals to overactive muscles, neurotransmitters in sweat glands that depend on acetylcholine for their activation may remain suppressed in the presence of botulinum toxin. Treatment of Frey's Syndrome through intradermal injection of botulinum toxin in or around a condition site may help prevent sweating for about 17 weeks, thereafter repeated injections may be needed to manage sweat secretion.
Genital hyperhydrosis is excessive sweating of the genital area, including the groin, inner thighs and buttocks. Genital hyperhidrosis is considered a hereditary condition, but may also be caused by stress, change in hormones, anxiety, exercise levels and high levels of humidity. Intradermal injection of botulinum toxin may treat genital hyperhydrosis by inhibiting exocytosis of acetylcholine, thereby suppressing secretion of sweat from sweat glands in or around the intradermal injection site. Effects from injections may last for about 3 to 17 months before repeat injections may be desired to treat genital hyperhydrosis.
Treatment of Cosmetic Conditions
Injection of type A and type B botulinum toxin in small quantities may be effectively used to treat cosmetic conditions such as, but not limited to, facial wrinkles, hyperkinetic skin lines, glabellar lines, crow's feet, marionette lines, skin disorders, nasolabial folds, blepharospasm, strabismus, hemifacial spasms and wound healing. Injecting overactive facial muscles with minute quantities of botulinum toxin can result in a decrease in acetylcholine release at the neuromuscular junction, thereby temporarily rendering the muscles unable to contract, and temporarily ending, for instance, spasmodic winking found in blepharospasms. Injection of small quantities of botulinum toxin may have a lasting effect for cosmetic purposes on average for a period of about 4 to 6 months.
Facial wrinkles are a slight ridge, crumpling, creasing or puckering in the smoothness of the surface of skin. Facial wrinkles may occur for a variety of reasons such as, but not limited to, aging, loss of elasticity in skin layers, muscle contraction, sun damage, smoking, dehydration and/or medication. Small doses of botulinum toxin may be injected into facial muscles to block the release of acetylcholine by nerve cells that signal muscle contraction. This interference results in a smoothing out of existing facial wrinkles thereby treating the wrinkles Treatment of botulinum toxin on facial wrinkles usually takes about 48 to 72 hours to take effect and the results remain for about 3 months before additional injections may be necessary to continue treating the wrinkles
Hyperkinetic skin lines are lines formed by hyperkinetic or overactive musculature, which are typically found along the cheeks around hyperkinetic muscles. Hyperkinetic skin lines are commonly associated with aging skin. Injection of small quantities of botulinum toxin to hyperkinetic muscles may reduce byperkinetic skin lines by temporarily inhibiting contraction of hyperkinetic muscles through acetylcholine inhibition. Treatment of hyperkinetic skin lines with botulinum toxin may last for about 3 months before retreatment of additional injections may be desired.
Glabellar lines are vertical lines between the eyebrows, which are formed by repeated contraction of the glabellar muscles. Over time the skin between the eyebrows loses elasticity, resulting in deep wrinkles between the eyebrows, commonly known as frown lines. Glabellar lines may be part of the aging process and the FDA has currently approved the use of Botulinum toxin type A for the treatment of glabellar lines. Injection of Botulinum toxin at the site of the glabellar lines, may result in relaxation of the muscles which have been repetitively contracted to create the glabellar lines. Repeat treatment may be necessary. The effects of treatment with botulinum toxin may last for about 3 months depending on factors such as, but not limited to, age, severity, and number of previous treatments.
Crow's feet are small wrinkles radiating outward from the outer corner of the eye, often occurring as the result of age. Crow's feet may be caused by contraction of the muscles that run around the circumference of the eye, while other causes of crow's feet may include, but are not limited to, aging, thin skin, muscle degeneration, tendon degeneration and sun damage. Injection of botulinum toxin to the muscles around the circumference of the eye may treat crow's feet through the inhibition of actetylcholine which is necessary to induce muscle contraction. Botulinum toxin may be less effective as a treatment for crow's feet which has been induced by conditions such as, but not limited to, age, thin skin, muscle degeneration, tendon degeneration and sun damage for instance, as these conditions are not related to underlying muscle activity around the eyes.
Marionette lines are vertical lines that laterally circumscribe the chin and may give the face an expression of being dissatisfied or grim. Marionette lines tend to appear as ligaments around the mouth and chin loosen and sag during the aging process. Botulinum toxin may be used around the chin and mouth to reduce or soften marionette lines caused by muscle contractions by inhibiting acetylcholine necessary for muscle contraction. Treatment of botulinum toxin for marionette lines may further include, but is not limited to, dermal fillers to have significant impact on deep marionette lines.
Skin disorders are abnormal conditions of the membranous tissue of the epidermis and dermis. Skin disorders may include, but are not limited to, dimples, warts, pimples, facial rashes, hives and xerosis. Botulinum toxin may be used to improve a variety of skin disorders as it may provide an anti-aging effect on the skin. Botulinum toxin is particularly useful for any skin disorder that may be caused by abnormal muscle contractions or related to sweat gland production. Dimples, for example, may be the result of overactivity of the mentalis muscle in the chin, which may be temporarily managed through botulinum toxin injections. In the chin area, special consideration to the dosing of botulinum toxin is important in order to prevent drooling or the drooping of muscles around the mouth.
Nasolabial folds are folds on the face that run from the corners of the nose to the mouth which become more pronounced with aging. Factors that may contribute to nasolabial folds include, but are not limited to, excess skin, skin thinning, excess cheek fat and ptosis of cheek fat. Injections of botulinum toxin may be used to treat nasolabial folds by relaxing the facial musculature. For example, injections of botulinum toxin may be at the lip elevator complex in the nasolabial fold and the levator labii superioris aleaque nasi muscle, one injection per side. Effects of treatment may last for up to 6 months before repeated injections may be desired.
Striae distensae are depressed lines or bands of thin reddened skin, which later become white, smooth, shiny and/or depressed. Striae distensae may occur in response to changes in weight or muscle mass and skin tension and are commonly referred to as stretch marks. Injection of Botulinum toxin may relax tense and stretched muscles resulting in improved muscle elasticity and a decrease in striae distensae.
Wound healing is an intricate process in which the skin repairs itself after injury. In normal skin the epidermis and dermis form a protective barrier against the external environment. Once the protective barrier is broken the normal process of wound healing includes inflammation, proliferation and skin remodeling. Increased metabolic activity and inflammation during the healing process may induce muscle contractions around the edges of the skin wound and may cause microtraumas around the scar during the normal healing process. Botulinum toxin may be an effective treatment to aid in would healing by decreasing muscle spasms during the healing process and may improve pain control, inflammation, blood flow and/or ischemia associated with wound healing.
Treatment of Head and Neck Conditions
Head and neck conditions such as but not limited to, achalasia, sialorrhea, rhinorrhea, cervical dystonia, focal dystonia, temporomandibular joint disorder, blepharospasms, strabismus, hemifacial spasm, vocal cord dysfunction, laryngeal dystonia, bulbar amyotrophic lateral sclerosis, masseter muscle reduction, epilepsy, obsessive-compulsive disorder. Injection of botulinum toxin at the site of head or neck pain may inhibit the release of the neurotransmitter acetylcholine at the neuromuscular junction thereby inhibiting muscle contractions, which are often the cause of pain. Besides reducing muscle tone, botulinum toxin tends to reduce pain in syndromes associated with muscle spasm and has been proposed as an analgesic, suggesting alternative non-cholinergic mechanisms of action that may affect head and neck conditions.
Achalasia is a condition of the esophagus, wherein the esophageal sphincter loses it contractile ability to normally prevent the back flow of food from the stomach to the esophagus. Achalasia may be caused by the degeneration of nerve cells that normally signal the brain to tell the esophageal sphincter to relax and continue contraction to keep the esophagus closed. Intrasphyncteric injection of botulinum toxin may be used to paralyze the muscles of the esophageal sphincter so that the muscles relax and continue to contract, which may be an effective therapy for several years after injection.
Sialorrhea is an excessive flow of saliva often resulting from neuromusclular dysfunction, hypersecretion, sensory dysfunction or anatomic dysfunction, resulting in poor oral and facial muscle control. Sialorrhea may be associated with a variety of conditions, including but not limited to acute inflammation of the mouth, mental retardation, mercurialism, pregnancy, teething, alcoholism, malnutrition and Parkinson's disease. Sialorrhea, as a result of Parkinson's disease, may occur because of a neuromuscular dysfunction that results in impaired or infrequent swallowing. Injection of botulinum toxin into the salivary glands of the head may block hypersecretion of saliva by inhibiting acetycholine release from nerve endings in the salivary glands. Botulinum toxin injections to treat Siallorhea as a result of Parkinson's disease may relax neuromuscular conditions and improve voluntary control over swallowing. Improvements may be noticed within 1 week of an injection and last for about 3 months. Repeated injections may necessarily for continued treatment.
Rhinorrhea is a condition where the nasal cavity is significantly filled with mucous fluid. Rhinorrhea frequently occurs as a common symptom of conditions such as, but not limited to, allergies, colds and hay fever. Topical application of botulinum toxin may act as a cholinergic blocking agent to treat Rhinorrhea, and may significantly reduce neurally induced rhinorrhea. Injections of botulinum toxin into the ganglion muscles may be a treatment option to reduce cholingerically induced rhinorrhea.
Cervical dystonia is a chronic neurological movement disorder that causes the neck to involuntarily turn to the left, right, upwards, and/or downwards. Both agonist and antagonist muscles contract simultaneously during cervical dystonia movements, resulting in involuntary movements. Most subjects first experience symptoms of cervical dystonia midlife. Cervical dystonia may be an inherited or acquired condition from exposure to various toxins and/or other diseases. Injections of botulinum toxin may be used to treat cervical dystonia by disabling the movement of antagonist muscles while the agonist muscles are allowed to move freely, resulting in relief from involuntary neck movements. Botulinum toxin injections may provide relief for about 3 to 4 months before repeat injections may be desired. Some preparations of botulinum toxin are approved by the FDA for clinical use in the United States for treating cervical dystonia.
Focal dystonia is a neurological condition which affects a muscle or group of muscles in a part of the body and causing an involuntary muscular contraction or twisting. Focal hand dystonia, for instance, results in fingers that either curl into the palm or extend outward without control. Injection of Botulinum toxin results in paralysis of striated muscle groups, which may treat involuntary muscle contractions in focal dystonia. The effects from treatment of botulinum toxin usually will be apparent about 2 weeks after injection and last for about 3 months before repeat injection may be desired.
Temporomandibular joint disorder is an umbrella term covering acute or chronic pain in the muscles of mastication and/or inflammation of the temporomandibular joint, which connect the mandible to the skull. Damage to the rotation or hinged action of the jawbone or the gliding actions of the mouth to open wide, for example, may be a cause of temporomandiular joint disorder. Temporomandibular joint disorder may result in significant pain combined with impairment of function. Symptoms of temporomandibular joint disorder may include, but are not limited to, difficulty biting and/or chewing, clicking, popping or grating sounds when opening and/or closing the mouth, dull aching pain in the face, earache, headache, and/or reduced ability to open and close the mouth. Injection of botulinum toxin to the orofacial musculature may result in weakening of hyperactive muscles that may cause temporomandibular joint disorder pain, and may also relieve head and neck pain resulting from temporomandibular joint disorder. Injections of botulinum toxin may need to be repeated about every 3 months to maintain relief of the symptoms.
Blepharospasms are spasms of the muscle of the eyelids, causing the eyes to shut tightly, which is often denoted by spasmodic winking Injection of Botulinum toxin weakens the muscles of the eyelids by blocking nerve impulses transmitted from the nerve endings of the muscles. For example, botulinum toxin may be injected intramuscularly into several sites above and/or below the eyes with a fine needle. Typically, results are seen about 1 to 14 days after the injection of botulinum toxin to treat blepharospasms and last for about 3 to 4 months.
Strabismus is a condition in which the eyes are not properly aligned with each other. Strabismus typically involves a lack of coordination between the extraocular muscles, which prevents bringing the gaze of each eye to the same point in space and preventing binocular vision, which may adversely affect depth perception. Strabismus may be a disorder of the brain in coordinating the eyes, or a disorder of relevant muscles' direction of motion. Botulinum toxin may be use to treat strabismus through injection into the extrocular muscles, temporarily prevention muscle contractions around the eye for several months, allowing for the opposing muscle to change the eye's position and potentially provide an improved eye alignment. Botulinum toxin may also be used in conjunction with surgery as an effective treatment for strabismus.
Hemifacial spasm is an involuntary twitching or contraction of facial muscles on one side of the face. Hemifacial spasm may be caused by an injury to a facial nerve and/or a condition such as, but not limited to, a tumor or blood vessel compressing the nerve, or Bell's palsy. Hemifacial spasms affect about 8 out of every 100,000 people in the United States, with an average age of onset of around 44 years. Injection of botulinum toxin into facial muscles may block the release of acetylcholine into the muscles and may also prevent muscle contraction thereby preventing the involuntary twitching associated with hemifacial spasms. Typically 1 to 3 injections of botulinum toxin may be used to treat hemifacial spasms, with effects seen around 3 days and last for about 3 months before repeated injections may be necessary.
Vocal cord dysfunction is a condition characterized by an abnormal adduction of the vocal cords during a respiratory cycle that produces airflow obstruction at the level of the larynx. Vocal cord dysfunction may be triggered suddenly or gradually and may be caused by conditions such as, but not limited to, gastroesophageal reflux disease, extra-esophageal reflux, exposure to inhaled allergens, post nasal drip, exercise and/or neurological conditions. Symptoms of vocal cord dysfunction may include, but are not limited to, shortness of breath, wheezing, coughing, tightness in the throat, skin discoloration, and noise during inhalation. Injection of botulinum toxin into the larynx may result in paralysis of vocal cord muscles, making it impossible for the vocal cords to move during respiration and thereby relieving effects from vocal cord dysfunction. Repeat injections of botulinum toxin may be required as the paralysis effect may wear off a few months after the injection.
Laryngeal dystonia is a voice disorder characterized by spasmodic contraction of laryngeal muscles, resulting in strained and strangle speech with break in rhythm. Laryngeal dysphonia may affect anyone, becomes most evident between the ages of 30 and 50, and is more prevalent in women than in men. Botulinum toxin injections of one or both of the voice box muscles may be an effective treatment to weaken the muscles that cause laryngeal dystonia. Injection dosages may depend on disease state and provide temporary relief from laryngeal dystonia for about 4 months.
Bulbar amyotrophic lateral sclerosis is a progressive neuromuscular weakening condition characterized by difficulty swallowing and chewing food, drooling, and facial tremors. Bulbar amyotrophic lateral sclerosis is the result of motor neurons that die and results in loss of muscle control by the brain nerves in the bulbar region of the brainstem, which control the throat, tongue, and jaw. Approximately 30,000 Americans have bulbar amytrophic lateral sclerosis, which typically occurs midlife. Injection of botulinum toxin into an effected area may help relieve symptoms of bulbar amyotrophic lateral sclerosis by blocking acetylcholine release from nerve ending and treating one or more aspects of a condition. As a non-limiting example, injection of botulinum toxin into the parotid glands may be used to treat drooling conditions and/or Sialorrhea. Injection of botulinum toxin may result in a decrease in saliva production from the parotid glands providing temporary relief of bulbar amyotrophic lateral sclerosis symptoms for about 2 to 3 months.
Masseter muscle reduction is the reduction in the size of a thick rectangular muscle in the cheek that functions to close the jaw. Botulinum toxin injection of the Masseter muscle may be used for aesthetic purposes to relax the masseter muscle and reduce a prominent mandibular angle in the face, resulting from the thick rectangular Masseter muscle. 1 to 3 injections of Botulinum toxin at the inferior masseter border may be used for treatment. The maximum reduction of masseter muscle size may be seen in about 1 to 2 months after injection, and aesthetic results may continue for up to 12 months before further injections may be desired.
Epilepsy is a brain disorder in which a person has repeated seizures over time. Seizures are episodes of disturbed brain activity that cause changes in attention and/or behavior. Intracranial injection of botulinum toxin may help relieve epilepsy seizures by inhibiting acetylcholine release at nerve ending and blocking nerve signals from the brain which normally create an epileptic seizure.
Obsessive-compulsive disorder is an anxiety disorder in which people have unwanted and repeated thoughts, feelings, ideas, sensations or behaviors that cause repetitive behaviors, which may cause major distress or interfere with every day activity. Family history and chemical imbalances in the brain may contribute to obsessive-compulsive disorder, and chemical imbalances from the brain, possibly including secretion levels of seratonin, may contribute to obsessive-compulsive disorder. Injection of botulinum toxin to inhibit imbalanced chemical secretions, such as hypersecretion of seratonin, may result in improvements in the symptoms for chemically induced obsessive-compulsive disorders. Injection dosages and location may vary depending on the severity of obsessive-compulsive disorder in a patient.
Treatment of Musculoskeletal Conditions of the Torso
Muscoloskeletal conditions, diseases and disorders are abnormal conditions of muscles and their associated ligaments, tendons, connective tissues and bones. Botulinum toxin may be use to treat musculoskeletal conditions of the torso, such as but not limited to, musculoskeletal condition, fibromyalgia, tremors, myoclonus, post stroke spasticity, juvenile cerebral palsy, smooth muscle spasms, arthritic pain, muscle contracture, muscle injury, writer's cramp, carpel tunnel syndrome, movement disorders, tics and tourette's syndrome. Botulinum toxin injection may be used to treat symptoms of musculoskeletal diseases, disorders and conditions by relaxing tense muscles, and inhibiting acetylcholine at nerve endings, thereby decreasing pain responses associated with musculoskeletal diseases, disorders and conditions.
Musculoskeletal conditions are pain that affects the muscles, ligaments and tendons, along with the bones. The causes of musculoskeletal conditions and pain may include, but are not limited to, wear and tear from daily activities, trauma to an area, auto accidents, falls, fractures, sprains, dislocations, direct blows to muscles, postural strain, repetitive movements, overuse and prolonged immobilization and disease related conditions. Subjects with musculoskeletal pain may have symptoms of pain, fatigue and sleep disturbance. Botulinum toxin may be injected at the location of musculoskeletal pain to reduce muscle spasms. Temporary muscle paralysis, improved blood flow and release of fibers normally under compression with botulinum toxin treatment may result in significant pain relief of musculoskeletal pain.
Fibromyalgia is a common syndrome in which a person has long-term, body-wide pain and tenderness in the joints, muscles, tendons, and other soft tissues. Causes of fibromyalgia include, but are not limited to, fatigue, sleep problems, headaches, depression, and anxiety. Botulinum toxin, when injected in small quantities, may cause selective weakening and paralysis of muscles, thereby alleviating spasms and pain associated with fibromyalgia. Injection of botulinum toxin may take up to 3 weeks for relief to be noted, and injections may be repeated every 3 to 4 months if desired.
A tremor is an involuntary muscle contraction and relaxation involving to-and-fro movements of one or more body parts. Tremors are common involuntary movements, which may affect the hands, arms, eyes, face, head, vocal folds, trunk and legs and may be a symptom of a neurological disorder. Botulinum toxin injections may be used to treat tremors and may be particularly effective to treat tremors of the head and voice. Injection of botulinum toxin may affect muscle efferents and afferents and may alter sensory feedback to the central nervous system, thereby allowing control of involuntary muscle contractions, which may cause tremors.
Myoclonus is a brief, involuntary twitching of a muscle or a group of muscles that may be caused by rapid contraction and relaxation of the muscles and may be seen in children, adolescents, and adults. Mynoclonus may develop in response to infection, head or spinal cord injury, stroke, stress, brain tumors, kidney or liver failure, lipid storage disease, chemical or drug poisoning, as a side effect of some drugs or other disorders. Injections of botulinum toxin may be used to treat myoclonus by inhibiting acetylcholine release at muscle junctions and preventing signals transmission, which result in involuntary muscle twitches in myoclonus.
Post stroke spasticity is a motor dysfunction arising from upper motor neuron lesions due to stroke. Post stroke spasticity often leaves subjects with recognizable antigravity postural patterns which may be characterized by shoulder adduction, elbow and wrist flexion in the upper limb, hip adduction, knee extension and ankle plantar flexion in the lower limb, which is thought to result from increased motor neuron activity in the antigravity muscles. Injection of botulinum toxin in or around the site of post stroke spasticity may effectively reduce muscle spasms, thereby decreasing muscle tone and increasing range of motion in muscles. Dosages of botulinum toxin to treat post stroke spasticity may be generally adjusted according to factors such as the severity of spasticity, number of muscles involved, age, and number of prior botulinum toxin treatments, for optimal treatment effects.
Juvenile cerebral palsy is a group of non-progressive, non-contagious motor conditions that cause physical disability in juvenile development, particularly in areas of body movement. Resulting limits in movement and posture are often accompanied by disturbances of sensation, depth perception, other sight-based perceptual problems, communication problems and impairment in juvenile cognition. Juveniles with cerebral palsy often have spastic muscles in or around the foot and tight muscles in the thighs. Injections of botulinum toxin in juveniles with cerebral palsy may result in relaxation of spastic muscles and relaxation of thigh muscles, making it comfortable for a juvenile patient to walk and/or sit. In some countries botulinum toxin has been approved for treatment of some cerebral palsy conditions in juveniles.
Smooth muscle spasms are a sudden involuntary contraction of a muscle or group of muscles which often results in moderate to severe pain. Smooth muscles are typically found in hollow organs including the stomach, intestines, blood vessels and the bladder. Injection of botulinum toxin to spasmic smooth muscles may help loosen and relax the muscle and prevent pain to a patient. Injection of botulinum toxin to smooth muscles may take effect within three days from the date of injection and last for about three months before repeat injections may be desired.
Arthritic pain is inflammation of a joint usually accompanied by pain, swelling and stiffness, resulting in infection, trauma, degenerative changes, metabolic disturbances, aging and other causes. Common forms of arthritic pain are osteoarthritis and rheumatoid arthritis, with indications including pain or swelling of a joint. Injection of botulinum toxin at a specific muscle may result in blocking contraction of muscles at the area of injection, which may result in decreased pain in arthritic joints and an increase in the range of motion, previously hindered by arthritic swelling. Treatment with botulinum toxin may reduce arthritic pain by about 50%.
Muscle contracture is shortening of the muscles and tendons, resulting in reduced flexibility, which may occur as a result of paralysis, muscular atrophy, spinal cord injury and some forms of muscular dystrophy, as a non-limiting example. Muscles contain receptors called spindles that monitor tension from the spinal cord to maintain muscle length. Injury to the spinal cord increases excitability of neural circuits that control muscle tension. Spastic muscles resulting from spinal cord injury resist muscle tension changes by contracting and lead to muscle contracture or shortening of the muscles. Injection of botulinum toxin to treat muscle contracture may relieve spasticity in muscles by blocking motor nerves and motorneurons that innervate muscles.
Muscle injury is damage that occurs by overstretched muscles or tendons which are pulled or torn. Botulinum toxin may help restore muscles and relieve pain from muscle injury by blocking motor and neural signals in the back which create spasms, injury and pain. Dosages of botulinum toxin for muscle injury may vary greatly, depending on the size and location of a muscle injury.
Writer's cramps are cramps or spasms that affect certain muscles of the hand and/or fingers. Symptoms of writer's cramp include loss of precision muscle coordination, camping pain with sustained use and muscle pain. Injection of botulinum toxin may be an effective treatment for writer's cramp. Injections in the forearm muscles may weaken cramped or spasmic muscles of the hand and/or fingers and result in temporary relief for writer's cramp, which may last for about 3 months.
Carpel tunnel syndrome is an entrapment of the median nerve traveling through the canal on the palmar side of the wrist connecting the forearm to the middle compartment of the palm, the carpel tunnel, which results in pain and numbness. The carpel tunnel is a narrow passageway with nine flexor tendons passing through it, if one or more flexor tendons swell, the narrowing canal often results in the median nerve becoming entrapped or compressed, resulting in pain. Injection of botulinum toxin into the carpel tunnel may relieve weakness and pain resulting from the compressed nerve in the tunnel, possibly by blocking the nerve signal, and be and effective treatment for about 3 months before further injections may be desired.
Movement disorders are a group of diseases and syndromes affecting the ability to produce and control movement. Examples of movement disorders include, but are not limited to, Parkinson's disease, Huntington's disease, Wilson's disease, tourette syndrome, dystonia, tic disorders, and restless leg syndrome. Movement is controlled through interaction of the brain centers, muscle reflexes, and neuronal signaling processes, injury or damage at any interaction point may cause a movement disorder. Treatment of movement disorders is disease specific, however, injection of Botulinum toxin may be a treatment for many movement disorders by inhibiting the release of neurotransmitters that cause muscle contraction. Treatment will typically last between about 3 to 4 months before repeated injections may be desired.
A tic is a condition in which a part of the body moves repeatedly, quickly, suddenly and uncontrollably. Tics can occur in any body part, such as the face, shoulders, hands or legs and can occur in children, adolescents and adults. Most tics are mild and hardly noticeable, however sometimes they may be chronic and do not go away. Injection of botulinum toxin to muscles with tic syndrome may weaken the muscle and relieve tic symptoms for about 3 to 12 months, before repeated injections may be desired.
Tourette's syndrome is an inherited neurological disorder with onset in childhood, typically characterized by multiple physical tics and some vocal tics. Genetic and environmental factors play a role in the etiology of tourette's syndrome, though the exact causes are unknown. Injection of botulinum toxin around motor tics may treat tics associated with tourette syndrome by blocking acetylcholine at nerve ending and suppressing neuronal signals that may create Tourette syndrome related tics. Injection sites may be patient specific depending on the tic location and require repeat injection within 2 to 3 months.
Treatment of Organ Conditions
Organs, such as the heart, bladder, pancreas, sex organs and thyroid may have conditions resulting from inflammation, muscle hyperactivity, or neuronal misfiring, creating discomfort and sometimes life-threatening illnesses for a patient. Botulinum toxin may be used to treat organ conditions such as but not limited to, thyroid disorder, pacreatitis, tachycardia, urinary retention, urinary incontinence, overactive bladder, detrusor-sphincter dyssnergia, idiopathic and neurogenic detrusor overactivity, vaginismus, prostatic enlargement, prostitis, benign prostatic hyperplasia, and autonomic hypersecretory disorders. Relief of organ conditions by injection of botulinum toxin may help ease pain and prevent recurrence of organ conditions through inhibition of acetylcholine release at neuromuscular junctures.
Thyroid disorder is a medical condition with impaired functioning of the thyroid, typically occurring as an overactive thyroid, hyperthyroidism, or an underactive thyroid, hypothyroidism. Imbalance in production of thyroid hormones arises from dysfunction of the thyroid gland, of the pituitary gland or of the hypothalamus. Botulinum toxin may be administered to a thyroid and reduce the effect of thyroid hormone on the thyroid. Injection, for instance, of botulinum toxin to the sympathetic ganglion that innervates the thyroid, may reduce the stimulatory effect of thyroid hormone secretion on the thyroid. Repeat injections of botulinum toxin to treat thyroid disorder may be desired about every 3 months.
Pancreatitis is a condition that occurs from inflammation of the pancreas. The pancreas is a gland located behind the stomach, which releases the hormones, insulin and glucagon, as well as digestive enzymes that aid in digestion and absorbtion of food. Botulinum toxin endoscopic injection into the pancrease may be an effective treatment for pancreatitis. Endoscopic injection of botulinum toxin into patients with recurrent pancreatitis my result in short-term relief for about 9 to 10 months and may also be used to determine if patients will benefit from other forms of endoscopic surgery such as pancreatic sphincter removal.
Tachycardia typically refers to a heart rate that exceeds the normal range of a resting heart rate, which may be dangerous depending on the speed and rhythm. Intraperitneal or intracardiac injection of botulinum toxin to heart muscles may help control tachycardia conditions by temporarily managing muscle contractile activity.
Urinary retention is a condition that causes urine to remain in the bladder even after urination. While anyone may experience urinary retention, it is most common in men in their 50's and 60's, which may be the result of progressive obstruction of the urethra by an enlarging prostate. In some cases, even with the sensation of a full bladder, urination may be impossible, causing discomfort and possible infection. Injection of botulinum toxin in the bladder may help relieve pain associated with muscle spasms found in patients with urinary retention.
Urinary incontinence is the unintentional loss of urine sufficient enough in frequency to cause physical and/or emotional discomfort. About 13 million Americans suffer from urinary incontinence, which may be caused by conditions including but not limited to: weakening of pelvic muscles during childbirth, hysterectomy or gynecological surgery, bladder dysfunction, menopause, neurological condition, and obesity. Injections of botulinum toxin to the urinary bladder may lead to relaxation of the bladder muscles and increase the bladders storage capacity, thereby decreasing urinary incontinence. The FDA has approved the use of botulinum toxin type A to treat urinary incontinence in people with some neurological conditions, such as spinal cord injury and multiple sclerosis, in which urinary incontinence may be a significant problem.
Overactive bladder is the strong, sudden need to urinate due to bladder spasms or contractions. Overactive bladders may result in leakage of urine because the bladder muscles contract at the wrong times. Causes of overactive bladder may include but are not limited to: bladder cancer, bladder inflammation, bladder outlet obstruction, bladder stones, infection and nervous system disease. Intravesical injection of botulinum toxin may be used to treat neurogenic and idiopathic overactive bladder in adults. To treat overactive bladder, 15 to 20 injections of botulinum toxin may be required, which may affect some of the nerves controlling the bladder, thereby providing an increased ability of a subject to squeeze the bladder to urinate when desired and less of a likelihood of the bladder squeezing when it should not.
Detrusor-sphincter dyssynergia is a condition in which there is a lack of coordination between the bladder and the external sphincter muscle, which creates a build-up of urinary pressure. Detrusor-sphincter dyssynergia may be caused by lesions between the brain stem and the lower part of the spinal cord, which results in damage to the nervous system. Botulinum toxin treatment into the external urethral sphincter may be an effective therapy for neurogenic disorders and bladder malfunction due to detrusor-sphincter dyssynergia. Dosages required for injection may vary depending on the severity of injury, with results that may range from about 2 months to 12 months.
Idiopathic and neurogenic detrusor overactivity is involuntary detrusor muscle contractions during the filling phase of the bladder, which may be spontaneous or provoked, and lead to involuntary bladder emptying. Idiopathic and neurogenic detrusor overactivity may lead to urinary incontinence or complete bladder emptying. Injection of botulinum toxin to the detrusor muscle of the bladder may result in weakening of the detrusor muscle and inhibit overactive contractions that push on and fill the bladder. Botulinum toxin treatment may result in less detrusor muscle pressure on the bladder and an increase in bladder compliance in cases where the baseline bladder compliance was abnormal prior to treatment. Treatments with Botulinum toxin for idiopathic and neurogenic detrusor activity may last for about 6 to 9 months.
Vaginismus is a condition that affects a women's ability to engage in any form of vaginal penetration. Vaginismus occurs as the result of a reflex by the pubococcygeus muscle that causes the vagina to tense suddenly, making vaginal penetration painful or impossible. Injection of botulinum toxin intravaginally may be an effective treatment of Vaginismus, to effectively paralyze the muscle causing intense muscle pain by blocking acetylcholine release in the innervated muscles and paralyzing nerve impulses. Tests to determine the areas of maximum reflex and resulting pain may be conducted prior to injection, for optimal treatment efficiency.
Prostatic enlargement is common as a man ages, as the prostate enlarges, the layer of tissue surrounding the prostate stops the prostate from expanding, causing the gland to press against the urethra like a clamp on a garden hose. The bladder wall becomes thicker and irritable. Intraprostatic injection of botulinum toxin may relax prostate muscles and reduce prostate volume for a period of up to about 18 months, wherein significant symptomatic improvement may be shown after treatment.
Prostatitis is an inflammation of the prostate gland, a common condition in adult males. Prostatitis is often caused by infection and may develop rapidly or slowly. Difficulties in urinating and pain from swelling may be symptoms of prosatitis. Injections of botulinum toxin to the prostate may be used to treat prostatitis. The inhibitory effect of botulinum toxin on motor end plates may result in prostate muscle relaxation, which may decrease inflammation and pain associated with prostatitis for a period of about 3 months, before repeat injections may be required.
Benign prostatic hyperplasia is an increase in size of the prostate, resulting from hyperplasia of prostatic cells, which form large discrete nodules in the prostate. These nodules may compress the urethral canal and cause partial or complete obstruction of the urethra, which interferes with the normal flow of urine. Symptoms such as urinary hesitancy, frequent urination, painful urination, urinary tract infection and urinary retention may be seen. Injection of botulinum toxin to the bladder may weaken tight muscles and block nerve signals, which may help improve urine flow and decrease residual urine left in the bladder in benign prostatic hyperplasia. Treatment of botulinum toxin may be an effective treatment for benign prostatic hyperplasia for about 3 months to 12 months, before repeat injections may be required.
Autonomic hypersecretory disorders are excessive production of a bodily fluid involuntarily, often controlled by the central nervous system. Autonomic hypersecretory disorders may occur anywhere in the body, including, but not limited to, excessive tears from tear glands, excessive sweating from sweat glands, and excessive gastric acid in the stomach. Injection of botulinum toxin at the site of a hypersecretory disorder may result in a decrease in hypersecretory activity by blocking acetylcholine release at nerve endings, which may be signaling the central nervous system to secrete excessively. Botulinum toxin has been shown to be an effective treatment for hypersecretory disorders, with improved symptoms typically lasting for about 3 months before repeat injections are required.
Treatment of Gastrointestinal Conditions
Gastrointestinal disorders are all diseases that pertain to the gastrointestinal tract including but not limited to disorders of the esophagus, stomach, first, second, and third part of the duodenum, jejunum, ileum, the ileo-cecal complex, large intestine (ascending, transverse, and descending colon), sigmoid colon, and rectum. Botulinum toxin may be an effective treatment for gastrointestinal disorders, such as but not limited to chronic anal fissure and peritoneal adhesion, as a potent neuromuscular blocking agent that inhibits acetylcholine release at nerve endings, thereby altering muscle function.
Chronic anal fissure is a painful tear or split in the distal anal canal, which persists for about 6 weeks. Botulinum toxin may be injected directly into the internal anal sphincter, which may inhibit the sphincter muscle from contraction for about a 3 month period, until the nerve ending regenerate. The 3 month period may allow fissures to heal and symptoms of chronic anal fissure to resolve. Repeated injections of Botulinum toxin may be made for a prolonged inhibitory effect on the sphincter, or may be followed by a sphinctereotomy surgery.
Peritoneal adhesion is part of the healing process from inflammation of the peritoneum, the thin tissue that lines the inner wall of the abdomen and covers most of the abdominal organs. Peritoneal adhesions may, by contraction, cause partial obstruction of the intestine and chronic or intermittent pain. Injection of botulinum toxin in or around the site of a peritoneal adhesion may relax muscles and reduce muscle tension in the peritoneal adhesion, resulting in decreased pain and improved peritoneal healing.
Treatment of Blood Conditions
Botulinum toxin may be used to treat several blood conditions and blood vessel conditions, such as, but not limited to: diabetes, diabetes neuropathy, hypercalcaemia, restonosis, Raynaud's syndrome, and vasospasms. Injection of botulinum toxin may inhibit acetylcholine at nerve ending in the location of an injection, thereby effecting both nerve and muscle responses to the inhibited nerve endings, which may result in effective treatment for blood condition.
Diabetes is a group of metabolic diseases in which a person has high blood sugar because his/her body does not produce enough insulin or because cells are not responding to insulin that is produced. Symptoms of diabetes may include but are not limited to: frequent urination, increased hunger and increased thirst. Diabetic neuropathy is damage to nerves in the body that occurs due to high blood sugar levels from diabetes. About 50% of the people with diabetes will develop nerve damage, which is more likely to develop if blood sugar levels are not well controlled. Diabetic neuropathy nerve damage may affect any part of the body and are important for managing major vital organs including the heart, bladder, stomach and intestines. Injection of Botulinum toxin may have an inhibitory effect on biochemical mediators of neuropathic pain by modulating afferent sensory fiber firing, and may be used to treat nerve pain which is associated with diabetes.
Hypercalcaemia is an elevated calcium level in the blood, which may be due to excessive skeletal calcium release, increased intestinal calcium absorption, overactivity of parathyroid glands or decreased renal calcium excretion. Symptoms of hypercalcaemia may include but are not limited to nausea, fatigue and muscle weakness. Injection of botulinum toxin to an overactive parathyroid gland may inhibit neural activity of the gland and result in reduction of hypercalcaemia induced from overactive parathyroid glands.
Restonosis is the reoccurrence of senosis, the narrowing of blood vessels, which leads to restricted blood flow. Restonosis typically pertains to an artery or other large blood vessel that has become narrowed, leading to restricted blood flow. Injection of botulinum toxin to vessels may reduce or eliminate blood vessel damage by expanding the inner diameter of the blood vessel and thereby treating restonosis. Botulinum toxin may dilate blood vessels when injected into blood vessels and be useful following a cardiovascular procedure, where narrowing of blood vessels may occur.
Raynaud's syndrome is a vasospastic disorder causing discoloration of the fingers, toes and occasionally other areas, which my result in pain and discoloration. Raynaud's is believed to be the result of vasospasms that decrease blood supply to the regions of the body. People in their 30's are most likely to develop Raynaud's syndrome, with the occurrence in women more prevelant than in men. Injection of botulinum toxin at the site of Raynaud's syndrome, for instance at the metacarpophalangeal joint in the hands to treat Raynaud's syndrome in the fingers, may impact nerves carrying pain signals and result in significant decrease in pain associated with Raynaud's syndrome. In addition ulcers and other skin conditions associated with Raynaud's syndrome may be effectively reduced with botulinum toxin treatment.
Vasospasms refer to a condition in which blood vessel spasms lead to vessel vasoconstriction, which may result in death. Endothelial cells normally induce relaxation of the smooth muscle cells, however aggregating platelets may induce contraction of smooth muscle cells which may outweigh normally induced relaxation of cells, thereby causing vasospasms and vessel vasoconstriction. Injection of botulinum toxin may reduce pain that occurs after a vasospasm has occurred, by direct injection into the head area. Botullinum toxin may also be used as a pretreatment when conditions for vasoconstriction are known, as treatment with botulinum toxin may maintain normal vessel diameters from constriction, possibly by inducing relaxation of smooth muscles surrounding vessel walls.
Fungal Diseases, Disorders and Conditions
The cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of the diseases, disorders and/or conditions caused by fungus. Fungal infections of the ingumentary system are common and include diseases, disorders and/or conditions such as, but not limited to athlete's foot, jock itch, ringworm of the body and/or scalp, bastomycosis, yeast infections, thrush, dandruff, seborrhoeic dermatitis, tinea versicolor, folliculitis, pityriasis versicolor, atopic dermatitis, psoriasis, confluent and reticulated papillomatosis, onychomycosis, and transient acantholytic dermatosis.
Athlete's Foot
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of athlete's foot. Athlete's foot, or tinea pedis, is a fungal infection of the foot which causes peeling, redness, itching, burning, and sometimes blisters and sores. Athlete's foot is caused by a microscopic fungus that lives on dead tissue of the hair, toenails, and outer skin layers. There are at least four kinds of fungus that can cause athlete's foot the most common of which is trichophyton rubrum. Athlete's foot is usually treated with topical formulations, but in severe cases it may be treated using oral formulations.
There are three types of athlete's foot. The most common type is interdigital, also called toe web infection, and usually occurs between the two smallest toes. Common symptoms of this type of athlete's foot are itching, burning, and scaling and the infection can spread to the sole of the foot. Moccassin-type infection of athlete's foot can begin with a minor irritation, dryness, itching, or scaly skin which may involve the entire sole of the foot and extend onto the sides of the foot. The least common type of athlete's foot is vesicular. Usually the condition begins with a sudden outbreak of fluid-filled blisters under the skin, most often on the underside of the foot.
Jock Itch
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of jock itch. Jock itch, or tinea cruris, is a common skin infection that is caused by a type of fungus called tinea. Jock itch appears as a red, itchy rash that is often ring-shaped in warm, moist areas of the body. Jock itch is usually treated with topical formulations such as creams and/or sprays.
Ringworm of the Body
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of ringworm of the body. Ringworm of the body, or tinea corpora, is a fungal infection of the skin that looks like a circular, red, flat sore and may be accompanied by scaly skin. There are many types of ringworm of the body including, but not limited to, facial ringworm or tinea faciei, blackdot ringworm or tinea capitis, ringworm of the hands or tinea manuum, ringworm of the nail, and onychomycosis, or tinea unguium. Ringworm of the body is commonly treated with topical formulations and severe cases may be treated with oral formulations.
Blastomvcosis
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of blastomycosis. Blastomycosis is caused by the fungus Blastomycoses dermatitidis which grows in soil as a mold producing conidia that infects human subjects through the air. This fungus grows as a budding yeast in human tissues and may also include symptoms common with mild respiratory infections.
Ringworm of the Scalp
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of ringworm of the scalp. Ringworm of the scalp, or tinea capitis, is an infection of the scalp which is often seen in school children which is primarily caused by dermatophytes in the Trichophyton and Microsporum genera that invade the hair shaft. Ringworm of the scalp is commonly treated with oral formulations.
Yeast Infection
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of yeast infections. Yeast infections of the skin, or cutaneous candidiasis, are caused by yeast-like fungi called candida. These infections occur when the yeast on the skin grow more actively and causes a red, scaling, itchy rash on the skin. Candida may also cause diaper rash in infants and cause infection of the nail. Treatment of yeast infection includes topical formulations, solutions and oral formulations.
Thrush
In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of thrush. Thrush is an infection of the mouth cause by candida often affecting babies, toddlers, older adults and people with weakened immune systems. Treatment of thrush includes the use of solutions and oral formulations.
Diseases, Disorders and Conditions Related to Malassezia fungi 
Malassezia, or Pityrosporum, is naturally found on the skin surface of many animals. There are numerous species of malasserzia fungus such as, but not limited to, malasserzia furfur, malasserzia pachydermatis, malasserzia globosa, malasserzia restricta, malasserzia slooffiae, malasserzia sympodialis, malasserzia nana, malasserzia yamatoensis, malasserzia dermatis, and malasserzia obtuse. Diseases, disorder and/or conditions caused by the malassezia fungus include, but are not limited to, dandruff, seborrhoeic dermatitis, tinea versicolor, folliculitis, pityriasis versicolor, atopic dermatisis, psoriasis, confluent and reticulated papillomatosis, onychomycosis, and transient acantholytic dermatosis. In one embodiment, the cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of diseases, disorder and/or conditions caused by the malassezia fungus.
VI. Kits and Devices
Kits
The invention provides a variety of kits for conveniently and/or effectively carrying out methods of the present invention. Typically kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
In one aspect, the present invention provides kits comprising the molecules (polynucleotides, primary constructs or mmRNA) of the invention. In one embodiment, the kit comprises one or more functional antibodies or function fragments thereof.
Said kits can be for protein production, comprising a first polynucleotide, primary construct or mmRNA comprising a translatable region. The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, a lipidoid or any delivery agent disclosed herein.
In one embodiment, the buffer solution may include sodium chloride, calcium chloride, phosphate and/or EDTA. In another embodiment, the buffer solution may include, but is not limited to, saline, saline with 2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5% Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodium chloride with 2 mM calcium and mannose (See e.g., U.S. Pub. No. 20120258046; herein incorporated by reference in its entirety). In a further embodiment, the buffer solutions may be precipitated or it may be lyophilized. The amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations. The components may also be varied in order to increase the stability of modified RNA in the buffer solution over a period of time and/or under a variety of conditions. In one aspect, the present invention provides kits for protein production, comprising: a polynucleotide, primary construct or mmRNA comprising a translatable region, provided in an amount effective to produce a desired amount of a protein encoded by the translatable region when introduced into a target cell; a second polynucleotide comprising an inhibitory nucleic acid, provided in an amount effective to substantially inhibit the innate immune response of the cell; and packaging and instructions.
In one aspect, the present invention provides kits for protein production, comprising a polynucleotide, primary construct or mmRNA comprising a translatable region, wherein the polynucleotide exhibits reduced degradation by a cellular nuclease, and packaging and instructions.
In one aspect, the present invention provides kits for protein production, comprising a polynucleotide, primary construct or mmRNA comprising a translatable region, wherein the polynucleotide exhibits reduced degradation by a cellular nuclease, and a mammalian cell suitable for translation of the translatable region of the first nucleic acid.
In one embodiment, the levels of Protein C may be measured by immunoassay. The assay may be purchased and is available from any number of suppliers including BioMerieux, Inc. (Durham, N.C.), Abbott Laboratories (Abbott Park, Ill.), Siemens Medical Solutions USA, Inc. (Malvern, Pa.), BIOPORTO® Diagnostics A/S (Gentofte, Denmark), USCN® Life Science Inc. (Houston, Tex.) or Roche Diagnostic Corporation (Indianapolis, Ind.). In this embodiment, the assay may be used to assess levels of Protein C or its activated form or a variant delivered as or in response to administration of a modified mRNA molecule.
Devices
The present invention provides for devices which may incorporate polynucleotides, primary constructs or mmRNA that encode polypeptides of interest. These devices contain in a stable formulation the reagents to synthesize a polynucleotide in a formulation available to be immediately delivered to a subject in need thereof, such as a human patient. Non-limiting examples of such a polypeptide of interest include a growth factor and/or angiogenesis stimulator for wound healing, a peptide antibiotic to facilitate infection control, and an antigen to rapidly stimulate an immune response to a newly identified virus.
Devices may also be used in conjunction with the present invention. In one embodiment, a device is used to assess levels of a protein which has been administered in the form of a modified mRNA. The device may comprise a blood, urine or other biofluidic test. It may be as large as to include an automated central lab platform or a small decentralized bench top device. It may be point of care or a handheld device. In this embodiment, for example, Protein C or APC may be quantitated before, during or after treatment with a modified mRNA encoding Protein C (its zymogen), APC or any variants thereof. Protein C, also known as autoprothrombin IIA and blood coagulation factor XIV is a zymogen, or precursor, of a serine protease which plays an important role in the regulation of blood coagulation and generation of fibrinolytic activity in vivo. It is synthesized in the liver as a single-chain polypeptide but undergoes posttranslational processing to give rise to a two-chain intermediate. The intermediate form of Protein C is converted via thrombin-mediated cleavage of a 12-residue peptide from the amino-terminus of the heavy chain to of the molecule to the active form, known as “activated protein C” (APC). The device may be useful in drug discovery efforts as a companion diagnostic test associated with Protein C, or APC treatment such as for sepsis or severe sepsis. In early studies it was suggested that APC had the ability to reduce mortality in severe sepsis. Following this line of work, clinical studies lead to the FDA approval of one compound, activated drotrecogin alfa (recombinant protein C). However, in late 2011, the drug was withdrawn from sale in all markets following results of the PROWESS-SHOCK study, which showed the study did not meet the primary endpoint of a statistically significant reduction in 28-day all-cause mortality in patients with septic shock. The present invention provides modified mRNA molecules which may be used in the diagnosis and treatment of sepsis, severe sepsis and septicemia which overcome prior issues or problems associated with increasing protein expression efficiencies in mammals.
In some embodiments the device is self-contained, and is optionally capable of wireless remote access to obtain instructions for synthesis and/or analysis of the generated polynucleotide, primary construct or mmRNA. The device is capable of mobile synthesis of at least one polynucleotide, primary construct or mmRNA and preferably an unlimited number of different polynucleotides, primary constructs or mmRNA. In certain embodiments, the device is capable of being transported by one or a small number of individuals. In other embodiments, the device is scaled to fit on a benchtop or desk. In other embodiments, the device is scaled to fit into a suitcase, backpack or similarly sized object. In another embodiment, the device may be a point of care or handheld device. In further embodiments, the device is scaled to fit into a vehicle, such as a car, truck or ambulance, or a military vehicle such as a tank or personnel carrier. The information necessary to generate a modified mRNA encoding polypeptide of interest is present within a computer readable medium present in the device.
In one embodiment, a device may be used to assess levels of a protein which has been administered in the form of a polynucleotide, primary construct or mmRNA. The device may comprise a blood, urine or other biofluidic test.
In some embodiments, the device is capable of communication (e.g., wireless communication) with a database of nucleic acid and polypeptide sequences. The device contains at least one sample block for insertion of one or more sample vessels. Such sample vessels are capable of accepting in liquid or other form any number of materials such as template DNA, nucleotides, enzymes, buffers, and other reagents. The sample vessels are also capable of being heated and cooled by contact with the sample block. The sample block is generally in communication with a device base with one or more electronic control units for the at least one sample block. The sample block preferably contains a heating module, such heating molecule capable of heating and/or cooling the sample vessels and contents thereof to temperatures between about −20 C and above +100 C. The device base is in communication with a voltage supply such as a battery or external voltage supply. The device also contains means for storing and distributing the materials for RNA synthesis.
Optionally, the sample block contains a module for separating the synthesized nucleic acids. Alternatively, the device contains a separation module operably linked to the sample block. Preferably the device contains a means for analysis of the synthesized nucleic acid. Such analysis includes sequence identity (demonstrated such as by hybridization), absence of non-desired sequences, measurement of integrity of synthesized mRNA (such has by microfluidic viscometry combined with spectrophotometry), and concentration and/or potency of modified RNA (such as by spectrophotometry).
In certain embodiments, the device is combined with a means for detection of pathogens present in a biological material obtained from a subject, e.g., the IBIS PLEX-ID system (Abbott, Abbott Park, Ill.) for microbial identification.
Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662; each of which is herein incorporated by reference in their entirety. Intradermal compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 (herein incorporated by reference in its entirety) and functional equivalents thereof. Jet injection devices which deliver liquid compositions to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Pat. Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97/37705 and WO 97/13537; each of which are herein incorporated by reference in their entirety. Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes may be used in the classical mantoux method of intradermal administration.
In some embodiments, the device may be a pump or comprise a catheter for administration of compounds or compositions of the invention across the blood brain barrier. Such devices include but are not limited to a pressurized olfactory delivery device, iontophoresis devices, multi-layered microfluidic devices, and the like. Such devices may be portable or stationary. They may be implantable or externally tethered to the body or combinations thereof.
Devices for administration may be employed to deliver the cosmetic polynucleotides, primary constructs or mmRNA of the present invention according to single, multi- or split-dosing regimens taught herein. Such devices are described below.
Method and devices known in the art for multi-administration to cells, organs and tissues are contemplated for use in conjunction with the methods and compositions disclosed herein as embodiments of the present invention. These include, for example, those methods and devices having multiple needles, hybrid devices employing for example lumens or catheters as well as devices utilizing heat, electric current or radiation driven mechanisms.
According to the present invention, these multi-administration devices may be utilized to deliver the single, multi- or split doses contemplated herein.
A method for delivering therapeutic agents to a solid tissue has been described by Bahrami et al. and is taught for example in US Patent Publication 20110230839, the contents of which are incorporated herein by reference in their entirety. According to Bahrami, an array of needles is incorporated into a device which delivers a substantially equal amount of fluid at any location in said solid tissue along each needle's length.
A device for delivery of biological material across the biological tissue has been described by Kodgule et al. and is taught for example in US Patent Publication 20110172610, the contents of which are incorporated herein by reference in their entirety. According to Kodgule, multiple hollow micro-needles made of one or more metals and having outer diameters from about 200 microns to about 350 microns and lengths of at least 100 microns are incorporated into the device which delivers peptides, proteins, carbohydrates, nucleic acid molecules, lipids and other pharmaceutically active ingredients or combinations thereof.
A delivery probe for delivering a therapeutic agent to a tissue has been described by Gunday et al. and is taught for example in US Patent Publication 20110270184, the contents of each of which are incorporated herein by reference in their entirety. According to Gunday, multiple needles are incorporated into the device which moves the attached capsules between an activated position and an inactivated position to force the agent out of the capsules through the needles.
A multiple-injection medical apparatus has been described by Assaf and is taught for example in US Patent Publication 20110218497, the contents of which are incorporated herein by reference in their entirety. According to Assaf, multiple needles are incorporated into the device which has a chamber connected to one or more of said needles and a means for continuously refilling the chamber with the medical fluid after each injection.
In one embodiment, the cosmetic polynucleotide, primary construct, or mmRNA is administered subcutaneously or intramuscularly via at least 3 needles to three different, optionally adjacent, sites simultaneously, or within a 60 minutes period (e.g., administration to 4, 5, 6, 7, 8, 9, or 10 sites simultaneously or within a 60 minute period). The split doses can be administered simultaneously to adjacent tissue using the devices described in U.S. Patent Publication Nos. 20110230839 and 20110218497, each of which is incorporated herein by reference in their entirety.
An at least partially implantable system for injecting a substance into a patient's body, in particular a penis erection stimulation system has been described by Forsell and is taught for example in US Patent Publication 20110196198, the contents of which are incorporated herein by reference in their entirety. According to Forsell, multiple needles are incorporated into the device which is implanted along with one or more housings adjacent the patient's left and right corpora cavernosa. A reservoir and a pump are also implanted to supply drugs through the needles.
A method for the transdermal delivery of a therapeutic effective amount of iron has been described by Berenson and is taught for example in US Patent Publication 20100130910, the contents of which are incorporated herein by reference in their entirety. According to Berenson, multiple needles may be used to create multiple micro channels in stratum corneum to enhance transdermal delivery of the ionic iron on an iontophoretic patch.
A method for delivery of biological material across the biological tissue has been described by Kodgule et al and is taught for example in US Patent Publication 20110196308, the contents of which are incorporated herein by reference in their entirety. According to Kodgule, multiple biodegradable microneedles containing a therapeutic active ingredient are incorporated in a device which delivers proteins, carbohydrates, nucleic acid molecules, lipids and other pharmaceutically active ingredients or combinations thereof.
A transdermal patch comprising a botulinum toxin composition has been described by Donovan and is taught for example in US Patent Publication 20080220020, the contents of which are incorporated herein by reference in their entirety. According to Donovan, multiple needles are incorporated into the patch which delivers botulinum toxin under stratum corneum through said needles which project through the stratum corneum of the skin without rupturing a blood vessel.
A small, disposable drug reservoir, or patch pump, which can hold approximately 0.2 to 15 mL of liquid formulations can be placed on the skin and deliver the formulation continuously subcutaneously using a small bore needed (e.g., 26 to 34 gauge). As non-limiting examples, the patch pump may be 50 mm by 76 mm by 20 mm spring loaded having a 30 to 34 gauge needle (BDTM Microinfuser, Franklin Lakes N.J.), 41 mm by 62 mm by 17 mm with a 2 mL reservoir used for drug delivery such as insulin (OMNIPOD®, Insulet Corporation Bedford, Mass.), or 43-60 mm diameter, 10 mm thick with a 0.5 to 10 mL reservoir (PATCHPUMP®, SteadyMed Therapeutics, San Francisco, Calif.). Further, the patch pump may be battery powered and/or rechargeable.
A cryoprobe for administration of an active agent to a location of cryogenic treatment has been described by Toubia and is taught for example in US Patent Publication 20080140061, the contents of which are incorporated herein by reference in their entirety. According to Toubia, multiple needles are incorporated into the probe which receives the active agent into a chamber and administers the agent to the tissue.
A method for treating or preventing inflammation or promoting healthy joints has been described by Stock et al and is taught for example in US Patent Publication 20090155186, the contents of which are incorporated herein by reference in their entirety. According to Stock, multiple needles are incorporated in a device which administers compositions containing signal transduction modulator compounds.
A multi-site injection system has been described by Kimmell et al. and is taught for example in US Patent Publication 20100256594, the contents of which are incorporated herein by reference in their entirety. According to Kimmell, multiple needles are incorporated into a device which delivers a medication into a stratum corneum through the needles.
A method for delivering interferons to the intradermal compartment has been described by Dekker et al. and is taught for example in US Patent Publication 20050181033, the contents of which are incorporated herein by reference in their entirety. According to Dekker, multiple needles having an outlet with an exposed height between 0 and 1 mm are incorporated into a device which improves pharmacokinetics and bioavailability by delivering the substance at a depth between 0.3 mm and 2 mm.
A method for delivering genes, enzymes and biological agents to tissue cells has described by Desai and is taught for example in US Patent Publication 20030073908, the contents of which are incorporated herein by reference in their entirety. According to Desai, multiple needles are incorporated into a device which is inserted into a body and delivers a medication fluid through said needles.
A method for treating cardiac arrhythmias with fibroblast cells has been described by Lee et al and is taught for example in US Patent Publication 20040005295, the contents of which are incorporated herein by reference in their entirety. According to Lee, multiple needles are incorporated into the device which delivers fibroblast cells into the local region of the tissue.
A method using a magnetically controlled pump for treating a brain tumor has been described by Shachar et al. and is taught for example in U.S. Pat. No. 7,799,012 (method) and U.S. Pat. No. 7,799,016 (device), the contents of which are incorporated herein by reference in their entirety. According Shachar, multiple needles were incorporated into the pump which pushes a medicating agent through the needles at a controlled rate.
Methods of treating functional disorders of the bladder in mammalian females have been described by Versi et al. and are taught for example in U.S. Pat. No. 8,029,496, the contents of which are incorporated herein by reference in their entirety. According to Versi, an array of micro-needles is incorporated into a device which delivers a therapeutic agent through the needles directly into the trigone of the bladder.
A micro-needle transdermal transport device has been described by Angel et al and is taught for example in U.S. Pat. No. 7,364,568, the contents of which are incorporated herein by reference in their entirety. According to Angel, multiple needles are incorporated into the device which transports a substance into a body surface through the needles which are inserted into the surface from different directions. The micro-needle transdermal transport device may be a solid micro-needle system or a hollow micro-needle system. As a non-limiting example, the solid micro-needle system may have up to a 0.5 mg capacity, with 300-1500 solid micro-needles per cm2 about 150-700 μm tall coated with a drug. The micro-needles penetrate the stratum corneum and remain in the skin for short duration (e.g., 20 seconds to 15 minutes). In another example, the hollow micro-needle system has up to a 3 mL capacity to deliver liquid formulations using 15-20 microneedles per cm2 being approximately 950 μm tall. The micro-needles penetrate the skin to allow the liquid formulations to flow from the device into the skin. The hollow micro-needle system may be worn from 1 to 30 minutes depending on the formulation volume and viscosity.
A device for subcutaneous infusion has been described by Dalton et al and is taught for example in U.S. Pat. No. 7,150,726, the contents of which are incorporated herein by reference in their entirety. According to Dalton, multiple needles are incorporated into the device which delivers fluid through the needles into a subcutaneous tissue.
A device and a method for intradermal delivery of vaccines and gene therapeutic agents through microcannula have been described by Mikszta et al. and are taught for example in U.S. Pat. No. 7,473,247, the contents of which are incorporated herein by reference in their entirety. According to Mitszta, at least one hollow micro-needle is incorporated into the device which delivers the vaccines to the subject's skin to a depth of between 0.025 mm and 2 mm.
A method of delivering insulin has been described by Pettis et al and is taught for example in U.S. Pat. No. 7,722,595, the contents of which are incorporated herein by reference in their entirety. According to Pettis, two needles are incorporated into a device wherein both needles insert essentially simultaneously into the skin with the first at a depth of less than 2.5 mm to deliver insulin to intradermal compartment and the second at a depth of greater than 2.5 mm and less than 5.0 mm to deliver insulin to subcutaneous compartment.
Cutaneous injection delivery under suction has been described by Kochamba et al. and is taught for example in U.S. Pat. No. 6,896,666, the contents of which are incorporated herein by reference in their entirety. According to Kochamba, multiple needles in relative adjacency with each other are incorporated into a device which injects a fluid below the cutaneous layer.
A device for withdrawing or delivering a substance through the skin has been described by Down et al and is taught for example in U.S. Pat. No. 6,607,513, the contents of which are incorporated herein by reference in their entirety. According to Down, multiple skin penetrating members which are incorporated into the device have lengths of about 100 microns to about 2000 microns and are about 30 to 50 gauge.
A device for delivering a substance to the skin has been described by Palmer et al and is taught for example in U.S. Pat. No. 6,537,242, the contents of which are incorporated herein by reference in their entirety. According to Palmer, an array of micro-needles is incorporated into the device which uses a stretching assembly to enhance the contact of the needles with the skin and provides a more uniform delivery of the substance.
A perfusion device for localized drug delivery has been described by Zamoyski and is taught for example in U.S. Pat. No. 6,468,247, the contents of which are incorporated herein by reference in their entirety. According to Zamoyski, multiple hypodermic needles are incorporated into the device which injects the contents of the hypodermics into a tissue as said hypodermics are being retracted.
A method for enhanced transport of drugs and biological molecules across tissue by improving the interaction between micro-needles and human skin has been described by Prausnitz et al. and is taught for example in U.S. Pat. No. 6,743,211, the contents of which are incorporated herein by reference in their entirety. According to Prausnitz, multiple micro-needles are incorporated into a device which is able to present a more rigid and less deformable surface to which the micro-needles are applied.
A device for intraorgan administration of medicinal agents has been described by Ting et al and is taught for example in U.S. Pat. No. 6,077,251, the contents of which are incorporated herein by reference in their entirety. According to Ting, multiple needles having side openings for enhanced administration are incorporated into a device which by extending and retracting said needles from and into the needle chamber forces a medicinal agent from a reservoir into said needles and injects said medicinal agent into a target organ.
A multiple needle holder and a subcutaneous multiple channel infusion port has been described by Brown and is taught for example in U.S. Pat. No. 4,695,273, the contents of which are incorporated herein by reference in their entirety. According to Brown, multiple needles on the needle holder are inserted through the septum of the infusion port and communicate with isolated chambers in said infusion port.
A dual hypodermic syringe has been described by Horn and is taught for example in U.S. Pat. No. 3,552,394, the contents of which are incorporated herein by reference in their entirety. According to Horn, two needles incorporated into the device are spaced apart less than 68 mm and may be of different styles and lengths, thus enabling injections to be made to different depths.
A syringe with multiple needles and multiple fluid compartments has been described by Hershberg and is taught for example in U.S. Pat. No. 3,572,336, the contents of which are incorporated herein by reference in their entirety. According to Hershberg, multiple needles are incorporated into the syringe which has multiple fluid compartments and is capable of simultaneously administering incompatible drugs which are not able to be mixed for one injection.
A surgical instrument for intradermal injection of fluids has been described by Eliscu et al. and is taught for example in U.S. Pat. No. 2,588,623, the contents of which are incorporated herein by reference in their entirety. According to Eliscu, multiple needles are incorporated into the instrument which injects fluids intradermally with a wider disperse.
An apparatus for simultaneous delivery of a substance to multiple breast milk ducts has been described by Hung and is taught for example in EP 1818017, the contents of which are incorporated herein by reference in their entirety. According to Hung, multiple lumens are incorporated into the device which inserts though the orifices of the ductal networks and delivers a fluid to the ductal networks.
A catheter for introduction of medications to the tissue of a heart or other organs has been described by Tkebuchava and is taught for example in WO2006138109, the contents of which are incorporated herein by reference in their entirety. According to Tkebuchava, two curved needles are incorporated which enter the organ wall in a flattened trajectory.
Devices for delivering medical agents have been described by Mckay et al. and are taught for example in WO2006118804, the content of which are incorporated herein by reference in their entirety. According to Mckay, multiple needles with multiple orifices on each needle are incorporated into the devices to facilitate regional delivery to a tissue, such as the interior disc space of a spinal disc.
A method for directly delivering an immunomodulatory substance into an intradermal space within a mammalian skin has been described by Pettis and is taught for example in WO2004020014, the contents of which are incorporated herein by reference in their entirety. According to Pettis, multiple needles are incorporated into a device which delivers the substance through the needles to a depth between 0.3 mm and 2 mm.
Methods and devices for administration of substances into at least two compartments in skin for systemic absorption and improved pharmacokinetics have been described by Pettis et al. and are taught for example in WO2003094995, the contents of which are incorporated herein by reference in their entirety. According to Pettis, multiple needles having lengths between about 300 μm and about 5 mm are incorporated into a device which delivers to intradermal and subcutaneous tissue compartments simultaneously.
A drug delivery device with needles and a roller has been described by Zimmerman et al. and is taught for example in WO2012006259, the contents of which are incorporated herein by reference in their entirety. According to Zimmerman, multiple hollow needles positioned in a roller are incorporated into the device which delivers the content in a reservoir through the needles as the roller rotates.
A drug delivery device such as a stent is known in the art and is taught for example in U.S. Pat. No. 8,333,799, U.S. Pub. Nos. US20060020329, US20040172127 and US20100161032; the contents of each of which are herein incorporated by reference in their entirety. Formulations of the polynucleotides, primary constructs, mmRNA described herein may be delivered using stents. Additionally, stents used herein may be able to deliver multiple polynucleotides, primary constructs and/or mmRNA and/or formulations at the same or varied rates of delivery. Non-limiting examples of manufacturers of stents include CORDIS® (Miami, Fla.) (CYPHER®), Boston Scientific Corporation (Natick, Mass.) (TAXUS®), Medtronic (Minneapolis, Minn.) (ENDEAVOUR®) and Abbott (Abbott Park, Ill.) (XIENCE V®).
Methods and Devices Utilizing Catheters and/or Lumens
Methods and devices using catheters and lumens may be employed to administer the mmRNA of the present invention on a single, multi- or split dosing schedule. Such methods and devices are described below.
A catheter-based delivery of skeletal myoblasts to the myocardium of damaged hearts has been described by Jacoby et al and is taught for example in US Patent Publication 20060263338, the contents of which are incorporated herein by reference in their entirety. According to Jacoby, multiple needles are incorporated into the device at least part of which is inserted into a blood vessel and delivers the cell composition through the needles into the localized region of the subject's heart.
An apparatus for treating asthma using neurotoxin has been described by Deem et al and is taught for example in US Patent Publication 20060225742, the contents of which are incorporated herein by reference in their entirety. According to Deem, multiple needles are incorporated into the device which delivers neurotoxin through the needles into the bronchial tissue.
A method for administering multiple-component therapies has been described by Nayak and is taught for example in U.S. Pat. No. 7,699,803, the contents of which are incorporated herein by reference in their entirety. According to Nayak, multiple injection cannulas may be incorporated into a device wherein depth slots may be included for controlling the depth at which the therapeutic substance is delivered within the tissue.
A surgical device for ablating a channel and delivering at least one therapeutic agent into a desired region of the tissue has been described by McIntyre et al and is taught for example in U.S. Pat. No. 8,012,096, the contents of which are incorporated herein by reference in their entirety. According to McIntyre, multiple needles are incorporated into the device which dispenses a therapeutic agent into a region of tissue surrounding the channel and is particularly well suited for transmyocardial revascularization operations.
Methods of treating functional disorders of the bladder in mammalian females have been described by Versi et al and are taught for example in U.S. Pat. No. 8,029,496, the contents of which are incorporated herein by reference in their entirety. According to Versi, an array of micro-needles is incorporated into a device which delivers a therapeutic agent through the needles directly into the trigone of the bladder.
A device and a method for delivering fluid into a flexible biological barrier have been described by Yeshurun et al. and are taught for example in U.S. Pat. No. 7,998,119 (device) and U.S. Pat. No. 8,007,466 (method), the contents of which are incorporated herein by reference in their entirety. According to Yeshurun, the micro-needles on the device penetrate and extend into the flexible biological barrier and fluid is injected through the bore of the hollow micro-needles.
A method for epicardially injecting a substance into an area of tissue of a heart having an epicardial surface and disposed within a torso has been described by Bonner et al and is taught for example in U.S. Pat. No. 7,628,780, the contents of which are incorporated herein by reference in their entirety. According to Bonner, the devices have elongate shafts and distal injection heads for driving needles into tissue and injecting medical agents into the tissue through the needles.
A device for sealing a puncture has been described by Nielsen et al and is taught for example in U.S. Pat. No. 7,972,358, the contents of which are incorporated herein by reference in their entirety. According to Nielsen, multiple needles are incorporated into the device which delivers a closure agent into the tissue surrounding the puncture tract.
A method for myogenesis and angiogenesis has been described by Chiu et al. and is taught for example in U.S. Pat. No. 6,551,338, the contents of which are incorporated herein by reference in their entirety. According to Chiu, 5 to 15 needles having a maximum diameter of at least 1.25 mm and a length effective to provide a puncture depth of 6 to 20 mm are incorporated into a device which inserts into proximity with a myocardium and supplies an exogeneous angiogenic or myogenic factor to said myocardium through the conduits which are in at least some of said needles.
A method for the treatment of prostate tissue has been described by Bolmsj et al. and is taught for example in U.S. Pat. No. 6,524,270, the contents of which are incorporated herein by reference in their entirety. According to Bolmsj, a device comprising a catheter which is inserted through the urethra has at least one hollow tip extendible into the surrounding prostate tissue. An astringent and analgesic medicine is administered through said tip into said prostate tissue.
A method for infusing fluids to an intraosseous site has been described by Findlay et al. and is taught for example in U.S. Pat. No. 6,761,726, the contents of which are incorporated herein by reference in their entirety. According to Findlay, multiple needles are incorporated into a device which is capable of penetrating a hard shell of material covered by a layer of soft material and delivers a fluid at a predetermined distance below said hard shell of material.
A device for injecting medications into a vessel wall has been described by Vigil et al. and is taught for example in U.S. Pat. No. 5,713,863, the contents of which are incorporated herein by reference in their entirety. According to Vigil, multiple injectors are mounted on each of the flexible tubes in the device which introduces a medication fluid through a multi-lumen catheter, into said flexible tubes and out of said injectors for infusion into the vessel wall.
A catheter for delivering therapeutic and/or diagnostic agents to the tissue surrounding a bodily passageway has been described by Faxon et al. and is taught for example in U.S. Pat. No. 5,464,395, the contents of which are incorporated herein by reference in their entirety. According to Faxon, at least one needle cannula is incorporated into the catheter which delivers the desired agents to the tissue through said needles which project outboard of the catheter.
Balloon catheters for delivering therapeutic agents have been described by Orr and are taught for example in WO2010024871, the contents of which are incorporated herein by reference in their entirety. According to Orr, multiple needles are incorporated into the devices which deliver the therapeutic agents to different depths within the tissue. In another aspect, drug-eluting balloons may be used to deliver the formulations described herein. The drug-eluting balloons may be used in target lesion applications such as, but are not limited to, in-stent restenosis, treating lesion in tortuous vessels, bifurcation lesions, femoral/popliteal lesions and below the knee lesions.
A device for delivering therapeutic agents (e.g., cosmetic polynucleotides, primary constructs or mmRNA) to tissue disposed about a lumin has been described by Perry et al. and is taught for example in U.S. Pat. Pub. US20100125239, the contents of which are herein incorporated by reference in their entirety. According to Perry, the catheter has a balloon which may be coated with a therapeutic agent by methods known in the art and described in Perry. When the balloon expands, the therapeutic agent will contact the surrounding tissue. The device may additionally have a heat source to change the temperature of the coating on the balloon to release the therapeutic agent to the tissue.
Methods and Devices Utilizing Electrical Current
Methods and devices utilizing electric current may be employed to deliver the mmRNA of the present invention according to the single, multi- or split dosing regimens taught herein. Such methods and devices are described below.
An electro collagen induction therapy device has been described by Marquez and is taught for example in US Patent Publication 20090137945, the contents of which are incorporated herein by reference in their entirety. According to Marquez, multiple needles are incorporated into the device which repeatedly pierce the skin and draw in the skin a portion of the substance which is applied to the skin first.
An electrokinetic system has been described by Etheredge et al. and is taught for example in US Patent Publication 20070185432, the contents of which are incorporated herein by reference in their entirety. According to Etheredge, micro-needles are incorporated into a device which drives by an electrical current the medication through the needles into the targeted treatment site.
An iontophoresis device has been described by Matsumura et al. and is taught for example in U.S. Pat. No. 7,437,189, the contents of which are incorporated herein by reference in their entirety. According to Matsumura, multiple needles are incorporated into the device which is capable of delivering ionizable drug into a living body at higher speed or with higher efficiency.
Intradermal delivery of biologically active agents by needle-free injection and electroporation has been described by Hoffmann et al and is taught for example in U.S. Pat. No. 7,171,264, the contents of which are incorporated herein by reference in their entirety. According to Hoffmann, one or more needle-free injectors are incorporated into an electroporation device and the combination of needle-free injection and electroporation is sufficient to introduce the agent into cells in skin, muscle or mucosa.
A method for electropermeabilization-mediated intracellular delivery has been described by Lundkvist et al. and is taught for example in U.S. Pat. No. 6,625,486, the contents of which are incorporated herein by reference in their entirety. According to Lundkvist, a pair of needle electrodes is incorporated into a catheter. Said catheter is positioned into a body lumen followed by extending said needle electrodes to penetrate into the tissue surrounding said lumen. Then the device introduces an agent through at least one of said needle electrodes and applies electric field by said pair of needle electrodes to allow said agent pass through the cell membranes into the cells at the treatment site.
A delivery system for transdermal immunization has been described by Levin et al. and is taught for example in WO2006003659, the contents of which are incorporated herein by reference in their entirety. According to Levin, multiple electrodes are incorporated into the device which applies electrical energy between the electrodes to generate micro channels in the skin to facilitate transdermal delivery.
A method for delivering RF energy into skin has been described by Schomacker and is taught for example in WO2011163264, the contents of which are incorporated herein by reference in their entirety. According to Schomacker, multiple needles are incorporated into a device which applies vacuum to draw skin into contact with a plate so that needles insert into skin through the holes on the plate and deliver RF energy.
VII. Definitions
At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “C1-6 alkyl” is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.
About: As used herein, the term “about” means+/−10% of the recited value.
Administered in combination: As used herein, the term “administered in combination” or “combined administration” means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.
Aesthetic presentation: As used herein, the phrase “aesthetic presentation” refers to an improved appearance relative to a previous appearance. For example, a change in aesthetic presentation may include the concealment of skin discoloration, scarring, and/or stretch marks.
Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically-engineered animal, or a clone.
Antigens of interest or desired antigens: As used herein, the terms “antigens of interest” or “desired antigens” include those proteins and other biomolecules provided herein that are immunospecifically bound by the antibodies and fragments, mutants, variants, and alterations thereof described herein. Examples of antigens of interest include, but are not limited to, insulin, insulin-like growth factor, hGH, tPA, cytokines, such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF beta, TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.
Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
Associated with: As used herein, the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the “associated” entities remain physically associated.
Bifunctional: As used herein, the term “bifunctional” refers to any substance, molecule or moiety which is capable of or maintains at least two functions. The functions may effect the same outcome or a different outcome. The structure that produces the function may be the same or different. For example, bifunctional modified RNAs of the present invention may encode a cytotoxic peptide (a first function) while those nucleosides which comprise the encoding RNA are, in and of themselves, cytotoxic (second function). In this example, delivery of the bifunctional modified RNA to a cancer cell would produce not only a peptide or protein molecule which may ameliorate or treat the cancer but would also deliver a cytotoxic payload of nucleosides to the cell should degradation, instead of translation of the modified RNA, occur.
Biocompatible: As used herein, the term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.
Biodegradable: As used herein, the term “biodegradable” means capable of being broken down into innocuous products by the action of living things.
Biologically active: As used herein, the phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, a cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA of the present invention may be considered biologically active if even a portion of the cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA is biologically active or mimics an activity considered biologically relevant.
Chemical terms: The following provides the definition of various chemical terms from “acyl” to “thiol.”
The term “acyl,” as used herein, represents a hydrogen or an alkyl group (e.g., a haloalkyl group), as defined herein, that is attached to the parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl (i.e., a carboxyaldehyde group), acetyl, propionyl, butanoyl and the like. Exemplary unsubstituted acyl groups include from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents as described herein.
The term “acylamino,” as used herein, represents an acyl group, as defined herein, attached to the parent molecular group though an amino group, as defined herein (i.e., —N(RN1)—C(O)—R, where R is H or an optionally substituted C1-6, C1-10, or C1-20 alkyl group and RN1 is as defined herein). Exemplary unsubstituted acylamino groups include from 1 to 41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to 21, from 2 to 7, from 2 to 13, from 2 to 21, or from 2 to 41 carbons). In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents as described herein, and/or the amino group is —NH2 or —NHRN1, wherein RN1 is, independently, OH, NO2, NH2, NRN22, SO2ORN2, SO2RN2, SORN2, alkyl, or aryl, and each RN2 can be H, alkyl, or aryl.
The term “acyloxy,” as used herein, represents an acyl group, as defined herein, attached to the parent molecular group though an oxygen atom (i.e., —O—C(O)—R, where R is H or an optionally substituted C1-6, C1-10, or C1-20 alkyl group). Exemplary unsubstituted acyloxy groups include from 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11 carbons). In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents as described herein, and/or the amino group is —NH2 or —NHRN1, wherein RN1 is, independently, OH, NO2, NH2, NRN22, SO2ORN2, SO2RN2, SORN2, alkyl, or aryl, and each RN2 can be H, alkyl, or aryl.
The term “alkaryl,” as used herein, represents an aryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. Exemplary unsubstituted alkaryl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C1-6 alk-C6-10 aryl, C1-10 alk-C6-10 aryl, or C1-20 alk-C6-10 aryl). In some embodiments, the alkylene and the aryl each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective groups. Other groups preceded by the prefix “alk-” are defined in the same manner, where “alk” refers to a C1-6 alkylene, unless otherwise noted, and the attached chemical structure is as defined herein.
The term “alkcycloalkyl” represents a cycloalkyl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein (e.g., an alkylene group of from 1 to 4, from 1 to 6, from 1 to 10, or form 1 to 20 carbons). In some embodiments, the alkylene and the cycloalkyl each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective group.
The term “alkenyl,” as used herein, represents monovalent straight or branched chain groups of, unless otherwise specified, from 2 to 20 carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one or more carbon-carbon double bonds and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyls include both cis and trans isomers. Alkenyl groups may be optionally substituted with 1, 2, 3, or 4 substituent groups that are selected, independently, from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituent groups described herein.
The term “alkenyloxy” represents a chemical substituent of formula —OR, where R is a C2-20 alkenyl group (e.g., C2-6 or C2-10 alkenyl), unless otherwise specified. Exemplary alkenyloxy groups include ethenyloxy, propenyloxy, and the like. In some embodiments, the alkenyl group can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein (e.g., a hydroxy group).
The term “alkheteroaryl” refers to a heteroaryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. Exemplary unsubstituted alkheteroaryl groups are from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to 12 carbons, such as C1-6 alk-C1-12 heteroaryl, C1-10 alk-C1-12 heteroaryl, or C1-20 alk-C1-12 heteroaryl). In some embodiments, the alkylene and the heteroaryl each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective group. Alkheteroaryl groups are a subset of alkheterocyclyl groups.
The term “alkheterocyclyl” represents a heterocyclyl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. Exemplary unsubstituted alkheterocyclyl groups are from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to 12 carbons, such as C1-6 alk-C1-12 heterocyclyl, C1-10 alk-C1-12 heterocyclyl, or C1-20 alk-C1-12 heterocyclyl). In some embodiments, the alkylene and the heterocyclyl each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective group.
The term “alkoxy” represents a chemical substituent of formula —OR, where R is a C1-20 alkyl group (e.g., C1-6 or C1-10 alkyl), unless otherwise specified. Exemplary alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein (e.g., hydroxy or alkoxy).
The term “alkoxyalkoxy” represents an alkoxy group that is substituted with an alkoxy group. Exemplary unsubstituted alkoxyalkoxy groups include between 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20 carbons, such as C1-6 alkoxy-C1-6 alkoxy, C1-10 alkoxy-C1-10 alkoxy, or C1-20 alkoxy-C1-20 alkoxy). In some embodiments, the each alkoxy group can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein.
The term “alkoxyalkyl” represents an alkyl group that is substituted with an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups include between 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20 carbons, such as C1-6 alkoxy-C1-6 alkyl, C1-10 alkoxy-C1-10 alkyl, or C1-20 alkoxy-C1-20 alkyl). In some embodiments, the alkyl and the alkoxy each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective group.
The term “alkoxycarbonyl,” as used herein, represents an alkoxy, as defined herein, attached to the parent molecular group through a carbonyl atom (e.g., —C(O)—OR, where R is H or an optionally substituted C1-6, C1-10, or C1-20 alkyl group). Exemplary unsubstituted alkoxycarbonyl include from 1 to 21 carbons (e.g., from 1 to 11 or from 1 to 7 carbons). In some embodiments, the alkoxy group is further substituted with 1, 2, 3, or 4 substituents as described herein.
The term “alkoxycarbonylalkoxy,” as used herein, represents an alkoxy group, as defined herein, that is substituted with an alkoxycarbonyl group, as defined herein (e.g., —O-alkyl-C(O)—OR, where R is an optionally substituted C1-6, C1-10, or C1-20 alkyl group). Exemplary unsubstituted alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g., from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31 carbons, such as C1-6 alkoxycarbonyl-C1-6 alkoxy, C1-10 alkoxycarbonyl-C1-10 alkoxy, or C1-20 alkoxycarbonyl-C1-20 alkoxy). In some embodiments, each alkoxy group is further independently substituted with 1, 2, 3, or 4 substituents, as described herein (e.g., a hydroxy group).
The term “alkoxycarbonylalkyl,” as used herein, represents an alkyl group, as defined herein, that is substituted with an alkoxycarbonyl group, as defined herein (e.g., -alkyl-C(O)—OR, where R is an optionally substituted C1-20, C1-10, or C1-6 alkyl group). Exemplary unsubstituted alkoxycarbonylalkyl include from 3 to 41 carbons (e.g., from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31 carbons, such as C1-6 alkoxycarbonyl-C1-6 alkyl, C1-10 alkoxycarbonyl-C1-10 alkyl, or C1-20 alkoxycarbonyl-C1-20 alkyl). In some embodiments, each alkyl and alkoxy group is further independently substituted with 1, 2, 3, or 4 substituents as described herein (e.g., a hydroxy group).
The term “alkyl,” as used herein, is inclusive of both straight chain and branched chain saturated groups from 1 to 20 carbons (e.g., from 1 to 10 or from 1 to 6), unless otherwise specified. Alkyl groups are exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- and tert-butyl, neopentyl, and the like, and may be optionally substituted with one, two, three, or, in the case of alkyl groups of two carbons or more, four substituents independently selected from the group consisting of: (1) C1-6 alkoxy; (2) C1-6 alkylsulfinyl; (3) amino, as defined herein (e.g., unsubstituted amino (i.e., —NH2) or a substituted amino (i.e., —N(RN1)2, where RN1 is as defined for amino); (4) C6-10 aryl-C1-6 alkoxy; (5) azido; (6) halo; (7) (C2-9heterocyclyl)oxy; (8) hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C1-7 spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO2RA′, where RA′ is selected from the group consisting of (a) C1-20 alkyl (e.g., C1-6 alkyl), (b) C2-20 alkenyl (e.g., C2-6 alkenyl), (c) C6-10 aryl, (d) hydrogen, (e) C1-6 alk-C6-10 aryl, (f) amino-C1-20 alkyl, (g) polyethylene glycol of —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, and (h) amino-polyethylene glycol of —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl; (15) —C(O)NRB′RC′, where each of RB′ and RC′ is, independently, selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl, and (d) C1-6 alk-C6-10 aryl; (16) —SO2RD′, where RD′ is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, (c) C1-6 alk-C6-10 aryl, and (d) hydroxy; (17) —SO2NRE′RF′, where each of RE′ and RF′ is, independently, selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl and (d) C1-6 alk-C6-10 aryl; (18) —C(O)RG′, where RG′ is selected from the group consisting of (a) C1-20 alkyl (e.g., C1-6 alkyl), (b) C2-20 alkenyl (e.g., C2-6 alkenyl), (c) C6-10 aryl, (d) hydrogen, (e) C1-6 alk-C6-10 aryl, (f) amino-C1-20 alkyl, (g) polyethylene glycol of —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, and (h) amino-polyethylene glycol of —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl; (19) —NRH′C(O)RI′, wherein RH′ is selected from the group consisting of (a1) hydrogen and (b1) C1-6 alkyl, and RI′ is selected from the group consisting of (a2) C1-20 alkyl (e.g., C1-6 alkyl), (b2) C2-20 alkenyl (e.g., C2-6 alkenyl), (c2) C6-10 aryl, (d2) hydrogen, (e2) C1-6 alk-C6-10 aryl, (f2) amino-C1-20 alkyl, (g2) polyethylene glycol of —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, and (h2) amino-polyethylene glycol of —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl; (20) —NRJ′C(O)ORK′, wherein RJ′ is selected from the group consisting of (a1) hydrogen and (b1) C1-6 alkyl, and RK′ is selected from the group consisting of (a2) C1-20 alkyl (e.g., C1-6 alkyl), (b2) C2-20 alkenyl (e.g., C2-6 alkenyl), (c2) C6-10 aryl, (d2) hydrogen, (e2) C1-6 alk-C6-10 aryl, (f2) amino-C1-20 alkyl, (g2) polyethylene glycol of —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, and (h2) amino-polyethylene glycol of —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl; and (21) amidine. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl can be further substituted with an oxo group to afford the respective aryloyl substituent.
The term “alkylene” and the prefix “alk-,” as used herein, represent a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene, and the like. The term “Cx-y alkylene” and the prefix “Cx-y alk-” represent alkylene groups having between x and y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., C1-6, C1-10, C2-20, C2-6, C2-10, or C2-20 alkylene). In some embodiments, the alkylene can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for an alkyl group.
The term “alkylsulfinyl,” as used herein, represents an alkyl group attached to the parent molecular group through an —S(O)— group. Exemplary unsubstituted alkylsulfinyl groups are from 1 to 6, from 1 to 10, or from 1 to 20 carbons. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein.
The term “alkylsulfinylalkyl,” as used herein, represents an alkyl group, as defined herein, substituted by an alkylsulfinyl group. Exemplary unsubstituted alkylsulfinylalkyl groups are from 2 to 12, from 2 to 20, or from 2 to 40 carbons. In some embodiments, each alkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein.
The term “alkynyl,” as used herein, represents monovalent straight or branched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from 2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bond and is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groups may be optionally substituted with 1, 2, 3, or 4 substituent groups that are selected, independently, from aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituent groups described herein.
The term “alkynyloxy” represents a chemical substituent of formula —OR, where R is a C2-20 alkynyl group (e.g., C2-6 or C2-10 alkynyl), unless otherwise specified. Exemplary alkynyloxy groups include ethynyloxy, propynyloxy, and the like. In some embodiments, the alkynyl group can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein (e.g., a hydroxy group).
The term “amidine,” as used herein, represents a —C(═NH)NH2 group.
The term “amino,” as used herein, represents —N(RN1)2, wherein each RN1 is, independently, H, OH, NO2, N(RN2)2, SO2ORN2, SO2RN2, SORN2, an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl, sulfoalkyl, heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), wherein each of these recited RN1 groups can be optionally substituted, as defined herein for each group; or two RN1 combine to form a heterocyclyl or an N-protecting group, and wherein each RN2 is, independently, H, alkyl, or aryl. The amino groups of the invention can be an unsubstituted amino (i.e., —NH2) or a substituted amino (i.e., —N(RN1)2). In a preferred embodiment, amino is —NH2 or —NHRN1, wherein RN1 is, independently, OH, NO2, NH2, NRN22, SO2ORN2, SO2RN2, SORN2, alkyl, carboxyalkyl, sulfoalkyl, or aryl, and each RN2 can be H, C1-20 alkyl (e.g., C1-6 alkyl), or C6-10 aryl.
The term “amino acid,” as described herein, refers to a molecule having a side chain, an amino group, and an acid group (e.g., a carboxy group of —CO2H or a sulfo group of —SO3H), wherein the amino acid is attached to the parent molecular group by the side chain, amino group, or acid group (e.g., the side chain). In some embodiments, the amino acid is attached to the parent molecular group by a carbonyl group, where the side chain or amino group is attached to the carbonyl group. Exemplary side chains include an optionally substituted alkyl, aryl, heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groups may be optionally substituted with one, two, three, or, in the case of amino acid groups of two carbons or more, four substituents independently selected from the group consisting of: (1) C1-6 alkoxy; (2) C1-6 alkylsulfinyl; (3) amino, as defined herein (e.g., unsubstituted amino (i.e., —NH2) or a substituted amino (i.e., —N(RN1)2, where RN1 is as defined for amino); (4) C6-10 aryl-C1-6 alkoxy; (5) azido; (6) halo; (7) (C2-9 heterocyclyl)oxy; (8) hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C1-7 spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO2RA′, where RA′ is selected from the group consisting of (a) C1-20 alkyl (e.g., C1-6 alkyl), (b) C2-20 alkenyl (e.g., C2-6 alkenyl), (c) C6-10 aryl, (d) hydrogen, (e) C1-6 alk-C6-10 aryl, (f) amino-C1-20 alkyl, (g) polyethylene glycol of —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, and (h) amino-polyethylene glycol of —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl; (15) —C(O)NRB′RC′, where each of RB′ and RC′ is, independently, selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl, and (d) C1-6 alk-C6-10 aryl; (16) —SO2RD′, where RD′ is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, (c) C1-6 alk-C6-10 aryl, and (d) hydroxy; (17) —SO2NRE′RF′, where each of RE′ and RF′ is, independently, selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl and (d) C1-6 alk-C6-10 aryl; (18) —C(O)RG′, where RG′ is selected from the group consisting of (a) C1-20 alkyl (e.g., C1-6 alkyl), (b) C2-20 alkenyl (e.g., C2-6 alkenyl), (c) C6-10 aryl, (d) hydrogen, (e) C1-6 alk-C6-10 aryl, (f) amino-C1-20 alkyl, (g) polyethylene glycol of —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, and (h) amino-polyethylene glycol of —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl; (19) —NRH′C(O)RI′, wherein RH′ is selected from the group consisting of (a1) hydrogen and (b1) C1-6 alkyl, and RI′ is selected from the group consisting of (a2) C1-20 alkyl (e.g., C1-6 alkyl), (b2) C2-20 alkenyl (e.g., C2-6 alkenyl), (c2) C6-10 aryl, (d2) hydrogen, (e2) C1-6 alk-C6-10 aryl, (f2) amino-C1-20 alkyl, (g2) polyethylene glycol of —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, and (h2) amino-polyethylene glycol of —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl; (20) —NRJ′C(O)ORK′, wherein RJ′ is selected from the group consisting of (a1) hydrogen and (b1) C1-6 alkyl, and RK′ is selected from the group consisting of (a2) C1-20 alkyl (e.g., C1-6 alkyl), (b2) C2-20 alkenyl (e.g., C2-6 alkenyl), (c2) C6-10 aryl, (d2) hydrogen, (e2) C1-6 alk-C6-10 aryl, (f2) amino-C1-20 alkyl, (g2) polyethylene glycol of —(CH2)s2(OCH2CH2)s1(CH2)s3OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C1-20 alkyl, and (h2) amino-polyethylene glycol of —NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl; and (21) amidine. In some embodiments, each of these groups can be further substituted as described herein.
The term “aminoalkoxy,” as used herein, represents an alkoxy group, as defined herein, substituted by an amino group, as defined herein. The alkyl and amino each can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for the respective group (e.g., CO2RA′, where RA′ is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, (c) hydrogen, and (d) C1-6 alk-C6-10 aryl, e.g., carboxy).
The term “aminoalkyl,” as used herein, represents an alkyl group, as defined herein, substituted by an amino group, as defined herein. The alkyl and amino each can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for the respective group (e.g., CO2RA′, where RA′ is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, (c) hydrogen, and (d) C1-6 alk-C6-10 aryl, e.g., carboxy).
The term “aryl,” as used herein, represents a mono-, bicyclic, or multicyclic carbocyclic ring system having one or two aromatic rings and is exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl, indanyl, indenyl, and the like, and may be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of: (1) C1-7 acyl (e.g., carboxyaldehyde); (2) C1-20 alkyl (e.g., C1-6 alkyl, C1-6 alkoxy-C1-6 alkyl, C1-6 alkylsulfinyl-C1-6 alkyl, amino-C1-6 alkyl, azido-C1-6 alkyl, (carboxyaldehyde)-C1-6 alkyl, halo-C1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C1-6 alkyl, nitro-C1-6 alkyl, or C1-6 thioalkoxy-C1-6 alkyl); (3) C1-20 alkoxy (e.g., C1-6 alkoxy, such as perfluoroalkoxy); (4) C1-6 alkylsulfinyl; (5) C6-10 aryl; (6) amino; (7) C1-6 alk-C6-10 aryl; (8) azido; (9) C3-8 cycloalkyl; (10) C1-6 alk-C3-8 cycloalkyl; (11) halo; (12) C1-12 heterocyclyl (e.g., C1-12 heteroaryl); (13) (C1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C1-20 thioalkoxy (e.g., C1-6 thioalkoxy); (17) —(CH2)—CO2RA′, where q is an integer from zero to four, and RA′ is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, (c) hydrogen, and (d) C1-6 alk-C6-10 aryl; (18) —(CH2)qCONRB′RC′, where q is an integer from zero to four and where RB′ and RC′ are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl, and (d) C1-6 alk-C6-10 aryl; (19) —(CH2)—SO2RD′, where q is an integer from zero to four and where RD′ is selected from the group consisting of (a) alkyl, (b) C6-10 aryl, and (c) alk-C6-10 aryl; (20) —(CH2)—SO2NRE′RF′, where q is an integer from zero to four and where each of RE′ and RF′ is, independently, selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl, and (d) C1-6 alk-C6-10 aryl; (21) thiol; (22) C6-10 aryloxy; (23) C3-8 cycloalkoxy; (24) C6-10 aryl-C1-6 alkoxy; (25) C1-6 alk-C1-12 heterocyclyl (e.g., C1-6 alk-C1-12 heteroaryl); (26) C2-20 alkenyl; and (27) C2-20 alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl or a C1-alkheterocyclyl can be further substituted with an oxo group to afford the respective aryloyl and (heterocyclyl)oyl substituent group.
The term “arylalkoxy,” as used herein, represents an alkaryl group, as defined herein, attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted alkoxyalkyl groups include from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C6-10 aryl-C1-6 alkoxy, C6-10 aryl-C1-10 alkoxy, or C6-10 aryl-C1-20 alkoxy). In some embodiments, the arylalkoxy group can be substituted with 1, 2, 3, or 4 substituents as defined herein
The term “aryloxy” represents a chemical substituent of formula —OR′, where R′ is an aryl group of 6 to 18 carbons, unless otherwise specified. In some embodiments, the aryl group can be substituted with 1, 2, 3, or 4 substituents as defined herein.
The term “aryloyl,” as used herein, represents an aryl group, as defined herein, that is attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted aryloyl groups are of 7 to 11 carbons. In some embodiments, the aryl group can be substituted with 1, 2, 3, or 4 substituents as defined herein.
The term “azido” represents an —N3 group, which can also be represented as —N═N═N.
The term “bicyclic,” as used herein, refer to a structure having two rings, which may be aromatic or non-aromatic. Bicyclic structures include spirocyclyl groups, as defined herein, and two rings that share one or more bridges, where such bridges can include one atom or a chain including two, three, or more atoms. Exemplary bicyclic groups include a bicyclic carbocyclyl group, where the first and second rings are carbocyclyl groups, as defined herein; a bicyclic aryl groups, where the first and second rings are aryl groups, as defined herein; bicyclic heterocyclyl groups, where the first ring is a heterocyclyl group and the second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g., heteroaryl) group; and bicyclic heteroaryl groups, where the first ring is a heteroaryl group and the second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g., heteroaryl) group. In some embodiments, the bicyclic group can be substituted with 1, 2, 3, or 4 substituents as defined herein for cycloalkyl, heterocyclyl, and aryl groups.
The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to an optionally substituted C3-12 monocyclic, bicyclic, or tricyclic structure in which the rings, which may be aromatic or non-aromatic, are formed by carbon atoms. Carbocyclic structures include cycloalkyl, cycloalkenyl, and aryl groups.
The term “carbamoyl,” as used herein, represents —C(O)—N(RN1)2, where the meaning of each RN1 is found in the definition of “amino” provided herein.
The term “carbamoylalkyl,” as used herein, represents an alkyl group, as defined herein, substituted by a carbamoyl group, as defined herein. The alkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein.
The term “carbamyl,” as used herein, refers to a carbamate group having the structure
—NRN1C(═O)OR or —OC(═O)N(RN1)2, where the meaning of each RN1 is found in the definition of “amino” provided herein, and R is alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as defined herein.
The term “carbonyl,” as used herein, represents a C(O) group, which can also be represented as C═O.
The term “carboxyaldehyde” represents an acyl group having the structure —CHO.
The term “carboxy,” as used herein, means —CO2H.
The term “carboxyalkoxy,” as used herein, represents an alkoxy group, as defined herein, substituted by a carboxy group, as defined herein. The alkoxy group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for the alkyl group.
The term “carboxyalkyl,” as used herein, represents an alkyl group, as defined herein, substituted by a carboxy group, as defined herein. The alkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein.
The term “cyano,” as used herein, represents an —CN group.
The term “cycloalkoxy” represents a chemical substituent of formula —OR, where R is a C3-8 cycloalkyl group, as defined herein, unless otherwise specified. The cycloalkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein. Exemplary unsubstituted cycloalkoxy groups are from 3 to 8 carbons. In some embodiment, the cycloalkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein.
The term “cycloalkyl,” as used herein represents a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group from three to eight carbons, unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl, and the like. When the cycloalkyl group includes one carbon-carbon double bond, the cycloalkyl group can be referred to as a “cycloalkenyl” group. Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, and the like. The cycloalkyl groups of this invention can be optionally substituted with: (1) C1-7 acyl (e.g., carboxyaldehyde); (2) C1-20 alkyl (e.g., C1-6 alkyl, C1-6 alkoxy-C1-6 alkyl, C1-6 alkylsulfinyl-C1-6 alkyl, amino-C1-6 alkyl, azido-C1-6 alkyl, (carboxyaldehyde)-C1-6 alkyl, halo-C1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C1-6 alkyl, nitro-C1-6 alkyl, or C1-6thioalkoxy-C1-6 alkyl); (3) C1-20 alkoxy (e.g., C1-6 alkoxy, such as perfluoroalkoxy); (4) C1-6 alkylsulfinyl; (5) C6-10 aryl; (6) amino; (7) C1-6 alk-C6-10 aryl; (8) azido; (9) C3-8 cycloalkyl; (10) C1-6 alk-C3-8 cycloalkyl; (11) halo; (12) C1-12 heterocyclyl (e.g., C1-12 heteroaryl); (13) (C1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C1-20 thioalkoxy (e.g., C1-6 thioalkoxy); (17) —(CH2)qCO2RA′, where q is an integer from zero to four, and RA′ is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, (c) hydrogen, and (d) C1-6 alk-C6-10 aryl; (18) —(CH2)qCONRB′RC′, where q is an integer from zero to four and where RB′ and RC′ are independently selected from the group consisting of (a) hydrogen, (b) C6-10 alkyl, (c) C6-10 aryl, and (d) C1-6 alk-C6-10 aryl; (19) —(CH2)qSO2RD′, where q is an integer from zero to four and where RD′ is selected from the group consisting of (a) C6-10 alkyl, (b) C6-10 aryl, and (c) C1-6 alk-C6-10 aryl; (20) —(CH2)—SO2NRE′RF′, where q is an integer from zero to four and where each of RE′ and RF′ is, independently, selected from the group consisting of (a) hydrogen, (b) C6-10 alkyl, (c) C6-10 aryl, and (d) C1-6 alk-C6-10 aryl; (21) thiol; (22) C6-10 aryloxy; (23) C3-8 cycloalkoxy; (24) C6-10 aryl-C1-6 alkoxy; (25) C1-6 alk-C1-12 heterocyclyl (e.g., C1-6 alk-C1-12 heteroaryl); (26) oxo; (27) C2-20 alkenyl; and (28) C2-20 alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl or a C1-alkheterocyclyl can be further substituted with an oxo group to afford the respective aryloyl and (heterocyclyl)oyl substituent group.
The term “diastereomer,” as used herein means stereoisomers that are not mirror images of one another and are non-superimposable on one another.
The term “effective amount” of an agent, as used herein, is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats cancer, an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of cancer, as compared to the response obtained without administration of the agent.
The term “enantiomer,” as used herein, means each individual optically active form of a compound of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.
The term “halo,” as used herein, represents a halogen selected from bromine, chlorine, iodine, or fluorine.
The term “haloalkoxy,” as used herein, represents an alkoxy group, as defined herein, substituted by a halogen group (i.e., F, Cl, Br, or I). A haloalkoxy may be substituted with one, two, three, or, in the case of alkyl groups of two carbons or more, four halogens. Haloalkoxy groups include perfluoroalkoxys (e.g., —OCF3), —OCHF2, —OCH2F, —OCCl3, —OCH2CH2Br, —OCH2CH(CH2CH2Br)CH3, and —OCHICH3. In some embodiments, the haloalkoxy group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups.
The term “haloalkyl,” as used herein, represents an alkyl group, as defined herein, substituted by a halogen group (i.e., F, Cl, Br, or I). A haloalkyl may be substituted with one, two, three, or, in the case of alkyl groups of two carbons or more, four halogens. Haloalkyl groups include perfluoroalkyls (e.g., —CF3), —CHF2, —CH2F, —CCl3, —CH2CH2Br, —CH2CH(CH2CH2Br)CH3, and —CHICH3. In some embodiments, the haloalkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups.
The term “heteroalkylene,” as used herein, refers to an alkylene group, as defined herein, in which one or two of the constituent carbon atoms have each been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkylene group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkylene groups.
The term “heteroaryl,” as used herein, represents that subset of heterocyclyls, as defined herein, which are aromatic: i.e., they contain 4n+2 pi electrons within the mono- or multicyclic ring system. Exemplary unsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In some embodiment, the heteroaryl is substituted with 1, 2, 3, or 4 substituents groups as defined for a heterocyclyl group.
The term “heterocyclyl,” as used herein represents a 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The 5-membered ring has zero to two double bonds, and the 6- and 7-membered rings have zero to three double bonds. Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term “heterocyclyl” also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbons and/or heteroatoms bridges two non-adjacent members of a monocyclic ring, e.g., a quinuclidinyl group. The term “heterocyclyl” includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three carbocyclic rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Examples of fused heterocyclyls include tropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, and the like, including dihydro and tetrahydro forms thereof, where one or more double bonds are reduced and replaced with hydrogens. Still other exemplary heterocyclyls include: 2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl; 2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g., 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl); 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g., 2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl); 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g., 2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl); 4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino 5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g., 1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl); 2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl); 1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g., 2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl); 1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g., 1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl); 1,6-dihydro-6-oxo-pyridazinyl (e.g., 1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl (e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl); 2,3-dihydro-2-oxo-1H-indolyl (e.g., 3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and 2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl); 1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl; 1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl); 2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g., 3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl); 2,3-dihydro-2-oxo-benzoxazolyl (e.g., 5-chloro-2,3-dihydro-2-oxo-benzoxazolyl); 2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl; 1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g., 2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl); 1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g., 1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl); 1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g., 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl); 1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g., 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl); 2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and 1,8-naphthylenedicarboxamido. Additional heterocyclics include 3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and 2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups also include groups of the formula




where

E′ is selected from the group consisting of —N— and —CH—; F′ is selected from the group consisting of —N═CH—, —NH—CH2—, —NH—C(O)—, —NH—, —CH═N—, —CH2—NH—, —C(O)—NH—, —CH═CH—, —CH2—, —CH2CH2—, —CH2O—, —OCH2—, —O—, and —S—; and G′ is selected from the group consisting of —CH— and —N—. Any of the heterocyclyl groups mentioned herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) C1-7 acyl (e.g., carboxyaldehyde); (2) C1-20 alkyl (e.g., C1-6 alkyl, C1-6 alkoxy-C1-6 alkyl, C1-6 alkylsulfinyl-C1-6 alkyl, amino-C1-6 alkyl, azido-C1-6 alkyl, (carboxyaldehyde)-C1-6 alkyl, halo-C1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C1-6 alkyl, nitro-C1-6 alkyl, or C1-6 thioalkoxy-C1-6 alkyl); (3) C1-20 alkoxy (e.g., C1-6 alkoxy, such as perfluoroalkoxy); (4) C1-6 alkylsulfinyl; (5) C6-10 aryl; (6) amino; (7) C1-6 alk-C6-10 aryl; (8) azido; (9) C3-8 cycloalkyl; (10) C1-6 alk-C3-8 cycloalkyl; (11) halo; (12) C1-12 heterocyclyl (e.g., C2-12 heteroaryl); (13) (C1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C1-20 thioalkoxy (e.g., C1-6 thioalkoxy); (17) —(CH2)—CO2RA′, where q is an integer from zero to four, and RA′ is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, (c) hydrogen, and (d) C1-6 alk-C6-10 aryl; (18) —(CH2)qCONRB′RC′, where q is an integer from zero to four and where RB′ and RC′ are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl, and (d) C1-6 alk-C6-10 aryl; (19) —(CH2)—SO2RD′, where q is an integer from zero to four and where RD′ is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, and (c) C1-6 alk-C6-10 aryl; (20) —(CH2)—SO2NRE′RF′, where q is an integer from zero to four and where each of RE′ and RF′ is, independently, selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl, and (d) C1-6 alk-C6-10 aryl; (21) thiol; (22) C6-10 aryloxy; (23) C3-8 cycloalkoxy; (24) arylalkoxy; (25) C1-6 alk-C1-12 heterocyclyl (e.g., C1-6 alk-C1-12 heteroaryl); (26) oxo; (27) (C1-12 heterocyclyl)imino; (28) C2-20 alkenyl; and (29) C2-20 alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C1-alkaryl or a C1-alkheterocyclyl can be further substituted with an oxo group to afford the respective aryloyl and (heterocyclyl)oyl substituent group.
The term “(heterocyclyl)imino,” as used herein, represents a heterocyclyl group, as defined herein, attached to the parent molecular group through an imino group. In some embodiments, the heterocyclyl group can be substituted with 1, 2, 3, or 4 substituent groups as defined herein.
The term “(heterocyclyl)oxy,” as used herein, represents a heterocyclyl group, as defined herein, attached to the parent molecular group through an oxygen atom. In some embodiments, the heterocyclyl group can be substituted with 1, 2, 3, or 4 substituent groups as defined herein.
The term “(heterocyclyl)oyl,” as used herein, represents a heterocyclyl group, as defined herein, attached to the parent molecular group through a carbonyl group. In some embodiments, the heterocyclyl group can be substituted with 1, 2, 3, or 4 substituent groups as defined herein.
The term “hydrocarbon,” as used herein, represents a group consisting only of carbon and hydrogen atoms.
The term “hydroxy,” as used herein, represents an —OH group.
The term “hydroxyalkenyl,” as used herein, represents an alkenyl group, as defined herein, substituted by one to three hydroxy groups, with the proviso that no more than one hydroxy group may be attached to a single carbon atom of the alkyl group, and is exemplified by dihydroxypropenyl, hydroxyisopentenyl, and the like.
The term “hydroxyalkyl,” as used herein, represents an alkyl group, as defined herein, substituted by one to three hydroxy groups, with the proviso that no more than one hydroxy group may be attached to a single carbon atom of the alkyl group, and is exemplified by hydroxymethyl, dihydroxypropyl, and the like.
The term “isomer,” as used herein, means any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention can have one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/trans isomers). According to the invention, the chemical structures depicted herein, and therefore the compounds of the invention, encompass all of the corresponding stereoisomers, that is, both the stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of compounds of the invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
The term “N-protected amino,” as used herein, refers to an amino group, as defined herein, to which is attached one or two N-protecting groups, as defined herein.
The term “N-protecting group,” as used herein, represents those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. N-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine, and the like; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl, and the like and silyl groups, such as trimethylsilyl, and the like. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).
The term “nitro,” as used herein, represents an —NO2 group.
The term “oxo” as used herein, represents ═O.
The term “perfluoroalkyl,” as used herein, represents an alkyl group, as defined herein, where each hydrogen radical bound to the alkyl group has been replaced by a fluoride radical. Perfluoroalkyl groups are exemplified by trifluoromethyl, pentafluoroethyl, and the like.
The term “perfluoroalkoxy,” as used herein, represents an alkoxy group, as defined herein, where each hydrogen radical bound to the alkoxy group has been replaced by a fluoride radical. Perfluoroalkoxy groups are exemplified by trifluoromethoxy, pentafluoroethoxy, and the like.
The term “spirocyclyl,” as used herein, represents a C2-7 alkylene diradical, both ends of which are bonded to the same carbon atom of the parent group to form a spirocyclic group, and also a C1-6 heteroalkylene diradical, both ends of which are bonded to the same atom. The heteroalkylene radical forming the spirocyclyl group can containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, the spirocyclyl group includes one to seven carbons, excluding the carbon atom to which the diradical is attached. The spirocyclyl groups of the invention may be optionally substituted with 1, 2, 3, or 4 substituents provided herein as optional substituents for cycloalkyl and/or heterocyclyl groups.
The term “stereoisomer,” as used herein, refers to all possible different isomeric as well as conformational forms which a compound may possess (e.g., a compound of any formula described herein), in particular all possible stereochemically and conformationally isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention.
The term “sulfoalkyl,” as used herein, represents an alkyl group, as defined herein, substituted by a sulfo group of —SO3H. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein.
The term “sulfonyl,” as used herein, represents an —S(O)2— group.
The term “thioalkaryl,” as used herein, represents a chemical substituent of formula —SR, where R is an alkaryl group. In some embodiments, the alkaryl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein.
The term “thioalkheterocyclyl,” as used herein, represents a chemical substituent of formula —SR, where R is an alkheterocyclyl group. In some embodiments, the alkheterocyclyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein.
The term “thioalkoxy,” as used herein, represents a chemical substituent of formula —SR, where R is an alkyl group, as defined herein. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein.
The term “thiol” represents an —SH group.
Compound: As used herein, the term “compound,” is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C═N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.
Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples prototropic tautomers include ketone—enol pairs, amide—imidic acid pairs, lactam—lactim pairs, amide—imidic acid pairs, enamine—imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium.
The compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
Condition: As used herein, the term “condition” refers to a disorder that presents with observable symptoms.
Conserved: As used herein, the term “conserved” refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
In some embodiments, two or more sequences are said to be “completely conserved” if they are 100% identical to one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another. In some embodiments, two or more sequences are said to be “conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be “conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of an oligonucleotide or polypeptide or may apply to a portion, region or feature thereof.
Cosmetic: As used herein, the term “cosmetic” refers to a substance which may be used to enhance, alter, modify and/or change the appearance of a cell, tissue and/or organism.
Cosmetic polypeptide: As used herein, a “cosmetic polypeptide” refers to a polypeptide that upon contact or administration enhances, alters, modifies and/or changes the phenotype or aesthetic presentation of a cell, tissue, system, and/or organism. A cosmetic polypeptide may be a therapeutic, e.g., it may comprise pharmaceutical compositions and/or formulations, or they may serve to provide only aesthetic benefits such as with lipsticks, fingernail and toenail polish, eye and facial make-up, cover-ups, concealers and the like.
Cosmetic protein: As used herein, a “cosmetic protein” refers to a protein which may be encoded by a cosmetic polypeptide, fragments and variants thereof
Cyclic or Cyclized: As used herein, the term “cyclic” refers to the presence of a continuous loop. Cyclic molecules need not be circular, only joined to form an unbroken chain of subunits. Cyclic molecules such as the engineered RNA or mRNA of the present invention may be single units or multimers or comprise one or more components of a complex or higher order structure.
Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing, suppressing the growth, division, or multiplication of a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof
Cytotoxic: As used herein, “cytotoxic” refers to killing or causing injurious, toxic, or deadly effect on a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof.
Delivery: As used herein, “delivery” refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.
Delivery Agent: As used herein, “delivery agent” refers to any substance which facilitates, at least in part, the in vivo delivery of a cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA to targeted cells.
Destabilized: As used herein, the term “destable,” “destabilize,” or “destabilizing region” means a region or molecule that is less stable than a starting, wild-type or native form of the same region or molecule.
Detectable label: As used herein, “detectable label” refers to one or more markers, signals, or moieties which are attached, incorporated or associated with another entity that is readily detected by methods known in the art including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like. Detectable labels may be located at any position in the peptides or proteins disclosed herein. They may be within the amino acids, the peptides, or proteins, or located at the N- or C-termini.
Disease: As used herein, the term “disease” refers to an abnormal condition affecting the body of an organism often showing specific bodily symptoms.
Disorder: As used herein, the term “disorder, “refers to a disruption of or an interference with normal functions or established systems of the body.
Digest: As used herein, the term “digest” means to break apart into smaller pieces or components. When referring to polypeptides or proteins, digestion results in the production of peptides.
Distal: As used herein, the term “distal” means situated away from the center or away from a point or region of interest.
Dosing regimen: As used herein, a “dosing regimen” is a schedule of administration or physician determined regimen of treatment, prophylaxis, or palliative care.
Dose splitting factor (DSF)-ratio of PUD of dose split treatment divided by PUD of total daily dose or single unit dose. The value is derived from comparison of dosing regimens groups.
Encoded protein cleavage signal: As used herein, “encoded protein cleavage signal” refers to the nucleotide sequence which encodes a protein cleavage signal.
Engineered: As used herein, embodiments of the invention are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.
Exosome: As used herein, “exosome” is a vesicle secreted by mammalian cells or a complex involved in RNA degradation.
Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or 3′ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
Feature: As used herein, a “feature” refers to a characteristic, a property, or a distinctive element.
Formulation: As used herein, a “formulation” includes at least a cosmetic polynucleotide, cosmetic primary construct or cosmetic mmRNA and a delivery agent.
Fragment: A “fragment,” as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.
Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
Heavy Chain: As used herein, when referring to neurotoxins, the phrase “heavy chain” describes the larger of the N-terminal or C-terminal chains.
Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). In accordance with the invention, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. In accordance with the invention, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.
Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between oligonucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
Inhibit expression of a gene: As used herein, the phrase “inhibit expression of a gene” means to cause a reduction in the amount of an expression product of the gene. The expression product can be an RNA transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene. Typically a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom. The level of expression may be determined using standard techniques for measuring mRNA or protein.
In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).
Isolated: As used herein, the term “isolated” refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances may have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. Substantially isolated: By “substantially isolated” is meant that the compound is substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the present disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art.
Light Chain: As used herein, when referring to neurotoxins, the phrase “light chain” describes the smaller of the N-terminal or C-terminal chains.
Linker: As used herein, a linker refers to a group of atoms, e.g., 10-1,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be attached to a modified nucleoside or nucleotide on the nucleobase or sugar moiety at a first end, and to a payload, e.g., a detectable or therapeutic agent, at a second end. The linker may be of sufficient length as to not interfere with incorporation into a nucleic acid sequence. The linker can be used for any useful purpose, such as to form mmRNA multimers (e.g., through linkage of two or more cosmetic polynucleotides, cosmetic primary constructs, or cosmetic mmRNA molecules) or mmRNA conjugates, as well as to administer a payload, as described herein. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomeric units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers, Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond (—S—S—) or an azo bond (—N═N—), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of a selectively cleavable bond include an amido bond can be cleaved for example by the use of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/or photolysis, as well as an ester bond can be cleaved for example by acidic or basic hydrolysis.
MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA) binding site represents a nucleotide location or region of a nucleic acid transcript to which at least the “seed” region of a miRNA binds.
Modified: As used herein “modified” refers to a changed state or structure of a molecule of the invention. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, the mRNA molecules of the present invention are modified by the introduction of non-natural nucleosides and/or nucleotides, e.g., as it relates to the natural ribonucleotides A, U, G, and C. Noncanonical nucleotides such as the cap structures are not considered “modified” although they differ from the chemical structure of the A, C, G, U ribonucleotides.
Mucus: As used herein, “mucus” refers to the natural substance that is viscous and comprises mucin glycoproteins.
Naturally occurring: As used herein, “naturally occurring” means existing in nature without artificial aid.
Non-human vertebrate: As used herein, a “non human vertebrate” includes all vertebrates except Homo sapiens, including wild and domesticated species. Examples of non-human vertebrates include, but are not limited to, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit, reindeer, sheep water buffalo, and yak.
Off-target: As used herein, “off target” refers to any unintended effect on any one or more target, gene, or cellular transcript.
Open reading frame: As used herein, “open reading frame” or “ORF” refers to a sequence which does not contain a stop codon in a given reading frame.
Operably linked: As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.
Paratope: As used herein, a “paratope” refers to the antigen-binding site of an antibody.
Patient: As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.
Optionally substituted: Herein a phrase of the form “optionally substituted X” (e.g., optionally substituted alkyl) is intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein said alkyl is optionally substituted”). It is not intended to mean that the feature “X” (e.g. alkyl) per se is optional.
Peptide: As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
Pharmaceutical composition: The phrase “pharmaceutical composition” refers to a composition that alters the etiology of a disease, disorder and/or condition.
Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
Pharmaceutically acceptable excipients: The phrase “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.
Pharmaceutically acceptable salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.
Pharmaceutically acceptable solvate: The term “pharmaceutically acceptable solvate,” as used herein, means a compound of the invention wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a “hydrate.”
Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one or more properties of a molecule or compound as it relates to the determination of the fate of substances administered to a living organism. Pharmacokinetics is divided into several areas including the extent and rate of absorption, distribution, metabolism and excretion. This is commonly referred to as ADME where: (A) Absorption is the process of a substance entering the blood circulation; (D) Distribution is the dispersion or dissemination of substances throughout the fluids and tissues of the body; (M) Metabolism (or Biotransformation) is the irreversible transformation of parent compounds into daughter metabolites; and (E) Excretion (or Elimination) refers to the elimination of the substances from the body. In rare cases, some drugs irreversibly accumulate in body tissue.
Phenotype: As used herein, “phenotype” refers to the set of observable characteristics of a subject, cell, tissue, organ and/or organism. For example, a change in phenotype may include the reduction of facial wrinkles, disappearance of age spots, disappearance of skin discoloration and the like.
Physicochemical: As used herein, “physicochemical” means of or relating to a physical and/or chemical property.
Polypeptide per unit drug (PUD): As used herein, a PUD or product per unit drug, is defined as a subdivided portion of total daily dose, usually 1 mg, pg, kg, etc., of a product (such as a polypeptide) as measured in body fluid or tissue, usually defined in concentration such as pmol/mL, mmol/mL, etc divided by the measure in the body fluid.
Preventing: As used herein, the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.
Prodrug: The present disclosure also includes prodrugs of the compounds described herein. As used herein, “prodrugs” refer to any substance, molecule or entity which is in a form predicate for that substance, molecule or entity to act as a therapeutic upon chemical or physical alteration. Prodrugs may by covalently bonded or sequestered in some way and which release or are converted into the active drug moiety prior to, upon or after administered to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include compounds wherein hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparation and use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference in their entirety.
Proliferate: As used herein, the term “proliferate” means to grow, expand or increase or cause to grow, expand or increase rapidly. “Proliferative” means having the ability to proliferate. “Anti-proliferative” means having properties counter to or inapposite to proliferative properties.
Protein cleavage site: As used herein, “protein cleavage site” refers to a site where controlled cleavage of the amino acid chain can be accomplished by chemical, enzymatic or photochemical means.
Protein cleavage signal: As used herein “protein cleavage signal” refers to at least one amino acid that flags or marks a polypeptide for cleavage.
Protein of interest: As used herein, the terms “proteins of interest” or “desired proteins” include those provided herein and fragments, mutants, variants, and alterations thereof
Proximal: As used herein, the term “proximal” means situated nearer to the center or to a point or region of interest.
Pseudouridine: As used herein, pseudouridine refers to the C-glycoside isomer of the nucleoside uridine. A “pseudouridine analog” is any modification, variant, isoform or derivative of pseudouridine. For example, pseudouridine analogs include but are not limited to 1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine, 1-methylpseudouridine (m1ψ), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), and 2′-O-methyl-pseudouridine (ψm).
Purified: As used herein, “purify,” “purified,” “purification” means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.
Sample: As used herein, the term “sample” or “biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.
Signal Sequences: As used herein, the phrase “signal sequences” refers to a sequence which can direct the transport or localization of a protein.
Single unit dose: As used herein, a “single unit dose” is a dose of any therapeutic administer in one dose/at one time/single route/single point of contact, i.e., single administration event.
Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.
Skin: The term “skin” is the thin layer of tissue forming the natural outer covering of the body of a subject and includes the epidermis and the dermis. The dermis is the thick layer of living tissue below the epidermis which is the surface epithelium of the skin.
Split dose: As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses.
Stable: As used herein “stable” refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
Stabilized: As used herein, the term “stabilize”, “stabilized,” “stabilized region” means to make or become stable.
Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
Substantially equal: As used herein as it relates to time differences between doses, the term means plus/minus 2%.
Substantially simultaneously: As used herein and as it relates to plurality of doses, the term means within 2 seconds.
Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition.
Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition but harbors a propensity to develop a disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (for example, cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
Synthetic: The term “synthetic” means produced, prepared, and/or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present invention may be chemical or enzymatic.
Targeted Cells: As used herein, “targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient.
Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
Therapeutically effective outcome: As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
Total daily dose: As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.
Transcription factor: As used herein, the term “transcription factor” refers to a DNA-binding protein that regulates transcription of DNA into RNA, for example, by activation or repression of transcription. Some transcription factors effect regulation of transcription alone, while others act in concert with other proteins. Some transcription factor can both activate and repress transcription under certain conditions. In general, transcription factors bind a specific target sequence or sequences highly similar to a specific consensus sequence in a regulatory region of a target gene. Transcription factors may regulate transcription of a target gene alone or in a complex with other molecules.
Treating: As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. For example, “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
Unmodified: As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.
EQUIVALENTS AND SCOPE
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.
Section and table headings are not intended to be limiting.
EXAMPLES
Example 1. Modified mRNA Production
Modified mRNAs (mmRNA) according to the invention may be made using standard laboratory methods and materials. The open reading frame (ORF) of the gene of interest may be flanked by a 5′ untranslated region (UTR) which may contain a strong Kozak translational initiation signal and/or an alpha-globin 3′ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. The modified mRNAs may be modified to reduce the cellular innate immune response. The modifications to reduce the cellular response may include pseudouridine (γ) and 5-methyl-cytidine (5meC, 5mc or m5C). (See, Kariko K et al. Immunity 23:165-75 (2005), Kariko K et al. Mol Ther 16:1833-40 (2008), Anderson B R et al. NAR (2010); each of which are herein incorporated by reference in their entireties).
The ORF may also include various upstream or downstream additions (such as, but not limited to, β-globin, tags, etc.) may be ordered from an optimization service such as, but limited to, DNA2.0 (Menlo Park, Calif.) and may contain multiple cloning sites which may have XbaI recognition. Upon receipt of the construct, it may be reconstituted and transformed into chemically competent E. coli. 
The methods described herein to make modified mRNA may be used to produce molecules of all sizes including long molecules. Modified mRNA using the described methods has been made for different sized molecules including glucosidase, alpha; acid (GAA) (3.2 kb), cystic fibrosis transmembrane conductance regulator (CFTR) (4.7 kb), Factor VII (7.3 kb), lysosomal acid lipase (45.4 kDa), glucocerebrosidase (59.7 kDa) and iduronate 2-sulfatase (76 kDa).
For the present invention, NEB DH5-alpha Competent E. coli are used. Transformations are performed according to NEB instructions using 100 ng of plasmid. The protocol is as follows:

    
    
        1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
        2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
        3. Place the mixture on ice for 30 minutes. Do not mix.
        4. Heat shock at 42° C. for exactly 30 seconds. Do not mix.
        5. Place on ice for 5 minutes. Do not mix.
        6. Pipette 950 μl of room temperature SOC into the mixture.
        7. Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or rotate.
        8. Warm selection plates to 37° C.
        9. Mix the cells thoroughly by flicking the tube and inverting.
        10. Spread 50-100 μl of each dilution onto a selection plate and incubate overnight at 37° C. Alternatively, incubate at 30° C. for 24-36 hours or 25° C. for 48 hours.
    
    


A single colony is then used to inoculate 5 ml of LB growth media using the appropriate antibiotic and then allowed to grow (250 RPM, 37° C.) for 5 hours. This is then used to inoculate a 200 ml culture medium and allowed to grow overnight under the same conditions.
To isolate the plasmid (up to 850 μg), a maxi prep is performed using the Invitrogen PURELINKTM HiPure Maxiprep Kit (Carlsbad, Calif.), following the manufacturer's instructions.
In order to generate cDNA for In Vitro Transcription (IVT), the plasmid (an Example of which is shown in FIG. 3) is first linearized using a restriction enzyme such as XbaI. A typical restriction digest with XbaI will comprise the following: Plasmid 1.0 μg; 10× Buffer 1.0 μl; XbaI 1.5 μl; dH2O up to 10 μl; incubated at 37° C. for 1 hr. If performing at lab scale (<5 μg), the reaction is cleaned up using Invitrogen's PURELINKTM PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions. Larger scale purifications may need to be done with a product that has a larger load capacity such as Invitrogen's standard PURELINKTM PCR Kit (Carlsbad, Calif.). Following the cleanup, the linearized vector is quantified using the NanoDrop and analyzed to confirm linearization using agarose gel electrophoresis.
As a non-limiting example, G-CSF may represent the polypeptide of interest. Sequences used in the steps outlined in Examples 1-5 are shown in Table 10. It should be noted that the start codon (ATG) has been underlined in each sequence of Table 10.







TABLE 10







G-CSF Sequences








SEQ



ID



NO
Description





5652
cDNAsequence:



ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGC



CCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCA



GGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAG



CTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGG



GCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTAC



AAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCT



CTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAG



GCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGC



CTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATC



TCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGAC



GTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGA



ACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCAT



GCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGT



CCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTA



CCGCGTTCTACGCCACCTTGCCCAGCCCTGA


 


5653
cDNA having T7 polymerase site, AfeI and 



Xba restriction site:



TAATACGACTCACTATA



GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG



AGCCACC



ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGC



CCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCA



GGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAG



CTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGG



GCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTAC



AAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCT



CTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAG



GCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGC



CTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATC



TCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGAC



GTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGA



ACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCAT



GCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGT



CCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTA



CCGCGTTCTACGCCACCTTGCCCAGCCCTGA



AGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCT



TCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCT



GAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA


 


5654
Optimized sequence; containing T7 polymerase 



site, AfeI and Xba restriction site



TAATACGACTCACTATA



GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG



AGCCACC



ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGC



CCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCA



AGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTC



ATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGG



CGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACA



AACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCT



TGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGG



CTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTT



GTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTC



GCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACG



TGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAA



CTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAAT



GCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT



CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTA



CCGGGTGCTGAGACATCTTGCGCAGCCGTGA



AGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCT



TCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCT



GAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA


 


5655
mRNA sequence (transcribed)



GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA



GAGCCACC



AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGG



CCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGU



CCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGC



AGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAU



UCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCG



ACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCG



GGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUG



UCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAG



CUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAG



CCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGA



CACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUC



UGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGC



AGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUU



UCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUU



CAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUC



UUGCGCAGCCGUGA



AGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCU



UCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUA



AAGCCUGAGUAGGAAG









Example 2: PCR for cDNA Production
PCR procedures for the preparation of cDNA are performed using 2×KAPA HIFITM HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This system includes 2×KAPA ReadyMix12.5 μl; Forward Primer (10 uM) 0.75 μl; Reverse Primer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH2O diluted to 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25 cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45 sec, then 72° C. for 5 min. then 4° C. to termination.
The reverse primer of the instant invention incorporates a poly-T120 for a poly-A120 in the mRNA. Other reverse primers with longer or shorter poly(T) tracts can be used to adjust the length of the poly(A) tail in the mRNA.
The reaction is cleaned up using Invitrogen's PURELINKTM PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Larger reactions will require a cleanup using a product with a larger capacity. Following the cleanup, the cDNA is quantified using the NANODROPTM and analyzed by agarose gel electrophoresis to confirm the cDNA is the expected size. The cDNA is then submitted for sequencing analysis before proceeding to the in vitro transcription reaction.
Example 3. In Vitro Transcription (IVT)
The in vitro transcription reaction generates mRNA containing modified nucleotides or modified RNA. The input nucleotide triphosphate (NTP) mix is made in-house using natural and un-natural NTPs.
A typical in vitro transcription reaction includes the following:


















1
Template cDNA
1.0
μg



2
10x transcription buffer (400 mM
2.0
μl




Tris-HCl pH 8.0, 190 mM MgCl2,




50 mM DTT, 10 mM Spermidine)



3
Custom NTPs (25 mM each)
7.2
μl



4
RNase Inhibitor
20
U



5
T7 RNA polymerase
3000
U











6
dH20
Up to 20.0 μl. and










7
Incubation at 37° C. for 3 hr-5 hrs.










The crude IVT mix may be stored at 4° C. overnight for cleanup the next day. 1 U of RNase-free DNase is then used to digest the original template. After 15 minutes of incubation at 37° C., the mRNA is purified using Ambion's MEGACLEARTM Kit (Austin, Tex.) following the manufacturer's instructions. This kit can purify up to 500 μg of RNA. Following the cleanup, the RNA is quantified using the NanoDrop and analyzed by agarose gel electrophoresis to confirm the RNA is the proper size and that no degradation of the RNA has occurred.
Example 4. Enzymatic Capping of mRNA
Capping of the mRNA is performed as follows where the mixture includes: IVT RNA 60 μg-180 μg and dH2O up to 72 μl. The mixture is incubated at 65° C. for 5 minutes to denature RNA, and then is transferred immediately to ice.
The protocol then involves the mixing of 10× Capping Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl2) (10.0 μl); 20 mM GTP (5.0 μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U); 2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyl transferase) (40 U); dH2O (Up to 28 μl); and incubation at 37° C. for 30 minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.
The mRNA is then purified using Ambion's MEGACLEARTM Kit (Austin, Tex.) following the manufacturer's instructions. Following the cleanup, the RNA is quantified using the NANODROPTM (ThermoFisher, Waltham, Mass.) and analyzed by agarose gel electrophoresis to confirm the RNA is the proper size and that no degradation of the RNA has occurred. The RNA product may also be sequenced by running a reverse-transcription-PCR to generate the cDNA for sequencing.
Example 5. PolyA Tailing Reaction
Without a poly-T in the cDNA, a poly-A tailing reaction must be performed before cleaning the final product. This is done by mixing Capped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl2)(12.0 μl); 20 mM ATP (6.0 μl); Poly-A Polymerase (20 U); dH2O up to 123.5 μl and incubation at 37° C. for 30 min. If the poly-A tail is already in the transcript, then the tailing reaction may be skipped and proceed directly to cleanup with Ambion's MEGACLEARTM kit (Austin, Tex.) (up to 500 μg). Poly-A Polymerase is preferably a recombinant enzyme expressed in yeast.
For studies performed and described herein, the poly-A tail is encoded in the IVT template to comprise 160 nucleotides in length. However, it should be understood that the processivity or integrity of the polyA tailing reaction may not always result in exactly 160 nucleotides. Hence polyA tails of approximately 160 nucleotides, e.g, about 150-165, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope of the invention.
Example 6. Natural 5′ Caps and 5′ Cap Analogues
5′-capping of modified RNA may be completed concomitantly during the in vitro-transcription reaction using the following chemical RNA cap analogs to generate the 5′-guanosine cap structure according to manufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap]; G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (New England BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs, Ipswich, Mass.). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2′-O methyl-transferase to generate: m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2′-O-methylation of the 5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-O methyl-transferase. Enzymes are preferably derived from a recombinant source.
When transfected into mammalian cells, the modified mRNAs have a stability of between 12-18 hours or more than 18 hours, e.g., 24, 36, 48, 60, 72 or greater than 72 hours.
Example 7. Capping
A. Protein Expression Assay
Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 5652; mRNA sequence fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site shown in SEQ ID NO: 5655 with a polyA tail approximately 160 nucleotides in length not shown in sequence) containing the ARCA (3′O-Me-m7G(5′)ppp(5′)G) cap analog or the Cap1 structure can be transfected into human primary keratinocytes at equal concentrations. 6, 12, 24 and 36 hours post-transfection the amount of G-CSF secreted into the culture medium can be assayed by ELISA. Synthetic mRNAs that secrete higher levels of G-CSF into the medium would correspond to a synthetic mRNA with a higher translationally-competent Cap structure.
B. Purity Analysis Synthesis
Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 5652; mRNA sequence fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site shown in SEQ ID NO: 5655 with a polyA tail approximately 160 nucleotides in length not shown in sequence) containing the ARCA cap analog or the Cap1 structure crude synthesis products can be compared for purity using denaturing Agarose-Urea gel electrophoresis or HPLC analysis. Synthetic mRNAs with a single, consolidated band by electrophoresis correspond to the higher purity product compared to a synthetic mRNA with multiple bands or streaking bands. Synthetic mRNAs with a single HPLC peak would also correspond to a higher purity product. The capping reaction with a higher efficiency would provide a more pure mRNA population.
C. Cytokine Analysis
Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 5652; mRNA sequence fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site shown in SEQ ID NO: 5655 with a polyA tail approximately 160 nucleotides in length not shown in sequence) containing the ARCA cap analog or the Cap1 structure can be transfected into human primary keratinocytes at multiple concentrations. 6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatory cytokines such as TNF-alpha and IFN-beta secreted into the culture medium can be assayed by ELISA. Synthetic mRNAs that secrete higher levels of pro-inflammatory cytokines into the medium would correspond to a synthetic mRNA containing an immune-activating cap structure.
D. Capping Reaction Efficiency
Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 5652; mRNA sequence fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site shown in SEQ ID NO: 5655 with a polyA tail approximately 160 nucleotides in length not shown in sequence) containing the ARCA cap analog or the Cap1 structure can be analyzed for capping reaction efficiency by LC-MS after capped mRNA nuclease treatment. Nuclease treatment of capped mRNAs would yield a mixture of free nucleotides and the capped 5′-5-triphosphate cap structure detectable by LC-MS. The amount of capped product on the LC-MS spectra can be expressed as a percent of total mRNA from the reaction and would correspond to capping reaction efficiency. The cap structure with higher capping reaction efficiency would have a higher amount of capped product by LC-MS.
Example 8. Agarose Gel Electrophoresis of Modified RNA or RT PCR Products
Individual modified RNAs (200-400 ng in a 20 μl volume) or reverse transcribed PCR products (200-400 ng) are loaded into a well on a non-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and run for 12-15 minutes according to the manufacturer protocol.
Example 9. Nanodrop Modified RNA Quantification and UV Spectral Data
Modified RNAs in TE buffer (1 μl) are used for Nanodrop UV absorbance readings to quantitate the yield of each modified RNA from an in vitro transcription reaction.
Example 10. Formulation of Modified mRNA Using Lipidoids
Modified mRNAs (mmRNA) are formulated for in vitro experiments by mixing the mmRNA with the lipidoid at a set ratio prior to addition to cells. In vivo formulation may require the addition of extra ingredients to facilitate circulation throughout the body. To test the ability of these lipidoids to form particles suitable for in vivo work, a standard formulation process used for siRNA-lipidoid formulations was used as a starting point. Initial mmRNA-lipidoid formulations may consist of particles composed of 42% lipidoid, 48% cholesterol and 10% PEG, with further optimization of ratios possible. After formation of the particle, mmRNA is added and allowed to integrate with the complex. The encapsulation efficiency is determined using a standard dye exclusion assays.
Materials and Methods for Examples 11-15
A. Lipid Synthesis
Six lipids, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 and DLin-MC3-DMA, were synthesized by methods outlined in the art in order to be formulated with modified RNA. DLin-DMA and precursors were synthesized as described in Heyes et. al, J. Control Release, 2005, 107, 276-287. DLin-K-DMA and DLin-KC2-DMA and precursors were synthesized as described in Semple et. al, Nature Biotechnology, 2010, 28, 172-176. 98N12-5 and precursor were synthesized as described in Akinc et. al, Nature Biotechnology, 2008, 26, 561-569.
C12-200 and precursors were synthesized according to the method outlined in Love et. al, PNAS, 2010, 107, 1864-1869. 2-epoxydodecane (5.10 g, 27.7 mmol, 8.2 eq) was added to a vial containing Amine 200 (0.723 g, 3.36 mmol, 1 eq) and a stirring bar. The vial was sealed and warmed to 80° C. The reaction was stirred for 4 days at 80° C. Then the mixture was purified by silica gel chromatography using a gradient from pure dichloromethane (DCM) to DCM:MeOH 98:2. The target compound was further purified by RP-HPLC to afford the desired compound.
DLin-MC3-DMA and precursors were synthesized according to procedures described in WO 2010054401 herein incorporated by reference in its entirety. A mixture of dilinoleyl methanol (1.5 g, 2.8 mmol, 1 eq), N,N-dimethylaminobutyric acid (1.5 g, 2.8 mmol, 1 eq), DIPEA (0.73 mL, 4.2 mmol, 1.5 eq) and TBTU (1.35 g, 4.2 mmol, 1.5 eq) in 10 mL of DMF was stirred for 10 h at room temperature. Then the reaction mixture was diluted in ether and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtrated and concentrated under reduced pressure. The crude product was purified by silica gel chromatography using a gradient DCM to DCM:MeOH 98:2. Subsequently the target compound was subjected to an additional RP-HPLC purification which was done using a YMC—Pack C4 column to afford the target compound.
B. Formulation of Modified RNA Nanoparticles
Solutions of synthesized lipid, 1,2-distearoyl-3-phosphatidylcholine (DSPC) (Avanti Polar Lipids, Alabaster, Ala.), cholesterol (Sigma-Aldrich, Taufkirchen, Germany), and α-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-w-methoxy-polyoxyethylene (PEG-c-DOMG) (NOF, Bouwelven, Belgium) were prepared at concentrations of 50 mM in ethanol and stored at −20° C. The lipids were combined to yield molar ratio of 50:10:38.5:1.5 (Lipid: DSPC: Cholesterol: PEG-c-DOMG) and diluted with ethanol to a final lipid concentration of 25 mM. Solutions of modified mRNA at a concentration of 1-2 mg/mL in water were diluted in 50 mM sodium citrate buffer at a pH of 3 to form a stock modified mRNA solution. Formulations of the lipid and modified mRNA were prepared by combining the synthesized lipid solution with the modified mRNA solution at total lipid to modified mRNA weight ratio of 10:1, 15:1, 20:1 and 30:1. The lipid ethanolic solution was rapidly injected into aqueous modified mRNA solution to afford a suspension containing 33% ethanol. The solutions were injected either manually (MI) or by the aid of a syringe pump (SP) (Harvard Pump 33 Dual Syringe Pump Harvard Apparatus Holliston, Mass.).
To remove the ethanol and to achieve the buffer exchange, the formulations were dialyzed twice against phosphate buffered saline (PBS), pH 7.4 at volumes 200-times of the primary product using a Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc. Rockford, Ill.) with a molecular weight cutoff (MWCO) of 10 kD. The first dialysis was carried at room temperature for 3 hours and then the formulations were dialyzed overnight at 4° C. The resulting nanoparticle suspension was filtered through 0.2 μm sterile filter (Sarstedt, Nümbrecht, Germany) into glass vials and sealed with a crimp closure.
C. Characterization of Formulations
A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) was used to determine the particle size, the polydispersity index (PDI) and the zeta potential of the modified mRNA nanoparticles in 1×PBS in determining particle size and 15 mM PBS in determining zeta potential.
Ultraviolet-visible spectroscopy was used to determine the concentration of modified mRNA nanoparticle formulation. 100 μL of the diluted formulation in 1×PBS was added to 900 μL of a 4:1 (v/v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution was recorded between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, Calif.). The modified RNA concentration in the nanoparticle formulation was calculated based on the extinction coefficient of the modified RNA used in the formulation and on the difference between the absorbance at a wavelength of 260 nm and the baseline value at a wavelength of 330 nm.
QUANT-ITTM RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.) was used to evaluate the encapsulation of modified RNA by the nanoparticle. The samples were diluted to a concentration of approximately 5 μg/mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of the diluted samples were transferred to a polystyrene 96 well plate, then either 50 μL of TE buffer or 50 μL of a 2% Triton X-100 solution was added. The plate was incubated at a temperature of 37° C. for 15 minutes. The RIBOGREEN® reagent was diluted 1:100 in TE buffer, 100 μL of this solution was added to each well. The fluorescence intensity was measured using a fluorescence plate reader (Wallac Victor 1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) at an excitation wavelength of  ̃480 nm and an emission wavelength of  ̃520 nm. The fluorescence values of the reagent blank were subtracted from that of each of the samples and the percentage of free modified RNA was determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).
D. In Vitro Incubation
Human embryonic kidney epithelial (HEK293) and hepatocellular carcinoma epithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) were seeded on 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany) and plates for HEK293 cells were precoated with collagen type1. HEK293 were seeded at a density of 30,000 and HepG2 were seeded at a density of 35,000 cells per well in 100 μl cell culture medium. For HEK293 the cell culture medium was DMEM, 10% FCS, adding 2 mM L-Glutamine, 1 mM Sodiumpyruvate and 1× non-essential amino acids (Biochrom AG, Berlin, Germany) and 1.2 mg/ml Sodiumbicarbonate (Sigma-Aldrich, Munich, Germany) and for HepG2 the culture medium was MEM (Gibco Life Technologies, Darmstadt, Germany), 10% FCS adding 2 mM L-Glutamine, 1 mM Sodiumpyruvate and 1× non-essential amino acids (Biochrom AG, Berlin, Germany. Formulations containing mCherry mRNA (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) were added in quadruplicates directly after seeding the cells and incubated. The mCherry cDNA with the T7 promoter, 5′untranslated region (UTR) and 3′ UTR used in in vitro transcription (IVT) is given in SEQ ID NO: 5657. The mCherry mRNA was modified with a 5meC at each cytosine and pseudouridine replacement at each uridine site.
Cells were harvested by transferring the culture media supernatants to a 96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg, Germany). Cells were trypsinized with 1⁄2 volume Trypsin/EDTA (Biochrom AG, Berlin, Germany), pooled with respective supernatants and fixed by adding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck, Darmstadt, Germany). Samples then were submitted to a flow cytometer measurement with a 532 nm excitation laser and the 610/20 filter for PE-Texas Red in a LSRII cytometer (Beckton Dickinson GmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI) of all events and the standard deviation of four independent wells are presented in for samples analyzed.
Example 11. Purification of Nanoparticle Formulations
Nanoparticle formulations of DLin-KC2-DMA and 98N12-5 in HEK293 and HepG2 were tested to determine if the mean fluorescent intensity (MFI) was dependent on the lipid to modified RNA ratio and/or purification. Three formulations of DLin-KC2-DMA and two formulations of 98N12-5 were produced using a syringe pump to the specifications described in Table 11. Purified samples were purified by SEPHADEXTM G-25 DNA grade (GE Healthcare, Sweden). Each formulation before and after purification (aP) was tested at concentration of 250 ng modified RNA per well in a 24 well plate. The percentage of cells that are positive for the marker for FL4 channel (% FL4-positive) when analyzed by the flow cytometer for each formulation and the background sample, and the MFI of the marker for the FL4 channel for each formulation and the background sample are shown in Table 12. The formulations which had been purified had a slightly higher MFI than those formulations tested before purification.







TABLE 11







Formulations










Formulation

Lipid/RNA
Mean size


#
Lipid
wt/wt
(nm)





NPA-001-1
DLin-KC2-DMA
10
155 nm





PDI: 0.08


NPA-001-1 aP
DLin-KC2-DMA
10
141 nm





PDI: 0.14


NPA-002-1
DLin-KC2-DMA
15
140 nm





PDI: 0.11


NPA-002-1 aP
DLin-KC2-DMA
15
125 nm





PDI: 0.12


NPA-003-1
DLin-KC2-DMA
20
114 nm





PDI: 0.08


NPA-003-1 aP
DLin-KC2-DMA
20
104 nm





PDI: 0.06


NPA-005-1
98N12-5
15
127 nm





PDI: 0.12


NPA-005-1 aP
98N12-5
15
134 nm





PDI: 0.17


NPA-006-1
98N12
20
126 nm





PDI: 0.08


NPA-006-1 aP
98N12
20
118 nm





PDI: 0.13
















TABLE 12







HEK293 and HepG2, 24-well, 250 ng Modified RNA/well












% FL4-positive

FL4 MFI














Formulation
HEK293
HepG2
HEK293
HepG2

















Untreated
0.33
0.40
0.25
0.30



NPA-001-1
62.42
5.68
1.49
0.41



NPA-001-ap
87.32
9.02
3.23
0.53



NPA-002-1
91.28
9.90
4.43
0.59



NPA-002-ap
92.68
14.02
5.07
0.90



NPA-003-1
87.70
11.76
6.83
0.88



NPA-003-ap
88.88
15.46
8.73
1.06



NPA-005-1
50.60
4.75
1.83
0.46



NPA-005-ap
38.64
5.16
1.32
0.46



NPA-006-1
54.19
13.16
1.30
0.60



NPA-006-ap
49.97
13.74
1.27
0.61










Example 12. Concentration Response Curve
Nanoparticle formulations of 98N12-5 (NPA-005) and DLin-KC2-DMA (NPA-003) were tested at varying concentrations to determine the MFI of FL4 or mCherry (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) over a range of doses. The formulations tested are outlined in Table 13. To determine the optimal concentration of nanoparticle formulations of 98N12-5, varying concentrations of formulated modified RNA (100 ng, 10 ng, 1.0 ng, 0.1 ng and 0.01 ng per well) were tested in a 24-well plate of HEK293, and the results of the FL4 MFI of each dose are shown in Table 14. Likewise, to determine the optimal concentration of nanoparticle formulations of DLin-KC2-DMA, varying concentrations of formulated modified RNA (250 ng 100 ng, 10 ng, 1.0 ng, 0.1 ng and 0.01 ng per well) were tested in a 24-well plate of HEK293, and the results of the FL4 MFI of each dose are shown in Table 15. Nanoparticle formulations of DLin-KC2-DMA were also tested at varying concentrations of formulated modified RNA (250 ng, 100 ng and 30 ng per well) in a 24 well plate of HEK293, and the results of the FL4 MFI of each dose are shown in Table 16. A dose of 1 ng/well for 98N12-5 and a dose of 10 ng/well for DLin-KC2-DMA were found to resemble the FL4 MFI of the background.
To determine how close the concentrations resembled the background, we utilized a flow cytometer with optimized filter sets for detection of mCherry expression, and were able to obtain results with increased sensitivity relative to background levels. Doses of 25 ng/well, 0.25 ng/well, 0.025 ng/well and 0.0025 ng/well were analyzed for 98N12-5 (NPA-005) and DLin-KC2-DMA (NPA-003) to determine the MFI of mCherry. As shown in Table 17, the concentration of 0.025 ng/well and lesser concentrations are similar to the background MFI level of mCherry which is about 386.125.







TABLE 13







Formulations











Formulation #
NPA-003
NPA-005







Lipid
DLin-KC2-DMA
98N12-5



Lipid/RNA
20
15



wt/wt



Mean size
114 nm
106 nm




PDI: 0.08
PDI: 0.12

















TABLE 14







HEK293, NPA-005, 24-well, n = 4










Formulation
FL4 MFI














Untreated control
0.246



NPA-005 100 ng
2.2175



NPA-005 10 ng
0.651



NPA-005 1.0 ng
0.28425



NPA-005 0.1 ng
0.27675



NPA-005 0.01 ng
0.2865

















TABLE 15







HEK293, NPA-003, 24-well, n = 4










Formulation
FL4 MFI














Untreated control
0.3225



NPA-003 250 ng
2.9575



NPA-003 100 ng
1.255



NPA-003 10 ng
0.40025



NPA-003 1 ng
0.33025



NPA-003 0.1 ng
0.34625



NPA-003 0.01 ng
0.3475

















TABLE 16







HEK293, NPA-003, 24-well, n = 4










Formulation
FL4 MFI














Untreated control
0.27425



NPA-003 250 ng
5.6075



NPA-003 100 ng
3.7825



NPA-003 30 ng
1.5525

















TABLE 17







Concentration and MFI










MFI mCherry










Formulation
NPA-003
NPA-005












  25 ng/well
11963.25
12256.75


 0.25 ng/well
1349.75
2572.75


0.025 ng/well
459.50
534.75


0.0025 ng/well 
310.75
471.75









Example 13. Manual Injection and Syringe Pump Formulations
Two formulations of DLin-KC2-DMA and 98N12-5 were prepared by manual injection (MI) and syringe pump injection (SP) and analyzed along with a background sample to compare the MFI of mCherry (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) of the different formulations. Table 18 shows that the syringe pump formulations had a higher MFI as compared to the manual injection formulations of the same lipid and lipid/RNA ratio.







TABLE 18







Formulations and MFI














Lipid/

Method



Formulation

RNA
Mean size
of formu-



#
Lipid
wt/wt
(nm)
lation
MFI















Untreated
N/A
N/A
N/A
N/A
674.67


Control







NPA-002
DLin-KC2-
15
140 nm
MI
10318.25



DMA

PDI: 0.11




NPA-002-2
DLin-KC2-
15
105 nm
SP
37054.75



DMA

PDI: 0.04




NPA-003
DLin-KC2-
20
114 nm
MI
22037.5



DMA

PDI: 0.08




NPA-003-2
DLin-KC2-
20
95 nm
SP
37868.75



DMA

PDI: 0.02




NPA-005
98N12-5
15
127 nm
MI
11504.75





PDI: 0.12




NPA-005-2
98N12-5
15
106 nm
SP
9343.75





PDI: 0.07




NPA-006
98N12-5
20
126 nm
MI
11182.25





PDI: 0.08




NPA-006-2
98N12-5
20
93 nm
SP
5167





PDI: 0.08









Example 14. Lipid Nanoparticle Formulations
Formulations of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 and DLin-MC3-DMA were incubated at a concentration of 60 ng/well or 62.5 ng/well in a plate of HEK293 and 62.5 ng/well in a plate of HepG2 cells for 24 hours to determine the MFI of mCherry (SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) for each formulation. The formulations tested are outlined in Table 19 below. As shown in Table 20 for the 60 ng/well and Tables 21, 22 and 23 for the 62.5 ng/well, the formulation of NPA-003 and NPA-018 have the highest mCherry MFI and the formulations of NPA-008, NPA-010 and NPA-013 are most the similar to the background sample mCherry MFI value.







TABLE 19







Formulations












Formulation

Lipid/RNA
Mean size



#
Lipid
wt/wt
(nm)






NPA-001
DLin-KC2-DMA
10
155 nm






PDI: 0.08



NPA-002
DLin-KC2-DMA
15
140 nm






PDI: 0.11



NPA-002-2
DLin-KC2-DMA
15
105 nm






PDI: 0.04



NPA-003
DLin-KC2-DMA
20
114 nm






PDI: 0.08



NPA-003-2
DLin-KC2-DMA
20
95 nm






PDI: 0.02



NPA-005
98N12-5
15
127 nm






PDI: 0.12



NPA-006
98N12-5
20
126 nm






PDI: 0.08



NPA-007
DLin-DMA
15
148 nm






PDI: 0.09



NPA-008
DLin-K-DMA
15
121 nm






PDI: 0.08



NPA-009
C12-200
15
138 nm






PDI: 0.15



NPA-010
DLin-MC3-DMA
15
126 nm






PDI: 0.09



NPA-012
DLin-DMA
20
86 nm






PDI: 0.08



NPA-013
DLin-K-DMA
20
104 nm






PDI: 0.03



NPA-014
C12-200
20
101 nm






PDI: 0.06



NPA-015
DLin-MC3-DMA
20
109 nm






PDI: 0.07
















TABLE 20







HEK293, 96-well, 60 ng Modified RNA/well










Formulation
MFI mCherry













Untreated
871.81



NPA-001
6407.25



NPA-002
14995



NPA-003
29499.5



NPA-005
3762



NPA-006
2676



NPA-007
9905.5



NPA-008
1648.75



NPA-009
2348.25



NPA-010
4426.75



NPA-012
11466



NPA-013
2098.25



NPA-014
3194.25



NPA-015
14524
















TABLE 21







HEK293, 62.5 ng/well










Formulation
MFI mCherry













Untreated
871.81



NPA-001
6407.25



NPA-002
14995



NPA-003
29499.5



NPA-005
3762



NPA-006
2676



NPA-007
9905.5



NPA-008
1648.75



NPA-009
2348.25



NPA-010
4426.75



NPA-012
11466



NPA-013
2098.25



NPA-014
3194.25



NPA-015
14524
















TABLE 22







HEK293, 62.5 ng/well










Formulation
MFI mCherry













Untreated
295



NPA-007
3504



NPA-012
8286



NPA-017
6128



NPA-003-2
17528



NPA-018
34142



NPA-010
1095



NPA-015
5859



NPA-019
3229
















TABLE 23







HepG2, 62.5 ng/well










Formulation
MFI mCherry










Study 1










Untreated
649.94



NPA-001
6006.25



NPA-002
8705



NPA-002-2
15860.25



NPA-003
15059.25



NPA-003-2
28881



NPA-005
1676



NPA-006
1473



NPA-007
15678



NPA-008
2976.25



NPA-009
961.75



NPA-010
3301.75



NPA-012
18333.25



NPA-013
5853



NPA-014
2257



NPA-015
16225.75







Study 2










Untreated control
656



NPA-007
16798



NPA-012
21993



NPA-017
20377



NPA-003-2
35651



NPA-018
40154



NPA-010
2496



NPA-015
19741



NPA-019
16373









Example 15. In Vivo Formulation Studies
Rodents (n=5) are administered intravenously, subcutaneously or intramuscularly a single dose of a formulation containing a modified mRNA and a lipid. The modified mRNA administered to the rodents is selected from G-CSF (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1), erythropoietin (EPO) (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1), Factor IX (mRNA sequence shown in SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) or mCherry (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1). The erythropoietin cDNA with the T7 promoter, 5′untranslated region (UTR) and 3′ UTR used in in vitro transcription (IVT) is given in SEQ ID NO: 5660 and SEQ ID NO: 5661.
Each formulation also contains a lipid which is selected from one of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, reLNP, ATUPLEX®, DACC and DBTC. The rodents are injected with 100 ug, 10 ug or 1 ug of the formulated modified mRNA and samples are collected at specified time intervals.
Serum from the rodents administered formulations containing human G-CSF modified mRNA are measured by specific G-CSF ELISA and serum from mice administered human factor IX modified RNA is analyzed by specific factor IX ELISA or chromogenic assay. The liver and spleen from the mice administered with mCherry modified mRNA are analyzed by immunohistochemistry (IHC) or fluorescence-activated cell sorting (FACS). As a control, a group of mice are not injected with any formulation and their serum and tissue are collected analyzed by ELISA, FACS and/or IHC.
A. Time Course
The rodents are administered formulations containing at least one modified mRNA to study the time course of protein expression for the administered formulation. The rodents are bled at specified time intervals prior to and after administration of the modified mRNA formulations to determine protein expression and complete blood count. Samples are also collected from the site of administration of rodents administered modified mRNA formulations subcutaneously and intramuscularly to determine the protein expression in the tissue.
B. Dose Response
The rodents are administered formulations containing at least one modified mRNA to determine dose response of each formulation. The rodents are bled at specified time intervals prior to and after administration of the modified mRNA formulations to determine protein expression and complete blood count. The rodents are also scarified to analyze the effect of the modified mRNA formulation on the internal tissue. Samples are also collected from the site of administration of rodents administered modified mRNA formulations subcutaneously and intramuscularly to determine the protein expression in the tissue.
C. Toxicity
The rodents are administered formulations containing at least one modified mRNA to study toxicity of each formulation. The rodents are bled at specified time intervals prior to and after administration of the modified mRNA formulations to determine protein expression and complete blood count. The rodents are also sacrificed to analyze the effect of the modified mRNA formulation on the internal tissue. Samples are also collected from the site of administration of rodents administered modified mRNA formulations subcutaneously and intramuscularly to determine the protein expression in the tissue.
Example 16. PLGA Microsphere Formulations
Optimization of parameters used in the formulation of PLGA microspheres may allow for tunable release rates and high encapsulation efficiencies while maintaining the integrity of the modified RNA encapsulated in the microspheres. Parameters such as, but not limited to, particle size, recovery rates and encapsulation efficiency may be optimized to achieve the optimal formulation.
A. Synthesis of PLGA Microspheres
Polylacticglycolic acid (PLGA) microspheres were synthesized using the water/oil/water double emulsification methods known in the art using PLGA (Lactel, Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA), polyvinylalcohol (PVA) (Sigma, Cat#348406-25G, MW 13-23 k) dichloromethane and water. Briefly, 0.1 ml of water (W1) was added to 2 ml of PLGA dissolved in dichloromethane (DCM) (O1) at concentrations ranging from 50-200 mg/ml of PLGA. The W1/O1 emulsion was homogenized (IKA Ultra-Turrax Homogenizer, T18) for 30 seconds at speed 4 ( ̃15,000 rpm). The W1/O1 emulsion was then added to 100 to 200 ml of 0.3 to 1% PVA (W2) and homogenized for 1 minute at varied speeds. Formulations were left to stir for 3 hours and then washed by centrifugation (20-25 min, 4,000 rpm, 4° C.). The supernatant was discarded and the PLGA pellets were resuspended in 5-10 ml of water, which was repeated 2×. Average particle size (represents 20-30 particles) for each formulation was determined by microscopy after washing. Table 24 shows that an increase in the PLGA concentration led to larger sized microspheres. A PLGA concentration of 200 mg/mL gave an average particle size of 14.8 μm, 100 mg/mL was 8.7 μm, and 50 mg/mL of PLGA gave an average particle size of 4.0 μm.







TABLE 24







Varied PLGA Concentration













Sam-
O1
PLGA Con-
W2
PVA Con-

Average


ple
Volume
centration
Volume
centration

Size


ID
(mL)
(mg/mL)
(mL)
(%)
Speed
(μm)
















1
2
200
100
0.3
5
14.8


2
2
100
100
0.3
5
8.7


3
2
50
100
0.3
5
4.0









Table 25 shows that decreasing the homogenization speed from 5 ( ̃20,000 rpm) to speed 4 ( ̃15,000 rpm) led to an increase in particle size from 14.8 μm to 29.7 μm.







TABLE 25







Varied Homogenization Speed













Sam-
O1
PLGA Con-
W2
PVA Con-

Average


ple
Volume
centration
Volume
centration

Size


ID
(mL)
(mg/mL)
(mL)
(%)
Speed
(μm)
















1
2
200
100
0.3
5
14.8


4
2
200
100
0.3
4
29.7









Table 26 shows that increasing the W2 volume (i.e. increasing the ratio of W2:O1 from 50:1 to 100:1), decreased average particle size slightly. Altering the PVA concentration from 0.3 to 1 wt % had little impact on PLGA microsphere size.







TABLE 26







Varied W2 Volume and Concentration















PLGA

PVA





O1
Con-
W2
Con-




Sample
Volume
centration
Volume
centration

Average


ID
(mL)
(mg/mL)
(mL)
(%)
Speed
Size (μm)
















1
2
200
100
0.3
5
14.8


5
2
200
200
0.3
5
11.7


6
2
200
190
0.3
5
11.4


7
2
200
190
1.0
5
12.3









B. Encapsulation of Modified mRNA
Modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) was dissolved in water at a concentration of 2 mg/ml (W3). Three batches of PLGA microsphere formulations were made as described above with the following parameters: 0.1 ml of W3 at 2 mg/ml, 1.6 ml of O1 at 200 mg/ml, 160 ml of W2 at 1%, and homogenized at a speed of 4 for the first emulsion (W3/O1) and homogenized at a speed of 5 for the second emulsion (W3/O1/W2). After washing by centrifugation, the formulations were frozen in liquid nitrogen and then lyophilized for 3 days. To test the encapsulation efficiency of the formulations, the lyophilized material was deformulated in DCM for 6 hours followed by an overnight extraction in water. The modified RNA concentration in the samples was then determined by OD260. Encapsulation efficiency was calculated by taking the actual amount of modified RNA and dividing by the starting amount of modified RNA. In the three batches tested, there was an encapsulation efficiency of 59.2, 49.8 and 61.3.
C. Integrity of Modified mRNA Encapsulated in PLGA Microspheres
Modified Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) was dissolved in water at varied concentrations (W4) to vary the weight percent loading in the formulation (mg modified RNA/mg PLGA*100) and to determine encapsulation efficiency. The parameters in Table 27 were used to make four different batches of PLGA microsphere formulations with a homogenization speed of 4 for the first emulsion (W4/O1) and a homogenization speed of 5 for the second emulsion (W4/O1/W2).







TABLE 27







Factor IX PLGA Microsphere Formulation Parameters

















Factor IX 
Factor IX 

PLGA

PVA
Weight %



W4 Vol
Conc
Amnt
O1 Vol
Conc
W2 Vol
Conc
(wt %)


ID
(uL)
(mg/ml)
(ug)
(ml)
(mg/ml)
(ml)
(%)
Loading


















A
100
2.0
200.0
2.0
200
200
1.0
0.05


B
100
4.0
400.0
2.0
200
200
1.0
0.10


C
400
2.0
800.0
2.0
200
200
1.0
0.20


D
400
4.0
1600.0
2.0
200
200
1.0
0.40









After lyophilization, PLGA microspheres were weighed out in 2 ml eppendorf tubes to correspond to  ̃10 ug of modified RNA. Lyophilization was found to not destroy the overall structure of the PLGA microspheres. To increase weight percent loading (wt %) for the PLGA microspheres, increasing amounts of modified RNA were added to the samples. PLGA microspheres were deformulated by adding 1.0 ml of DCM to each tube and then shaking the samples for 6 hours. For modified RNA extraction, 0.5 ml of water was added to each sample and the samples were shaken overnight before the concentration of modified RNA in the samples was determined by OD260. To determine the recovery of the extraction process, unformulated Factor IX modified RNA (SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) (deformulation control) was spiked into DCM and was subjected to the deformulation process. Table 28 shows the loading and encapsulation efficiency for the samples. All encapsulation efficiency samples were normalized to the deformulation control.







TABLE 28







Weight Percent Loading and Encapsulation Efficiency











Theoretical
Actual modified




modified RNA
RNA
Encapsulation


ID
loading (wt %)
loading (wt %)
Efficiency (%)













A
0.05
0.06
97.1


B
0.10
0.10
85.7


C
0.20
0.18
77.6


D
0.40
0.31
68.1


Control
—
—
100.0









D. Release Study of Modified mRNA Encapsulated in PLGA Microspheres
PLGA microspheres formulated with Factor IX modified RNA (SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) were deformulated as described above and the integrity of the extracted modified RNA was determined by automated electrophoresis (Bio-Rad Experion). The extracted modified mRNA was compared against unformulated modified mRNA and the deformulation control in order to test the integrity of the encapsulated modified mRNA. As shown in FIG. 4, the majority of modRNA was intact for batch ID A, B, C and D, for the deformulated control (Deform control) and the unformulated control (Unform control).
E. Protein Expression of Modified mRNA Encapsulated in PLGA Microspheres
PLGA microspheres formulated with Factor IX modified RNA (SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) were deformulated as described above and the protein expression of the extracted modified RNA was determined by an in vitro transfection assay. HEK293 cells were reverse transfected with 250 ng of Factor IX modified RNA complexed with RNAiMAX (Invitrogen) in triplicate.
Factor IX modified RNA was diluted in nuclease-free water to a concentration of 25 ng/μl and RNAiMAX was diluted 13.3× in serum-free EMEM. Equal volumes of diluted modified RNA and diluted RNAiMAX were mixed together and were allowed to stand for 20 to 30 minutes at room temperature. Subsequently, 20 μl of the transfection mix containing 250 ng of Factor IX modified RNA was added to 80 μl of a cell suspension containing 30,000 cells. Cells were then incubated for 16 h in a humidified 37° C./5% CO2 cell culture incubator before harvesting the cell culture supernatant. Factor IX protein expression in the cell supernatant was analyzed by an ELISA kit specific for Factor IX (Molecular Innovations, Cat # HFIXKT-TOT) and the protein expression is shown in Table 29. In all PLGA microsphere batches tested, Factor IX modified RNA remained active and expressed Factor IX protein after formulation in PLGA microspheres and subsequent deformulation.







TABLE 29







Protein Expression











Factor IX Protein



Sample
Expression (ng/ml)






Batch A
0.83



Batch B
1.83



Batch C
1.54



Batch D
2.52



Deformulated Control
4.34



Unformulated Control
3.35









F. Release Study of Modified mRNA Encapsulated in PLGA Microspheres
PLGA micropsheres formulated with Factor IX modified RNA (SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) were resuspended in water to a PLGA microsphere concentration of 24 mg/ml. After resuspension, 150 ul of the PLGA microsphere suspension was aliquoted into eppendorf tubes. Samples were kept incubating and shaking at 37° C. during the course of the study. Triplicate samples were pulled at 0.2, 1, 2, 8, 14, and 21 days. To determine the amount of modified RNA released from the PLGA microspheres, samples were centrifuged, the supernatant was removed, and the modified RNA concentration in the supernatant was determined by OD 260. The percent release, shown in Table 30, was calculated based on the total amount of modified RNA in each sample. After 31 days, 96% of the Factor IX modified RNA was released from the PLGA microsphere formulations.







TABLE 30







Percent Release










Time (days)
% Release













0
0.0



0.2
27.0



1
37.7



2
45.3



4
50.9



8
57.0



14
61.8



21
75.5



31
96.4









G. Particle Size Reproducibility of PLGA Microspheres
Three batches of Factor IX modified RNA (SEQ ID NO: 5659 polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) PLGA microspheres were made using the same conditions described for Batch D, shown in Table 27, (0.4 ml of W4 at 4 mg/ml, 2.0 ml of O1 at 200 mg/ml, 200 ml of W2 at 1%, and homogenized at a speed of 5 for the W4/O1/W2 emulsion). To improve the homogeneity of the PLGA microsphere suspension, filtration was incorporated prior to centrifugation. After stirring for 3 hours and before centrifuging, all formulated material was passed through a 100 μm nylon mesh strainer (Fisherbrand Cell Strainer, Cat #22-363-549) to remove larger aggregates. After washing and resuspension with water, 100-200 μl of a PLGA microspheres sample was used to measure particle size of the formulations by laser diffraction (Malvern Mastersizer2000). The particle size of the samples is shown in Table 31.







TABLE 31







Particle Size Summary
















Volume







Weighted



ID
D10 (μm)
D50 (μm)
D90 (μm)
Mean (um)
Filtration















Control
19.2
62.5
722.4
223.1
No


A
9.8
31.6
65.5
35.2
Yes


B
10.5
32.3
66.9
36.1
Yes


C
10.8
35.7
79.8
41.4
Yes









Results of the 3 PLGA microsphere batches using filtration were compared to a PLGA microsphere batch made under the same conditions without filtration. The inclusion of a filtration step before washing reduced the mean particle size and demonstrated a consistent particle size distribution between 3 PLGA microsphere batches.
H. Serum Stability of Factor IX PLGA Microspheres
Factor IX mRNA RNA (SEQ ID NO: 5659 polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) in buffer (TE) or 90% serum (Se), or Factor IX mRNA in PLGA in buffer, 90% serum or 1% serum was incubated in buffer, 90% serum or 1% serum at an mRNA concentration of 50 ng/ul in a total volume of 70 ul. The samples were removed at 0, 30, 60 or 120 minutes. RNases were inactivated with proteinase K digestion for 20 minutes at 55° C. by adding 25 ul of 4× proteinase K buffer (0.4 ml 1M TRIS-HCl pH 7.5, 0.1 ml 0.5M EDTA, 0.12 ml 5M NaCl, and 0.4 ml 10% SDS) and 8 ul of proteinase K at 20 mg/ml. The Factor IX mRNA was precipitated (add 250 ul 95% ethanol for 1 hour, centrifuge for 10 min at 13 k rpm and remove supernatant, add 200 ul 70% ethanol to the pellet, centrifuge again for 5 min at 13 k rpm and remove supernatant and resuspend the pellet in 70 ul water) or extracted from PLGA microspheres (centrifuge 5 min at 13 k rpm and remove supernatant, wash pellet with 1 ml water, centrifuge 5 min at 13 k rpm and remove supernatant, add 280 ul dichloromethane to the pellet and shake for 15 minutes, add 70 ul water and then shake for 2 hours and remove the aqueous phase) before being analyzed by bioanalyzer. PLGA microspheres protect Factor IX modified mRNA from degradation in 90% and 1% serum over 2 hours. Factor IX modified mRNA completely degrades in 90% serum at the initial time point.
Example 17. Lipid Nanoparticle In Vivo Studies
G-CSF (cDNA with the T7 promoter, 5′ Untranslated region (UTR) and 3′UTR used in in vitro transcription is given in SEQ ID NO: 5654. mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap 1; fully modified with 5-methylcytosine and pseudouridine) and Factor IX (cDNA with the T7 promoter, 5′ UTR and 3′UTR used in in vitro transcription is given in SEQ ID NO: 5662. mRNA sequence shown in SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap 1; fully modified with 5-methylcytosine and pseudouridine) modified mRNA were formulated as lipid nanoparticles (LNPs) using the syringe pump method. The LNPs were formulated at a 20:1 weight ratio of total lipid to modified mRNA with a final lipid molar ratio of 50:10:38.5:1.5 (DLin-KC2-DMA: DSPC: Cholesterol: PEG-c-DOMG). Formulations, listed in Table 32, were characterized by particle size, zeta potential, and encapsulation.







TABLE 32







Formulations











Formulation #
NPA-029-1
NPA-030-1






Modified mRNA
Factor IX
G-CSF



Mean size
91 nm
106 nm




PDI: 0.04
PDI: 0.06



Zeta at pH 7.4
1.8 mV
0.9 mV



Encaps.
92%
100%



(RiboGr)









LNP formulations were administered to mice (n=5) intravenously at a modified mRNA dose of 100, 10, or 1 ug. Mice were sacrificed at 8 hrs after dosing. Serum was collected by cardiac puncture from mice that were administered with G-CSF or Factor IX modified mRNA formulations. Protein expression was determined by ELISA.
There was no significant body weight loss (<5%) in the G-CSF or Factor IX dose groups. Protein expression for G-CSF or Factor IX dose groups was determined by ELISA from a standard curve. Serum samples were diluted (about 20-2500× for G-CSF and about 10-250× for Factor IX) to ensure samples were within the linear range of the standard curve. As shown in Table 33, G-CSF protein expression determined by ELISA was approximately 17, 1200, and 4700 ng/ml for the 1, 10, and 100 ug dose groups, respectively. As shown in Table 34, Factor IX protein expression determined by ELISA was approximately 36, 380, and 3000-11000 ng/ml for the 1, 10, and 100 ug dose groups, respectively.







TABLE 33







G-CSF Protein Expression










Dose (ug)
Conc (ng/ml)
Dilution Factor
Sample Volume













1
17.73
 20x
  5 ul


10
1204.82
2500x
0.04 ul


100
4722.20
2500x
0.04 ul
















TABLE 34







Factor IX Protein Expression










Dose (ug)
Conc (ng/ml)
Dilution Factor
Sample Volume













 1
36.05
10x
5 ul


10
383.04
10x
5 ul


100*
3247.75
50x
1 ul


100*
11177.20
250x 
0.2 ul 









As shown in Table 35, the LNP formulations described above have about a 10,000-100,000-fold increase in protein production compared to an administration of an intravenous (IV)-lipoplex formulation for the same dosage of modified mRNA and intramuscular (IM) or subcutaneous (SC) administration of the same dose of modified mRNA in saline. As used in Table 35, the symbol “ ̃” means about.







TABLE 35







Protein Production













Serum Concentration (pg/ml)



G-CSF
Dose (ug)
8-12 hours after administration






IM
100
~20-80



SC
100
~10-40



IV (Lipoplex)
100
~30



IV (LNP)
100
~5,000,000



IV (LNP)
10
~1,000,000



IV (LNP)
1
~20,000








Serum Concentration (ng/ml)



Factor IX
Dose (ug)
8-12 hours after administration















IM
2 × 100
~1.6
ng/ml











IV (LNP)
100
~3,000-10,000 ng/ml












IV (LNP)
10
~400
ng/ml



IV (LNP)
1
~40
ng/ml









Materials and Methods for Examples 18-23
G-CSF (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap 1; fully modified with 5-methylcytosine and pseudouridine) and EPO (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap 1; fully modified with 5-methylcytosine and pseudouridine) modified mRNA were formulated as lipid nanoparticles (LNPs) using the syringe pump method. The LNPs were formulated at a 20:1 weight ratio of total lipid to modified mRNA with a final lipid molar ratio of 50:10:38.5:1.5 (DLin-KC2-DMA: DSPC: Cholesterol: PEG-c-DOMG). Formulations, listed in Table 36, were characterized by particle size, zeta potential, and encapsulation.







TABLE 36







Formulations











Formulation #
NPA-030-2
NPA-060-1







Modified mRNA
G-CSF
EPO



Mean size
84 nm
85 nm




PDI: 0.04
PDI: 0.03



Zeta at pH 7.4
0.8 mV
1.5 mV



Encapsulation
95%
98%



(RiboGreen)










Example 18. Lipid Nanoparticle In Vivo Studies with Modified mRNA
LNP formulations, shown in Table 36 (above), were administered to rats (n=5) intravenously (IV), intramuscularly (IM) or subcutaneously (SC) at a single modified mRNA dose of 0.05 mg/kg. A control group of rats (n=4) was untreated. The rats were bled at 2 hours, 8 hours, 24 hours, 48 hours and 96 hours and after they were administered with G-CSF or EPO modified mRNA formulations to determine protein expression using ELISA. The rats administered EPO modified mRNA intravenously were also bled at 7 days.
As shown in Table 37, EPO protein expression in the rats intravenously administered modified EPO mRNA was detectable out to 5 days. G-CSF in the rats intravenously administered modified G-CSF mRNA was detectable to 7 days. Subcutaneous and intramuscular administration of EPO modified mRNA was detectable to at least 24 hours and G-CSF modified mRNA was detectable to at least 8 hours. In Table 37, “OSC” refers to values that were outside the standard curve and “NT” means not tested.







TABLE 37







G-CSF and EPO Protein Expression














EPO Serum
G-CSF Serum





Concentration
Concentration



Route
Time
(pg/ml)
(pg/ml)

















IV
2
hours
36,981.0
31,331.9



IV
8
hours
62,053.3
70,532.4



IV
24
hours
42,077.0
5,738.6



IV
48
hours
5,561.5
233.8



IV
5
days
0.0
60.4



IV
7
days
0.0
NT



IM
2
hours
1395.4
1620.4



IM
8
hours
8974.6
7910.4



IM
24
hours
4678.3
893.3



IM
48
hours
NT
OSC



IM
5
days
NT
OSC



SC
2
hours
386.2
80.3



SC
8
hours
985.6
164.2



SC
24
hours
544.2
OSC



SC
48
hours
NT
OSC



SC
5
days
NT
OSC












Untreated
All bleeds
0
0










Example 19. Time Course In Vivo Study
LNP formulations, shown in Table 36 (above), were administered to mice (n=5) intravenously (IV) at a single modified mRNA dose of 0.5, 0.05 or 0.005 mg/kg. The mice were bled at 8 hours, 24 hours, 72 hours and 6 days after they were administered with G-CSF or EPO modified mRNA formulations to determine protein expression using ELISA.
As shown in Table 38, EPO and G-CSF protein expression in the mice administered with the modified mRNA intravenously was detectable out to 72 hours for the mice dosed with 0.005 mg/kg and 0.05 mg/kg of modified mRNA and out to 6 days for the mice administered the EPO modified mRNA. In Table 38, “>” means greater than and “ND” means not detected.







TABLE 38







Protein Expression












EPO Serum
G-CSF Serum




Concentration
Concentration


Dose (mg/kg)
Time
(pg/ml)
(pg/ml)














0.005
8
hours
12,508.3
11,550.6


0.005
24
hours
6,803.0
5,068.9


0.005
72
hours
ND
ND


0.005
6
days
ND
ND


0.05
8
hours
92,139.9
462,312.5


0.05
24
hours
54,389.4
80,903.8


0.05
72
hours
ND
ND


0.05
6
days
ND
ND


0.5
8
hours
498,515.3
>1,250,000


0.5
24
hours
160,566.3
495,812.5


0.5
72
hours
3,492.5
1,325.6


0.5
6
days
21.2
ND









Example 20. LNP Formulations In Vivo Study in Rodents
A. LNP Formulations in Mice
LNP formulations, shown in Table 36 (above), were administered to mice (n=4) intravenously (IV) at a single modified mRNA dose 0.05 mg/kg or 0.005 mg/kg. There was also 3 control groups of mice (n=4) that were untreated. The mice were bled at 2 hours, 8 hours, 24 hours, 48 hours and 72 hours after they were administered with G-CSF or EPO modified mRNA formulations to determine the protein expression. Protein expression of G-CSF and EPO were determined using ELISA.
As shown in Table 39, EPO and G-CSF protein expression in the mice was detectable at least out to 48 hours for the mice that received a dose of 0.005 mg/kg modified RNA and 72 hours for the mice that received a dose of 0.05 mg/kg modified RNA. In Table 39, “OSC” refers to values that were outside the standard curve and “NT” means not tested.







TABLE 39







Protein Expression in Mice












EPO Serum
G-CSF Serum




Concentration
Concentration


Dose (mg/kg)
Time
(pg/ml)
(pg/ml)













0.005
 2 hours
OSC
3,447.8


0.005
 8 hours
1,632.8
11,454.0


0.005
24 hours
1,141.0
4,960.2


0.005
48 hours
137.4
686.4


0.005
72 hours
0
NT


0.05
 2 hours
10,027.3
20,951.4


0.05
 8 hours
56,547.2
70,012.8


0.05
24 hours
25,027.3
19,356.2


0.05
48 hours
1,432.3
1,963.0


0.05
72 hours
82.2
47.3









B. LNP Formulations in Rats
LNP formulations, shown in Table 36 (above), are administered to rats (n=4) intravenously (IV) at a single modified mRNA dose 0.05 mg/kg. There is also a control group of rats (n=4) that are untreated. The rats are bled at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, 7 days and 14 days after they were administered with G-CSF or EPO modified mRNA formulations to determine the protein expression. Protein expression of G-CSF and EPO are determined using ELISA.
Example 21. Early Time Course Study of LNPs
LNP formulations, shown in Table 36 (above), are administered to mammals intravenously (IV), intramuscularly (IM) or subcutaneously (SC) at a single modified mRNA dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg. A control group of mammals are not treated. The mammals are bled at 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours and/or 2 hours after they are administered with the modified mRNA LNP formulations to determine protein expression using ELISA. The mammals are also bled to determine the complete blood count such as the granulocyte levels and red blood cell count.
Example 22. Non-Human Primate In Vivo Study
LNP formulations, shown in Table 36 (above), were administered to non-human primates (NHP) (cynomolgus monkey) (n=2) as a bolus intravenous injection (IV) over approximately 30 seconds using a hypodermic needle, which may be attached to a syringe/abbocath or butterfly if needed. The NHP were administered a single modified mRNA IV dose of 0.05 mg/kg of EPO or G-CSF or 0.005 mg/kg of EPO in a dose volume of 0.5 mL/kg. The NHPs were bled 5-6 days before dosing with the modified mRNA LNP formulations to determine protein expression in the serum and a baseline complete blood count. After administration with the modified mRNA formulation the NHP were bled at 8, 24, 48 and 72 hours to determined protein expression. At 24 and 72 hours after administration the complete blood count of the NHP was also determined. Protein expression of G-CSF and EPO was determined by ELISA. Urine from the NHPs was collected over the course of the entire experiment and analyzed to evaluate clinical safety. Samples were collected from the NHPs after they were administered with G-CSF or EPO modified mRNA formulations to determine protein expression using ELISA. Clinical chemistry, hematology, urinalysis and cytokines of the non-human primates were also analyzed.
As shown in Table 40, EPO protein expression in the NHPs administered 0.05 mg/kg is detectable out to 72 hours and the 0.005 mg/kg dosing of the EPO formulation is detectable out to 48 hours. In Table 40, the “<” means less than a given value. G-CSF protein expression was seen out to 24 hours after administration with the modified mRNA formulation. Preliminarily, there was an increase in granulocytes and reticulocytes levels seen in the NHP after administration with the modified mRNA formulations.







TABLE 40







Protein Expression in Non-Human Primates















Female NHP
Male NHP
Average





Serum
Serum
Serum





Concen-
Concen-
Conen-


Modified
Dose

tration
tration
tration


mRNA
(mg/kg)
Time
(pg/ml)
(pg/ml)
(pg/ml)















G-CSF
0.05
Pre-bleed
0
0
0




 8 hours
3289
1722
2,506




24 hours
722
307
515




48 hours
0
0
0




72 hours
0
0
0


EPO
0.05
Pre-bleed
0
0
0




 8 hours
19,858
7,072
13,465




24 hours
18,178
4,913
11,546




48 hours
5,291
498
2,895




72 hours
744
60
402


EPO
0.005
Pre-bleed
0
0
0




 8 hours
523
250
387




24 hours
302
113
208




48 hours
<7.8
<7.8
<7.8




72 hours
0
0
0









Example 23. Non-Human Primate In Vivo Study for G-CSF and EPO
LNP formulations, shown in Table 36 (above), were administered to non-human primates (NHP) (cynomolgus monkey) (n=2) as intravenous injection (IV). The NHP were administered a single modified mRNA IV dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg of G-CSF or EPO in a dose volume of 0.5 mL/kg. The NHPs were bled before dosing with the modified mRNA LNP formulations to determine protein expression in the serum and a baseline complete blood count. After administration with the G-CSF modified mRNA formulation the NHP were bled at 8, 24, 48 and 72 hours to determined protein expression. After administration with the EPO modified mRNA formulation the NHP were bled at 8, 24, 48, 72 hours and 7 days to determined protein expression.
Samples collected from the NHPs after they were administered with G-CSF or EPO modified mRNA formulations were analyzed by ELISA to determine protein expression. Neutrophil and reticulocyte count was also determined pre-dose, 24 hours, 3 days, 7 days, 14 days and 18 days after administration of the modified G-CSF or EPO formulation.
As shown in Table 41, G-CSF protein expression was not detected beyond 72 hours. In Table 41, “<39” refers to a value below the lower limit of detection of 39 pg/ml.







TABLE 41







G-CSF Protein Expression














Female NHP
Male NHP





Serum G-CSF
Serum G-CSF


Modified
Dose

Concentration
Concentration


mRNA
(mg/kg)
Time
(pg/ml)
(pg/ml)














G-CSF
0.5
Pre-bleed
<39
<39




 8 hours
43,525
43,594




24 hours
11,374
3,628




48 hours
1,100
833




72 hours
<39
306


G-CSF
0.05
Pre-bleed
<39
<39




 8 hours
3,289
1,722




24 hours
722
307




48 hours
<39
<39




72 hours
<39
<39


G-CSF
0.005
Pre-bleed
<39
<39




 8 hours
559
700




24 hours
155
<39




48 hours
<39
<39




72 hours
<39
<39









As shown in Table 42, EPO protein expression was not detected beyond 7 days. In Table 42, “<7.8” refers to a value below the lower limit of detection of 7.8 pg/ml.







TABLE 42







EPO Protein Expression














Female NHP
Male NHP





Serum EPO
Serum EPO


Modified
Dose

Concentration
Concentration


mRNA
(mg/kg)
Time
(pg/ml)
(pg/ml)














EPO
0.5
Pre-bleed
<7.8
<7.8




 8 hours
158,771
119,086




24 hours
133,978
85,825




48 hours
45,250
64,793




72 hours
15,097
20,407




7 days
<7.8
<7.8


EPO
0.05
Pre-bleed
<7.8
<7.8




 8 hours
19,858
7,072




24 hours
18,187
4,913




48 hours
5,291
498




72 hours
744
60




7 days
<7.8
<7.8


EPO
0.005
Pre-bleed
<7.8
<7.8




 8 hours
523
250




24 hours
302
113




48 hours
11
29




72 hours
<7.8
<7.8




7 days
<7.8
<7.8









As shown in Table 43, there was an increase in neutrophils in all G-CSF groups relative to pre-dose levels.







TABLE 43







Pharmacologic Effect of G-CSF mRNA in NHP














Male NHP (G-CSF)
Female NHP (G-CSF)
Male NHP (EPO)
Female NHP (EPO)


Dose (mg/kg)
Time
Neutrophils (109/L)
Neutrophils (109/L)
Neutrophils (109/L)
Neutrophils (109/L)















0.5
Pre-dose
1.53
1.27
9.72
1.82



24 hours 
14.92
13.96
7.5
11.85



3 days
9.76
13.7
11.07
5.22



7 days
2.74
3.81
11.8
2.85



14/18 days   
2.58
1.98
7.16
2.36


0.05
Pre-dose
13.74
3.05
0.97
2.15



24 hours 
19.92
29.91
2.51
2.63



3 days
7.49
10.77
1.73
4.08



7 days
4.13
3.8
1.23
2.77



14/18 days   
3.59
1.82
1.53
1.27


0.005
Pre-dose
1.52
2.54
5.46
5.96



24 hours 
16.44
8.6
5.37
2.59



3 days
3.74
1.78
6.08
2.83



7 days
7.28
2.27
3.51
2.23



14/18 days   
4.31
2.28
1.52
2.54









As shown in Table 44, there was an increase in reticulocytes in all EPO groups 3 days to 14/18 days after dosing relative to reticulocyte levels 24 hours after dosing.







TABLE 44







Pharmacologic Effect of EPO mRNA on Neutrophil Count














Male NHP (G-CSF)
Female NHP (G-CSF)
Male NHP (EPO)
Female NHP (EPO)


Dose (mg/kg)
Time
Neutrophils (1012/L)
Neutrophils (1012/L)
Neutrophils (1012/L)
Neutrophils (1012/L)















0.5
Pre-dose
0.067
0.055
0.107
0.06



24 hours 
0.032
0.046
0.049
0.045



3 days
0.041
0.017
0.09
0.064



7 days
0.009
0.021
0.35
0.367



14/18 days   
0.029
0.071
0.066
0.071


0.05
Pre-dose
0.055
0.049
0.054
0.032



24 hours 
0.048
0.046
0.071
0.04



3 days
0.101
0.061
0.102
0.105



7 days
0.157
0.094
0.15
0.241



14/18 days   
0.107
0.06
0.067
0.055


0.005
Pre-dose
0.037
0.06
0.036
0.052



24 hours 
0.037
0.07
0.034
0.061



3 days
0.037
0.054
0.079
0.118



7 days
0.046
0.066
0.049
0.087



14/18 days   
0.069
0.057
0.037
0.06









As shown in Tables 45-47, the administration of EPO modified RNA had an effect on other erythropoetic parameters including hemoglobin (HGB), hematocrit (HCT) and red blood cell (RBC) count.







TABLE 45







Pharmacologic Effect of EPO mRNA on Hemoglobin














Male NHP (G-CSF)
Female NHP (G-CSF)
Male NHP (EPO)
Female NHP (EPO)


Dose (mg/kg)
Time
HGB (g/L)
HGB (g/L)
HGB (g/L)
HGB (g/L)















0.5
Pre-dose
133
129
134
123



24 hours 
113
112
127
108



3 days
118
114
126
120



7 days
115
116
140
134



14/18 days   
98
113
146
133


0.05
Pre-dose
137
129
133
133



24 hours 
122
117
123
116



3 days
126
115
116
120



7 days
126
116
126
121



14/18 days   
134
123
133
129


0.005
Pre-dose
128
129
132
136



24 hours 
117
127
122
128



3 days
116
127
125
130



7 days
116
129
119
127



14/18 days   
118
129
128
129
















TABLE 46







Pharmacologic Effect of EPO mRNA on Hematocrit














Male NHP (G-CSF)
Female NHP (G-CSF)
Male NHP (EPO)
Female NHP (EPO)


Dose (mg/kg)
Time
HCT (L/L)
HCT (L/L)
HCT (L/L)
HCT (L/L)















0.5
Pre-dose
0.46
0.43
0.44
0.4



24 hours 
0.37
0.38
0.4
0.36



3 days
0.39
0.38
0.41
0.39



7 days
0.39
0.38
0.45
0.45



14/18 days   
0.34
0.37
0.48
0.46


0.05
Pre-dose
0.44
0.44
0.45
0.43



24 hours 
0.39
0.4
0.43
0.39



3 days
0.41
0.39
0.38
0.4



7 days
0.42
0.4
0.45
0.41



14/18 days   
0.44
0.4
0.46
0.43


0.005
Pre-dose
0.42
0.42
0.48
0.45



24 hours 
0.4
0.42
0.42
0.43



3 days
0.4
0.41
0.44
0.42



7 days
0.39
0.42
0.41
0.42



14/18 days   
0.41
0.42
0.42
0.42
















TABLE 47







Pharmacologic Effect of EPO mRNA on Red Blood Cells














Male NHP (G-CSF)
Female NHP (G-CSF)
Male NHP (EPO)
Female NHP (EPO)


Dose (mg/kg)
Time
RBC (1012/L)
RBC (1012/L)
RBC (1012/L)
RBC (1012/L)















0.5
Pre-dose
5.57
5.57
5.43
5.26



24 hours 
4.66
4.96
5.12
4.69



3 days
4.91
4.97
5.13
5.15



7 days
4.8
5.04
5.55
5.68



14/18 days   
4.21
4.92
5.83
5.72


0.05
Pre-dose
5.68
5.64
5.57
5.84



24 hours 
4.96
5.08
5.25
5.18



3 days
5.13
5.04
4.81
5.16



7 days
5.17
5.05
5.37
5.31



14/18 days   
5.43
5.26
5.57
5.57


0.005
Pre-dose
5.67
5.36
6.15
5.72



24 hours 
5.34
5.35
5.63
5.35



3 days
5.32
5.24
5.77
5.42



7 days
5.25
5.34
5.49
5.35



14/18 days   
5.37
5.34
5.67
5.36









As shown in Tables 48 and 49, the administration of modified RNA had an effect on serum chemistry parameters including alanine transaminase (ALT) and aspartate transaminase (AST).







TABLE 48







Pharmacologic Effect of EPO mRNA on Alanine Transaminase














Male NHP (G-CSF)
Female NHP (G-CSF)
Male NHP (EPO)
Female NHP (EPO)


Dose (mg/kg)
Time
ALT (U/L)
ALT (U/L)
ALT (U/L)
ALT (U/L)















0.5
Pre-dose
29
216
50
31



2 days
63
209
98
77



4 days
70
98
94
87



7 days
41
149
60
59



14 days 
43
145
88
44


0.05
Pre-dose
58
53
56
160



2 days
82
39
95
254



4 days
88
56
70
200



7 days
73
73
64
187



14 days 
50
31
29
216


0.005
Pre-dose
43
51
45
45



2 days
39
32
62
48



4 days
48
58
48
50



7 days
29
55
21
48



14 days 
44
46
43
51
















TABLE 49







Pharmacologic Effect of EPO mRNA on Aspartate Transaminase














Male NHP (G-CSF)
Female NHP (G-CSF)
Male NHP (EPO)
Female NHP (EPO)


Dose (mg/kg)
Time
AST (U/L)
AST (U/L)
AST (U/L)
AST (U/L)















0.5
Pre-dose
32
47
59
20



2 days
196
294
125
141



4 days
67
63
71
60



7 days
53
68
56
47



14 days 
47
67
82
44


0.05
Pre-dose
99
33
74
58



2 days
95
34
61
80



4 days
69
42
48
94



7 days
62
52
53
78



14 days 
59
20
32
47


0.005
Pre-dose
35
54
39
40



2 days
70
34
29
25



4 days
39
36
43
55



7 days
28
31
55
31



14 days 
39
20
35
54









As shown in Table 50, the administration of lipid nanoparticle-formulated modified RNA at high doses (0.5 mg/kg) caused an increase in cytokines, interferon-alpha (IFN-alpha) after administration of modified mRNA.







TABLE 50







Pharmacologic Effect of EPO mRNA on Alanine Transaminase














Male NHP
Female NHP
Male NHP
Female NHP




(G-CSF)
(G-CSF)
(EPO)
(EPO)


Dose

IFN-alpha
IFN-alpha
IFN-alpha
IFN-alpha


(mg/kg)
Time
(pg/mL)
(pg/mL)
(pg/mL)
(pg/mL)















0.5
Pre-dose
0
0
0
0



Day 1 +
503.8
529.2
16.79
217.5



8 hr



4 days
0
0
0
0


0.05
Pre-dose
0
0
0
0



Day 1 +
0
0
0
0



8 hr



4 days
0
0
0
0


0.005
Pre-dose
0
0
0
0



Day 1 +
0
0
0
0



8 hr



4 days
0
0
0
0









Example 24. Study of Intramuscular and/or Subcutaneous Administration in Non-Human Primates
Formulations containing modified EPO mRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) or G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) in saline were administered to non-human primates (Cynomolgus monkey) (NHP) intramuscularly (IM) or subcutaneously (SC). The single modified mRNA dose of 0.05 mg/kg or 0.005 mg/kg was in a dose volume of 0.5 mL/kg. The non-human primates are bled 5-6 days prior to dosing to determine serum protein concentration and a baseline complete blood count. After administration with the modified mRNA formulation the NHP are bled at 8 hours, 24 hours, 48 hours, 72 hours, 7 days and 14 days to determined protein expression. Protein expression of G-CSF and EPO is determined by ELISA. At 24 hours, 72 hours, 7 days and 14 days after administration the complete blood count of the NHP is also determined. Urine from the NHPs is collected over the course of the entire experiment and analyzed to evaluate clinical safety. Tissue near the injection site is also collected and analyzed to determine protein expression.
Example 25. Modified mRNA Trafficking
In order to determine localization and/or trafficking of the modified mRNA, studies may be performed as follows.
LNP formulations of siRNA and modified mRNA are formulated according to methods known in the art and/or described herein. The LNP formulations may include at least one modified mRNA which may encode a protein such as G-CSF, EPO, Factor VII, and/or any protein described herein. The formulations may be administered locally into muscle of mammals using intramuscular or subcutaneous injection. The dose of modified mRNA and the size of the LNP may be varied to determine the effect on trafficking in the body of the mammal and/or to assess the impact on a biologic reaction such as, but not limited to, inflammation. The mammal may be bled at different time points to determine the expression of protein encoded by the modified mRNA administered present in the serum and/or to determine the complete blood count in the mammal.
For example, modified mRNA encoding Factor VII, expressed in the liver and secreted into the serum, may be administered intramuscularly and/or subcutaneously. Coincident or prior to modified mRNA administration, siRNA is administered to knock out endogenous Factor VII. Factor VII arising from the intramuscular and/or subcutaneous injection of modified mRNA is administered is measured in the blood. Also, the levels of Factor VII is measured in the tissues near the injection site. If Factor VII is expressed in blood then there is trafficking of the modified mRNA. If Factor VII is expressed in tissue and not in the blood than there is only local expression of Factor VII.
Example 26. Formulations of Multiple Modified mRNA
LNP formulations of modified mRNA are formulated according to methods known in the art and/or described herein or known in the art. The LNP formulations may include at least one modified mRNA which may encode a protein such as G-CSF, EPO, thrombopoietin and/or any protein described herein. The at least one modified mRNA may include 1, 2, 3, 4 or 5 modified mRNA molecules. The formulations containing at least one modified mRNA may be administered intravenously, intramuscularly or subcutaneously in a single or multiple dosing regimens. Biological samples such as, but not limited to, blood and/or serum may be collected and analyzed at different time points before and/or after administration of the at least one modified mRNA formulation. An expression of a protein in a biological sample of 50-200 pg/ml after the mammal has been administered a formulation containing at least one modified mRNA encoding said protein would be considered biologically effective.
Example 27. Polyethylene Glycol Ratio Studies
A. Formulation and Characterization of PEG LNPs
Lipid nanoparticles (LNPs) were formulated using the syringe pump method. The LNPs were formulated at a 20:1 weight ratio of total lipid to modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine). The molar ratio ranges of the formulations are shown in Table 51.







TABLE 51







Molar Ratios












DLin-


PEG-c-



KC2-DMA
DSPC
Cholesterol
DOMG















Mole Percent
50.0
10.0
37-38.5
1.5-3


(mol %)









Two types of PEG lipid, 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DMG, NOF Cat # SUNBRIGHT® GM-020) and 1,2-Distearoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DSG, NOF Cat # SUNBRIGHT® GS-020), were tested at 1.5 or 3.0 mol %. After the formation of the LNPs and the encapsulation of the modified G-CSF mRNA, the LNP formulations were characterized by particle size, zeta potential and encapsulation percentage and the results are shown in Table 52.







TABLE 52







Characterization of LNP Formulations









Formulation No.












NPA-071-1
NPA-072-1
NPA-073-1
NPA-074-1















Lipid
PEG-DMG
PEG-DMG
PEG-DSA
PEG-DSA



1.5%
3%
1.5%
3%


Mean Size
95 nm
85 nm
95 nm
75 nm



PDI: 0.01
PDI: 0.06
PDI: 0.08
PDI: 0.08


Zeta at pH 7.4
−1.1 mV
−2.6 mV
1.7 mV
0.7 mV


Encapsulation
88%
89%
98%
95%


(RiboGreen)









B. In Vivo Screening of PEG LNPs
Formulations of the PEG LNPs described in Table 52 were administered to mice (n=5) intravenously at a dose of 0.5 mg/kg. Serum was collected from the mice at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours and 8 days after administration of the formulation. The serum was analyzed by ELISA to determine the protein expression of G-CSF and the expression levels are shown in Table 53. LNP formulations using PEG-DMG gave substantially higher levels of protein expression than LNP formulations with PEG-DSA.







TABLE 53







Protein Expression











Formulation

Protein Expression


Lipid
No.
Time
(pg/ml)













PEG-DMG, 1.5%
NPA-071-1
 2 hours
114,102




 8 hours
357,944




24 hours
104,832




48 hours
6,697




72 hours
980




 8 days
0


PEG-DMG, 3%
NPA-072-1
 2 hours
154,079




 8 hours
354,994




24 hours
164,311




48 hours
13,048




72 hours
1,182




 8 days
13


PEG-DSA, 1.5%
NPA-073-1
 2 hours
3,193




 8 hours
6,162




24 hours
446




48 hours
197




72 hours
124




 8 days
5


PEG-DSA, 3%
NPA-074-1
 2 hours
259




 8 hours
567




24 hours
258




48 hours
160




72 hours
328




 8 days
33









Example 27. Cationic Lipid Formulation Studies
A. Formulation and Characterization of Cationic Lipid Nanoparticles
Lipid nanoparticles (LNPs) were formulated using the syringe pump method. The LNPs were formulated at a 20:1 weight ratio of total lipid to modified mRNA. The final lipid molar ratio ranges of cationic lipid, DSPC, cholesterol and PEG-c-DOMG are outlined in Table 54.







TABLE 54







Molar Ratios












Cationic


PEG-c-



Lipid
DSPC
Cholesterol
DOMG

















Mole Percent
50.0
10.0
38.5
1.5



(mol %)










A 25 mM lipid solution in ethanol and modified RNA in 50 mM citrate at a pH of 3 were mixed to create spontaneous vesicle formation. The vesicles were stabilized in ethanol before the ethanol was removed and there was a buffer exchange by dialysis. The LNPs were then characterized by particle size, zeta potential, and encapsulation percentage. Table 55 describes the characterization of LNPs encapsulating EPO modified mRNA (mRNA sequence shown in SEQ ID NO: 5658 polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) or G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) using DLin-MC3-DMA, DLin-DMA or C12-200 as the cationic lipid.







TABLE 55







Characterization of Cationic Lipid Formulations









Formulation No.














NPA-
NPA-
NPA-
NPA-
NPA-
NPA-



071-1
072-1
073-1
074-1
075-1
076-1





Lipid
DLin-
DLin-
DLin-
DLin-
C12-200
C12-200



MC3-
MC3-
DMA
DMA





DMA
DMA






Modified
EPO
G-CSF
EPO
G-CSF
EPO
G-CSF


RNA








Mean Size
89 nm
96 nm
70 nm
73 nm
97 nm
103 nm



PDI: 0.07
PDI: 0.08
PDI: 0.04
PDI: 0.06
PDI: 0.05
PDI: 0.09


Zeta at pH 7.4
−1.1 mV
−1.4 mV
−1.6 mV
−0.4 mV
1.4 mV
0.9 mV


Encapsulation
100%
100%
99%
100%
88%
98%


(RiboGreen)









B. In Vivo Screening of Cationic LNP Formulations
Formulations of the cationic lipid formulations described in Table 55 were administered to mice (n=5) intravenously at a dose of 0.5 mg/kg. Serum was collected from the mice at 2 hours, 24 hours, 72 hours and/or 7 days after administration of the formulation. The serum was analyzed by ELISA to determine the protein expression of EPO or G-CSF and the expression levels are shown in Table 56.







TABLE 56







Protein Expression















Protein



Modified
Formulation

Expression



mRNA
No.
Time
(pg/ml)
















EPO
NPA-071-1
 2 hours
304,190.0





24 hours
166,811.5





72 hours
1,356.1





 7 days
20.3



EPO
NPA-073-1
 2 hours
73,852.0





24 hours
75,559.7





72 hours
130.8



EPO
NPA-075-1
 2 hours
413,010.2





24 hours
56,463.8



G-CSF
NPA-072-1
 2 hours
62,113.1





24 hours
53,206.6



G-CSF
NPA-074-1
24 hours
25,059.3



G-CSF
NPA-076-1
 2 hours
219,198.1





24 hours
8,470.0










Toxcity was seen in the mice administered the LNPs formulations with the cationic lipid C12-200 (NPA-075-1 and NPA-076-1) and they were sacrificed at 24 hours because they showed symptoms such as scrubby fur, cowering behavior and weight loss of greater than 10%. C12-200 was expected to be more toxic but also had a high level of expression over a short period. The cationic lipid DLin-DMA (NPA-073-1 and NPA-074-1) had the lowest expression out of the three cationic lipids tested. DLin-MC3-DMA (NPA-071-1 and NPA-072-1) showed good expression up to day three and was above the background sample out to day 7 for EPO formulations.
Example 29. Method of Screening for Protein Expression
A. Electrospray Ionization
A biological sample which may contain proteins encoded by modified RNA administered to the subject is prepared and analyzed according to the manufacturer protocol for electrospray ionization (ESI) using 1, 2, 3 or 4 mass analyzers. A biologic sample may also be analyzed using a tandem ESI mass spectrometry system.
Patterns of protein fragments, or whole proteins, are compared to known controls for a given protein and identity is determined by comparison.
B. Matrix-Assisted Laser Desorption/Ionization
A biological sample which may contain proteins encoded by modified RNA administered to the subject is prepared and analyzed according to the manufacturer protocol for matrix-assisted laser desorption/ionization (MALDI).
Patterns of protein fragments, or whole proteins, are compared to known controls for a given protein and identity is determined by comparison.
C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry
A biological sample, which may contain proteins encoded by modified RNA, may be treated with a trypsin enzyme to digest the proteins contained within. The resulting peptides are analyzed by liquid chromatography-mass spectrometry-mass spectrometry (LC/MS/MS). The peptides are fragmented in the mass spectrometer to yield diagnostic patterns that can be matched to protein sequence databases via computer algorithms. The digested sample may be diluted to achieve 1 ng or less starting material for a given protein. Biological samples containing a simple buffer background (e.g. water or volatile salts) are amenable to direct in-solution digest; more complex backgrounds (e.g. detergent, non-volatile salts, glycerol) require an additional clean-up step to facilitate the sample analysis.
Patterns of protein fragments, or whole proteins, are compared to known controls for a given protein and identity is determined by comparison.
Example 30. Lipid Nanoparticle In Vivo Studies
mCherry mRNA (mRNA sequence shown in SEQ ID NO: 5663; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) was formulated as a lipid nanoparticle (LNP) using the syringe pump method. The LNP was formulated at a 20:1 weight ratio of total lipid to modified mRNA with a final lipid molar ratio of 50:10:38.5:1.5 (DLin-KC2-DMA: DSPC: Cholesterol: PEG-c-DOMG). The mCherry formulation, listed in Table 57, was characterized by particle size, zeta potential, and encapsulation.







TABLE 57







mCherry Formulation










Formulation #
NPA-003-5







Modified mRNA
mCherry



Mean size
105 nm




PDI: 0.09



Zeta at pH 7.4
1.8 mV



Encaps. (RiboGr)
100%










The LNP formulation was administered to mice (n=5) intravenously at a modified mRNA dose of 100 ug. Mice were sacrificed at 24 hrs after dosing. The liver and spleen from the mice administered with mCherry modified mRNA formulations were analyzed by immunohistochemistry (IHC), western blot, or fluorescence-activated cell sorting (FACS).
Histology of the liver showed uniform mCherry expression throughout the section, while untreated animals did not express mCherry. Western blots were also used to confirm mCherry expression in the treated animals, whereas mCherry was not detected in the untreated animals. Tubulin was used as a control marker and was detected in both treated and untreated mice, indicating that normal protein expression in hepatocytes was unaffected.
FACS and IHC were also performed on the spleens of mCherry and untreated mice. All leukocyte cell populations were negative for mCherry expression by FACS analysis. By IHC, there were also no observable differences in the spleen in the spleen between mCherry treated and untreated mice.
Example 31. Syringe Pump In Vivo Studies
mCherry modified mRNA (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) is formulated as a lipid nanoparticle (LNP) using the syringe pump method. The LNP is formulated at a 20:1 weight ratio of total lipid to modified mRNA with a final lipid molar ratio of 50:10:38.5:1.5 (DLin-KC2-DMA: DSPC: Cholesterol: PEG-c-DOMG). The mCherry formulation is characterized by particle size, zeta potential, and encapsulation.
The LNP formulation is administered to mice (n=5) intravenously at a modified mRNA dose of 10 or 100 ug. Mice are sacrificed at 24 hrs after dosing. The liver and spleen from the mice administered with mCherry modified mRNA formulations are analyzed by immunohistochemistry (IHC), western blot, and/or fluorescence-activated cell sorting (FACS).
Example 32. In Vitro and In Vivo Expression
A. In Vitro Expression in Human Cells Using Lipidoid Formulations
The ratio of mmRNA to lipidoid used to test for in vitro transfection is tested empirically at different lipidoid:mmRNA ratios. Previous work using siRNA and lipidoids have utilized 2.5:1, 5:1, 10:1, and 15:1 lipidoid:siRNA wt:wt ratios. Given the longer length of mmRNA relative to siRNA, a lower wt:wt ratio of lipidoid to mmRNA may be effective. In addition, for comparison mmRNA were also formulated using RNAIMAXTM (Invitrogen, Carlsbad, Calif.) or TRANSIT-mRNA (Mirus Bio, Madison, Wis.) cationic lipid delivery vehicles.
The ability of lipidoid-formulated Luciferase (IVT cDNA sequence as shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site), green fluorescent protein (GFP) (IVT cDNA sequence as shown in SEQ ID NO: 5666; mRNA sequence shown in SEQ ID NO: 5667, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site), G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1), and EPO mRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) to express the desired protein product can be confirmed by luminescence for luciferase expression, flow cytometry for GFP expression, and by ELISA for G-CSF and Erythropoietin (EPO) secretion.
B. In Vivo Expression Following Intravenous Injection
Systemic intravenous administration of the formulations are created using various different lipidoids including, but not limited to, 98N12-5, C12-200, and MD1.
Lipidoid formulations containing mmRNA are injected intravenously into animals. The expression of the modified mRNA (mmRNA)-encoded proteins are assessed in blood and/or other organs samples such as, but not limited to, the liver and spleen collected from the animal. Conducting single dose intravenous studies will also allow an assessment of the magnitude, dose responsiveness, and longevity of expression of the desired product.
In one embodiment, lipidoid based formulations of 98N12-5, C12-200, MD1 and other lipidoids, are used to deliver luciferase, green fluorescent protein (GFP), mCherry fluorescent protein, secreted alkaline phosphatase (sAP), human G-CSF, human Factor IX, or human Erythropoietin (EPO) mmRNA into the animal. After formulating mmRNA with a lipid, as described previously, animals are divided into groups to receive either a saline formulation, or a lipidoid-formulation which contains one of a different mmRNA selected from luciferase, GFP, mCherry, sAP, human G-CSF, human Factor IX, and human EPO. Prior to injection into the animal, mmRNA-containing lipidoid formulations are diluted in PBS. Animals are then administered a single dose of formulated mmRNA ranging from a dose of 10 mg/kg to doses as low as 1 ng/kg, with a preferred range to be 10 mg/kg to 100 ng/kg, where the dose of mmRNA depends on the animal body weight such as a 20 gram mouse receiving a maximum formulation of 0.2 ml (dosing is based no mmRNA per kg body weight). After the administration of the mmRNA-lipidoid formulation, serum, tissues, and/or tissue lysates are obtained and the level of the mmRNA-encoded product is determined at a single and/or a range of time intervals. The ability of lipidoid-formulated Luciferase, GFP, mCherry, sAP, G-CSF, Factor IX, and EPO mmRNA to express the desired protein product is confirmed by luminescence for the expression of Luciferase, flow cytometry for the expression of GFP and mCherry expression, by enzymatic activity for sAP, or by ELISA for the section of G-CSF, Factor IX and/or EPO.
Further studies for a multi-dose regimen are also performed to determine the maximal expression of mmRNA, to evaluate the saturability of the mmRNA-driven expression (by giving a control and active mmRNA formulation in parallel or in sequence), and to determine the feasibility of repeat drug administration (by giving mmRNA in doses separated by weeks or months and then determining whether expression level is affected by factors such as immunogenicity). An assessment of the physiological function of proteins such as G-CSF and EPO are also determined through analyzing samples from the animal tested and detecting increases in granulocyte and red blood cell counts, respectively. Activity of an expressed protein product such as Factor IX, in animals can also be assessed through analysis of Factor IX enzymatic activity (such as an activated partial thromboplastin time assay) and effect of clotting times.
C. In Vitro Expression Following Intramuscular and/or Subcutaneous Injection
The use of lipidoid formulations to deliver oligonucleotides, including mRNA, via an intramuscular route or a subcutaneous route of injection needs to be evaluated as it has not been previously reported. Intramuscular and/or subcutaneous injection of mmRNA are evaluated to determine if mmRNA-containing lipidoid formulations are capabable to produce both localized and systemic expression of a desired proteins.
Lipidoid formulations of 98N12-5, C12-200, and MD1 containing mmRNA selected from luciferase, green fluorescent protein (GFP), mCherry fluorescent protein, secreted alkaline phosphatase (sAP), human G-CSF, human factor IX, or human Erythropoietin (EPO) mmRNA are injected intramuscularly and/or subcutaneously into animals. The expression of mmRNA-encoded proteins are assessed both within the muscle or subcutaneous tissue and systemically in blood and other organs such as the liver and spleen. Single dose studies allow an assessment of the magnitude, dose responsiveness, and longevity of expression of the desired product.
Animals are divided into groups to receive either a saline formulation or a formulation containing modified mRNA. Prior to injection mmRNA-containing lipidoid formulations are diluted in PBS. Animals are administered a single intramuscular dose of formulated mmRNA ranging from 50 mg/kg to doses as low as 1 ng/kg with a preferred range to be 10 mg/kg to 100 ng/kg. A maximum dose for intramuscular administration, for a mouse, is roughly 1 mg mmRNA or as low as 0.02 ng mmRNA for an intramuscular injection into the hind limb of the mouse. For subcutaneous administration, the animals are administered a single subcutaneous dose of formulated mmRNA ranging from 400 mg/kg to doses as low as 1 ng/kg with a preferred range to be 80 mg/kg to 100 ng/kg. A maximum dose for subcutaneous administration, for a mouse, is roughly 8 mg mmRNA or as low as 0.02 ng mmRNA.
For a 20 gram mouse the volume of a single intramuscular injection is maximally 0.025 ml and a single subcutaneous injection is maximally 0.2 ml. The optimal dose of mmRNA administered is calculated from the body weight of the animal. At various points in time points following the administration of the mmRNA-lipidoid, serum, tissues, and tissue lysates is obtained and the level of the mmRNA-encoded product is determined. The ability of lipidoid-formulated luciferase, green fluorescent protein (GFP), mCherry fluorescent protein, secreted alkaline phosphatase (sAP), human G-CSF, human factor IX, or human Erythropoietin (EPO) mmRNA to express the desired protein product is confirmed by luminescence for luciferase expression, flow cytometry for GFP and mCherry expression, by enzymatic activity for sAP, and by ELISA for G-CSF, Factor IX and Erythropoietin (EPO) secretion.
Additional studies for a multi-dose regimen are also performed to determine the maximal expression using mmRNA, to evaluate the saturability of the mmRNA-driven expression (achieved by giving a control and active mmRNA formulation in parallel or in sequence), and to determine the feasibility of repeat drug administration (by giving mmRNA in doses separated by weeks or months and then determining whether expression level is affected by factors such as immunogenicity). Studies utilizing multiple subcutaneous or intramuscular injection sites at one time point, are also utilized to further increase mmRNA drug exposure and improve protein production. An assessment of the physiological function of proteins, such as GFP, mCherry, sAP, human G-CSF, human factor IX, and human EPO, are determined through analyzing samples from the tested animals and detecting a change in granulocyte and/or red blood cell counts. Activity of an expressed protein product such as Factor IX, in animals can also be assessed through analysis of Factor IX enzymatic activity (such as an activated partial thromboplastin time assay) and effect of clotting times.
Example 33. Bifunctional mmRNA
Using the teachings and synthesis methods described herein, modified RNAs are designed and synthesized to be bifunctional, thereby encoding one or more cytotoxic protein molecules as well as be synthesized using cytotoxic nucleosides.
Administration of the bifunctional modified mRNAs is effected using either saline or a lipid carrier. Once administered, the bifunctional modified mRNA is translated to produce the encoded cytotoxic peptide. Upon degradation of the delivered modified mRNA, the cytotoxic nucleosides are released which also effect therapeutic benefit to the subject.
Example 34. Modified mRNA Transfection
A. Reverse Transfection
For experiments performed in a 24-well collagen-coated tissue culture plate, Keratinocytes are seeded at a cell density of 1×105. For experiments performed in a 96-well collagen-coated tissue culture plate, Keratinocytes are seeded at a cell density of 0.5×105. For each modified mRNA (mmRNA) to be transfected, modified mRNA: RNAIMAXTM is prepared as described and mixed with the cells in the multi-well plate within a period of time, e.g., 6 hours, of cell seeding before cells had adhered to the tissue culture plate.
B. Forward Transfection
In a 24-well collagen-coated tissue culture plate, Keratinocytes are seeded at a cell density of 0.7×105. For experiments performed in a 96-well collagen-coated tissue culture plate, Keratinocytes are seeded at a cell density of 0.3×105. Keratinocytes are grown to a confluency of >70% for over 24 hours. For each modified mRNA (mmRNA) to be transfected, modified mRNA: RNAIMAXTM is prepared as described and transfected onto the cells in the multi-well plate over 24 hours after cell seeding and adherence to the tissue culture plate.
C. Modified mRNA Translation Screen: G-CSF ELISA
Keratinocytes are grown in EPILIFE medium with Supplement S7 from Invitrogen (Carlsbad, Calif.) at a confluence of >70%. One set of keratinocytes were reverse transfected with 300 ng of the chemically modified mRNA (mmRNA) complexed with RNAIMAXTM from Invitrogen. Another set of keratinocytes are forward transfected with 300 ng modified mRNA complexed with RNAIMAXTM from Invitrogen. The modified mRNA: RNAIMAXTM complex is formed by first incubating the RNA with Supplement-free EPILIFE® media in a 5× volumetric dilution for 10 minutes at room temperature.
In a second vial, RNAIMAXTM reagent was incubated with Supplement-free EPILIFE® Media in 10× volumetric dilution for 10 minutes at room temperature. The RNA vial was then mixed with the RNAIMAXTM vial and incubated for 20-30 minutes at room temperature before being added to the cells in a drop-wise fashion. Secreted human Granulocyte-Colony Stimulating Factor (G-CSF) concentration in the culture medium is measured at 18 hours post-transfection for each of the chemically modified mRNA in triplicate.
Secretion of Human G-CSF from transfected human keratinocytes is quantified using an ELISA kit from Invitrogen or R&D Systems (Minneapolis, Minn.) following the manufacturers recommended instructions.
D. Modified mRNA Dose and Duration: G-CSF ELISA
Keratinocytes are grown in EPILIFE® medium with Supplement S7 from Invitrogen at a confluence of >70%. Keratinocytes are reverse transfected with either 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng modified mRNA complexed with the RNAIMAXTM from Invitrogen (Carlsbad, Calif.). The modified mRNA:RNAIMAXTM complex is formed as described. Secreted human G-CSF concentration in the culture medium is measured at 0, 6, 12, 24, and 48 hours post-transfection for each concentration of each modified mRNA in triplicate. Secretion of human G-CSF from transfected human keratinocytes is quantified using an ELISA kit from Invitrogen or R&D Systems following the manufacturers recommended instructions.
Example 35. Split Dose Studies
Studies utilizing multiple subcutaneous or intramuscular injection sites at one time point were designed and performed to investigate ways to increase mmRNA drug exposure and improve protein production. In addition to detection of the expressed protein product, an assessment of the physiological function of proteins was also determined through analyzing samples from the animal tested.
Surprisingly, it has been determined that split dosing of mmRNA produces greater protein production and phenotypic responses than those produced by single unit dosing or multi-dosing schemes.
The design of a single unit dose, multi-dose and split dose experiment involved using human erythropoietin (EPO) mmRNA (mRNA shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) administered in buffer alone. The dosing vehicle (F. buffer) consisted of 150 mM NaCl, 2 mM CaCl2, 2 mM Na+-phosphate (1.4 mM monobasic sodium phosphate; 0.6 mM dibasic sodium phosphate), and 0.5 mM EDTA, pH 6.5. The pH was adjusted using sodium hydroxide and the final solution was filter sterilized. The mmRNA was modified with 5meC at each cytosine and pseudouridine replacement at each uridine site.
Animals (n=5) were injected IM (intramuscular) for the single unit dose of 100 ug. For multi-dosing, two schedules were used, 3 doses of 100 ug and 6 doses of 100 ug. For the split dosing scheme, two schedules were used, 3 doses at 33.3 ug and 6 doses of 16.5 ug mmRNA. Control dosing involved use of buffer only at 6 doses. Control mmRNA involved the use of luciferase mmRNA (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) dosed 6 times at 100 ug. Blood and muscle tissue were evaluated 13 hrs post injection.
Human EPO protein was measured in mouse serum 13 h post I.M. single, multi- or split dosing of the EPO mmRNA in buffer. Seven groups of mice (n=5 mice per group) were treated and evaluated. The results are shown in Table 58.







TABLE 58







Split dose study

















Avg.
Poly-







pmol/
peptide







mL
per unit
Dose




Dose of
Total
human 
drug
Splitting


Group
Treatment
mmRNA
Dose
EPO
(pmol/ug)
Factor
















1
Human EPO
1 × 
100 ug
14.3
.14
1



mmRNA
100 ug






2
Human EPO
3 × 
300 ug
82.5
.28
2



mmRNA
100 ug






3
Human EPO
6 × 
600 ug
273.0
.46
3.3



mmRNA
100 ug






4
Human EPO
3 ×
100 ug
104.7
1.1
7.9



mmRNA
33.3 ug 






5
Human EPO
6 × 
100 ug
127.9
1.3
9.3



mmRNA
16.5 ug 






6
Luciferase
6 × 
600 ug 
0
—
—



mmRNA
100 ug






7
Buffer Alone
—
—
0
—
—









The splitting factor is defined as the product per unit drug divided by the single dose product per unit drug (PUD). For example for treatment group 2 the value 0.28 or product (EPO) per unit drug (mmRNA) is divided by the single dose product per unit drug of 0.14. The result is 2. Likewise, for treatment group 4, the value 1.1 or product (EPO) per unit drug (mmRNA) is divided by the single dose product per unit drug of 0.14. The result is 7.9. Consequently, the dose splitting factor (DSF) may be used as an indicator of the efficacy of a split dose regimen. For any single administration of a total daily dose, the DSF should be equal to 1. Therefore any DSF greater than this value in a split dose regimen is an indication of increased efficacy.
To determine the dose response trends, impact of injection site and impact of injection timing, studies are performed. In these studies, varied doses of 1 ug, 5 ug, 10 ug, 25 ug, 50 ug, and values in between are used to determine dose response outcomes. Split dosing for a 100 ug total dose includes three or six doses of 1.6 ug, 4.2 ug, 8.3 ug, 16.6 ug, or values and total doses equal to administration of the total dose selected.
Injection sites are chosen from the limbs or any body surface presenting enough area suitable for injection. This may also include a selection of injection depth to target the dermis (Intradermal), epidermis (Epidermal), subcutaneous tissue (SC) or muscle (IM). Injection angle will vary based on targeted delivery site with injections targeting the intradermal site to be 10-15 degree angles from the plane of the surface of the skin, between 20-45 degrees from the plane of the surface of the skin for subcutaneous injections and angles of between 60-90 degrees for injections substantially into the muscle.
Example 36. Quantification in Exosomes
The quantity and localization of the mmRNA of the present invention can be determined by measuring the amounts (initial, timecourse, or residual basis) in isolated exosomes. In this study, since the mmRNA are typically codon-optimized and distinct in sequence from endogenous mRNA, the levels of mmRNA are quantitated as compared to endogenous levels of native or wild type mRNA by using the methods of Gibbings, PCT/IB2009/005878, the contents of which are incorporated herein by reference in their entirety.
In these studies, the method is performed by first isolating exosomes or vesicles preferably from a bodily fluid of a patient previously treated with a polynucleotide, primary construct or mmRNA of the invention, then measuring, in said exosomes, the polynucleotide, primary construct or mmRNA levels by one of mRNA microarray, qRT-PCR, or other means for measuring RNA in the art including by suitable antibody or immunohistochemical methods.
Example 37. Effect of Modified mRNA on Cellular Viability, Cytotoxicity and Apoptosis
This experiment demonstrates cellular viability, cytotoxicity and apoptosis for distinct modified mRNA in-vitro transfected Human Keratinocyte cells. Keratinocytes are grown in EPILIFE® medium with Human Keratinocyte Growth Supplement in the absence of hydrocortisone from Invitrogen (Carlsbad, Calif.) at a confluence of >70%. Keratinocytes are reverse transfected with 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, 1500 ng, 3000 ng, or 6000 ng of modified mRNA complexed with RNAIMAXTM from Invitrogen. The modified mRNA:RNAIMAXTM complex is formed. Secreted human G-CSF concentration in the culture medium is measured at 0, 6, 12, 24, and 48 hours post-transfection for each concentration of each modified m RNA in triplicate. Secretion of human G-CSF from transfected human keratinocytes is quantified using an ELISA kit from Invitrogen or R&D Systems following the manufacturers recommended instructions.
Cellular viability, cytotoxicity and apoptosis is measured at 0, 12, 48, 96, and 192 hours post-transfection using the APOTOX-GLOTM kit from Promega (Madison, Wis.) according to manufacturer instructions.
Example 38. Detection of a Cellular Innate Immune Response to Modified mRNA Using an ELISA Assay
An enzyme-linked immunosorbent assay (ELISA) for Human Tumor Necrosis Factor-α (TNF-α), Human Interferon-β (IFN-β) and Human Granulocyte-Colony Stimulating Factor (G-CSF) secreted from in vitro-transfected Human Keratinocyte cells is tested for the detection of a cellular innate immune response. Keratinocytes are grown in EPILIFE® medium with Human Keratinocyte Growth Supplement in the absence of hydrocortisone from Invitrogen (Carlsbad, Calif.) at a confluence of >70%. Secreted TNF-α keratinocytes are reverse transfected with 0 ng, 93.75 ng, 1 87.5 ng, 375 ng, 750 ng, 1500 ng or 3000 ng of the chemically modified mRNA (mmRNA) complexed with RNAIMAXTM from Invitrogen as described in triplicate. Secreted TNF-α in the culture medium is measured 24 hours post-transfection for each of the chemically modified mRNA using an ELISA kit from Invitrogen according to the manufacturer protocols.
Secreted IFN-β in the same culture medium is measured 24 hours post-transfection for each of the chemically modified mRNA using an ELISA kit from Invitrogen according to the manufacturer protocols. Secreted human G-CSF concentration in the same culture medium is measured at 24 hours post-transfection for each of the chemically modified mRNA. Secretion of human G-CSF from transfected human keratinocytes is quantified using an ELISA kit from Invitrogen or R&D Systems (Minneapolis, Minn.) following the manufacturers recommended instructions. These data indicate which modified mRNA (mmRNA) are capable eliciting a reduced cellular innate immune response in comparison to natural and other chemically modified polynucleotides or reference compounds by measuring exemplary type 1 cytokines TNF-α and IFN-β.
Example 39. Human Granulocyte—Colony Stimulating Factor (G-CSF) Modified mRNA-Induced Cell Proliferation Assay
Human keratinocytes are grown in EPILIFE® medium with Supplement S7 from Invitrogen at a confluence of >70% in a 24-well collagen-coated TRANSWELL® (Coming, Lowell, Mass.) co-culture tissue culture plate. Keratinocytes are reverse transfected with 750 ng of the indicated chemically modified mRNA (mmRNA) complexed with RNAIMAX from Invitrogen as described in triplicate. The modified mRNA:RNAIMAX complex is formed as described. Keratinocyte media is exchanged 6-8 hours post-transfection. 42-hours post-transfection, the 24-well TRANSWELL® plate insert with a 0.4 μm-pore semi-permeable polyester membrane is placed into the human G-CSF modified mRNA-transfected keratinocyte containing culture plate
Human myeloblast cells, Kasumi-1 cells or KG-1 (0.2×105 cells), are seeded into the insert well and cell proliferation is quantified 42 hours post-co-culture initiation using the CyQuant Direct Cell Proliferation Assay (Invitrogen, Carlsbad, Calif.) in a 100-120 μl volume in a 96-well plate. Modified mRNA-encoding human G-CSF-induced myeloblast cell proliferation is expressed as a percent cell proliferation normalized to untransfected keratinocyte/myeloblast co-culture control wells. Secreted human G-CSF concentration in both the keratinocyte and myeloblast insert co-culture wells is measured at 42 hours post-co-culture initiation for each modified mRNA in duplicate. Secretion of human G-CSF is quantified using an ELISA kit from Invitrogen following the manufacturer recommended instructions.
Transfected human G-CSF modified mRNA in human keratinocyte feeder cells and untransfected human myeloblast cells are detected by RT-PCR. Total RNA from sample cells is extracted and lysed using RNEASY® kit (Qiagen, Valencia, Calif.) according to the manufacturer instructions. Extracted total RNA is submitted to RT-PCR for specific amplification of modified mRNA-G-CSF using PROTOSCRIPT® M-MuLV Taq RT-PCR kit (New England BioLabs, Ipswich, Mass.) according to the manufacturer instructions with human G-CSF-specific primers. RT-PCR products are visualized by 1.2% agarose gel electrophoresis.
Example 40: Co-Culture Assay
Modified mRNA comprised of chemically-distinct modified nucleotides encoding human Granulocyte-Colony Stimulating Factor (G-CSF) may stimulate the cellular proliferation of a transfection incompetent cell in a co-culture environment. The co-culture includes a highly transfectable cell type such as a human keratinocyte and a transfection incompetent cell type such as a white blood cell (WBC). The modified mRNA encoding G-CSF are transfected into the highly transfectable cell allowing for the production and secretion of G-CSF protein into the extracellular environment where G-CSF acts in a paracrine-like manner to stimulate the white blood cell expressing the G-CSF receptor to proliferate. The expanded WBC population may be used to treat immune-compromised patients or partially reconstitute the WBC population of an immunosuppressed patient and thus reduce the risk of opportunistic infections.
In another example, a highly transfectable cell such as a fibroblast are transfected with certain growth factors support and simulate the growth, maintenance, or differentiation of poorly transfectable embryonic stem cells or induced pluripotent stem cells.
Example 41: Detection Assays of Human IgG Antibodies
A. ELISA Detection of Human IgG Antibodies
This example describes an ELISA for Human IgG from Chinese Hamster Ovary's (CHO) and Human Embryonic Kidney (HEK, HER-2 Negative) 293 cells transfected with human IgG modified mRNA (mmRNA). The Human Embryonic Embryonic Kidney (HEK) 293 are grown in CD 293 Medium with Supplement of L-Glutamine from Invitrogen until they reach a confluence of 80-90%. The CHO cells are grown in CD CHO Medium with Supplement of L-Glutamine, Hypoxanthine and Thymidine. In one aspect, 2×106 cells are transfected with 24 μg modified mRNA complexed with RNAIMAXTM from Invitrogen in a 75 cm2 culture flask from Corning in 7 ml of medium. In another aspect, 80,000 cells are transfected with 1 μg modified mRNA complexed with RNAIMAXTM from Invitrogen in a 24-well plate. The modified mRNA:RNAIMAXTM complex is formed by incubating in a vial the mmRNA with either the CD 293 or CD CHO medium in a 5× volumetric dilution for 10 minutes at room temperature. In a second vial, RNAIMAXTM reagent is incubated with CD 293 medium or CD CHO medium in a 10× volumetric dilution for 10 minutes at room temperature. The mmRNA vial is then mixed with the RNAIMAXTM vial and incubated for 20-30 minutes at room temperature before it is added to the CHO or HEK cells in a drop-wise fashion. The culture supernatants are stored at 4 degrees Celsius. The concentration of the secreted human IgG in the culture medium in the 24 μg mmRNA transfections is measured at 12, 24, 36 hours post-transfection and the 1 μg mmRNA transfection is measured at 36 hours. Secretion of Trastuzumab from transfected HEK 293 cells is quantified using an ELISA kit from Abcam (Cambridge, Mass.) following the manufacturers recommended instructions. The data shows that a Humanized IgG antibody (such as Trastuzumab) mmRNA is capable of being translated in HEK Cells and that Trastuzumab is secreted out of the cells and released into the extracellular environment. Furthermore, the data demonstrate that transfection of cells with mmRNA encoding Trastuzumab for the production of secreted protein can be scaled up to a bioreactor or large cell culture conditions.
B. Western Detection of Modified mRNA Produced Human IgG Antibody
A Western Blot of CHO-K1 cells is co-transfected with 1 μg each of Heavy and Light Chain of Trastuzumab modified mRNA (mmRNA). CHO cells are grown using standard protocols in 24-well plates. The cell supernatants or cell lysates are collected 24 hours post-transfection, separated on a 12% SDS-Page gel and transferred onto a nitrocellulose membrane using the IBOT® by Invitrogen (Carlsbad, Calif.). The cells are incubated with a first conjugation of a rabbit polyclonal antibody to Human IgG conjugated to DYLIGHT594 (ab96904, abcam, Cambridge, Mass.) and a second conjugation of a goat polyclonal antibody to Rb IgG which is conjugated to alkaline phosphatase. After incubation, the antibody is detected using Novex® alkaline phosphatase chromogenic substrate by Invitrogen (Carlsbad, Calif.).
C. Cell Immuno Staining of Modified mRNA Produced Trastuzumab and Rituximab
CHO-K1 cells are co-transfected with 500 ng each of Heavy and Light Chain of either Trastuzumab or Rituximab. Cells are grown in F-12K Medium from GIBCO® (Grand Island, N.Y.) and 10% FBS. Cells are fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS for 5-10 minutes at room temperature and cells are washed 3 times with room temperature PBS. Trastuzumab and Rituximab staining is performed using rabbit polyclonal antibody to Human IgG conjugated to DYLIGHT®594 (ab96904, abcam, Cambridge, Mass.) according to the manufacture's recommended dilutions. Nuclear DNA staining is performed with DAPI dye from Invitrogen (Carlsbad, Calif.). The protein for Trastuzumab and Rituximab is translated and localized to the cytoplasm upon modified mRNA transfections. Pictures are taken 13 hours post-transfection.
D. Binding Immunoblot Assay for Modified mRNA Produced Trastuzumab and Rituximab
Trastuzumab and Rituximab are detected using a binding immunoblot detection assay. Varying concentrations (100 ng/ul to 0 ng/ul) of the ErB2 peptide (ab40048, abeam, Cambridge, Mass.), antigen for Trastuzumab and the CD20 peptide (ab97360, abeam, Cambridge, Mass.), antigen for Rituximab are run on a 12% SDS-Page gel and transferred onto a membrane using the iBlot from Invitrogen. The membranes are incubated for 1 hour with their respective cell supernatants from CHO-K1 cells which are co-transfected with 500 ng each of Heavy and Light Chain of either Trastuzumab or Rituximab. The membranes are blocked with 1% BSA and a secondary anti-human IgG antibody conjugated to alkaline phosphatase (abcam, Cambridge, Mass.) is added. Antibody detection is conducted using the NOVEX alkaline phosphatase chromogenic substrate by Invitrogen (Carlsbad, Calif.). The data shows that a humanized IgG antibodies generated from modified mRNA is capable of recognizing and binding to their respective antigens.
E. Cell Proliferation Assay
The SK-BR-3 cell line, an adherent cell line derived from a human breast adenocarcinoma, which overexpresses the HER2/neu receptor can be used to compare the anti-proliferative properties of modified mRNA (mmRNA) generated Trastuzumab. Varying concentrations of purified Trastuzumab generated from modified mRNA and trastuzumab are be added to cell cultures, and their effects on cell growth are be assessed in triplicate cytotoxicity and viability assays.
Example 42: Bulk Transfection of Modified mRNA into Cell Culture
A. Cationic Lipid Delivery Vehicles
RNA transfections are carried out using RNAIMAXTM (Invitrogen, Carlsbad, Calif.) or TRANSIT-mRNA (Mirus Bio, Madison, Wis.) cationic lipid delivery vehicles. RNA and reagent are first diluted in Opti-MEM basal media (Invitrogen, Carlsbad, Calif.). 100 ng/uL RNA is diluted 5× and 5 μL of RNAIMax per μg of RNA is diluted 10×. The diluted components are pooled and incubated 15 minutes at room temperature before they are dispensed to culture media. For TRANSIT-mRNA transfections, 100 ng/uL RNA is diluted 10× in Opti-MEM and BOOST reagent is added (at a concentration of 2 μL per μg of RNA), TRANSIT-mRNA is added (at a concentration of 2 μL per μg of RNA), and then the RNA-lipid complexes are delivered to the culture media after a 2-minute incubation at room temperature. RNA transfections are performed in Nutristem xenofree hES media (Stemgent, Cambridge, Mass.) for RiPS derivations, Dermal Cell Basal Medium plus Keratinocyte Growth Kit (ATCC) for keratinocyte experiments, and Opti-MEM plus 2% FBS for all other experiments. Successful introduction of a modified mRNA (mmRNA) into host cells can be monitored using various known methods, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Successful transfection of a modified mRNA can also be determined by measuring the protein expression level of the target polypeptide by e.g., Western Blotting or immunocytochemistry. Similar methods may be followed for large volume scale-up to multi-liter (5-10,000 L) culture format following similar RNA-lipid complex ratios.
B. Electroporation Delivery of Exogenous Synthetic mRNA Transcripts
Electroporation parameters are optimized by transfecting MRC-5 fibroblasts with in vitro synthetic modified mRNA (mmRNA) transcripts and measuring transfection efficiency by quantitative RT-PCR with primers designed to specifically detect the exogenous transcripts. Discharging a 150 uF capacitor charged to F into 2.5×106 cells suspended in 50 μl of Opti-MEM (Invitrogen, Carlsbad, Calif.) in a standard electroporation cuvette with a 2 mm gap is sufficient for repeated delivery in excess of 10,000 copies of modified mRNA transcripts per cell, as determined using the standard curve method, while maintaining high viability (>70%). Further experiments may reveal that the voltage required to efficiently transfect cells with mmRNA transcripts can depend on the cell density during electroporation. Cell density may vary from 1×106 cell/50 μl to a density of 2.5×106 cells/50 μl and require from 110V to 145V to transfect cells with similar efficiencies measured in transcript copies per cell. Large multi-liter (5-10,000 L) electroporation may be performed similar to large volume flow electroporation strategies similar to methods described with the above described constraints (Li et al., 2002; Geng et al., 2010).
Example 43: In Vivo Delivery Using Lipoplexes
A. Human EPO Modified RNA Lipoplex
A formulation containing 100 μg of modified human erythropoietin mRNA (mRNA shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) (EPO; fully modified 5-methylcytosine; N1-methyl-pseudouridine) was lipoplexed with 30% by volume of RNAIMAXTM (Lipoplex-h-Epo-46; Generation 2 or Gen2) in 50-70 uL delivered intramuscularly to four C57/BL6 mice. Other groups consisted of mice receiving an injection of the lipoplexed modified luciferase mRNA (Lipoplex-luc) (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) which served as a control containing 100 μg of modified luciferase mRNA was lipoplexed with 30% by volume of RNAiMAXTM or mice receiving an injection of the formulation buffer as negative control at a dose volume of 65 ul. 13 hours after the intramuscular injection, serum was collected from each mouse to measure the amount of human EPO protein in the mouse serum by human EPO ELISA and the results are shown in Table 59.







TABLE 59







Human EPO Production (IM Injection Route)










Formualtion
Average














Lipoplex-h-Epo-46
251.95



Lipoplex-Luc
0



Formulation Buffer
0










B. Human G-CSF Modified RNA Lipoplex
A formulation containing 100 μg of one of the two types of modified human G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) (G-CSF fully modified with 5-methylcytosine and pseudouridine (G-CSF) or G-CSF fully modified with 5-methylcytosine and N1-methyl-pseudouridine (G-CSF-N1) lipoplexed with 30% by volume of RNAIMAXTM and delivered in 150 uL intramuscularly (I.M), in 150 uL subcutaneously (S.C) and in 225 uL intravenously (I.V) to C57/BL6 mice. Three control groups were administered either 100 μg of modified luciferase mRNA (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) intramuscularly (Luc-unsp I.M.) or 150 μg of modified luciferase mRNA intravenously (Luc-unsp I.V.) or 150 uL of the formulation buffer intramuscularly (Buffer I.M.). 6 hours after administration of a formulation, serum was collected from each mouse to measure the amount of human G-CSF protein in the mouse serum by human G-CSF ELISA and the results are shown in Table 60.
These results demonstrate that both 5-methylcytosine/pseudouridine and 5-methylcytosine/N1-methyl-pseudouridine modified human G-CSF mRNA can result in specific human G-CSF protein expression in serum when delivered via I.V. or I.M. route of administration in a lipoplex formulation.







TABLE 60







Human G-CSF in Serum (I.M., I.V., S.C. Injection Route)











Formulation
Route
G-CSF (pg/ml)















G-CSF
I.M.
85.6



G-CSF N1
I.M.
40.1



G-CSF
S.C.
3.9



G-CSF N1
S.C.
0.0



G-CSF
I.V.
31.0



G-CSF N1
I.V.
6.1



Luc-unsp
I.M.
0.0



Luc-unsp
I.V.
0.0



Buffer
I.M.
0.0










C. Human G-CSF Modified RNA Lipoplex Comparison
A formulation containing 100 μg of either modified human G-CSF mRNA lipoplexed with 30% by volume of RNAIMAXTM with a 5-methylcytosine (5mc) and a pseudouridine (ψ) modification (G-CSF-Gen1-Lipoplex), modified human G-CSF mRNA with a 5mc and ψ modification in saline (G-CSF-Gen1-Saline), modified human G-CSF mRNA with a N1-5-methylcytosine (N1-5mc) and a w modification lipoplexed with 30% by volume of RNAIMAXTM (G-CSF-Gen2-Lipoplex), modified human G-CSF mRNA with a N1-5mc and ψ modification in saline (G-CSF-Gen2-Saline), modified luciferase with a 5mc and ψ modification lipoplexed with 30% by volume of RNAIMAXTM (Luc-Lipoplex), or modified luciferase mRNA with a 5mc and ψ modification in saline (Luc-Saline) was delivered intramuscularly (I.M.) or subcutaneously (S.C.) and a control group for each method of administration was giving a dose of 80 uL of the formulation buffer (F. Buffer) to C57/BL6 mice. 13 hours post injection serum and tissue from the site of injection were collected from each mouse and analyzed by G-CSF ELISA to compare human G-CSF protein levels. The results of the human G-CSF protein in mouse serum from the intramuscular administration, and the subcutaneous administration results are shown in Table 61.
These results demonstrate that 5-methylcytosine/pseudouridine and 5-methylcytosine/N1-methyl-pseudouridine modified human G-CSF mRNA can result in specific human G-CSF protein expression in serum when delivered via I.M. or S.C. route of administration whether in a saline formulation or in a lipoplex formulation. As shown in Table 61, 5-methylcytosine/N1-methyl-pseudouridine modified human G-CSF mRNA generally demonstrates increased human G-CSF protein production relative to 5-methylcytosine/pseudouridine modified human G-CSF mRNA.







TABLE 61







Human G-CSF Protein in Mouse Serum









G-CSF (pg/ml)









Formulation
I.M. Injection Route
S.C. Injenction Route












G-CSF-Gen1-Lipoplex
13.988
42.855


G-CSF-Gen1-saline
9.375
4.614


G-CSF-Gen2-lipoplex
75.572
32.107


G-CSF-Gen2-saline
20.190
45.024


Luc lipoplex
0
3.754


Luc saline
0.0748
0


F. Buffer
4.977
2.156









D. mCherry Modified RNA Lipoplex Comparison
Intramuscular and Subcutaneous Administration
A formulation containing 100 μg of either modified mCherry mRNA (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) lipoplexed with 30% by volume of RNAIMAXTM or modified mCherry mRNA in saline is delivered intramuscularly and subcutaneously to mice. A formulation buffer is also administered to a control group of mice either intramuscularly or subcutaneously. The site of injection on the mice may be collected 17 hours post injection for sectioning to determine the cell type(s) responsible for producing protein.
Intravitreal Administration
A formulation containing 10 μg of either modified mCherry mRNA lipoplexed with RNAIMAXTM, modified mCherry mRNA in a formulation buffer, modified luciferase mRNA lipoplexed with RNAMAXTM, modified luciferase mRNA in a formulation buffer can be administered by intravitreal injection (IVT) in rats in a dose volume of 5 μl/eye. A formulation buffer is also administered by IVT to a control group of rats in a dose volume of 5 μl/eye. Eyes from treated rats can be collected after 18 hours post injection for sectioning and lysating to determine whether mmRNA can be effectively delivered in vivo to the eye and result in protein production, and to also determine the cell type(s) responsible for producing protein in vivo.
Intranasal Administration
A formulation containing 100 μg of either modified mCherry mRNA lipoplexed with 30% by volume of RNAIMAXTM, modified mCherry mRNA in saline, modified luciferase mRNA lipoplexed with 30% by volume of RNAIMAXTM or modified luciferase mRNA in saline is delivered intranasally. A formulation buffer is also administered to a control group intranasally. Lungs may be collected about 13 hours post instillation for sectioning (for those receiving mCherry mRNA) or homogenization (for those receiving luciferase mRNA). These samples will be used to determine whether mmRNA can be effectively delivered in vivo to the lungs and result in protein production, and to also determine the cell type(s) responsible for producing protein in vivo.
Example 44: In Vivo Delivery Using Varying Lipid Ratios
Modified mRNA was delivered to C57/BL6 mice to evaluate varying lipid ratios and the resulting protein expression. Formulations of 100 μg modified human EPO mRNA (mRNA shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) lipoplexed with 10%, 30% or 50% RNAIMAXTM, 100 μg modified luciferase mRNA (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) lipoplexed with 10%, 30% or 50% RNAIMAXTM or a formulation buffer were administered intramuscularly to mice in a single 70 μl dose. Serum was collected 13 hours post injection to undergo a human EPO ELISA to determine the human EPO protein level in each mouse. The results of the human EPO ELISA, shown in Table 62, show that modified human EPO expressed in the muscle is secreted into the serum for each of the different percentage of RNAIMAXTM.







TABLE 62







Human EPO Protein in Mouse Serum (IM Injection Route)










Formulation
EPO (pg/ml)














Epo + 10% RNAiMAX
11.4



Luc + 10% RNAiMAX
0



Epo + 30% RNAiMAX
27.1



Luc + 30% RNAiMAX
0



Epo + 50% RNAiMAX
19.7



Luc + 50% RNAiMAX
0



F. Buffer
0










Example 45: Intramuscular and Subcutaneous In Vivo Delivery in Mammals
Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) formulated in saline was delivered to either C57/BL6 mice or Sprague-Dawley rats to evaluate the dose dependency on human EPO production. Rats were intramuscularlly injected with 50 μl of the modified human EPO mRNA (h-EPO), modified luciferase mRNA (Luc) (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) or the formulation buffer (F.Buffer) as described in the dosing chart Table 63.
Mice were intramuscularly or subcutaneously injected with 50 μl of the modified human EPO mRNA (h-EPO), modified luciferase mRNA (Luc) or the formulation buffer (F.Buffer) as described in the dosing chart Table 64. 13 hours post injection blood was collected and serum was analyzed to determine the amount human EPO for each mouse or rat. The average and geometric mean in pg/ml for the rat study are also shown in Table 63.







TABLE 63







Rat Study













Dose
Avg.
Geometric-



Group
(ug)
pg/ml
mean pg/ml

















h-EPO
G#1
150
67.7
67.1



h-EPO
G#2
100
79.4
66.9



h-EPO
G#3
50
101.5
85.4



h-EPO
G#4
10
46.3
31.2



h-EPO
G#5
1
28.7
25.4



Luc
G#6
100
24.5
22.4



F. Buffer
G#7
—
18.7
18.5

















TABLE 64







Mouse Study

















Average Level



Route
Treatment
Group
Dose
in serum pg/ml

















IM
h-EPO
1
100 μg
96.2



IM
h-EPO
2
 50 μg
63.5



IM
h-EPO
3
 25 μg
18.7



IM
h-EPO
4
 10 μg
25.9



IM
h-EPO
5
 1 μg
2.6



IM
Luc
6
100 μg
0



IM
F. Buffer
7
—
1.0



SC
h-EPO
1
100 μg
72.0



SC
Luc
2
100 μg
26.7



SC
F. Buffer
3
—
17.4










Example 46: Duration of Activity after Intramuscular In Vivo Delivery in Rats
Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) formulated in formulation buffer was delivered to Sprague-Dawley rats to determine the duration of the dose response. Rats were intramuscularly injected with 50 μl of the modified human EPO mRNA (h-EPO), modified luciferase mRNA (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) (Luc) or the formulation buffer (F.Buffer) as described in the dosing chart Table 65. The rats were bled 2, 6, 12, 24, 48 and 72 hours after the intramuscular injection to determine the concentration of human EPO in serum at a given time. The average and geometric mean in pg/ml for this study are also shown in Table 65.







TABLE 65







Dosing Chart













Dose
Avg.
Geometric-



Group
(ug)
pg/ml
mean (pg/ml)

















h-EPO
 2 hour
100
59.6
58.2



h-EPO
 6 hour
100
68.6
55.8



h-EPO
12 hour
100
87.4
84.5



h-EPO
24 hour
100
108.6
95.3



h-EPO
48 hour
100
77.9
77.0



h-EPO
72 hour
100
80.1
75.8



Luc
24, 48 and
100
37.2
29.2




72 hour



F. Buffer
24, 48 and
—
48.9
10.4




72 hour










Example 47: Routes of Administration
Further studies were performed to investigate dosing using different routes of administration. Following the protocol outlined in Example 35, 4 mice per group were dosed intramuscularly (I.M.), intravenously (IV) or subcutaneously (S.C.) by the dosing chart outlined in Table 66. Serum was collected 13 hours post injection from all mice, tissue was collected from the site of injection from the intramuscular and subcutaneous group and the spleen, liver and kidneys were collected from the intravenous group. The results from the intramuscular group and the subcutaneous group results are shown in Table 67.







TABLE 66







Dosing Chart
















Total
Dosing


Group
Treatment
Route
Dose of mmRNA
Dose
Vehicle















1
Lipoplex-human EPO mmRNA
I.M.
4 × 100 ug + 30% Lipoplex
4 × 70 ul
Lipoplex


2
Lipoplex-human EPO mmRNA
I.M.
4 × 100 ug
4 × 70 ul
Buffer


3
Lipoplex-human EPO mmRNA
S.C.
4 × 100 ug + 30% Lipoplex
4 × 70 ul
Lipoplex


4
Lipoplex-human EPO mmRNA
S.C.
4 × 100 ug
4 × 70 ul
Buffer


5
Lipoplex-human EPO mmRNA
I.V.
200 ug + 30% Lipoplex
  140 ul
Lipoplex


6
Lipoplexed-Luciferase mmRNA
I.M.
100 ug + 30% Lipoplex
4 × 70 ul
Lipoplex


7
Lipoplexed-Luciferase mmRNA
I.M.
100 ug
4 × 70 ul
Buffer


8
Lipoplexed-Luciferase mmRNA
S.C.
100 ug + 30% Lipoplex
4 × 70 ul
Lipoplex


9
Lipoplexed-Luciferase mmRNA
S.C.
100 ug
4 × 70 ul
Buffer


10
Lipoplexed-human EPO mmRNA
I.V.
200 ug + 30% Lipoplex
  140 ul
Lipoplex


11
Formulation Buffer
I.M.
4× multi dosing
4 × 70 ul
Buffer
















TABLE 67







Human EPO Protein in Mouse Serum (I.M. Injection Route)










EPO (pg/ml)










Formulation
I.M. Injection Route
S.C. Injection Route












Epo-Lipoplex
67.115
2.154


Luc-Lipoplex
0
0


Epo-Saline
100.891
11.37


Luc-Saline
0
0


Formulation Buffer
0
0









Example 48. Rapidly Eliminated Lipid Nanoparticle (reLNP) Studies
A. Formulation of Modified RNA reLNPs
Solutions of synthesized lipid, 1,2-distearoyl-3-phosphatidylcholine (DSPC) (Avanti Polar Lipids, Alabaster, Ala.), cholesterol (Sigma-Aldrich, Taufkirchen, Germany), and α-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-w-methoxy-polyoxyethylene (PEG-c-DOMG) (NOF, Bouwelven, Belgium) are prepared and stored at −20° C. The synthesized lipid is selected from DLin-DMA with an internal ester, DLin-DMA with a terminal ester, DLin-MC3-DMA-internal ester, and DLin-MC3-DMA with a terminal ester. The reLNPs are combined to yield a molar ratio of 50:10:38.5:1.5 (reLNP: DSPC: Cholesterol: PEG-c-DOMG). Formulations of the reLNPs and modified mRNA are prepared by combining the lipid solution with the modified mRNA solution at total lipid to modified mRNA weight ratio of 10:1, 15:1, 20:1 and 30:1.
B. Characterization of Formulations
A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) is used to determine the particle size, the polydispersity index (PDI) and the zeta potential of the modified mRNA nanoparticles in 1×PBS in determining particle size and 15 mM PBS in determining zeta potential.
Ultraviolet-visible spectroscopy is used to determine the concentration of modified mRNA nanoparticle formulation. After mixing, the absorbance spectrum of the solution is recorded between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, Calif.). The modified RNA concentration in the nanoparticle formulation is calculated based on the extinction coefficient of the modified RNA used in the formulation and on the difference between the absorbance at a wavelength of 260 nm and the baseline value at a wavelength of 330 nm.
QUANT-ITTM RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.) is used to evaluate the encapsulation of modified RNA by the nanoparticle. The samples are diluted, transferred to a polystyrene 96 well plate, then either a TE buffer or a 2% Triton X-100 solution is added. The plate is incubated and the RIBOGREEN® reagent is diluted in TE buffer, and of this solution is added to each well. The fluorescence intensity is measured using a fluorescence plate reader (Wallac Victor 1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) The fluorescence values of the reagent blank are subtracted from each of the samples and the percentage of free modified RNA is determined by dividing the fluorescence intensity of the intact sample by the fluorescence value of the disrupted sample.
C. In Vitro Incubation
Human embryonic kidney epithelial (HEK293) and hepatocellular carcinoma epithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) are seeded on 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany) and plates for HEK293 cells are precoated with collagen type1. HEK293 are seeded at a density of about 30,000 and HepG2 are seeded at a density of about 35,000 cells per well in 100 μl cell culture medium. Formulations containing mCherry mRNA (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) are added directly after seeding the cells and incubated. The mCherry cDNA with the T7 promoter, 5′untranslated region (UTR) and 3′ UTR used in in vitro transcription (IVT) is given in SEQ ID NO: 5657.
Cells are harvested by transferring the culture media supernatants to a 96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg, Germany). Cells are trypsinized with 1⁄2 volume Trypsin/EDTA (Biochrom AG, Berlin, Germany), pooled with respective supernatants and fixed by adding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck, Darmstadt, Germany). Samples are then submitted to a flow cytometer measurement with an excitation laser and a filter for PE-Texas Red in a LSRII cytometer (Beckton Dickinson GmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI) of all events and the standard deviation of four independent wells are presented in for samples analyzed.
D. In Vivo Formulation Studies
Mice are administered intravenously a single dose of a formulation containing a modified mRNA and a reLNP. The modified mRNA administered to the mice is selected from G-CSF (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1), Factor IX (mRNA shown in SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) or mCherry (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1).
The mice are injected with 100 ug, 10 ug or 1 ug of the formulated modified mRNA and are sacrificed 8 hours after they are administered the formulation. Serum from the mice administered formulations containing human G-CSF modified mRNA are measured by specific G-CSF ELISA and serum from mice administered human Factor IX modified RNA is analyzed by specific factor IX ELISA or chromogenic assay. The liver and spleen from the mice administered with mCherry modified mRNA are analyzed by immunohistochemistry (IHC) or fluorescence-activated cell sorting (FACS). As a control, a group of mice are not injected with any formulation and their serum and tissue are collected analyzed by ELISA, FACS and/or IHC.
Example 49. In Vitro Transfection of VEGF-A
Human vascular endothelial growth factor-isoform A (VEGF-A) modified mRNA (mRNA sequence shown in SEQ ID NO: 5668; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) was transfected via reverse transfection in Human Keratinocyte cells in 24 multi-well plates. Human Keratinocytes cells were grown in EPILIFE® medium with Supplement S7 from Invitrogen (Carlsbad, Calif.) until they reached a confluence of 50-70%. The cells were transfected with 0, 46.875, 93.75, 187.5, 375, 750, and 1500 ng of modified mRNA (mmRNA) encoding VEGF-A which had been complexed with RNAIMAXTM from Invitrogen (Carlsbad, Calif.). The RNA:RNAIMAXTM complex was formed by first incubating the RNA with Supplement-free EPILIFE® media in a 5× volumetric dilution for 10 minutes at room temperature. In a second vial, RNAIMAXTM reagent was incubated with Supplement-free EPILIFE® Media in a 10× volumetric dilution for 10 minutes at room temperature. The RNA vial was then mixed with the RNAIMAXTM vial and incubated for 20-30 minutes at room temperature before being added to the cells in a drop-wise fashion.
The fully optimized mRNA encoding VEGF-A (mRNA sequence shown in SEQ ID NO: 5668; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) transfected with the Human Keratinocyte cells included modifications during translation such as natural nucleoside triphosphates (NTP), pseudouridine at each uridine site and 5-methylcytosine at each cytosine site (pseudo-U/5mC), and N1-methyl-pseudouridine at each uridine site and 5-methylcytosine at each cytosine site (N1-methyl-Pseudo-U/5mC). Cells were transfected with the mmRNA encoding VEGF-A and secreted VEGF-A concentration (pg/ml) in the culture medium was measured at 6, 12, 24, and 48 hours post-transfection for each of the concentrations using an ELISA kit from Invitrogen (Carlsbad, Calif.) following the manufacturers recommended instructions. These data, shown in Table 68, show that modified mRNA encoding VEGF-A is capable of being translated in Human Keratinocyte cells and that VEGF-A is transported out of the cells and released into the extracellular environment.







TABLE 68







VEGF-A Dosing and Protein Secretion












6 hours
12 hours
24 hours
48 hours


Dose (ng)
(pg/ml)
(pg/ml)
(pg/ml)
(pg/ml)










VEGF-A Dose Containing Natural NTPs











46.875
10.37
18.07
33.90
67.02


93.75
9.79
20.54
41.95
65.75


187.5
14.07
24.56
45.25
64.39


375
19.16
37.53
53.61
88.28


750
21.51
38.90
51.44
61.79


1500
36.11
61.90
76.70
86.54







VEGF-A Dose Containing Pseudo-U/5mC











46.875
10.13
16.67
33.99
72.88


93.75
11.00
20.00
46.47
145.61


187.5
16.04
34.07
83.00
120.77


375
69.15
188.10
448.50
392.44


750
133.95
304.30
524.02
526.58


1500
198.96
345.65
426.97
505.41







VEGF-A Dose Containing N1-methyl-Pseudo-U/5mC











46.875
0.03
6.02
27.65
100.42


93.75
12.37
46.38
121.23
167.56


187.5
104.55
365.71
1025.41
1056.91


375
605.89
1201.23
1653.63
1889.23


750
445.41
1036.45
1522.86
1954.81


1500
261.61
714.68
1053.12
1513.39









Example 50. In Vivo Studies of Factor IX
Human Factor IX mmRNA (Gen1; fully modified 5-methycytosine and pseudouridine) formulated in formulation buffer was delivered to mice via intramuscular injection. The results demonstrate that Factor IX protein was elevated in serum as measured 13 hours after administration.
In this study, mice (N=5 for Factor IX, N=3 for Luciferase or Buffer controls) were intramuscularly injected with 50 μl of the Factor IX mmRNA (mRNA sequence shown in SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1), Luciferase (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) or the formulation buffer (F.Buffer) at 2×100 ug/mouse. The mice were bled at 13 hours after the intramuscular injection to determine the concentration of human the polypeptide in serum in pg/mL. The results revealed that administration of Factor IX mmRNA resulted in levels of 1600 pg/mL at 13 hours as compared to less than 100 pg/mL of Factor IX for either Luciferase or buffer control administration.
Example 51. Multi-Site Administration: Intramuscular and Subcutaneous
Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) modified as either Gen1 or Gen2 (5-methylcytosine (5mc) and a pseudouridine (ψ) modification, G-CSF-Gen1; or N1-5-methylcytosine (N1-5mc) and a w modification, G-CSF-Gen2) and formulated in formulation buffer were delivered to mice via intramuscular (IM) or subcutaneous (SC) injection. Injection of four doses or 2×50 ug (two sites) daily for three days (24 hrs interval) was performed. The fourth dose was administered 6 hrs before blood collection and CBC analysis. Controls included Luciferase (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) or the formulation buffer (F.Buffer). The mice were bled at 72 hours after the first mRNA injection (6 hours after the last modified mRNA dose) to determine the effect of mRNA-encoded human G-CSF on the neutrophil count. The dosing regimen is shown in Table 69 as are the resulting neutrophil counts (thousands/uL). In Table 69, an asterisk (*) indicates statistical significance at p<0.05.
For intramuscular administration, the data reveal a four fold increase in neutrophil count above control at day 3 for the Gen1 G-CSF mRNA and a two fold increase for the Gen2 G-CSF mmRNA. For subcutaneous administration, the data reveal a two fold increase in neutrophil count above control at day 3 for the Gen2 G-CSF mRNA.
These data demonstrate that both 5-methylcytidine/pseudouridine and 5-methylcytidine/N1-methyl-pseudouridine-modified mRNA can be biologically active, as evidenced by specific increases in blood neutrophil counts.







TABLE 69







Dosing Regimen


















Dose
















Treat-


Dose
Vol. (μl/
Dosing
Neutrophil


Gr. 
ment
Route
N=
(μg/mouse)
mouse)
Vehicle
Thous/uL

















1
G-CSF 
I.M
5
2 × 50 ug 
50
F. buffer
 840*



(Gen1)


(four doses)





2
G-CSF 
S.0
5
2 × 50 ug 
50
F. buffer
430



(Gen1)


(four doses)





3
G-CSF 
I.M
5
2 × 50 ug 
50
F. buffer
 746*



(Gen2)


(four doses)





4
G-CSF 
S.C
5
2 × 50 ug 
50
F. buffer
683



(Gen2)


(four doses)





5
Luc 
I.M.
5
2 × 50 ug 
50
F. buffer
201



(Gen1)


(four doses)





6
Luc 
S.C.
5
2 × 50 ug 
50
F. buffer
307



(Gen1)


(four doses)





7
Luc 
I.M
5
2 × 50 ug 
50
F. buffer
336



(Gen2)


(four doses)





8
Luc 
S.C
5
2 × 50 ug 
50
F. buffer
357



(Gen2)


(four doses)





9
F. Buffer
I.M
4
0 (four
50
F. buffer
245






doses)





10
F. Buffer
S.C.
4
0 (four
50
F. buffer
509






doses)





11
Untreated
—
4


—
312









Example 52. Intravenous Administration
Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) modified with 5-methylcytosine (5mc) and a pseudouridine (ψ) modification (Gen1); or having no modifications and formulated in 10% lipoplex (RNAiMax) were delivered to mice at a dose of 50 ug RNA and in a volume of 100 ul via intravenous (IV) injection at days 0, 2 and 4. Neutrophils were measured at days 1, 5 and 8. Controls included non-specific mammalian RNA or the formulation buffer alone (F.Buffer). The mice were bled at days 1, 5 and 8 to determine the effect of modified mRNA-encoded human G-CSF to increase neutrophil count. The dosing regimen is shown in Table 70 as are the resulting neutrophil counts (thousands/uL; K/uL).
For intravenous administration, the data reveal a four to five fold increase in neutrophil count above control at day 5 with G-CSF modified mRNA but not with unmodified G-CSF mRNA or non-specific controls. Blood count returned to baseline four days after the final injection. No other changes in leukocyte populations were observed.
In Table 70, an asterisk (*) indicates statistical significance at p<0.001 compared to buffer.
These data demonstrate that lipoplex-formulated 5-methylcytidine/pseudouridine-modified mRNA can be biologically active, when delivered through an I.V. route of administration as evidenced by specific increases in blood neutrophil counts. No other cell subsets were significantly altered. Unmodified G-CSF mRNA similarly administered showed no pharmacologic effect on neutrophil counts.







TABLE 70







Dosing Regimen

















Dose








Vol.
Dosing
Neutrophil


Gr.
Day
Treatment
N=
(μl/mouse)
Vehicle
K/uL
















1
1
G-CSF (Gen1)
5
100
10% lipoplex
2.91


2
5
G-CSF (Gen1)
5
100
10% lipoplex
5.32*


3
8
G-CSF (Gen1)
5
100
10% lipoplex
2.06


4
1
G-CSF
5
100
10% lipoplex
1.88




(no modification)






5
5
G-CSF
5
100
10% lipoplex
1.95




(no modification)






6
8
G-CSF
5
100
10% lipoplex
2.09




(no modification)






7
1
RNA control
5
100
10% lipoplex
2.90


8
5
RNA control
5
100
10% lipoplex
1.68


9
8
RNA control
4
100
10% lipoplex
1.72


10
1
F. Buffer
4
100
10% lipoplex
2.51


11
5
F. Buffer
4
100
10% lipoplex
1.31


12
8
F. Buffer
4
100
10% lipoplex
1.92









Example 53. Saline Formulation: Intramuscular Administration
Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) and human EPO mmRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1); G-CSF modified mRNA (modified with 5-methylcytosine (5mc) and pseudouridine (ψ)) and EPO modified mRNA (modified with N1-5-methylcytosine (N1-5mc) and ψ modification), were formulated in formulation buffer (150 mM sodium chloride, 2 mM calcium chloride, 2 mM phosphate, 0.5 mM EDTA at a pH of 6.5) and delivered to mice via intramuscular (IM) injection at a dose of 100 ug.
Controls included Luciferase (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) or the formulation buffer (F.Buffer). The mice were bled at 13 hours after the injection to determine the concentration of the human polypeptide in serum in pg/mL. (G-CSF groups measured human G-CSF in mouse serum and EPO groups measured human EPO in mouse serum). The data are shown in Table 71.







TABLE 71







Dosing Regimen















Dose Vol.
Dosing
Average Protein


Group
Treatment
N=
(μl/mouse)
Vehicle
Product pg/mL, serum















G-CSF
G-CSF
5
50
Saline
19.8


G-CSF
Luciferase
5
50
Saline
0.5


G-CSF
F. buffer
5
50
F. buffer
0.5


EPO
EPO
5
50
Saline
191.5


EPO
Luciferase
5
50
Saline
15.0


EPO
F. buffer


F. buffer
4.8









B. Dose Response
Human EPO modified mRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) were formulated in formulation buffer and delivered to mice via intramuscular (IM) injection.
Controls included Luciferase (mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine and pseudouridine) or the formulation buffer (F.Buffer). The mice were bled at 13 hours after the injection to determine the concentration of the human polypeptide in serum in pg/mL. The dose and expression are shown in Table 72.







TABLE 72







Dosing Regimen and Expression












Dose Vol.
Average Protein



Treatment
(μl/mouse)
Product pg/mL, serum















EPO
100
96.2



EPO
50
63.5



EPO
25
18.7



EPO
10
25.9



EPO
1
2.6



Luciferase
100
0.0



F. buffer
100
1.0










Example 54. EPO Multi-Dose/Multi-Administration
Studies utilizing multiple intramuscular injection sites at one time point were designed and performed.
The design of a single multi-dose experiment involved using human erythropoietin (EPO) mmRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) or G-CSF mmRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) administered in formulation buffer. The dosing vehicle (F. buffer) was used as a control. The EPO and G-CSF modified mRNA were modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site.
Animals (n=5), Sprague-Dawley rats, were injected IM (intramuscular) for the single unit dose of 100 ug (delivered to one thigh). For multi-dosing 6 doses of 100 ug (delivered to two thighs) were used for both EPO and G-CSF mmRNA. Control dosing involved use of buffer at a single dose. Human EPO blood levels were evaluated 13 hrs post injection.
Human EPO protein was measured in rat serum 13 hrs post intramuscular injection. Five groups of rats were treated and evaluated. The results are shown in Table 73.







TABLE 73







Multi-dose study















Avg. Pg/mL




Dose of
Total
human EPO,


Group
Treatment
mmRNA
Dose
serum














1
Human EPO mmRNA
1 × 100 ug
100 ug
143


2
Human EPO mmRNA
6 × 100 ug
600 ug
256


3
G-CSF mmRNA
1 × 100 ug
100 ug
43


4
G-CSF mmRNA
6 × 100 ug
600 ug
58


5
Buffer Alone
—
—
20









Example 55. Signal Sequence Exchange Study
Several variants of mmRNAs encoding human Granulocyte colony stimulating factor (G-CSF) (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) were synthesized using modified nucleotides pseudouridine and 5-methylcytosine (pseudo-U/5mC). These variants included the G-CSF constructs encoding either the wild-type N terminal secretory signal peptide sequence (MAGPATQSPMKLMALQLLLWHSALWTVQEA; SEQ ID NO: 95), no secretory signal peptide sequence, or secretory signal peptide sequences taken from other mRNAs. These included sequences where the wild type G-CSF signal peptide sequence was replaced with the signal peptide sequence of either: human α-1-anti trypsin (AAT) (MMPSSVSWGILLLAGLCCLVPVSLA; SEQ ID NO: 94), human Factor IX (FIX) (MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKR; SEQ ID NO: 96), human Prolactin (Prolac) (MKGSLLLLLVSNLLLCQSVAP; SEQ ID NO: 97), or human Albumin (Alb) (MKWVTFISLLFLFSSAYSRGVFRR; SEQ ID NO: 98).
250 ng of modified mRNA encoding each G-CSF variant was transfected into HEK293A (293A in the table), mouse myoblast (MM in the table) (C2C12, CRL-1772, ATCC) and rat myoblast (RM in the table) (L6 line, CRL-1458, ATCC) cell lines in a 24 well plate using 1 ul of Lipofectamine 2000 (Life Technologies), each well containing 300,000 cells. The supernatants were harvested after 24 hrs and the secreted G-CSF protein was analyzed by ELISA using the Human G-CSF ELISA kit (Life Technologies). The data shown in Table 74 reveal that cells transfected with G-CSF mmRNA encoding the Albumin signal peptide secrete at least 12 fold more G-CSF protein than its wild type counterpart.







TABLE 74







Signal Peptide Exchange













293A
MM
RM



Signal peptides
(pg/ml)
(pg/ml)
(pg/ml)
















G-CSF Natural
9650
3450
6050



α-1-anti trypsin
9950
5000
8475



Factor IX
11675
6175
11675



Prolactin
7875
1525
9800



Albumin
122050
81050
173300



No Signal peptide
0
0
0










Example 56. Cytokine Study: PBMC
PBMC Isolation and Culture
50 mL of human blood from two donors was received from Research Blood Components (lots KP30928 and KP30931) in sodium heparin tubes. For each donor, the blood was pooled and diluted to 70 mL with DPBS (SAFC Bioscience 59331C, lot 071M8408) and split evenly between two 50 mL conical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot 10074400) was gently dispensed below the blood layer. The tubes were centrifuged at 2000 rpm for 30 minutes with low acceleration and braking. The tubes were removed and the buffy coat PBMC layers were gently transferred to a fresh 50 mL conical and washed with DPBS. The tubes were centrifuged at 1450 rpm for 10 minutes.
The supernatant was aspirated and the PBMC pellets were resuspended and washed in 50 mL of DPBS. The tubes were centrifuged at 1250 rpm for 10 minutes. This wash step was repeated, and the PBMC pellets were resuspended in 19 mL of Optimem I (Gibco 11058, lot 1072088) and counted. The cell suspensions were adjusted to a concentration of 3.0×10^6 cells/mL live cells.
These cells were then plated on five 96 well tissue culture treated round bottom plates (Costar 3799) per donor at 50 uL per well. Within 30 minutes, transfection mixtures were added to each well at a volume of 50 uL per well. After 4 hours post transfection, the media was supplemented with 10 uL of Fetal Bovine Serum (Gibco 10082, lot 1012368)
Transfection Preparation
mmRNA encoding human G-CSF (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) (containing either (1) natural NTPs, (2) 100% substitution with 5-methyl cytidine and pseudouridine, or (3) 100% substitution with 5-methyl cytidine and N1-methyl pseudouridine; mmRNA encoding luciferase (IVT cDNA sequence shown in SEQ ID NO: 5665; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) (containing either (1) natural NTPs or (2) 100% substitution with 5-methyl cytidine and pseudouridine) and TLR agonist R848 (Invivogen tlr1-r848) were diluted to 38.4 ng/uL in a final volume of 2500 uL Optimem I.
Separately, 432 uL of Lipofectamine 2000 (Invitrogen 11668-027, lot 1070962) was diluted with 13.1 mL Optimem I. In a 96 well plate nine aliquots of 135 uL of each mmRNA, positive control (R-848) or negative control (Optimem I) was added to 135 uL of the diluted Lipofectamine 2000. The plate containing the material to be transfected was incubated for 20 minutes. The transfection mixtures were then transferred to each of the human PBMC plates at 50 uL per well. The plates were then incubated at 37 C. At 2, 4, 8, 20, and 44 hours each plate was removed from the incubator, and the supernatants were frozen.
After the last plate was removed, the supernatants were assayed using a human G-CSF ELISA kit (Invitrogen KHC2032) and human IFN-alpha ELISA kit (Thermo Scientific 41105-2). Each condition was done in duplicate.
Results:
The ability of unmodified and modified mRNA (mmRNAs) to produce the encoded protein was assessed (G-CSF production) over time as was the ability of the mRNA to trigger innate immune recognition as measured by interferon-alpha production. Use of in vitro PBMC cultures is an accepted way to measure the immunostimulatory potential of oligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102; herein incorporated by reference in its entirety).
Results were interpolated against the standard curve of each ELISA plate using a four parameter logistic curve fit. Shown in Tables 75 and 76 are the average from 2 separate PBMC donors of the G-CSF and IFN-alpha production over time as measured by specific ELISA.
In the G-CSF ELISA, background signal from the Lipofectamine 2000 untreated condition was subtracted at each timepoint. The data demonstrated specific production of human G-CSF protein by human peripheral blood mononuclear is seen with G-CSF mRNA containing natural NTPs, 100% substitution with 5-methyl cytidine and pseudouridine, or 100% substitution with 5-methyl cytidine and N1-methyl pseudouridine. Production of G-CSF was significantly increased through the use of modified mRNA relative to unmodified mRNA, with the 5-methyl cytidine and N1-methyl pseudouridine containing G-CSF mmRNA showing the highest level of G-CSF production. With regards to innate immune recognition, unmodified mRNA resulted in substantial IFN-alpha production, while the modified mRNA largely prevented interferon-alpha production. G-CSF mRNA fully modified with 5-methyl cytidine and N1-methyl-pseudouridine did not significantly increase cytokines whereas G-CSF mRNA fully modified with 5-methyl cytidine and pseudouridine induced IFN-alpha, TNF-alpha and IP10. Many other cytokines were not affected by either modification.







TABLE 75







G-CSF Signal


G-CSF signal - 2 Donor Average












pg/mL
2 Hr
4 Hr
8 Hr
20 Hr
44 Hr















G-CSF (5mC/pseudouridine)
120.3
136.8
421.0
346.1
431.8


G-CSF (5mC/N1-methyl-
256.3
273.7
919.3
1603.3
1843.3


pseudouridine)


G-CSF(Natural-no
63.5
92.6
129.6
258.3
242.4


modification)


Luciferase
4.5
153.7
33.0
186.5
58.0


(5mC/pseudouridine)
















TABLE 76







IFN-alpha signal


IFN-alpha signal - 2 donor average












pg/mL
2 Hr
4 Hr
8 Hr
20 Hr
44 Hr















G-CSF (5mC/pseudouridine)
21.1
2.9
3.7
22.7
4.3


G-CSF (5mC/N1-methyl-
0.5
0.4
3.0
2.3
2.1


pseudouridine)


G-CSF(Natural)
0.0
2.1
23.3
74.9
119.7


Luciferase
0.4
0.4
4.7
1.0
2.4


(5mC/pseudouridine)


R-848
39.1
151.3
278.4
362.2
208.1


Lpf. 2000 control
0.8
17.2
16.5
0.7
3.1









Example 57. Chemical Modification Ranges of Modified mRNA
Modified nucleotides such as, but not limited to, the chemical modifications 5-methylcytosine and pseudouridine have been shown to lower the innate immune response and increase expression of RNA in mammalian cells. Surprisingly, and not previously known, the effects manifested by the chemical modifications can be titrated when the amount of chemical modification is less than 100%. Previously, it was believed that full modification was necessary and sufficient to elicit the beneficial effects of the chemical modifications and that less than 100% modification of an mRNA had little effect. However, it has now been shown that the benefits of chemical modification can be derived using less than complete modification and that the effects are target, concentration and modification dependent.
A. Modified RNA Transfected in PBMC
960 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) and pseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with 0.8 uL of Lipofectamine 2000 into peripheral blood mononuclear cells (PBMC) from three normal blood donors (D1, D2, D3). The G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) was completely modified with 5mC and pseudoU (100% modification), not modified with 5mC and pseudoU (0% modification) or was partially modified with 5mC and pseudoU so the mRNA would contain 50% modification, 25% modification, 10% modification, %5 modification, 1% modification or 0.1% modification. A control sample of Luciferase (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified 5meC and pseudoU) was also analyzed for G-CSF expression. For TNF-alpha and IFN-alpha control samples of Lipofectamine2000, LPS, R-848, Luciferase (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified 5mC and pseudo), and P(I)P(C) were also analyzed. The supernatant was harvested and run by ELISA 22 hours after transfection to determine the protein expression. The expression of G-CSF is shown in Table 77 and the expression of IFN-alpha and TNF-alpha is shown in Table 78. The expression of IFN-alpha and TNF-alpha may be a secondary effect from the transfection of the G-CSF mRNA. Tables 77 and 78 show that the amount of chemical modification of G-CSF, IFN-alpha and TNF-alpha is titratable when the mRNA is not fully modified and the titratable trend is not the same for each target.







TABLE 77







G-CSF Expression









G-CSF Expression (pg/ml)











D1
D2
D3
















100% modification
270.3
151.6
162.2



50% modification
45.6
19.8
26.3



25% modification
23.6
10.8
8.9



10% modification
39.4
12.9
12.9



5% modification
70.9
26.8
26.3



1% modification
70.3
26.9
66.9



0.1% modification
67.5
25.2
28.7



Luciferase
14.5
3.1
10.0

















TABLE 78







IFN-alpha and TNF-alpha Expression










IFN-alpha Expression
TNF-alpha



(pg/ml)
Expression (pg/ml)














D1
D2
D3
D1
D2
D3
















100% 
76.8
6.8
15.1
5.6
1.4
21.4


modification








50%
22.0
5.5
257.3
4.7
1.7
12.1


modification








25%
64.1
14.9
549.7
3.9
0.7
10.1


modification








10%
150.2
18.8
787.8
6.6
0.9
13.4


modification








 5%
143.9
41.3
1009.6
2.5
1.8
12.0


modification








 1%
189.1
40.5
375.2
9.1
1.2
25.7


modification








0.1% 
261.2
37.8
392.8
9.0
2.
13.7


modification








 0%
230.3
45.1
558.3
10.9
1.4
10.9


modification








LF 200
0
0
1.5
45.8
2.8
53.6


LPS
0
0
1.0
114.5
70.0 
227.0


R-848
39.5
11.9
183.5
389.3
256.6
410.6


Luciferase
9.1
0
3.9
4.5
2.7
13.6


P(I)P(C)
1498.1
216.8
238.8
61.2
4.4
69.1









B. Modified RNA Transfected in HEK293
Human embryonic kidney epithelial (HEK293) cells were seeded on 96-well plates at a density of 30,000 cells per well in 100 ul cell culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) formulated with RNAiMAXTM (Invitrogen, Carlsbad, Calif.) was added to a well. The G-CSF was completely modified with 5mC and pseudoU (100% modification), not modified with 5mC and pseudoU (0% modification) or was partially modified with 5mC and pseudoU so the mRNA would contain 75% modification, 50% modification or 25% modification. Control samples (AK 5/2, mCherry (SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified 5mC and pseudoU) and untreated) were also analyzed. The half-life of G-CSF mRNA fully modified with 5-methylcytosine and pseudouridine is approximately 8-10 hours. The supernatants were harvested after 16 hours and the secreted G-CSF protein was analyzed by ELISA. Table 79 shows that the amount of chemical modification of G-CSF is titratable when the mRNA is not fully modified.







TABLE 79







G-CSF Expression









G-CSF Expression (ng/ml)














100% modification
118.4



75% modification
101.9



50% modification
105.7



25% modification
231.1



0% modification
270.9



AK 5/2
166.8



mCherry
0



Untreated
0










Example 58: In Vivo Delivery of Modified mRNA (mmRNA)
Modified RNA was delivered to C57/BL6 mice intramuscularly, subcutaneously, or intravenously to evaluate the bio-distribution of modified RNA using luciferase. A formulation buffer used with all delivery methods contained 150 mM sodium chloride, 2 mM calcium chloride, 2 mM Na+-phosphate which included 1.4 mM monobasic sodium phosphate and 0.6 mM of dibasic sodium phosphate, and 0.5 mM ethylenediaminetetraacetic acid (EDTA) was adjusted using sodium hydroxide to reach a final pH of 6.5 before being filtered and sterilized. A 1× concentration was used as the delivery buffer. To create the lipoplexed solution delivered to the mice, in one vial 50 μg of RNA was equilibrated for 10 minutes at room temperature in the delivery buffer and in a second vial 10 μl RNAiMAXTM was equilibrated for 10 minutes at room temperature in the delivery buffer. After equilibrium, the vials were combined and delivery buffer was added to reach a final volume of 100 μl which was then incubated for 20 minutes at room temperature. Luciferin was administered by intraperitoneal injection (IP) at 150 mg/kg to each mouse prior to imaging during the plateau phase of the luciferin exposure curve which was between 15 and 30 minutes. To create luciferin, 1 g of D-luciferin potassium or sodium salt was dissolved in 66.6 ml of distilled phosphate buffer solution (DPBS), not containing Mg2+ or Ca2+, to make a 15 mg/ml solution. The solution was gently mixed and passed through a 0.2 μm syringe filter, before being purged with nitrogen, aliquoted and frozen at −80° C. while being protected from light as much as possible. The solution was thawed using a waterbath if luciferin was not dissolved, gently mixed and kept on ice on the day of dosing.
Whole body images were taken of each mouse 2, 8 and 24 hours after dosing. Tissue images and serum was collected from each mouse 24 hours after dosing. Mice administered doses intravenously had their liver, spleen, kidneys, lungs, heart, peri-renal adipose tissue and thymus imaged. Mice administered doses intramuscularly or subcutaneously had their liver, spleen, kidneys, lungs, peri-renal adipose tissue, and muscle at the injection site. From the whole body images the bioluminescence was measured in photon per second for each route of administration and dosing regimen.
A. Intramuscular Administration
Mice were intramuscularly (I.M.) administered either modified luciferase mRNA fully modified with 5-methylcytosine and pseudouridine (Naked-Luc), lipoplexed modified luciferase mRNA fully modified with 5-methylcytosine and pseudouridine (Lipoplex-luc) (IVT cDNA sequence shown in SEQ ID NO: 5664; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site), lipoplexed modified granulocyte colony-stimulating factor (G-CSF) mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) (Lipoplex-Cytokine) or the formation buffer at a single dose of 50 μg of modified RNA in an injection volume of 50 μl for each formulation in the right hind limb and a single dose of 5 μg of modified RNA in an injection volume of 50 μl in the left hind limb. The bioluminescence average for the luciferase expression signals for each group at 2, 8 and 24 hours after dosing are shown in Table 80. The bioluminescence showed a positive signal at the injection site of the 5 μg and 50 μg modified RNA formulations containing and not containing lipoplex.







TABLE 80







In vivo Biophotoic Imaging (I.M. Injection Route)










Dose
Bioluminescence (photon/sec)











Formulation
(ug)
2 hours
8 hours
24 hours














Naked-Luc
5
224,000
683,000
927,000


Lipolplex-Luc
5
579,000
639,000
186,000


Lipoplex-G-CSF
5
64,600
85,600
75,100


Formulation Buffer
5
102,000
86,000
90,700


Naked-Luc
50
446,000
766,000
509,000


Lipolplex-Luc
50
374,000
501,000
332,000


Lipoplex-G-CSF
50
49,400
74,800
74,200


Formulation Buffer
50
59,300
69,200
63,600









B. Subcutaneous Administration
Mice were subcutaneously (S.C.) administered either modified luciferase mRNA (Naked-Luc), lipoplexed modified luciferase mRNA (Lipoplex-luc), lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or the formation buffer at a single dose of 50 μg of modified mRNA in an injection volume of 100 μl for each formulation. The bioluminescence average for the luciferase expression signals for each group at 2, 8 and 24 hours after dosing are shown in Table 81. The bioluminescence showed a positive signal at the injection site of the 50 μg modified mRNA formulations containing and not containing lipoplex.







TABLE 81







In vivo Biophotoic Imaging (S.C. Injection Route)









Bioluminescence (photon/sec)












Formulation
2 hours
8 hours
24 hours
















Naked-Luc
3,700,000
8,060,000
2,080,000



Lipolplex-Luc
3,960,000
1,700,000
1,290,000



Lipoplex-G-CSF
123,000
121,000
117,000



Formulation Buffer
116,000
127,000
123,000










C. Intravenous Administration
Mice were intravenously (I.V.) administered either modified luciferase mRNA (Naked-Luc), lipoplexed modified luciferase mRNA (Lipoplex-luc), lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or the formation buffer at a single dose of 50 μg of modified mRNA in an injection volume of 100 μl for each formulation. The bioluminescence average for the luciferase expression signal in the spleen from each group at 2 hours after dosing is shown in Table 82. The bioluminescence showed a positive signal in the spleen of the 50 μg modified mRNA formulations containing lipoplex.







TABLE 82







In vivo Biophotoic Imaging (I.V. Injection Route)











Bioluminescence (photon/



Formulation
sec) of the Spleen







Naked-Luc
58,400



Lipolplex-Luc
65,000



Lipoplex-G-CSF
57,100



Formulation Buffer
58,300










Example 59. Buffer Formulation Studies
G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with N1-pseudouridine and 5-methylcytosine) or Factor IX modified mRNA (mRNA sequence shown in SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with N1-pseudouridine and 5-methylcytosine) in a buffer solution is administered intramuscularly to rats in an injection volume of 50 μl (n=5) at a modified mRNA dose of 200 ug per rat as described in Table 83. The modified mRNA is lyophilized in water for 1-2 days. It is then reconstituted in the buffers listed below to a target concentration of 6 mg/ml. Concentration is determined by OD 260. Samples are diluted to 4 mg/ml in the appropriate buffer before dosing.
To precipitate the modified mRNA, 3M sodium acetate, pH 5.5 and pure ethanol are added at 1/10th the total volume and 4 times the total volume of modified mRNA, respectively. The material is placed at −80 C for a minimum of 1 hour. The material is then centrifuged for 30 minutes at 4000 rpm, 4 C. The supernatant is removed and the pellet is centrifuged and washed 3× with 75% ethanol. Finally, the pellet is reconstituted with buffer to a target concentration of 6 mg/ml. Concentration is determined by OD 260. Samples are diluted to 4 mg/ml in the appropriate buffer before dosing. All samples are prepared by lyophilization unless noted below.







TABLE 83







Buffer Dosing Groups










Group
Treatment
Buffer
Dose (ug/rat)





1
G-CSF
0.9% Saline
200



Factor IX
0.9% Saline
200


2
G-CSF
0.9% Saline + 2 mM
200




Calcium



Factor IX
0.9% Saline + 2 mM
200




Calcium


3
G-CSF
Lactated Ringer's
200



Factor IX
Lactated Ringer's
200


4
G-CSF
5% Sucrose
200



Factor IX
5% Sucrose
200


5
G-CSF
5% Sucrose + 2 mM
200




Calcium



Factor IX
5% Sucrose + 2 mM
200




Calcium


6
G-CSF
5% Mannitol
200



Factor IX
5% Mannitol
200


7
G-CSF
5% Mannitol + 2 mM
200




Calcium



Factor IX
5% Mannitol + 2 mM
200




Calcium


8
G-CSF
0.9% saline (precipitation)
200



Factor IX
0.9% saline (precipitation)
200









Serum samples are collected from the rats at various time intervals and analyzed for G-CSF or Factor IX protein expression using G-CSF or Factor IX ELISA.
Example 60. Multi-Dose Study
Sprague-Dawley rats (n=8) are injected intravenously eight times (twice a week) over 28 days. The rats are injected with 0.5 mg/kg, 0.05 mg/kg, 0.005 mg/kg or 0.0005 mg/kg of human G-CSF modified mRNA of luciferase modified mRNA formulated in a lipid nanoparticle, 0.5 mg/kg of human G-CSF modified mRNA in saline, 0.2 mg/kg of the human G-CSF protein Neupogen or non-translatable human G-CSF modified mRNA formulated in a lipid nanoparticle. Serum is collected during predetermined time intervals to evaluate G-CSF protein expression (8, 24 and 72 hours after the first dose of the week), complete blood count and white blood count (24 and 72 hours after the first dose of the week) and clinical chemistry (24 and 72 hours after the first dose of the week). The rats are sacrificed at day 29, 4 days after the final dosing, to determine the complete blood count, white blood count, clinical chemistry, protein expression and to evaluate the effect on the major organs by histopathology and necropsy. Further, an antibody assay is performed on the rats on day 29.
Example 61. LNP In Vivo Study
Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence, 5′ cap, Cap1; fully modified with 5-methylcytosine and pseudouridine was formulated as a lipid nanoparticle (LNP) using the syringe pump method. The LNP was formulated at a 20:1 weight ratio of total lipid to modified mRNA with a final lipid molar ratio of 50:10:38.5:1.5 (DLin-KC2-DMA: DSPC: Cholesterol: PEG-DMG). As shown in Table 84, the luciferase LNP formulation was characterized by particle size, zeta potential, and encapsulation.







TABLE 84







Luciferase Formulation










Formulation
NPA-098-1







Modified mRNA
Luciferase



Mean size
 135 nm




PDI: 0.08



Zeta at pH 7.4
−0.6 mV



Encaps.
91%



(RiboGr)










As outlined in Table 85, the luciferase LNP formulation was administered to Balb-C mice (n=3) intramuscularly, intravenously and subcutaneously and a luciferase modified RNA formulated in PBS was administered to mice intravenously.







TABLE 85







Luciferase Formulations
















Con-
Injection
Amount of



Formu-


centration
Volume
modified
Dose


lation
Vehicle
Route
(mg/ml)
(ul)
RNA (ug)
(mg/kg)





Luc-LNP
PBS
IV
0.20
50
10
0.50


Luc-LNP
PBS
IM
0.20
50
10
0.50


Luc-LNP
PBS
SC
0.20
50
10
0.50


Luc-PBS
PBS
IV
0.20
50
10
0.50









The mice administered the luciferase LNP formulation intravenously and intramuscularly were imaged at 2, 8, 24, 48, 120 and 192 hours and the mice administered the luciferase LNP formulation subcutaneously were imaged at 2, 8, 24, 48 and 120 hours to determine the luciferase expression as shown in Table 86. In Table 86, “NT” means not tested. Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse.







TABLE 86







Luciferase Expression










Route
Average Expression (photon/second)















of





192


Formulation
Admin
2 hours
8 hours
24 hours
48 hours
120 hours
hours





Luc-LNP
IV
1.62E+08
3.00E+09
7.77E+08
4.98E+08
1.89E+08
6.08E+07


Luc-LNP
IM
4.85E+07
4.92E+08
9.02E+07
3.17E+07
1.22E+07
2.38E+06


Luc-LNP
SC
1.85E+07
9.79E+08
3.09E+08
4.94E+07
1.98E+06
NT


Luc-PBS
IV
3.61E+05
5.64E+05
3.19E+05
NT
NT
NT









One mouse administered the LNP formulation intravenously was sacrificed at 8 hours to determine the luciferase expression in the liver and spleen. Also, one mouse administered the LNP formulation intramuscular was sacrificed at 8 hours to determine the luciferase expression of the muscle around the injection site and in the liver and spleen. As shown in Table 87, expression was seen in the both the liver and spleen after intravenous and intramuscular administration and in the muscle around the intramuscular injection site.







TABLE 87







Luciferase Expression in Tissue









Expression



(photon/second)














Luciferase LNP: IV Administration




Liver
7.984E+08



Spleen
3.951E+08



Luciferase LNP: IM Administration



Muscle around the injection site
3.688E+07



Liver
1.507E+08



Spleen
1.096E+07










Example 62. Cytokine Study: PBMC
A. PBMC Isolation and Culture
50 mL of human blood from two donors was received from Research Blood Components (lots KP30928 and KP30931) in sodium heparin tubes. For each donor, the blood was pooled and diluted to 70 mL with DPBS (SAFC Bioscience 59331C, lot 071M8408) and split evenly between two 50 mL conical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot 10074400) was gently dispensed below the blood layer. The tubes were centrifuged at 2000 rpm for 30 minutes with low acceleration and braking. The tubes were removed and the buffy coat PBMC layers were gently transferred to a fresh 50 mL conical and washed with DPBS. The tubes were centrifuged at 1450 rpm for 10 minutes.
The supernatant was aspirated and the PBMC pellets were resuspended and washed in 50 mL of DPBS. The tubes were centrifuged at 1250 rpm for 10 minutes. This wash step was repeated, and the PBMC pellets were resuspended in 19 mL of Optimem I (Gibco 11058, lot 1072088) and counted. The cell suspensions were adjusted to a concentration of 3.0×10^6 cells/mL live cells.
These cells were then plated on five 96 well tissue culture treated round bottom plates (Costar 3799) per donor at 50 uL per well. Within 30 minutes, transfection mixtures were added to each well at a volume of 50 uL per well. After 4 hours post transfection, the media was supplemented with 10 uL of Fetal Bovine Serum (Gibco 10082, lot 1012368).
B. Transfection Preparation
Modified mRNA encoding human G-CSF (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) (containing either (1) natural NTPs, (2) 100% substitution with 5-methyl cytidine and pseudouridine, or (3) 100% substitution with 5-methyl cytidine and N1-methyl pseudouridine; mRNA encoding luciferase (IVT cDNA sequence shown in SEQ ID NO: 5654; mRNA sequence shown in SEQ ID NO: 5665, polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine and pseudouridine replacement at each uridine site) (containing either (1) natural NTPs or (2) 100% substitution with 5-methyl cytidine and pseudouridine) and TLR agonist R848 (Invivogen tlr1-r848) were diluted to 38.4 ng/uL in a final volume of 2500 uL Optimem I.
Separately, 110 uL of Lipofectamine 2000 (Invitrogen 11668-027, lot 1070962) was diluted with 6.76 mL Optimem I. In a 96 well plate nine aliquots of 135 uL of each mRNA, positive control (R-848) or negative control (Optimem I) was added to 135 uL of the diluted Lipofectamine 2000. The plate containing the material to be transfected was incubated for 20 minutes. The transfection mixtures were then transferred to each of the human PBMC plates at 50 uL per well. The plates were then incubated at 37° C. At 2, 4, 8, 20, and 44 hours each plate was removed from the incubator, and the supernatants were frozen.
After the last plate was removed, the supernatants were assayed using a human G-CSF ELISA kit (Invitrogen KHC2032) and human IFN-alpha ELISA kit (Thermo Scientific 41105-2). Each condition was done in duplicate.
C. Protein and Innate Immune Response Analysis
The ability of unmodified and modified mRNA to produce the encoded protein was assessed (G-CSF production) over time as was the ability of the mRNA to trigger innate immune recognition as measured by interferon-alpha production. Use of in vitro PBMC cultures is an accepted way to measure the immunostimulatory potential of oligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102).
Results were interpolated against the standard curve of each ELISA plate using a four parameter logistic curve fit. Shown in Tables 88 and 89 are the average from 3 separate PBMC donors of the G-CSF, interferon-alpha (IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) production over time as measured by specific ELISA.
In the G-CSF ELISA, background signal from the Lipofectamine 2000 (LF2000) untreated condition was subtracted at each time point. The data demonstrated specific production of human G-CSF protein by human peripheral blood mononuclear is seen with G-CSF mRNA containing natural NTPs, 100% substitution with 5-methyl cytidine and pseudouridine, or 100% substitution with 5-methyl cytidine and N1-methyl pseudouridine. Production of G-CSF was significantly increased through the use of 5-methyl cytidine and N1-methyl pseudouridine modified mRNA relative to 5-methyl cytidine and pseudouridine modified mRNA.
With regards to innate immune recognition, while both modified mRNA chemistries largely prevented IFN-alpha and TNF-alpha production relative to positive controls (R848, p(I)p(C)), significant differences did exist between the chemistries. 5-methyl cytidine and pseudouridine modified mRNA resulted in low but detectable levels of IFN-alpha and TNF-alpha production, while 5-methyl cytidine and N1-methyl pseudouridine modified mRNA resulted in no detectable IFN-alpha and TNF-alpha production.
Consequently, it has been determined that, in addition to the need to review more than one cytokine marker of the activation of the innate immune response, it has surprisingly been found that combinations of modifications provide differing levels of cellular response (protein production and immune activation). The modification, N1-methyl pseudouridine, in this study has been shown to convey added protection over the standard combination of 5-methylcytidine/pseudouridine explored by others resulting in twice as much protein and almost 150 fold reduction in immune activation (TNF-alpha).
Given that PBMC contain a large array of innate immune RNA recognition sensors and are also capable of protein translation, it offers a useful system to test the interdependency of these two pathways. It is known that mRNA translation can be negatively affected by activation of such innate immune pathways (Kariko et al. Immunity (2005) 23:165-175; Warren et al. Cell Stem Cell (2010) 7:618-630). Using PBMC as an in vitro assay system it is possible to establish a correlation between translation (in this case G-CSF protein production) and cytokine production (in this case exemplified by IFN-alpha and TNF-alpha protein production). Better protein production is correlated with lower induction of innate immune activation pathway, and new chemistries can be judged favorably based on this ratio (Table 90).
In this study, the PC Ratio for the two chemical modifications, pseudouridine and N1-methyl pseudouridine, both with 5-methy cytosine was 4742/141=34 as compared to 9944/1=9944 for the cytokine IFN-alpha. For the cytokine, TNF-alpha, the two chemistries had PC Ratios of 153 and 1243, respectively suggesting that for either cytokine, the N1-methyl-pseudouridine is the superior modification. In Tables 88 and 89, “NT” means not tested.







TABLE 88





G-CSF


G-CSF: 3 Donor Average (pg/ml)


















G-CSF
4742



5-methylcytosine/



pseudouridine



G-CSF
9944



5-methylcytosine/



N1-methyl-pseudouridine



Luciferase
18



LF2000
16

















TABLE 89







IFN-alpha and TNF-alpha










IFN-alpha: 3
TNF-alpha: 3



Donor Average
Donor Average



(pg/ml)
(pg/ml)















G-CSF
141
31



5-methylcytosine/



pseudouridine



G-CSF
1
8



5-methylcytosine/



N1-methyl-pseudouridine



P(I)P(C)
1104
NT



R-848
NT
1477



LF2000
17
25

















TABLE 90







G-CSF to Cytokine Ratios










G-CSF/IFN-alpha (ratio)
G-CSF/TNF-alpha (ratio)













5-methyl-

5-methyl-



5-methyl-
cytosine/
5-methyl-
cytosine/



cytosine/
N1-methyl-
cytosine/
N1-methyl-



pseudouridine
pseudouridine
pseudouridine
pseudouridine















PC
34
9944
153
1243


Ratio









Example 63. In Vitro PBMC Studies: Percent Modification
480 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) and pseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with 0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells (PBMC) from three normal blood donors (D1, D2, and D3). The G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) was completely modified with 5mC and pseudoU (100% modification), not modified with 5mC and pseudoU (0% modification) or was partially modified with 5mC and pseudoU so the mRNA would contain 75% modification, 50% modification or 25% modification. A control sample of Luciferase (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified 5meC and pseudoU) was also analyzed for G-CSF expression. For TNF-alpha and IFN-alpha control samples of Lipofectamine2000, LPS, R-848, Luciferase (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified 5mC and pseudo), and P(I)P(C) were also analyzed. The supernatant was harvested and run by ELISA 22 hours after transfection to determine the protein expression. The expression of G-CSF is shown in Table 91 and the expression of IFN-alpha and TNF-alpha is shown in Table 92. The expression of IFN-alpha and TNF-alpha may be a secondary effect from the transfection of the G-CSF mRNA. Tables 91 and 92 show that the amount of chemical modification of G-CSF, interferon alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) is titratable when the mRNA is not fully modified and the titratable trend is not the same for each target.
By using PBMC as an in vitro assay system it is possible to establish a correlation between translation (in this case G-CSF protein production) and cytokine production (in this case exemplified by IFN-alpha protein production). Better protein production is correlated with lower induction of innate immune activation pathway, and the percentage modification of a chemistry can be judged favorably based on this ratio (Table 93). As calculated from Tables 91 and 92 and shown in Table 93, full modification with 5-methylcytidine and pseudouridine shows a much better ratio of protein cytokine production than without any modification (natural G-CSF mRNA) (100-fold for IFN-alpha and 27-fold for TNF-alpha). Partial modification shows a linear relationship with increasingly less modification resulting in a lower protein cytokine ratio.







TABLE 91







G-CSF Expression









G-CSF Expression (pg/ml)











D1
D2
D3
















100% modification
1968.9
2595.6
2835.7



75% modification
566.7
631.4
659.5



50% modification
188.9
187.2
191.9



25% modification
139.3
126.9
102.0



0% modification
194.8
182.0
183.3



Luciferase
90.2
0.0
22.1

















TABLE 92







IFN-alpha and TNF-alpha Expression










IFN-alpha Expression (pg/ml)
TNF-alpha Expression (pg/ml)














D1
D2
D3
D1
D2
D3
















100% modification
336.5
78.0
46.4
115.0
15.0
11.1


 75% modification 
339.6
107.6
160.9
107.4
21.7
11.8


 50% modification 
478.9
261.1
389.7
49.6
24.1
10.4


 25% modification
564.3
400.4
670.7
85.6
26.6
19.8


 0% modification
1421.6
810.5
1260.5
154.6
96.8
45.9


LPS
0.0
0.6
0.0
0.0
12.6
4.3


R-848
0.5
3.0
14.1
655.2
989.9
420.4


P(I)P(C)
130.8
297.1
585.2
765.8
2362.7
1874.4


Lipid only
1952.2
866.6
855.8
248.5
82.0
60.7
















TABLE 93







PC Ratio and Effect of Percentage of Modification













Average
Average
Average
G-CSF/IFN-
G-CSF/TNF-


%
G-CSF
IFN-a
TNF-a
alpha
alpha


Modification
(pg/ml)
(pg/ml)
(pg/ml)
(PC ratio)
(PC ratio)















100
2466
153
47
16
52


75
619
202
47
3.1
13


50
189
376
28
0.5
6.8


25
122
545
44
0.2
2.8


0
186
1164
99
0.16
1.9









Example 64. Modified RNA Transfected in PBMC
500 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) and pseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with 0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells (PBMC) from three normal blood donors (D1, D2, and D3). The G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) was completely modified with 5mC and pseudoU (100% modification), not modified with 5mC and pseudoU (0% modification) or was partially modified with 5mC and pseudoU so the mRNA would contain 50% modification, 25% modification, 10% modification, %5 modification, 1% modification or 0.1% modification. A control sample of mCherry (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified 5meC and pseudouridine), G-CSF fully modified with 5-methylcytosine and pseudouridine (Control G-CSF) and an untreated control was also analyzed for expression of G-CSF, tumor necrosis factor-alpha (TNF-alpha) and interferon-alpha (IFN-alpha). The supernatant was harvested 6 hours and 18 hours after transfection and run by ELISA to determine the protein expression. The expression of G-CSF, IFN-alpha, and TNF-alpha for Donor 1 is shown in Table 94, Donor 2 is shown in Table 95 and Donor 3 is shown in Table 96.
Full 100% modification with 5-methylcytidine and pseudouridine resulted in the most protein translation (G-CSF) and the least amount of cytokine produced across all three human PBMC donors. Decreasing amounts of modification results in more cytokine production (IFN-alpha and TNF-alpha), thus further highlighting the importance of fully modification to reduce cytokines and to improve protein translation (as evidenced here by G-CSF production).







TABLE 94







Donor 1











G-CSF
IFN-
TNF-



(pg/mL)
alpha (pg/mL)
alpha (pg/mL)














6 hours 
18 hours
6 hours 
18 hours
6 hours
18 hours
















100% Mod
1815
2224
1
13
0
0


 75% Mod
591
614
0
89
0
0


 50% Mod
172
147
0
193
0
0


 25% Mod
111
92
2
219
0
0


 10% Mod
138
138
7
536
18
0


 1% Mod
199
214
9
660
18
3


 0.1% Mod 
222
208
10
597
0
6


 0% Mod
273
299
10
501
10
0


Control 
957
1274
3
123
18633
1620


G-CSF








mCherry
0
0
0
10
0
0


Untreated
N/A
N/A
0
0
1
1
















TABLE 95







Donor 2











G-CSF
IFN-
TNF-



(pg/mL)
alpha (pg/mL)
alpha (pg/mL)














6 hours
18 hours
6 hours
18 hours
6 hours 
18 hours
















100% Mod 
2184
2432
0
7
0
11


75% Mod
935
958
3
130
0
0


50% Mod
192
253
2
625
7
23


25% Mod
153
158
7
464
6
6


10% Mod
203
223
25
700
22
39


 1% Mod
288
275
27
962
51
66


0.1% Mod 
318
288
33
635
28
5


 0% Mod
389
413
26
748
1
253


Control 
1461
1634
1
59
481
814


G-CSF








mCherry
0
7
0
1
0
0


Untreated
N/A
N/A
1
0
0
0
















TABLE 96







Donor 3











G-CSF 
IFN-
TNF-



(pg/mL)
alpha (pg/mL)
alpha (pg/mL)














6 hours
18 hours
6 hours
18 hours
6 hours
18 hours
















100% Mod 
6086
7549
7
658
11
11


75% Mod
2479
2378
23
752
4
35


50% Mod
667
774
24
896
22
18


25% Mod
480
541
57
1557
43
115


10% Mod
838
956
159
2755
144
123


 1% Mod
1108
1197
235
3415
88
270


0.1% Mod 
1338
1177
191
2873
37
363


 0% Mod
1463
1666
215
3793
74
429


Control 
3272
3603
16
1557
731
9066


G-CSF








mCherry
0
0
2
645
0
0


Untreated
N/A
N/A
1
1
0
8









Example 65. Innate Immune Response Study in BJ Fibroblasts
A. Single Transfection
Human primary foreskin fibroblasts (BJ fibroblasts) were obtained from American Type Culture Collection (ATCC) (catalog # CRL-2522) and grown in Eagle's Minimum Essential Medium (ATCC, catalog #30-2003) supplemented with 10% fetal bovine serum at 37° C., under 5% CO2. BJ fibroblasts were seeded on a 24-well plate at a density of 300,000 cells per well in 0.5 ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine (Gen1) or fully modified with 5-methylcytosine and N1-methyl-pseudouridine (Gen2) having Cap0, Cap1 or no cap was transfected using Lipofectamine 2000 (Invitrogen, catalog #11668-019), following manufacturer's protocol. Control samples of poly I:C (PIC), Lipofectamine 2000 (Lipo), natural luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) and natural G-CSF mRNA were also transfected. The cells were harvested after 18 hours, the total RNA was isolated and DNASE® treated using the RNeasy micro kit (catalog #74004) following the manufacturer's protocol. 100 ng of total RNA was used for cDNA synthesis using High Capacity cDNA Reverse Transcription kit (catalog #4368814) following the manufacturer's protocol. The cDNA was then analyzed for the expression of innate immune response genes by quantitative real time PCR using SybrGreen in a Biorad CFX 384 instrument following manufacturer's protocol. Table 97 shows the expression level of innate immune response transcripts relative to house-keeping gene HPRT (hypoxanthine phosphoribosytransferase) and is expressed as fold-induction relative to HPRT. In the table, the panel of standard metrics includes: RIG-I is retinoic acid inducible gene 1, IL6 is interleukin-6, OAS-1 is oligoadenylate synthetase 1, IFNb is interferon-beta, AIM2 is absent in melanoma-2, IFIT-1 is interferon-induced protein with tetratricopeptide repeats 1, PKR is protein kinase R, TNFa is tumor necrosis factor alpha and IFNa is interferon alpha.







TABLE 97







Innate Immune Response Transcript Levels

















RIG-

OAS-








Formulation
I
IL6
1
IFNb
AIM2
IFIT-1
PKR
TNFa
IFNa



















Natural
71.5
20.6
20.778
11.404
0.251
151.218
16.001 
0.526
0.067


Luciferase











Natural G-
73.3
47.1
19.359
13.615
0.264
142.011
11.667
1.185
0.153


CSF











PIC
30.0
2.8
8.628
1.523
0.100
71.914
10.326
0.264
0.063


G-CSF
0.81
0.22
0.080
0.009
0.008
2.220
1.592
0.090
0.027


Gen1-UC











G-CSF
0.54
0.26
0.042
0.005
0.008
1.314
1.568
0.088
0.038


Genl-Cap0











G-CSF
0.58
0.30 
0.035
0.007
0.006
1.510
1.371
0.090
0.040


Genl-Cap1











G-CSF
0.21
0.20
0.002
0.007
0.007
0.603
0.969
0.129
0.005


Gen2-UC











G-CSF
0.23
0.21
0.002
0.0014 
0.007
0.648
1.547
0.121
0.035


Gen2-Cap0











G-CSF
0.27
0.26
0.011
0.004
0.005
0.678
1.557
0.099
0.037


Gen2-Cap1











Lipo
0.27
0.53 
0.001
0
0.007
0.954
1.536
0.158
0.064









B. Repeat Transfection
Human primary foreskin fibroblasts (BJ fibroblasts) were obtained from American Type Culture Collection (ATCC) (catalog # CRL-2522) and grown in Eagle's Minimum Essential Medium (ATCC, catalog #30-2003) supplemented with 10% fetal bovine serum at 37° C., under 5% CO2. BJ fibroblasts were seeded on a 24-well plate at a density of 300,000 cells per well in 0.5 ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) unmodified, fully modified with 5-methylcytosine and pseudouridine (Gen1) or fully modified with 5-methylcytosine and N1-methyl-pseudouridine (Gen2) was transfected daily for 5 days following manufacturer's protocol. Control samples of Lipofectamine 2000 (L2000) and mCherry mRNA (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytidine and pseudouridine) were also transfected daily for 5 days. The results are shown in Table 98.
Unmodified mRNA showed a cytokine response in interferon-beta (IFN-beta) and interleukin-6 (IL-6) after one day. mRNA modified with at least pseudouridine showed a cytokine response after 2-3 days whereas mRNA modified with 5-methylcytosine and N1-methyl-pseudouridine showed a reduced response after 3-5 days.







TABLE 98







Cytokine Response










Formulation
Transfection
IFN-beta (pg/ml)
IL-6 (pg/ml)













G-CSF unmodified
6 hours
0
3596



Day 1
1363
15207



Day 2
238
12415



Day 3
225
5017



Day 4
363
4267



Day 5
225
3094


G-CSF Gen 1
6 hours
0
3396



Day 1
38
3870



Day 2
1125
16341



Day 3
100
25983



Day 4
75
18922



Day 5
213
15928


G-CSF Gen 2
6 hours
0
3337



Day 1
0
3733



Day 2
150
974



Day 3
213
4972



Day 4
1400
4122



Day 5
350
2906


mCherry
6 hours
0
3278



Day 1
238
3893



Day 2
113
1833



Day 3
413
25539



Day 4
413
29233



Day 5
213
20178


L2000
6 hours
0
3270



Day 1
13
3933



Day 2
388
567



Day 3
338
1517



Day 4
475
1594



Day 5
263
1561









Example 66. In Vivo Detection of Innate Immune Response
In an effort to distinguish the importance of different chemical modification of mRNA on in vivo protein production and cytokine response in vivo, female BALB/C mice (n=5) are injected intramuscularly with G-CSF mRNA (G-CSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence) with a 5′cap of Cap1, G-CSF mRNA fully modified with 5-methylcytosine and pseudouridine (G-CSF mRNA 5mc/pU), G-CSF mRNA fully modified with 5-methylcytosine and N1-methyl-pseudouridine with (G-CSF mRNA 5mc/N1pU) or without a 5′ cap (G-CSF mRNA 5mc/N1 pU no cap) or a control of either R848 or 5% sucrose as described in Table 99.







TABLE 99







Dosing Chart












Formulation
Route
Dose (ug/mouse)
Dose (ul)







G-CSF mRNA
I.M.
200
50



unmod



G-CSF mRNA
I.M.
200
50



5mc/pU



G-CSF mRNA
I.M.
200
50



5mc/N1pU



G-CSF mRNA
I.M.
200
50



5mc/N1pU no cap



R848
I.M.
 75
50



5% sucrose
I.M.
—
50



Untreated
I.M.
—
—










Blood is collected at 8 hours after dosing. Using ELISA the protein levels of G-CSF, TNF-alpha and IFN-alpha is determined by ELISA. 8 hours after dosing, muscle is collected from the injection site and quantitative real time polymerase chain reaction (QPCR) is used to determine the mRNA levels of RIG-I, PKR, AIM-2, IFIT-1, OAS-2, MDA-5, IFN-beta, TNF-alpha, IL-6, G-CSF, CD45 in the muscle.
Example 67. In Vivo Detection of Innate Immune Response Study
Female BALB/C mice (n=5) were injected intramuscularly with G-CSF mRNA (G-CSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence) with a 5′cap of Cap1, G-CSF mRNA fully modified with 5-methylcytosine and pseudouridine (G-CSF mRNA 5mc/pU), G-CSF mRNA fully modified with 5-methylcytosine and N1-methyl-pseudouridine with (G-CSF mRNA 5mc/N1pU) or without a 5′ cap (G-CSF mRNA 5mc/N1 pU no cap) or a control of either R848 or 5% sucrose as described in Table 100. Blood is collected at 8 hours after dosing and using ELISA the protein levels of G-CSF and interferon-alpha (IFN-alpha) is determined by ELISA and are shown in Table 100.
As shown in Table 100, unmodified, 5mc/pU, and 5mc/N1pU modified G-CSF mRNA resulted in human G-CSF expression in mouse serum. The uncapped 5mC/N1pU modified G-CSF mRNA showed no human G-CSF expression in serum, highlighting the importance of having a 5′ cap structure for protein translation.
As expected, no human G-CSF protein was expressed in the R848, 5% sucrose only, and untreated groups. Importantly, significant differences were seen in cytokine production as measured by mouse IFN-alpha in the serum. As expected, unmodified G-CSF mRNA demonstrated a robust cytokine response in vivo (greater than the R848 positive control). The 5mc/pU modified G-CSF mRNA did show a low but detectable cytokine response in vivo, while the 5mc/N1pU modified mRNA showed no detectable IFN-alpha in the serum (and same as vehicle or untreated animals).
Also, the response of 5mc/N1pU modified mRNA was the same regardless of whether it was capped or not. These in vivo results reinforce the conclusion that 1) that unmodified mRNA produce a robust innate immune response, 2) that this is reduced, but not abolished, through 100% incorporation of 5mc/pU modification, and 3) that incorporation of 5mc/N1pU modifications results in no detectable cytokine response.
Lastly, given that these injections are in 5% sucrose (which has no effect by itself), these result should accurately reflect the immunostimulatory potential of these modifications.
From the data it is evident that N1pU modified molecules produce more protein while concomitantly having little or no effect on IFN-alpha expression. It is also evident that capping is required for protein production for this chemical modification. The Protein: Cytokine Ratio of 748 as compared to the PC Ratio for the unmodified mRNA (PC=9) means that this chemical modification is far superior as related to the effects or biological implications associated with IFN-alpha.







TABLE 100







Human G-CSF and Mouse IFN-alpha in serum















Dose

G-CSF
IFN-alpha





(ug/
Dose
protein
expression
PC


Formulation
Route
mouse)
(ul)
(pg/ml)
(pg/ml)
Ratio
















G-CSF mRNA 
I.M.
200
50
605.6
67.01
9


unmod








G-CSF mRNA 
I.M.
200
50
356.5
8.87
40


5mc/pU








G-CSF mRNA5mc/
I.M.
200
50
748.1
0
748


N1pU








G-CSF mRNA5mc/
IM.
200
50
6.5
0
6.5


N1pU no cap








R848
I.M.
75
50
3.4
40.97
.08


5% sucrose
I.M.
—
50
0
1.49
0


Untreated
I.M.
—
—
0
0
0









Example 68: In Vivo Delivery of Modified RNA
Protein production of modified mRNA was evaluated by delivering modified G-CSF mRNA or modified Factor IX mRNA to female Sprague Dawley rats (n=6). Rats were injected with 400 ug in 100 ul of G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine (G-CSF Gen1), G-CSF mRNA fully modified with 5-methylcytosine and N1-methyl-pseudouridine (G-CSF Gen2) or Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine (Factor IX Gen1) reconstituted from the lyophilized form in 5% sucrose. Blood was collected 8 hours after injection and the G-CSF protein level in serum was measured by ELISA. Table 101 shows the G-CSF protein levels in serum after 8 hours.
These results demonstrate that both G-CSF Gen 1 and G-CSF Gen 2 modified mRNA can produce human G-CSF protein in a rat following a single intramuscular injection, and that human G-CSF protein production is improved when using Gen 2 chemistry over Gen 1 chemistry.







TABLE 101







G-CSF Protein in Rat Serum (I.M. Injection Route)










Formulation
G-CSF protein (pg/ml)














G-CSF Gen1
19.37



G-CSF Gen2
64.72



Factor IX Gen 1
2.25










Example 69. Chemical Modification: In Vitro Studies
A. In Vitro Screening in PBMC
500 ng of G-CSF (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) mRNA fully modified with the chemical modification outlined Tables 104 and 105 was transfected with 0.4 uL Lipofectamine 2000 into peripheral blood mononuclear cells (PBMC) from three normal blood donors. Control samples of LPS, R848, P(I)P(C) and mCherry (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) were also analyzed. The supernatant was harvested and stored frozen until analyzed by ELISA to determine the G-CSF protein expression, and the induction of the cytokines interferon-alpha (IFN-α) and tumor necrosis factor alpha (TNF-α). The protein expression of G-CSF is shown in Table 102, the expression of IFN-α and TNF-α is shown in Table 103.
The data in Table 102 demonstrates that many, but not all, chemical modifications can be used to productively produce human G-CSF in PBMC. Of note, 100% N1-methyl-pseudouridine substitution demonstrates the highest level of human G-CSF production (almost 10-fold higher than pseudouridine itself). When N1-methyl-pseudouridine is used in combination with 5-methylcytidine a high level of human G-CSF protein is also produced (this is also higher than when pseudouridine is used in combination with 5 methylcytidine).
Given the inverse relationship between protein production and cytokine production in PBMC, a similar trend is also seen in Table 103, where 100% substitution with N1-methyl-pseudouridine results no cytokine induction (similar to transfection only controls) and pseudouridine shows detectable cytokine induction which is above background.
Other modifications such as N6-methyladenosine and α-thiocytidine appear to increase cytokine stimulation.







TABLE 102







Chemical Modifications and G-CSF Protein Expression









G-CSF Protein Expression



(pg/ml)











Donor
Donor
Donor


Chemical Modifications
1
2
3













Pseudouridine
2477
1,909
1,498


5-methyluridine
318
359
345


N1-methyl-pseudouridine
21,495
16,550
12,441


2-thiouridine
932
1,000
600


4-thiouridine
5
391
218


5-methoxyuridine
2,964
1,832
1,800


5-methylcytosine and pseudouridine (1st set)
2,632
1,955
1,373


5-methylcytosine and N1-methyl-
10,232
7,245
6,214


pseudouridine (1st set)


2′Fluoroguanosine
59
186
177


2′Fluorouridine
118
209
191


5-methylcytosine and pseudouridine (2nd set)
1,682
1,382
1,036


5-methylcytosine and N1-methyl-
9,564
8,509
7,141


pseudouridine (2nd set)


5-bromouridine
314
482
291


5-(2-carbomethoxyvinyl)uridine
77
286
177


5-[3(1-E-propenylamino)uridine
541
491
550


α-thiocytidine
105
264
245


5-methylcytosine and pseudouridine (3rd set)
1,595
1,432
955


N1-methyladenosine
182
177
191


N6-methyladenosine
100
168
200


5-methylcytidine
291
277
359


N4-acetylcytidine
50
136
36


5-formylcytidine
18
205
23


5-methylcytosine and pseudouridine (4th set)
264
350
182


5-methylcytosine and N1-methyl-
9,505
6,927
5,405


pseudouridine (4th set)


LPS
1,209
786
636


mCherry
5
168
164


R848
709
732
636


P(I)P(C)
5
186
182
















TABLE 103







Chemical Modifications and Cytokine Expression










IFN-α Expression
TNF-α Expression



(pg/ml)
(pg/ml)














Donor
Donor
Donor
Donor
Donor
Donor


Chemical Modifications
1
2
3
1
2
3
















Pseudouridine
120
77
171
36
81
126


5-methyluridine
245
135
334
94
100
157


N1-methyl-pseudouridine
26
75
138
101
106
134


2-thiouridine
100
108
154
133
133
141


4-thiouridine
463
258
659
169
126
254


5-methoxyuridine
0
64
133
39
74
111


5-methylcytosine and
88
94
148
64
89
121


pseudouridine (1st set)








5-methylcytosine and N1-
0
60
136
54
79
126


methyl-pseudouridine (1st








set)








2′Fluoroguanosine
107
97
194
91
94
141


2′Fluorouridine
158
103
178
164
121
156


5-methylcytosine and
133
92
167
99
111
150


pseudouridine (2nd set)








5-methylcytosine and N1-
0
66
140
54
97
149


methyl-pseudouridine (2nd








set)








5-bromouridine
95
86
181
87
106
157


5-(2-
0
61
130
40
81
116


carbomethoxyvinyl)uridine








5-[3(1-E-
0
58
132
71
90
119


propenylamino)uridine








α-thiocytidine
1,138
565
695
300
273
277


5-methylcytosine and
88
75
150
84
89
130


pseudouridine (3rd set)








N1-methyladenosine
322
255
377
256
157
294


N6-methyladenosine
1,935
1,065
1,492
1,080
630
857


5-methylcytidine
643
359
529
176
136
193


N4-acetylcytidine
789
593
431
263
67
207


5-formylcytidine
180
93
88
136
30
40


5-methylcytosine and
131
28
18
53
24
29


pseudouridine (4th set)








5-methylcytosine and N1-
0
0
0
36
14
13


methyl-pseudouridine (4th








set)








LPS
0
67
146
7,004
3,974
4,020


mCherry
100
75
143
67
100
133


R848
674
619
562
11,179
8,546
9,907


P(I)P(C)
470
117
362
249
177
197









B. In Vitro Screening in HeLa Cells
The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate (Corning, Manassas, Va.). The cells were grown at 37° G in 5% CO2 atmosphere overnight. Next day, 83 ng of Luciferase modified RNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) with the chemical modification described in Table 104, were diluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as transfection reagent and 0.2 ul were diluted in 10 ul final volume of OPTI-MEM. After 5 minutes of incubation at room temperature, both solutions were combined and incubated an additional 15 minute at room temperature. Then the 20 ul combined solution was added to the 100 ul cell culture medium containing the HeLa cells and incubated at room temperature.
After 18 to 22 hours of incubation cells expressing luciferase were lysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.) according to manufacturer instructions. Aliquots of the lysates were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The lysate volumes were adjusted or diluted until no more than 2 mio relative light units (RLU) per well were detected for the strongest signal producing samples and the RLUs for each chemistry tested are shown in Table 104. The plate reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of the plates without reagent was about 200 relative light units per well.
These results demonstrate that many, but not all, chemical modifications can be used to productively produce human G-CSF in HeLa cells. Of note, 100% N1-methyl-pseudouridine substitution demonstrates the highest level of human G-CSF production.







TABLE 104







Relative Light Units of Luciferase










Chemical Modification
RLU














N6-methyladenosine (m6a)
534



5-methylcytidine (m5c)
138,428



N4-acetylcytidine (ac4c)
235,412



5-formylcytidine (f5c)
436



5-methylcytosine/pseudouridine, test A1
48,659



5-methylcytosine/N1-methyl-pseudouridine, test A1
190,924



Pseudouridine
655,632



1-methylpseudouridine (m1u)
1,517,998



2-thiouridine (s2u)
3387



5-methoxyuridine (mo5u)
253,719



5-methylcytosine/pseudouridine, test B1
317,744



5-methylcytosine/N1-methyl-pseudouridine, test B1
265,871



5-Bromo-uridine
43,276



5 (2 carbovinyl) uridine
531



5 (3-1E propenyl Amino) uridine
446



5-methylcytosine/pseudouridine, test A2
295,824



5-methylcytosine/N1-methyl-pseudouridine, test A2
233,921



5-methyluridine
50,932



α-Thio-cytidine
26,358



5-methylcytosine/pseudouridine, test B2
481,477



5-methylcytosine/N1-methyl-pseudouridine, test B2
271,989



5-methylcytosine/pseudouridine, test A3
438,831



5-methylcytosine/N1-methyl-pseudouridine, test A3
277,499



Unmodified Luciferase
234,802










C. In Vitro Screening in Rabbit Reticulocyte Lysates
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) was modified with the chemical modification listed in Table 105 and were diluted in sterile nuclease-free water to a final amount of 250 ng in 10 ul. The diluted luciferase was added to 40 ul of freshly prepared Rabbit Reticulocyte Lysate and the in vitro translation reaction was done in a standard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific, Waltham, Mass.) at 30° C. in a dry heating block. The translation assay was done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit (Promega, Madison, Wis.) according to the manufacturer's instructions. The reaction buffer was supplemented with a one-to-one blend of provided amino acid stock solutions devoid of either Leucine or Methionine resulting in a reaction mix containing sufficient amounts of both amino acids to allow effective in vitro translation.
After 60 minutes of incubation, the reaction was stopped by placing the reaction tubes on ice. Aliquots of the in vitro translation reaction containing luciferase modified RNA were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The volumes of the in vitro translation reactions were adjusted or diluted until no more than 2 mio relative light units (RLUs) per well were detected for the strongest signal producing samples and the RLUs for each chemistry tested are shown in Table 105. The plate reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of the plates without reagent was about 200 relative light units per well.
These cell-free translation results very nicely correlate with the protein production results in HeLa, with the same modifications generally working or not working in both systems. One notable exception is 5-formylcytidine modified luciferase mRNA which worked in the cell-free translation system, but not in the HeLa cell-based transfection system. A similar difference between the two assays was also seen with 5-formylcytidine modified G-CSF mRNA.







TABLE 105







Relative Light Units of Luciferase










Chemical Modification
RLU














N6-methyladenosine (m6a)
398



5-methylcytidine (m5c)
152,989



N4-acetylcytidine (ac4c)
60,879



5-formylcytidine (f5c)
55,208



5-methylcytosine/pseudouridine, test A1
349,398



5-methylcytosine/N1-methyl-pseudouridine, test A1
205,465



Pseudouridine
587,795



1-methylpseudouridine (m1u)
589,758



2-thiouridine (s2u)
708



5-methoxyuridine (mo5u)
288,647



5-methylcytosine/pseudouridine, test B1
454,662



5-methylcytosine/N1-methyl-pseudouridine, test B1
223,732



5-Bromo-uridine
221,879



5 (2 carbovinyl) uridine
225



5 (3-1E propenyl Amino) uridine
211



5-methylcytosine/pseudouridine, test A2
558,779



5-methylcytosine/N1-methyl-pseudouridine, test A2
333,082



5-methyluridine
214,680



α-Thio-cytidine
123,878



5-methylcytosine/pseudouridine, test B2
487,805



5-methylcytosine/N1-methyl-pseudouridine, test B2
154,096



5-methylcytosine/pseudouridine, test A3
413,535



5-methylcytosine/N1-methyl-pseudouridine, test A3
292,954



Unmodified Luciferase
225,986










Example 70. Chemical Modification: In Vivo Studies
A. In Vivo Screening of G-CSF Modified mRNA
Balb-C mice (n=4) are intramuscularly injected in each leg with modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1), fully modified with the chemical modifications outlined in Table 106, is formulated in 1×PBS. A control of luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with pseudouridine and 5-methylcytosine) and a control of PBS are also tested. After 8 hours serum is collected to determine G-CSF protein levels cytokine levels by ELISA.







TABLE 106







G-CSF










mRNA
Chemical Modifications







G-CSF
Pseudouridine



G-CSF
5-methyluridine



G-CSF
2-thiouridine



G-CSF
4-thiouridine



G-CSF
5-methoxyuridine



G-CSF
2′-fluorouridine



G-CSF
5-bromouridine



G-CSF
5-[3(1-E-propenylamino)uridine)



G-CSF
alpha-thio-cytidine



G-CSF
5-methylcytidine



G-CSF
N4-acetylcytidine



G-CSF
Pseudouridine and 5-methylcytosine



G-CSF
N1-methyl-pseudouridine and 5-methylcytosine



Luciferase
Pseudouridine and 5-methylcytosine



PBS
None










B. In Vivo Screening of Luciferase Modified mRNA
Balb-C mice (n=4) were subcutaneously injected with 200 ul containing 42 to 103 ug of modified luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1), fully modified with the chemical modifications outlined in Table 107, was formulated in 1×PBS. A control of PBS was also tested. The dosages of the modified luciferase mRNA is also outlined in Table 107. 8 hours after dosing the mice were imaged to determine luciferase expression. Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse.
As demonstrated in Table 107, all luciferase mRNA modified chemistries demonstrated in vivo activity, with the exception of 2′-fluorouridine. In addition 1-methyl-pseudouridine modified mRNA demonstrated very high expression of luciferase (5-fold greater expression than pseudouridine containing mRNA).







TABLE 107







Luciferase Screening













Dose
Dose
Luciferase



Chemical
(ug) of
volume
expression


mRNA
Modifications
mRNA
(ml)
(photon/second)














Luciferase
5-methylcytidine
83
0.72
1.94E+07


Luciferase
N4-acetylcytidine
76
0.72
1.11E07


Luciferase
Pseudouridine
95
1.20
1.36E+07


Luciferase
1-
103
0.72
7.40E+07



methylpseudouridine


Luciferase
5-methoxyuridine
95
1.22
3.32+07


Luciferase
5-methyluridine
94
0.86
7.42E+06


Luciferase
5-bromouridine
89
1.49
3.75E+07


Luciferase
2′-fluoroguanosine
42
0.72
5.88E+05


Luciferase
2′-fluorocytidine
47
0.72
4.21E+05


Luciferase
2′-flurorouridine
59
0.72
3.47E+05


PBS
None
—
0.72
3.16E+05









Example 71. In Vivo Screening of Combination Luciferase Modified mRNA
Balb-C mice (n=4) were subcutaneously injected with 200 ul of 100 ug of modified luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1), fully modified with the chemical modifications outlined in Table 108, was formulated in 1×PBS. A control of PBS was also tested. 8 hours after dosing the mice were imaged to determine luciferase expression. Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse.
As demonstrated in Table 108, all luciferase mRNA modified chemistries (in combination) demonstrated in vivo activity. In addition the presence of N1-methyl-pseudouridine in the modified mRNA (with N4-acetylcytidine or 5 methylcytidine) demonstrated higher expression than when the same combinations where tested using with pseudouridine. Taken together, these data demonstrate that N1-methyl-pseudouridine containing luciferase mRNA results in improved protein expression in vivo whether used alone (Table 107) or when used in combination with other modified nucleotides (Table 108).







TABLE 108







Luciferase Screening Combinations











Luciferase




expression




(photon/


mRNA
Chemical Modifications
second)





Luciferase
N4-acetylcytidine/pseudouridine
4.18E+06


Luciferase
N4-acetylcytidine/N1-methyl-pseudouridine
2.88E+07


Luciferase
5-methylcytidine/5-methoxyuridine
3.48E+07


Luciferase
5-methylcytidine/5-methyluridine
1.44E+07


Luciferase
5-methylcytidine/where 50% of the uridine
2.39E+06



is replaced with 2-thiouridine


Luciferase
5-methylcytidine/pseudouridine
2.36E+07


Luciferase
5-methylcytidine/N1-methyl-pseudouridine
4.15E+07


PBS
None
3.59E+05









Example 72. Innate Immune Response in BJ Fibroblasts
Human primary foreskin fibroblasts (BJ fibroblasts) are obtained from American Type Culture Collection (ATCC) (catalog #CRL-2522) and grown in Eagle's Minimum Essential Medium (ATCC, cat #30-2003) supplemented with 10% fetal bovine serum at 37° C., under 5% CO2. BJ fibroblasts are seeded on a 24-well plate at a density of 130,000 cells per well in 0.5 ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine (Gen1) or fully modified with 5-methylcytosine and N1-methyl-pseudouridine (Gen2) is transfected using Lipofectamine 2000 (Invitrogen, cat #11668-019), following manufacturer's protocol. Control samples of Lipofectamine 2000 and unmodified G-CSF mRNA (natural G-CSF) are also transfected. The cells are transfected for five consecutive days. The transfection complexes are removed four hours after each round of transfection.
The culture supernatant is assayed for secreted G-CSF (R&D Systems, catalog #DCS50), tumor necrosis factor-alpha (TNF-alpha) and interferon alpha (IFN-alpha) by ELISA every day after transfection following manufacturer's protocols. The cells are analyzed for viability using CELL TITER GLO® (Promega, catalog #G7570) 6 hrs and 18 hrs after the first round of transfection and every alternate day following that. At the same time from the harvested cells, total RNA is isolated and treated with DNASE® using the RNAEASY micro kit (catalog #74004) following the manufacturer's protocol. 100 ng of total RNA is used for cDNA synthesis using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, cat #4368814) following the manufacturer's protocol. The cDNA is then analyzed for the expression of innate immune response genes by quantitative real time PCR using SybrGreen in a Biorad CFX 384 instrument following the manufacturer's protocol.
Example 73. In Vitro Transcription with Wild-Type T7 Polymerase
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) and G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) were fully modified with different chemistries and chemistry combinations listed in Tables 109-112 using wild-type T7 polymerase as previously described.
The yield of the translation reactions was determined by spectrophometric measurement (OD260) and the yield for Luciferase is shown in Table 109 and G-CSF is shown in Table 111.
The luciferase and G-CSF modified mRNA were also subjected to an enzymatic capping reaction and each modified mRNA capping reaction was evaluated for yield by spectrophometic measurement (OD260) and correct size assessed using bioanalyzer. The yield from the capping reaction for luciferase is shown in Table 110 and G-CSF is shown in Table 112.







TABLE 109







In vitro transcription chemistry for Luciferase










Chemical Modification
Yield (mg)














N6-methyladenosine
0.99



5-methylcytidine
1.29



N4-acetylcytidine
1.0



5-formylcytidine
0.55



Pseudouridine
2.0



N1-methyl-pseudouridine
1.43



2-thiouridine
1.56



5-methoxyuridine
2.35



5-methyluridine
1.01



α-Thio-cytidine
0.83



5-Br-uridine (5Bru)
1.96



5 (2 carbomethoxyvinyl) uridine
0.89



5 (3-1E propenyl Amino) uridine
2.01



N4-acetylcytidine/pseudouridine
1.34



N4-acetylcytidine/N1-methyl-pseudouridine
1.26



5-methylcytidine/5-methoxyuridine
1.38



5-methylcytidine/5-bromouridine
0.12



5-methylcytidine/5-methy luridine
2.97



5-methylcytidine/half of the uridines
1.59



are modified with 2-thiouridine



5-methylcytidine/2-thiouridine
0.90



5-methylcytidine/pseudouridine
1.83



5-methylcytidine/N1 methyl pseudouridine
1.33

















TABLE 110







Capping chemistry and yield for Luciferase modified mRNA










Chemical Modification
Yield (mg)














5-methylcytidine
1.02



N4-acetylcytidine
0.93



5-formylcytidine
0.55



Pseudouridine
2.07



N1-methyl-pseudouridine
1.27



2-thiouridine
1.44



5-methoxyuridine
2



5-methyluridine
0.8



α-Thio-cytidine
0.74



5-Br-uridine (5Bru)
1.29



5 (2 carbomethoxyvinyl) uridine
0.54



5 (3-1E propenyl Amino) uridine
1.39



N4-acetylcytidine/pseudouridine
0.99



N4-acetylcytidine/N1-methyl-pseudouridine
1.08



5-methylcytidine/5-methoxyuridine
1.13



5-methylcytidine/5-methyluridine
1.08



5-methylcytidine/half of the uridines
1.2



are modified with 2-thiouridine



5-methylcytidine/2-thiouridine
1.27



5-methylcytidine/pseudouridine
1.19



5-methylcytidine/N1 methyl pseudouridine
1.04

















TABLE 111







In vitro transcription chemistry


and yield for G-CSF modified mRNA










Chemical Modification
Yield (mg)














N6-methyladenosine
1.57



5-methylcytidine
2.05



N4-acetylcytidine
3.13



5-formylcytidine
1.41



Pseudouridine
4.1



N1-methyl-pseudouridine
3.24



2-thiouridine
3.46



5-methoxyuridine
2.57



5-methyluridine
4.27



4-thiouridine
1.45



2′-F-uridine
0.96



α-Thio-cytidine
2.29



2′-F-guanosine
0.6



N-1-methyladenosine
0.63



5-Br-uridine (5Bru)
1.08



5 (2 carbomethoxyvinyl) uridine
1.8



5 (3-1E propenyl Amino) uridine
2.09



N4-acetylcytidine/pseudouridine
1.72



N4-acetylcytidine/N1-methyl-pseudouridine
1.37



5-methylcytidine/5-methoxyuridine
1.85



5-methylcytidine/5-methyluridine
1.56



5-methylcytidine/half of the uridines
1.84



are modified with 2-thiouridine



5-methylcytidine/2-thiouridine
2.53



5-methylcytidine/pseudouridine
0.63



N4-acetylcytidine/2-thiouridine
1.3



N4-acetylcytidine/5-bromouridine
1.37



5-methylcytidine/N1 methyl pseudouridine
1.25



N4-acetylcytidine/pseudouridine
2.24

















TABLE 112







Capping chemistry and yield for G-CSF modified mRNA










Chemical Modification
Yield (mg)














N6-methyladenosine
1.04



5-methylcytidine
1.08



N4-acetylcytidine
2.73



5-formylcytidine
0.95



Pseudouridine
3.88



N1-methyl-pseudouridine
2.58



2-thiouridine
2.57



5-methoxyuridine
2.05



5-methyluridine
3.56



4-thiouridine
0.91



2′-F-uridine
0.54



α-Thio-cytidine
1.79



2′-F-guanosine
0.14



5-Br-uridine (5Bru)
0.79



5 (2 carbomethoxyvinyl) uridine
1.28



5 (3-1E propenyl Amino) uridine
1.78



N4-acetylcytidine/pseudouridine
0.29



N4-acetylcytidine/N1-methyl-pseudouridine
0.33



5-methylcytidine/5-methoxyuridine
0.91



5-methylcytidine/5-methyluridine
0.61



5-methylcytidine/half of the uridines
1.24



are modified with 2-thiouridine



5-methylcytidine/pseudouridine
1.08



N4-acetylcytidine/2-thiouridine
1.34



N4-acetylcytidine/5-bromouridine
1.22



5-methylcytidine/N1 methyl pseudouridine
1.56










Example 74. In Vitro Transcription with Mutant T7 Polymerase
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) and G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) were fully modified with different chemistries and chemistry combinations listed in Tables 113-116 using a mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.).
The yield of the translation reactions was determined by spectrophometric measurement (OD260) and the yield for Luciferase is shown in Table 113 and G-CSF is shown in Table 115.
The luciferase and G-CSF modified mRNA were also subjected to an enzymatic capping reaction and each modified mRNA capping reaction was evaluated for yield by spectrophometic measurement (OD260) and correct size assessed using bioanalyzer. The yield from the capping reaction for luciferase is shown in Table 114 and G-CSF is shown in Table 115.







TABLE 113







In vitro transcription chemistry and


yield for Luciferase modified mRNA








Chemical Modification
Yield (ug)











2′Fluorocytosine
71.4


2′Fluorouridine
57.5


5-methylcytosine/pseudouridine, test A
26.4


5-methylcytosine/N1-methyl-pseudouridine, test A
73.3


N1-acetylcytidine/2-fluorouridine
202.2


5-methylcytidine/2-fluorouridine
131.9


2-fluorocytosine/pseudouridine
119.3


2-fluorocytosine/N1-methyl-pseudouridine
107.0


2-fluorocytosine/2-thiouridine
34.7


2-fluorocytosine/5-bromouridine
81.0


2-fluorocytosine/2-fluorouridine
80.4


2-fluoroguanine/5-methylcytosine
61.2


2-fluoroguanine/5-methylcytosine/pseudouridine
65.0


2-fluoroguanine/5-methylcytidine/N1-methyl-pseudouridine
41.2


2-fluoroguanine/pseudouridine
79.1


2-fluoroguanine/N1-methyl-pseudouridine
74.6


5-methylcytidine/pseudouridine, test B
91.8


5-methylcytidine/N1 methyl pseudouridine, test B
72.4


2′fluoroadenosine
190.98
















TABLE 114







Capping chemistry and yield for Luciferase modified mRNA








Chemical Modification
Yield (ug)











2′Fluorocytosine
19.2


2′Fluorouridine
16.7


5-methylcytosine/pseudouridine, test A
7.0


5-methylcytosine/N1-methyl-pseudouridine, test A
21.5


N1-acetylcytidine/2-fluorouridine
47.5


5-methylcytidine/2- fluorouridine
53.2


2-fluorocytosine/pseudouridine
58.4


2-fluorocytosine/N1-methyl-pseudouridine
26.2


2-fluorocytosine/2-thiouridine
12.9


2-fluorocytosine/5-bromouridine
26.5


2-fluorocytosine/2-fluorouridine
35.7


2-fluoroguanine/5-methylcytosine
24.7


2-fluoroguanine/5-methylcytosine/pseudouridine
32.3


2-fluoroguanine/5-methylcytidine/N1-methyl-pseudouridine
31.3


2-fluoroguanine/pseudouridine
20.9


2-fluoroguanine/N1-methyl-pseudouridine
29.8


5-methylcytidine/pseudouridine, test B
58.2


5-methylcytidine/N1-methyl-pseudouridine, test B
44.4
















TABLE 115







In vitro transcription chemistry


and yield for G-CSF modified mRNA








Chemical Modification
Yield (ug)











2′Fluorocytosine
56.5


2′Fluorouridine
79.4


5-methylcytosine/pseudouridine, test A
21.2


5-methylcytosine/N1-methyl-pseudouridine, test A
77.1


N1-acetylcytidine/2-fluorouridine
168.6


5-methylcytidine/2-fluorouridine
134.7


2-fluorocytosine/pseudouridine
97.8


2-fluorocytosine/N1-methyl-pseudouridine
103.1


2-fluorocytosine/2-thiouridine
58.8


2-fluorocytosine/5-bromouridine
88.8


2-fluorocytosine/2-fluorouridine
93.9


2-fluoroguanine/5-methylcytosine
97.3


2-fluoroguanine/5-methylcytosine/pseudouridine
96.0


2-fluoroguanine/5-methylcytidine/N1-methyl-pseudouridine
82.0


2-fluoroguanine/pseudouridine
68.0


2-fluoroguanine/N1-methyl-pseudouridine
59.3


5-methylcytidine/pseudouridine, test B
58.7


5-methylcytidine/N1-methyl-pseudouridine, test B
78.0
















TABLE 116







Capping chemistry and yield for G-CSF modified mRNA








Chemical Modification
Yield (ug)











2′Fluorocytosine
16.9


2′Fluorouridine
17.0


5-methylcytosine/pseudouridine, test A
10.6


5-methylcytosine/N1-methyl-pseudouridine, test A
22.7


N1-acetylcytidine/2-fluorouridine
19.9


5-methylcytidine/2-fluorouridine
21.3


2-fluorocytosine/pseudouridine
65.2


2-fluorocytosine/N1-methyl-pseudouridine
58.9


2-fluorocytosine/2-thiouridine
41.2


2-fluorocytosine/5-bromouridine
35.8


2-fluorocytosine/2-fluorouridine
36.7


2-fluoroguanine/5-methylcytosine
36.6


2-fluoroguanine/5-methylcytosine/pseudouridine
37.3


2-fluoroguanine/5-methylcytidine/N1-methyl-pseudouridine
30.7


2-fluoroguanine/pseudouridine
29.0


2-fluoroguanine/N1-methyl-pseudouridine
22.7


5-methylcytidine/pseudouridine, test B
60.4


5-methylcytidine/N1-methyl-pseudouridine, test B
33.0









Example 75. 2′O-Methyl and 2′Fluoro Compounds
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) were produced as fully modified versions with the chemistries in Table 117 and transcribed using mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.). 2′ fluoro-containing mRNA were made using Durascribe T7, however, 2′Omethyl-containing mRNA could not be transcribed using Durascribe T7.
Incorporation of 2′Omethyl modified mRNA might possibly be accomplished using other mutant T7 polymerases (Nat Biotechnol. (2004) 22:1155-1160; Nucleic Acids Res. (2002) 30:e138) or U.S. Pat. No. 7,309,570, the contents of each of which are incorporated herein by reference in their entirety. Alternatively, 2′OMe modifications could be introduced post-transcriptionally using enzymatic means.
Introduction of modifications on the 2′ group of the sugar has many potential advantages. 2′OMe substitutions, like 2′ fluoro substitutions are known to protect against nucleases and also have been shown to abolish innate immune recognition when incorporated into other nucleic acids such as siRNA and anti-sense (incorporated in its entirety, Crooke, ed. Antisense Drug Technology, 2nd edition; Boca Raton: CRC press).
The 2′Fluoro-modified mRNA were then transfected into HeLa cells to assess protein production in a cell context and the same mRNA were also assessed in a cell-free rabbit reticulocyte system. A control of unmodified luciferase (natural luciferase) was used for both transcription experiments, a control of untreated and mock transfected (Lipofectamine 2000 alone) were also analyzed for the HeLa transfection and a control of no RNA was analyzed for the rabbit reticulysates.
For the HeLa transfection experiments, the day before transfection, 20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate (Corning, Manassas, Va.). The cells were grown at 37° G in 5% CO2 atmosphere overnight. Next day, 83 ng of the 2′fluoro-containing luciferase modified RNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) with the chemical modification described in Table 117, were diluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as transfection reagent and 0.2 ul were diluted in 10 ul final volume of OPTI-MEM. After 5 minutes of incubation at room temperature, both solutions were combined and incubated an additional 15 minute at room temperature. Then the 20 ul combined solution was added to the 100 ul cell culture medium containing the HeLa cells and incubated at room temperature. After 18 to 22 hours of incubation cells expressing luciferase were lysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.) according to manufacturer instructions. Aliquots of the lysates were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The lysate volumes were adjusted or diluted until no more than 2 mio relative light units (RLU) per well were detected for the strongest signal producing samples and the RLUs for each chemistry tested are shown in Table 117. The plate reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of the plates without reagent was about 200 relative light units per well.
For the rabbit reticulocyte lysate assay, 2′-fluoro-containing luciferase mRNA were diluted in sterile nuclease-free water to a final amount of 250 ng in 10 ul and added to 40 ul of freshly prepared Rabbit Reticulocyte Lysate and the in vitro translation reaction was done in a standard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific, Waltham, Mass.) at 30° C. in a dry heating block. The translation assay was done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit (Promega, Madison, Wis.) according to the manufacturer's instructions. The reaction buffer was supplemented with a one-to-one blend of provided amino acid stock solutions devoid of either Leucine or Methionine resulting in a reaction mix containing sufficient amounts of both amino acids to allow effective in vitro translation. After 60 minutes of incubation, the reaction was stopped by placing the reaction tubes on ice.
Aliquots of the in vitro translation reaction containing luciferase modified RNA were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The volumes of the in vitro translation reactions were adjusted or diluted until no more than 2 mio relative light units (RLUs) per well were detected for the strongest signal producing samples and the RLUs for each chemistry tested are shown in Table 118. The plate reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of the plates without reagent was about 160 relative light units per well.
As can be seen in Table 117 and 118, multiple 2′Fluoro-containing compounds are active in vitro and produce luciferase protein.







TABLE 117







HeLa Cells












Concentration
Volume
Yield



Chemical Modification
(ug/ml)
(ul)
(ug)
RLU














2′Fluoroadenosine
381.96
500
190.98
388.5


2′Fluorocytosine
654.56
500
327.28
2420


2′Fluoroguanine
541.795
500
270.90
11,705.5


2′Flurorouridine
944.005
500
472.00
6767.5


Natural luciferase
N/A
N/A
N/A
133,853.5


Mock
N/A
N/A
N/A
340


Untreated
N/A
N/A
N/A
238
















TABLE 118







Rabbit Reticulysates










Chemical Modification
RLU














2′Fluoroadenosine
162



2′Fluorocytosine
208



2′Fluoroguanine
371,509



2′Flurorouridine
258



Natural luciferase
2,159,968



No RNA
156










Example 76. Luciferase in HeLa Cells Using a Combination of Modifications
To evaluate using of 2′fluoro-modified mRNA in combination with other modification a series of mRNA were transcribed using either wild-type T7 polymerase (non-fluoro-containing compounds) or using mutant T7 polymerases (fluyoro-containing compounds) as described in Example 75. All modified mRNA were tested by in vitro transfection in HeLa cells.
The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate (Corning, Manassas, Va.). The cells were grown at 37° G in 5% CO2 atmosphere overnight. Next day, 83 ng of Luciferase modified RNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) with the chemical modification described in Table 119, were diluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as transfection reagent and 0.2 ul were diluted in 10 ul final volume of OPTI-MEM. After 5 minutes of incubation at room temperature, both solutions were combined and incubated an additional 15 minute at room temperature. Then the 20 ul combined solution was added to the 100 ul cell culture medium containing the HeLa cells and incubated at room temperature.
After 18 to 22 hours of incubation cells expressing luciferase were lysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.) according to manufacturer instructions. Aliquots of the lysates were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The lysate volumes were adjusted or diluted until no more than 2 mio relative light units (RLU) per well were detected for the strongest signal producing samples and the RLUs for each chemistry tested are shown in Table 119. The plate reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of the plates without reagent was about 200 relative light units per well.
As evidenced in Table 119, most combinations of modifications resulted in mRNA which produced functional luciferase protein, including all the non-flouro containing compounds and many of the combinations containing 2′fluro modifications.







TABLE 119







Luciferase








Chemical Modification
RLU











N4-acetylcytidine/pseudouridine
113,796


N4-acetylcytidine/N1-methyl-pseudouridine
316,326


5-methylcytidine/5-methoxyuridine
24,948


5-methylcytidine/5-methyluridine
43,675


5-methylcytidine/half of the uridines modified with 50%
41,601


2-thiouridine


5-methylcytidine/2-thiouridine
1,102


5-methylcytidine/pseudouridine
51,035


5-methylcytidine/N1-methyl-pseudouridine
152,151


N4-acetylcytidine/2′Fluorouridine triphosphate
288


5-methylcytidine/2′Fluorouridine triphosphate
269


2′Fluorocytosine triphosphate/pseudouridine
260


2′Fluorocytosine triphosphate/N1-methyl-pseudouridine
412


2′Fluorocytosine triphosphate/2-thiouridine
427


2′Fluorocytosine triphosphate/5-bromouridine
253


2′Fluorocytosine triphosphate/2′Fluorouridine triphosphate
184


2′Fluoroguanine triphosphate/5-methylcytidine
321


2′Fluoroguanine triphosphate/5-methylcytidine/Pseudouridine
207


2′Fluoroguanine/5-methylcytidine/N1 methylpsuedouridine
235


2′Fluoroguanine/pseudouridine
218


2′Fluoroguanine/N1-methylpsuedouridine
247


5-methylcytidine/pseudouridine, test A
13,833


5-methylcytidine/N1-methyl-pseudouridine, test A
598


2′Fluorocytosine triphosphate
201


2′Fluorouridine triphosphate
305


5-methylcytidine/pseudouridine, test B
115,401


5-methylcytidine/N1-methyl-pseudouridine, test B
21,034


Natural luciferase
30,801


Untreated
344


Mock
262









Example 77. G-CSF In Vitro Transcription
To assess the activity of all our different chemical modifications in the context of a second open reading frame, we replicated experiments previously conducted using luciferase mRNA, with human G-CSF mRNA. G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) were fully modified with the chemistries in Tables 120 and 121 using wild-type T7 polymerase (for all non-fluoro-containing compounds) or mutant T7 polymerase (for all fluoro-containing compounds). The mutant T7 polymerase was obtained commercially (Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.).
The modified RNA in Tables 120 and 121 were transfected in vitro in HeLa cells or added to rabbit reticulysates (250 ng of modified mRNA) as indicated. A control of untreated, mock transfected (transfection reagent alone), G-CSF fully modified with 5-methylcytosine and N1-methyl-pseudouridine or luciferase control (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine were also analyzed. The expression of G-CSF protein was determined by ELISA and the values are shown in Tables 120 and 121. In Table 120, “NT” means not tested.
As shown in Table 120, many, but not all, chemical modifications resulted in human G-CSF protein production. These results from cell-based and cell-free translation systems correlate very nicely with the same modifications generally working or not working in both systems. One notable exception is 5-formylcytidine modified G-CSF mRNA which worked in the cell-free translation system, but not in the HeLa cell-based transfection system. A similar difference between the two assays was also seen with 5-formylcytidine modified luciferase mRNA.
As demonstrated in Table 121, many, but not all, G-CSF mRNA modified chemistries (when used in combination) demonstrated in vivo activity. In addition the presence of N1-methyl-pseudouridine in the modified mRNA (with N4-acetylcytidine or 5 methylcytidine) demonstrated higher expression than when the same combinations where tested using with pseudouridine. Taken together, these data demonstrate that N1-methyl-pseudouridine containing G-CSF mRNA results in improved protein expression in vitro.







TABLE 120







G-CSF Expression











G-CSF protein



G-CSF protein
(pg/ml) Rabbit



(pg/ml)
reticulysates


Chemical Modification
HeLa cells
cells












Pseudouridine
1,150,909
147,875


5-methyluridine
347,045
147,250


2-thiouridine
417,273
18,375


N1-methyl-pseudouridine
NT
230,000


4-thiouridine
107,273
52,375


5-methoxyuridine
1,715,909
201,750


5-methylcytosine/pseudouridine,
609,545
119,750


Test A


5-methylcytosine/N1-methyl-
1,534,318
110,500


pseudouridine, Test A


2′-Fluoro-guanosine
11,818
0


2′-Fluoro-uridine
60,455
0


5-methylcytosine/pseudouridine,
358,182
57,875


Test B


5-methylcytosine/N1-methyl-
1,568,636
76,750


pseudouridine, Test B


5-Bromo-uridine
186,591
72,000


5-(2carbomethoxyvinyl) uridine
1,364
0


5-[3(1-E-propenylamino) uridine
27,955
32,625


α-thio-cytidine
120,455
42,625


5-methylcytosine/pseudouridine,
882,500
49,250


Test C


N1-methyl-adenosine
4,773
0


N6-methyl-adenosine
1,591
0


5-methyl-cytidine
646,591
79,375


N4-acetylcytidine
39,545
8,000


5-formyl-cytidine
0
24,000


5-methylcytosine/pseudouridine,
87,045
47,750


Test D


5-methylcytosine/N1-methyl-
1,168,864
97,125


pseudouridine, Test D


Mock
909
682


Untreated
0
0


5-methylcytosine/N1-methyl-
1,106,591
NT


pseudouridine, Control


Luciferase control
NT
0
















TABLE 121







Combination Chemistries in HeLa cells











G-CSF protein




(pg/ml)



Chemical Modification
HeLa cells














N4-acetylcytidine/pseudouridine
537,273



N4-acetylcytidine/N1-methyl-pseudouridine
1,091,818



5-methylcytidine/5-methoxyuridine
516,136



5-methylcytidine/5-bromouridine
48,864



5-methylcytidine/5-methyluridine
207,500



5-methylcytidine/2-thiouridine
33,409



N4-acetylcytidine/5-bromouridine
211,591



N4-acetylcytidine/2-thiouridine
46,136



5-methylcytosine/pseudouridine
301,364



5-methylcytosine/N1-methyl-pseudouridine
1,017,727



N4-acetylcytidine/2′Fluorouridine triphosphate
62,273



5-methylcytidine/2′Fluorouridine triphosphate
49,318



2′Fluorocytosine triphosphate/pseudouridine
7,955



2′Fluorocytosine triphosphate/N1-methyl-
1,364



pseudouridine



2′Fluorocytosine triphosphate/2-thiouridine
0



2′Fluorocytosine triphosphate/5-bromouridine
1,818



2′Fluorocytosine triphosphate/2′Fluorouridine
909



triphosphate



2′Fluoroguanine triphosphate/5-methylcytidine
0



2′Fluoroguanine triphosphate/5-methylcytidine/
0



pseudouridine



2′Fluoroguanine triphosphate/5-methylcytidine/
1,818



N1 methylpseudouridine



2′Fluoroguanine triphosphate/pseudouridine
1,136



2′Fluoroguanine triphosphate/2′Fluorocytosine
0



triphosphate/N1-methyl-pseudouridine



5-methylcytidine/pseudouridine
617,727



5-methylcytidine/N1-methyl-pseudouridine
747,045



5-methylcytidine/pseudouridine
475,455



5-methylcytidine/N1-methyl-pseudouridine
689,091



5-methylcytosine/N1-methyl-pseudouridine,
848,409



Control 1



5-methylcytosine/N1-methyl-pseudouridine,
581,818



Control 2



Mock
682



Untreated
0



Luciferase 2′Fluorocytosine triphosphate
0



Luciferase 2′Fluorouridine triphosphate
0










Example 78. Screening of Chemistries
The tables listed in below (Tables 122-124) summarize much of the in vitro and in vitro screening data with the different compounds presented in the previous examples. A good correlation exists between cell-based and cell-free translation assays. The same chemistry substitutions generally show good concordance whether tested in the context of luciferase or G-CSF mRNA. Lastly, N1-methyl-pseudouridine containing mRNA show a very high level of protein expression with little to no detectable cytokine stimulation in vitro and in vivo, and is superior to mRNA containing pseudouridine both in vitro and in vivo.
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) and G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) were modified with naturally and non-naturally occurring chemistries described in Tables 122 and 123 or combination chemistries described in Table 124 and tested using methods described herein.
In Tables 122 and 123, “*” refers to in vitro transcription reaction using a mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.); “**” refers to the second result in vitro transcription reaction using a mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.); “***” refers to production seen in cell free translations (rabbit reticulocyte lysates); the protein production of HeLa is judged by “+,” “+/−” and “−”; when referring to G-CSF PBMC “++++” means greater than 6,000 pg/ml G-CSF, “+++” means greater than 3,000 pg/ml G-CSF, “++” means greater than 1,500 pg/ml G-CSF, “+” means greater than 300 pg/ml G-CSF, “+1-” means 150-300 pg/ml G-CSF and the background was about 110 pg/ml; when referring to cytokine PBMC “++++” means greater than 1,000 pg/ml interferon-alpha (IFN-alpha), “+++” means greater than 600 pg/ml IFN-alpha, “++” means greater than 300 pg/ml IFN-alpha, “+” means greater than 100 pg/ml IFN-alpha, “−” means less than 100 pg/ml and the background was about 70 pg/ml; and “NT” means not tested. In Table 123, the protein production was evaluated using a mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.).







TABLE 122







Naturally Occurring

















IVT
Protein
Protein
Protein
Cytokines
In Vivo
In Vivo


Common Name
IVT
(G-
(Luc;
(G-CSF;
(G-CSF;
(G-CSF;
Protein
Protein


(symbol)
(Luc)
CSF)
HeLa)
HeLa)
PBMC)
PBMC)
(Luc)
(G-CSF)





1-methyladenosine
Fail
Pass
NT
−
+/−
++
NT
NT


(m1A)










N6-methyladenosine
Pass
Pass
−
−
+/−
++++
NT
NT


(m6A)










2′-O-methyladenosine
Fail*
Not
NT
NT
NT
NT
NT
NT


(Am)

Done








5-methylcytidine
Pass
Pass
+
+
+
++
+
NT


(m5C)










2′-O-methylcytidine
Fail*
Not
NT
NT
NT
NT
NT
NT


(Cm)

Done








2-thiocytidine (s2C)
Fail
Fail
NT
NT
NT
NT
NT
NT


N4-acetylcytidine
Pass
Pass
+
+
+/−
+++
+
NT


(ac4C)










5-formylcytidine
Pass
Pass
−***
−***
−
+
NT
NT


(f5C)










2′-O-methylguanosine
Fail*
Not
NT
NT
NT
NT
NT
NT


(Gm)

Done








inosine (I)
Fail
Fail
NT
NT
NT
NT
NT
NT


pseudouridine (Y)
Pass
Pass
+
+
++
+
+
NT


5-methyluridine
Pass
Pass
+
+
+/−
+
NT
NT


(m5U)










2′-O-methyluridine
Fail*
Not
NT
NT
NT
NT
NT
NT


(Um)

Done








1-methylpseudouridine
Pass
Pass
+
Not 
++++
−
+
NT


(m1Y)



Done






2-thiouridine (s2U)
Pass
Pass
−
+
+
+
NT
NT


4-thiouridine (s4U)
Fail
Pass

+
+/−
++
NT
NT


5-methoxyuridine
Pass
Pass
+
+
++
−
+
NT


(mo5U)










3-methyluridine
Fail
Fail
NT
NT
NT
NT
NT
NT


(m3U)
























TABLE 123







Non-Naturally Occurring



















Protein
Protein


In Vivo




IVT
Protein
(G-
(G-
Cytokines
In Vivo
Protein



IVT
(G-
(Luc;
CSF;
CSF;
(G-CSF;
Protein
(G-


Common Name
(Luc)
CSF)
HeLa)
HeLa)
PBMC)
PBMC)
(Luc)
CSF)





2′-F-ara-guanosine
Fail
Fail
NT
NT
NT
NT
NT
NT


2′-F-ara-adenosine
Fail
Fail
NT
NT
NT
NT
NT
NT


2′-F-ara-cytidine
Fail
Fail
NT
NT
NT
NT
NT
NT


2′-F-ara-uridine
Fail
Fail
NT
NT
NT
NT
NT
NT


2′-F-guanosine
Fail/
Pass/ 
+**
+/−
−
+
+
NT



Pass** 
Fail**








2′-F-adenosine
Fail/
Fail/
−**
NT
NT
NT
NT
NT



Pass** 
Fail**








2′-F-cytidine
Fail/
Fail/
+**
NT
NT
NT
+
NT



Pass** 
Pass**








2′-F-uridine
Fail/
Pass/
+**
+
+/−
+
−
NT



Pass** 
Pass**








2′-OH-ara-guanosine
Fail
Fail
NT
NT
NT
NT
NT
NT


2′-OH-ara-adenosine
Not
Not
NT
NT
NT
NT
NT
NT



Done
Done








2′-OH-ara-cytidine
Fail
Fail
NT
NT
NT
NT
NT
NT


2′-OH-ara-uridine
Fail
Fail
NT
NT
NT
NT
NT
NT


5-Br-Uridine
Pass
Pass
+
+
+
+
+



5-(2-
Pass
Pass
−
−
+/−
−




carbomethoxyvinyl)










Uridine










5-[3-(1-E-
Pass
Pass
−
+
+
−




Propenylamino)










Uridine (aka Chem 5)










N6-(19-Amino-
Fail
Fail
NT
NT
NT
NT
NT
NT


pentaoxanonadecyl) A










2-Dimethylamino
Fail
Fail
NT
NT
NT
NT
NT
NT


guanosine










6-Aza-cytidine
Fail
Fail
NT
NT
NT
NT
NT
NT


a-Thio-cytidine
Pass
Pass
+
+
+/−
+++
NT
NT


Pseudo-isocytidine 
NT
NT
NT
NT
NT
NT
NT
NT


5-Iodo-uridine
NT
NT
NT
NT
NT
NT
NT
NT


a-Thio-uridine
NT
NT
NT
NT
NT
NT
NT
NT


6-Aza-uridine
NT
NT
NT
NT
NT
NT
NT
NT


Deoxy-thymidine
NT
NT
NT
NT
NT
NT
NT
NT


a-Thio guanosine
NT
NT
NT
NT
NT
NT
NT
NT


8-Oxo-guanosine
NT
NT
NT
NT
NT
NT
NT
NT


O6-Methyl-guanosine
NT
NT
NT
NT
NT
NT
NT
NT


7-Deaza-guanosine
NT
NT
NT
NT
NT
NT
NT
NT


6-Chloro-purine
NT
NT
NT
NT
NT
NT
NT
NT


a-Thio-adenosine
NT
NT
NT
NT
NT
NT
NT
NT


7-Deaza-adenosine
NT
NT
NT
NT
NT
NT
NT
NT


5-iodo-cytidine
NT
NT
NT
NT
NT
NT
NT
NT









In Table 124, the protein production of HeLa is judged by “+,” “+/−” and “−”; when referring to G-CSF PBMC “++++” means greater than 6,000 pg/ml G-CSF, “+++” means greater than 3,000 pg/ml G-CSF, “++” means greater than 1,500 pg/ml G-CSF, “+” means greater than 300 pg/ml G-CSF, “+/−” means 150-300 pg/ml G-CSF and the background was about 110 pg/ml; when referring to cytokine PBMC “++++” means greater than 1,000 pg/ml interferon-alpha (IFN-alpha), “+++” means greater than 600 pg/ml IFN-alpha, “++” means greater than 300 pg/ml IFN-alpha, “+” means greater than 100 pg/ml IFN-alpha, “−” means less than 100 pg/ml and the background was about 70 pg/ml; “WT” refers to the wild type T7 polymerase, “MT” refers to mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.) and “NT” means not tested.







TABLE 124







Combination Chemistry




















IVT
Protein
Protein
Protein
Cytokines
In Vivo



Uridine

IVT
(G-
(Luc;
(G-CSF;
(G-CSF;
(G-CSF;
Protein


Cytidine analog
analog
Purine
Luc
CSF)
HeLa)
HeLa)
PBMC)
PBMC)
(Luc)





N4-acetylcytidine
pseudouridine
A,G
Pass
Pass
+
+
NT
NT
+





WT
WT







N4-acetylcytidine
N1-methyl-
A,G
Pass
Pass
+
+
NT
NT
+



pseudouridine

WT
WT







5-methylcytidine
5-
A,G
Pass
Pass
+
+
NT
NT
+



methoxyuridine

WT
WT







5-methylcytidine
5-
A,G
Pass
Pass
Not
+
NT
NT




bromouridine

WT
WT
Done






5-methylcytidine
5-
A,G
Pass
Pass
+
+
NT
NT
+



methyluridine

WT
WT







5-methylcytidine
50% 2-
A,G
Pass
Pass
+
NT
NT
NT
+



thiouridine;

WT
WT








50% uridine










5-methylcytidine
100% 2-
A,G 
Pass
Pass
−
+
NT
NT




thiouridine

WT
WT







5-methylcytidine
pseudouridine
A,G
Pass
Pass
+
+
++
+
+





WT
WT







5-methylcytidine
N1-methyl-
A,G
Pass
Pass
+
+
++++
−
+



pseudouridine

WT
WT







N4-acetylcytidine
2-
A,G
Not
Pass
Not
+
NT
NT
NT



thiouridine

Done
WT
Done






N4-acetylcytidine
5-
A,G
Not
Pass
Not
+
NT
NT
NT



bromouridine

Done
WT
Done






N4-acetylcytidine
2
A,G
Pass
Pass
−
+
NT
NT
NT



Fluorouridine











triphosphate










5-methylcytidine
2
A,G
Pass
Pass
−
+
NT
NT
NT



Fluorouridine











triphosphate










2 Fluorocytosine
pseudouridine
A,G
Pass
Pass
−
+
NT
NT
NT


triphosphate











2 Fluorocytosine
N1-methyl-
A,G
Pass
Pass
−
+/−
NT
NT
NT


triphosphate
pseudouridine










2 Fluorocytosine
2-
A,G
Pass
Pass
−
−
NT
NT
NT


triphosphate
thiouridine










2 Fluorocytosine
5-
A,G
Pass
Pass
−
+/−
NT
NT
NT


triphosphate
bromouridine










2 Fluorocytosine
2
A,G
Pass
Pass
−
+/−
NT
NT
NT


triphosphate
Fluorouridine











triphosphate










5-methylcytidine
uridine
A,2
Pass
Pass
−
−
NT
NT
NT




Fluoro











GTP









5-methylcytidine
pseudouridine
A,2
Pass
Pass
−
−
NT
NT
NT




Fluoro











GTP









5-methylcytidine
N1-methyl-
A,2
Pass
Pass
−
+/−
NT
NT
NT



pseudouridine
Fluoro











GTP









2 Fluorocytosine
pseudouridine
A,2
Pass
Pass
−
+/−
NT
NT
NT


triphosphate

Fluoro











GTP









2 Fluorocytosine
N1-methyl-
A,2
Pass
Pass
−
−
NT
NT
NT


triphosphate
pseudouridine
Fluoro











GTP









Example 79. 2′Fluoro Chemistries in PBMC
The ability of G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) to trigger innate an immune response was determined by measuring interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) production. Use of in vitro PBMC cultures is an accepted way to measure the immunostimulatory potential of oligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102) and transfection methods are described herein. Shown in Table 125 are the average from 2 or 3 separate PBMC donors of the interferon-alpha (IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) production over time as measured by specific ELISA. Controls of R848, P(I)P(C), LPS and Lipofectamine 2000 (L2000) were also analyzed.
With regards to innate immune recognition, while both modified mRNA chemistries largely prevented IFN-alpha and TNF-alpha production relative to positive controls (R848, P(I)P(C)), 2′fluoro compounds reduce IFN-alpha and TNF-alpha production even lower than other combinations and N4-acetylcytidine combinations raised the cytokine profile.







TABLE 125







IFN-alpha and TNF-alpha










IFN-alpha:
TNF-alpha:



3 Donor
2 Donor



Average
Average



(pg/ml)
(pg/ml)













L2000
1
361


P(I)P(C)
482
544


R848
45
8,235


LPS
0
6,889


N4-acetylcytidine/pseudouridine
694
528


N4-acetylcytidine/N1-methyl-pseudouridine
307
283


5-methylcytidine/5-methoxyuridine
0
411


5-methylcytidine/5-bromouridine
0
270


5-methylcytidine/5-methyluridine
456
428


5-methylcytidine/2-thiouridine
274
277


N4-acetylcytidine/2-thiouridine
0
285


N4-acetylcytidine/5-bromouridine
44
403


5-methylcytidine/pseudouridine
73
332


5-methylcytidine/N1-methyl-pseudouridine
31
280


N4-acetylcytidine/2′fluorouridine triphosphate
35
32


5-methylcytodine/2′fluorouridine triphosphate
24
0


2′fluorocytidine triphosphate/N1-methyl-
0
11


pseudouridine


2′fluorocytidine triphosphate/2-thiouridine
0
0


2′fluorocytidine/triphosphate5-bromouridine
12
2


2′fluorocytidine triphosphate/2′fluorouridine
11
0


triphosphate


2′fluorocytidine triphosphate/5-methylcytidine
14
23


2′fluorocytidine triphosphate/5-
6
21


methylcytidine/pseudouridine


2′fluorocytidine triphosphate/5-
3
15


methylcytidine/N1-methyl-pseudouridine


2′fluorocytidine triphosphate/pseudouridine
0
4


2′fluorocytidine triphosphate/N1-methyl-
6
20


pseudouridine


5-methylcytidine/pseudouridine
82
18


5-methylcytidine/N1-methyl-pseudouridine
35
3









Example 80. Modified mRNA with a Tobacco Etch Virus 5′UTR
A 5′ untranslated region (UTR) may be provided as a flanking region. Multiple 5′ UTRs may be included in the flanking region and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical modifications, before and/or after codon optimization.
The 5′ UTR may comprise the 5′UTR from the tobacco etch virus (TEV). Variants of 5′ UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.
Example 81. Expression of PLGA Formulated mRNA
A. Synthesis and Characterization of Luciferase PLGA Microspheres
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl pseudouridine, modified with 25% of uridine replaced with 2-thiouridine and 25% of cytosine replaced with 5-methylcytosine, fully modified with N1-methyl pseudouridine, or fully modified with pseudouridine was reconstituted in 1×TE buffer and then formulated in PLGA microspheres. PLGA microspheres were synthesized using the water/oil/water double emulsification methods known in the art using PLGA-ester cap (Lactel, Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA), polyvinylalcohol (PVA) (Sigma, Cat#348406-25G, MW 13-23 k) dichloromethane and water. Briefly, 0.4 ml of mRNA in TE buffer at 4 mg/ml (W1) was added to 2 ml of PLGA dissolved in dichloromethane (DCM) (O1) at a concentration of 200 mg/ml of PLGA. The W1/O1 emulsion was homogenized (IKA Ultra-Turrax Homogenizer, T18) for 30 seconds at speed 5 ( ̃19,000 rpm). The W1/O1 emulsion was then added to 250 ml 1% PVA (W2) and homogenized for 1 minute at speed 5 ( ̃19,000 rpm). Formulations were left to stir for 3 hours, then passed through a 100 μm nylon mesh strainer (Fisherbrand Cell Strainer, Cat #22-363-549) to remove larger aggregates, and finally washed by centrifugation (10 min, 9,250 rpm, 4° C.). The supernatant was discarded and the PLGA pellets were resuspended in 5-10 ml of water, which was repeated 2×. After washing and resuspension with water, 100-200 μl of a PLGA microspheres sample was used to measure particle size of the formulations by laser diffraction (Malvern Mastersizer2000). The washed formulations were frozen in liquid nitrogen and then lyophilized for 2-3 days.
After lyophilization,  ̃10 mg of PLGA MS were weighed out in 2 ml eppendorf tubes and deformulated by adding 1 ml of DCM and letting the samples shake for 2-6 hrs. The mRNA was extracted from the deformulated PLGA micropsheres by adding 0.5 ml of water and shaking the sample overnight. Unformulated luciferase mRNA in TE buffer (unformulated control) was spiked into DCM and went through the deformulation process (deformulation control) to be used as controls in the transfection assay. The encapsulation efficiency, weight percent loading and particle size are shown in Table 126. Encapsulation efficiency was calculated as mg of mRNA from deformulation of PLGA microspheres divided by the initial amount of mRNA added to the formulation. Weight percent loading in the formulation was calculated as mg of mRNA from deformulation of PLGA microspheres divided by the initial amount of PLGA added to the formulation.







TABLE 126







PLGA Characteristics















Theoretical
Actual





Encapsulation
mRNA
mRNA
Particle




Efficiency
Loading
Loading
Size


Chemical Modifications
Sample ID
(%)
(wt %)
(wt %)
(D50, um)















Fully modified with 5-
43-66A
45.8
0.4
0.18
33.4


methylcytosine and N1-
43-66B
29.6

0.12
27.7


methyl pseudouridine
43-66C
25.5

0.10
27.1


25% of uridine replaced
43-67A
34.6
0.4
0.14
29.9


with 2-thiouridine and
43-67B
22.8

0.09
30.2


25% of cytosine replaced
43-67C
23.9

0.10
25.1


with 5-methylcytosine


Fully modified with N1-
43-69A
55.8
0.4
0.22
40.5


methyl pseudouridine
43-69B
31.2

0.12
41.1



43-69C
24.9

0.10
46.1


Fully modified with
43-68-1
49.3
0.4
0.20
34.8


pseudouridine
43-68-2
37.4

0.15
35.9



43-68-3
45.0

0.18
36.5









B. Protein Expression of Modified mRNA Encapsulated in PLGA Microspheres
The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate (Corning, Manassas, Va.). The cells were grown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 83 ng of the deformulated luciferase mRNA PLGA microsphere samples, deformulated luciferase mRNA control (Deform control), or unformulated luciferase mRNA control (Unfomul control) was diluted in a 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as a transfection reagent and 0.2 ul was diluted in a 10 ul final volume of OPTI-MEM. After 5 min of incubation at room temperature, both solutions were combined and incubated an additional 15 min at room temperature. Then 20 ul of the combined solution was added to 100 ul of cell culture medium containing the HeLa cells. The plates were then incubated as described before.
After an 18 to 22 hour incubation, cells expressing luciferase were lysed with 100 ul Passive Lysis Buffer (Promega, Madison, Wis.) according to manufacturer instructions. Aliquots of the lysates were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The background signal of the plates without reagent was about 200 relative light units per well. The plate reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.).
Cells were harvested and the bioluminescence (in relative light units, RLU) for each sample is shown in Table 127. Transfection of these samples confirmed that the varied chemistries of luciferase mRNA is still able to express luciferase protein after PLGA microsphere formulation.







TABLE 127







Chemical Modifications











Chemical

Biolum.



Modifications
Sample ID
(RLU)















Fully modified with
Deform contol
164266.5



5-methylcytosine
Unformul control
113714



and N1-methyl
43-66A
25174



pseudouridine
43-66B
25359




43-66C
20060



25% of uridine
Deform contol
90816.5



replaced with 2-
Unformul control
129806



thiouridine and 25%
43-67A
38329.5



of cytosine replaced
43-67B
8471.5



with 5-
43-67C
10991.5



methylcytosine



Fully modified with
Deform contol
928093.5



N1-methyl
Unformul control
1512273.5



pseudouridine
43-69A
1240299.5




43-69B
748667.5




43-69C
1193314



Fully modified with
Deform contol
154168



pseudouridine
Unformul control
151581




43-68-1
120974.5




43-68-2
107669




43-68-3
97226










Example 82. In Vitro Studies of Factor IX
A. Serum-Free Media
Human Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 5659; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) was transfected in serum-free media. The cell culture supernatant was collected and subjected to trypsin digestion before undergoing 2-dimensional HPLC separation of the peptides. Matrix-assisted laser desorption/ionization was used to detect the peptides. 8 peptides were detected and 7 of the detected peptides are unique to Factor IX. These results indicate that the mRNA transfected in the serum-free media was able to express full-length Factor IX protein.
B. Human Embryonic Kidney (HEK) 293A Cells
250 ng of codon optimized Human Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 5659; fully modified with 5-methylcytosine and pseudouridine; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) was transfected into HEK 293A cells (150, 000 cells/well) using Lipofectamine 2000 in DMEM in presence of 10% FBS. The transfection complexes were removed 3 hours after transfection. Cells were harvested at 3, 6, 9, 12, 24, 48 and 72 hours after transfection. Total RNA was isolated and used for cDNA synthesis. The cDNA was subjected to analysis by quantitative Real-Time PCR using codon optimized Factor IX specific primer set. Human hypoxanthine phosphoribosyltransfersase 1 (HPRT) level was used for normalization. The data is plotted as a percent of detectable mRNA considering the mRNA level as 100% at the 3 hour time point. The half-life of Factor IX modified mRNA fully modified with 5-methylcytosine and pseudouridine in human embryonic kidney 293 (HEK293) cells is about 8-10 hours.
Example 83. Saline Formulation: Subcutaneous Administration
Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) and human EPO modified mRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine), were formulated in saline and delivered to mice via intramuscular (IM) injection at a dose of 100 ug.
Controls included Luciferase (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine)) or the formulation buffer (F.Buffer). The mice were bled at 13 hours after the injection to determine the concentration of the human polypeptide in serum in pg/mL. (G-CSF groups measured human G-CSF in mouse serum and EPO groups measured human EPO in mouse serum). The data are shown in Table 128.
mRNA degrades rapidly in serum in the absence of formulation suggesting the best method to deliver mRNA to last longer in the system is by formulating the mRNA. As shown in Table 128, mRNA can be delivered subcutaneously using only a buffer formulation.







TABLE 128







Dosing Regimen















Average Protein




Dose Vol.
Dosing
Product pg/mL,


Group
Treatment
(μl/mouse)
Vehicle
serum














G-CSF
G-CSF
100
F. buffer
45


G-CSF
Luciferase
100
F. buffer
0


G-CSF
F. buffer
100
F. buffer
2.2


EPO
EPO
100
F. buffer
72.03


EPO
Luciferase
100
F. buffer
26.7


EPO
F. buffer
100
F. buffer
13.05









Example 84. Intravitreal Delivery
mCherry modified mRNA (mRNA sequence shown in SEQ ID NO: 5656; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) and luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) formulated in saline was delivered intravitreally in rats as described in Table 129. The sample was compared against a control of saline only delivered intravitreally.







TABLE 129







Dosing Chart











Dose Level
Dose
Treatment












(μg modified
volume
Right Eye
Left Eye


Group No.
RNA/eye)
(μL/eye)
(OD)
(OS)














Control
0
5
Delivery
Delivery





buffer only
buffer only


Modified RNA in
10
5
mCherry
Luciferase


delivery buffer









The formulation will be administered to the left or right eye of each animal on day 1 while the animal is anesthetized. On the day prior to administration gentamicin ophthalmic ointment or solution was applied to both eyes twice. The gentamicin ophthalmic ointment or solution was also applied immediately following the injection and on the day following the injection. Prior to dosing, mydriatic drops (1% tropicamide and/or 2.5% phenylephrine) are applied to each eye.
18 hours post dosing the eyes receiving the dose of mCherry and delivery buffer are enucleated and each eye was separately placed in a tube containing 10 mL 4% paraformaldehyde at room temperature for overnight tissue fixation. The following day, eyes will be separately transferred to tubes containing 10 mL of 30% sucrose and stored at 21° C. until they were processed and sectioned. The slides prepared from different sections were evaluated under F-microscopy. Positive expression was seen in the slides prepared with the eyes administered mCherry modified mRNA and the control showed no expression.
Example 85. In Vivo Cytokine Expression Study
Mice were injected intramuscularly with 200 ug of G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 160 nucleotides not shown in sequence) which was unmodified with a 5′cap, Cap1 (unmodified), fully modified with 5-methylcytosine and pseudouridine and a 5′cap, Cap1 (Gen1) or fully modified with 5-methylcytosine and N1-methyl-pseudouridine and a 5′cap, Cap1 (Gen2 cap) or no cap (Gen2 uncapped). Controls of R-848, 5% sucrose and untreated mice were also analyzed. After 8 hours serum was collected from the mice and analyzed for interferon-alpha (IFN-alpha) expression. The results are shown in Table 130.







TABLE 130







IFN-alpha Expression










Formulation
IFN-alpha (pg/ml)














G-CSF unmodified
67.012



G-CSF Gen1
8.867



G-CSF Gen2 cap
0



G-CSF Gen2 uncapped
0



R-848
40.971



5% sucrose
1.493



Untreated
0










Example 86. In Vitro Expression of VEGF Modified mRNA
HEK293 cells were transfected with modified mRNA (mmRNA) VEGF-A (mRNA sequence shown in SEQ ID NO: 5668; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) which had been complexed with Lipofectamine2000 from Invitrogen (Carlsbad, Calif.) at the concentration shown in Table 131. The protein expression was detected by ELISA and the protein (pg/ml) is shown in Table 131.







TABLE 131







Protein Expression















Amount
10
2.5
625
156
39
10
2
610


Transfected 
ng 
ng
pg
pg
pg 
pg
pg
fg





Protein
10495
10038
2321.23
189.6
0
0
0
0


(pg/ml)









Example 87. In Vitro Screening in HeLa Cells of GFP
The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO2 atmosphere overnight. Next day, 37.5 ng or 75 ng of Green Fluroescent protein (GFP) modified RNA (mRNA sequence shown in SEQ ID NO: 5667; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) with the chemical modification described in Table 132, were diluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as transfection reagent and 0.2 ul were diluted in 10 ul final volume of OPTI-MEM. After 5 minutes of incubation at room temperature, both solutions were combined and incubated an additional 15 minute at room temperature. Then the 20 ul combined solution was added to the 100 ul cell culture medium containing the HeLa cells and incubated at room temperature.
After an 18 to 22 hour incubation cells expressing luciferase were lysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.) according to manufacturer instructions. Aliquots of the lysates were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The median fluorescence intensity (MFI) was determined for each chemistry and is shown in Table 132.
These results demonstrate that GFP fully modified with N1-methyl-pseudouridine and 5-methylcytosine produces more protein in HeLa cells compared to the other chemistry. Additionally the higher dose of GFP administered to the cells resulted in the highest MFI value.







TABLE 132







Mean Fluorescence Intensity










37.5 ng
75 ng


Chemistry
MFI
MFI












No modifications
97400
89500


5-methylcytosine/pseudouridine
324000
715000


5-methylcytosine/N1-methyl-pseudouridine
643000
1990000









Example 88. Homogenization
Different luciferase mRNA solutions (as described in Table 132 where “X” refers to the solution containing that component) (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) were evaluated to test the percent yield of the different solutions, the integrity of the mRNA by bioanalyzer, and the protein expression of the mRNA by in vitro transfection. The mRNA solutions were prepared in water, 1×TE buffer at 4 mg/ml as indicated in Table 133, and added to either dichloromethane (DCM) or DCM containing 200 mg/ml of poly(lactic-co-glycolic acid) (PLGA) (Lactel, Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA) to achieve a final mRNA concentration of 0.8 mg/ml. The solutions requiring homogenization were homogenized for 30 seconds at speed 5 (approximately 19,000 rpm) (IKA Ultra-Turrax Homogenizer, T18). The mRNA samples in water, dicloromethane and poly(lactic-co-glycolic acid) (PLGA) were not recoverable (NR). All samples, except the NR samples, maintained integrity of the mRNA as determined by bioanalyzer (Bio-rad Experion).
The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate (Corning, Manassas, Va.). The cells were grown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 250 ng of luciferase mRNA from the recoverable samples was diluted in a 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as a transfection reagent and 0.2 ul was diluted in a 10 ul final volume of OPTI-MEM. After 5 minutes of incubation at room temperature, both solutions were combined and incubated an additional 15 minutes at room temperature. Then 20 ul of the combined solution was added to 100 ul of cell culture medium containing the HeLa cells. The plates were then incubated as described before. Controls luciferase mRNA (luciferase mRNA formulated in saline) (Control) and untreated cells (Untreat.) were also evaluated. Cells were harvested and the bioluminescence average (in photons/second) (biolum. (p/s)) for each signal is also shown in Table 133. The recoverable samples all showed activity of luciferase mRNA when analyzed.
After an 18 to 22 hour incubation, cells expressing luciferase were lysed with 100 ul Passive Lysis Buffer (Promega, Madison, Wis.) according to manufacturer instructions. Aliquots of the lysates were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The background signal of the plates without reagent was about 200 relative light units per well. The plate reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.).
Cells were harvested and the bioluminescence average (in relative light units, RLU) (biolum. (RLU)) for each signal is also shown in Table 133. The recoverable samples all showed activity of luciferase mRNA when analyzed.







TABLE 133







Solutions














Solution 

1x TE

DCM/

Yield
Biolum.


No.
Water
Buffer
DCM
PLGA
Homogenizer
(%)
(RLU)

















1
X




96
5423780


2

X


X
95
4911950


3
X



X
92
2367230


4

X


X
90
4349410


5
X

X

X
66
4145340


6

X
X

X
71
3834440


7
X


X
X
NR
n/a


8

X

X
X
24
3182080


9
X


X

NR
n/a


10

X

X

79
3276800


11
X

X


79
5563550


12

X
X


79
4919100


Control 






2158060


Untreat.






3530









Example 89. TE Buffer and Water Evaluation
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) was reconstituted in water or TE buffer as outlined in Table 134 and then formulated in PLGA microspheres. PLGA microspheres were synthesized using the water/oil/water double emulsification methods known in the art using PLGA (Lactel, Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA), polyvinylalcohol (PVA) (Sigma, Cat#348406-25G, MW 13-23 k) dichloromethane and water. Briefly, 0.2 to 0.6 ml of mRNA in water or TE buffer at a concentration of 2 to 6 mg/ml (W1) was added to 2 ml of PLGA dissolved in dichloromethane (DCM) (O1) at a concentration of 100 mg/ml of PLGA. The W1/O1 emulsion was homogenized (IKA Ultra-Turrax Homogenizer, T18) for 30 seconds at speed 5 ( ̃19,000 rpm). The W1/O1 emulsion was then added to 250 ml 1% PVA (W2) and homogenized for 1 minute at speed 5 ( ̃19,000 rpm). Formulations were left to stir for 3 hours, then passed through a 100 μm nylon mesh strainer (Fisherbrand Cell Strainer, Cat #22-363-549) to remove larger aggregates, and finally washed by centrifugation (10 min, 9,250 rpm, 4° C.). The supernatant was discarded and the PLGA pellets were resuspended in 5-10 ml of water, which was repeated 2×. The washed formulations were frozen in liquid nitrogen and then lyophilized for 2-3 days. After lyophilization,  ̃10 mg of PLGA MS were weighed out in 2 ml eppendorf tubes and deformulated by adding 1 ml of DCM and letting the samples shake for 2-6 hrs. mRNA was extracted from the deformulated PLGA micropsheres by adding 0.5 ml of water and shaking the sample overnight.
Unformulated luciferase mRNA in water or TE buffer (deformulation controls) was spiked into DCM and went through the deformulation process to be used as controls in the transfection assay.
The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate (Corning, Manassas, Va.). The cells were grown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 100 ng of the deformulated luciferase mRNA samples was diluted in a 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as a transfection reagent and 0.2 ul was diluted in a 10 ul final volume of OPTI-MEM. After 5 minutes of incubation at room temperature, both solutions were combined and incubated an additional 15 minutes at room temperature. Then 20 ul of the combined solution was added to 100 ul of cell culture medium containing the HeLa cells. The plates were then incubated as described before.
After 18 to 22 hour incubation, cells expressing luciferase were lysed with 100 ul Passive Lysis Buffer (Promega, Madison, Wis.) according to manufacturer instructions. Aliquots of the lysates were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The background signal of the plates without reagent was about 200 relative light units per well. The plate reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). To determine the activity of the luciferase mRNA from each formulation, the relative light units (RLU) for each formulation was divided by the RLU of the appropriate mRNA deformulation control (mRNA in water or TE buffer). Table 134 shows the activity of the luciferase mRNA. The activity of the luciferase mRNA in the PLGA microsphere formulations (Form.) was substantially improved by formulating in TE buffer versus water.







TABLE 134







Formulations


















Theoretical 
Actual





mRNA
W1 Solvent 
Total
mRNA
mRNA

Activity (% of



conc.
volume
mRNA
Loading
Loading

deformulation


Form.
(mg/ml)
(ul)
(ug)
(wt %)
(wt %)
W1 Solvent
control)

















PLGA A
4
400
1600
0.80
0.14
Water
12.5%


PLGA B
4
200
800
0.40
0.13
Water
 1.3%


PLGA C
4
600
2400
1.20
0.13
Water
12.1%


PLGA D
2
400
800
0.40
0.07
Water
 1.3%


PLGA E
6
400
2400
1.20
0.18
TE Buffer
38.9%


PLGA F
4
400
1600
0.80
0.16
TE Buffer
39.7%


PLGA G
4
400
1600
0.80
0.10
TE Buffer
26.6%









Example 90. Chemical Modifications on mRNA
The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution (Life Technologies, Grand Island, N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO2 atmosphere overnight. The next day, 83 ng of Luciferase modified RNA (mRNA sequence shown SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) with the chemical modification described in Table 135, were diluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as transfection reagent and 0.2 ul were diluted in 10 ul final volume of OPTI-MEM. After 5 minutes of incubation at room temperature, both solutions were combined and incubated an additional 15 minute at room temperature. Then the 20 ul combined solution was added to the 100 ul cell culture medium containing the HeLa cells and incubated at room temperature.
After 18 to 22 hours of incubation cells expressing luciferase were lysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.) according to manufacturer instructions. Aliquots of the lysates were transferred to white opaque polystyrene 96-well plates (Corning, Manassas, Va.) and combined with 100 ul complete luciferase assay solution (Promega, Madison, Wis.). The lysate volumes were adjusted or diluted until no more than 2 mio relative light units (RLU) per well were detected for the strongest signal producing samples and the RLUs for each chemistry tested are shown in Table 135. The plate reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of the plates without reagent was about 200 relative light units per well.







TABLE 135







Chemical Modifications










Sample
RLU














Untreated
336



Unmodified Luciferase
33980



5-methylcytosine and pseudouridine
1601234



5-methylcytosine and N1-methyl-pseudouridine
421189



25% cytosines replaced with 5-methylcytosine
222114



and 25% of uridines replaced with 2-thiouridine



N1-methyl-pseudouridine
3068261



Pseudouridine
140234



N4-Acetylcytidine
1073251



5-methoxyuridine
219657



5-Bromouridine
6787



N4-Acetylcytidineand N1-methyl-pseudouridine
976219



5-methylcytosine and 5-methoxyuridine
66621



5-methylcytosine and 2′fluorouridine
11333










Example 91. Intramuscular and Subcutaneous Administration of Modified mRNA
Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine (5mC/pU), fully modified with 5-methylcytosine and N1-methyl-pseudouridine (5mC/N1mpU), fully modified with pseudouridine (pU), fully modified with N1-methyl-pseudouridine (N1mpU) or modified where 25% of the cytosines replaced with 5-methylcytosine and 25% of the uridines replaced with 2-thiouridine (5mC/s2U) formulated in PBS (pH 7.4) was administered to Balb-C mice intramuscularly or subcutaneously at a dose of 2.5 mg/kg. The mice were imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours and 144 hours for intramuscular delivery and 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours and 120 hours for subcutaneous delivery. Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The average total flux (photons/second) for intramuscular administration is shown in Table 136 and the average total flux (photons/second) for subcutaneous administration is shown in Table 137. The background signal was 3.79E+05 (p/s). The peak expression for intramuscular administration was seen between 24 and 48 hours for all chemistry and expression was still detected at 144 hours. For subcutaneous delivery the peak expression was seen at 2-8 hours and expression was detected at 72 hours.







TABLE 136







Intramuscular Administration













5mC/
5mC/
5mC/





pU
N1mpU
s2U
pU
N1mpU



Flux
Flux
Flux
Flux
Flux



(p/s)
(p/s)
(p/s)
(p/s)
(p/s)
















 2 hours
1.98E+07
4.65E+06
4.68E+06
2.33E+06
3.66E+07


 8 hours
1.42E+07
3.64E+06
3.78E+06
8.07E+06
7.21E+07


24 hours
2.92E+07
1.22E+07
3.35E+07
1.01E+07
1.75E+08


48 hours
2.64E+07
1.01E+07
5.06E+07
7.46E+06
3.42E+08


72 hours
2.18E+07
8.59E+06
3.42E+07
4.08E+06
5.83E+07


96 hours
2.75E+07
2.70E+06
2.38E+07
4.35E+06
7.15E+07


120 hours 
2.19E+07
1.60E+06
1.54E+07
1.25E+06
3.87E+07


144 hours 
9.17E+06
2.19E+06
1.14E+07
1.86E+06
5.04E+07
















TABLE 137







Subcutaneous Administration













5mC/
5mC/
5mC/





pU
N1mpU
s2U
pU
N1mpU



Flux
Flux
Flux
Flux
Flux



(p/s)
(p/s)
(p/s)
(p/s)
(p/s)
















 2 hours
5.26E+06
4.54E+06
9.34E+06
2.43E+06
2.80E+07


 8 hours
2.32E+06
8.75E+05
8.15E+06
2.12E+06
3.09E+07


24 hours
2.67E+06
5.49E+06
3.80E+06
2.24E+06
1.48E+07


48 hours
1.22E+06
1.77E+06
3.07E+06
1.58E+06
1.24E+07


72 hours
1.12E+06
8.00E+05
8.53E+05
4.80E+05
2.29E+06


96 hours
5.16E+05
5.33E+05
4.30E+05
4.30E+05
6.62E+05


120 hours 
3.80E+05
4.09E+05
3.21E+05
6.82E+05
5.05E+05









Example 92. Osmotic Pump Study
Prior to implantation, an osmotic pump (ALZET® Osmotic Pump 2001D, DURECT Corp. Cupertino, Calif.) is loaded with the 0.2 ml of 1×PBS (pH 7.4) (PBS loaded pump) or 0.2 ml of luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) at 1 mg/ml in 1×PBS (pH 7.4) (Luciferase loaded pump) and incubated overnight in 1×PBS (pH 7.4) at 37° C.
Balb-C mice (n=3) are implanted subcutaneously with either the PBS loaded pump or the luciferase loaded pump and imaged at 2 hours, 8 hours and 24 hours. As a control a PBS loaded pump is implanted subcutaneously and the mice are injected subcutaneously with luciferase modified mRNA in 1×PBS (PBS loaded pump; SC Luciferase) or an osmotic pump is not implanted and the mice are injected subcutaneously with luciferase modified mRNA in 1×PBS (SC Luciferase). The luciferase formulations are outlined in Table 138.







TABLE 138







Luciferase Formulations














Conc
Inj. Vol.
Amt
Dose


Group
Vehicle
(mg/ml)
(ul)
(ug)
(mg/kg)















PBS loaded pump;
PBS
1.00
50
50
2.5


SC Luciferase


Luciferase loaded
PBS
1.00
—
200
10.0


pump


PBS loaded pump
PBS
—
—
—
—


SC Luciferase
PBS
1.00
50
50
2.5









Example 93. External Osmotic Pump Study
An external osmotic pump (ALZET® Osmotic Pump 2001D, DURECT Corp. Cupertino, Calif.) is loaded with the 0.2 ml of 1×PBS (pH 7.4) (PBS loaded pump) or 0.2 ml of luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) at 1 mg/ml in 1×PBS (pH 7.4) (luciferase loaded pump) and incubated overnight in 1×PBS (pH 7.4) at 37° C.
Using a catheter connected to the external PBS loaded pump or the luciferase loaded pump Balb-C mice (n=3) are administered the formulation. The mice are imaged at 2 hours, 8 hours and 24 hours. As a control an external PBS loaded pump is used and the mice are injected subcutaneously with luciferase modified mRNA in 1×PBS (PBS loaded pump; SC Luciferase) or the external pump is not used and the mice are only injected subcutaneously with luciferase modified mRNA in 1×PBS (SC Luciferase). Twenty minutes prior to imaging, mice are injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals are then anesthetized and images are acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence is measured as total flux (photons/second) of the entire mouse. The luciferase formulations are outlined in Table 139 and the average total flux (photons/second).







TABLE 139







Luciferase Formulations














Conc
Inj. Vol.
Amt
Dose


Group
Vehicle
(mg/ml)
(ul)
(ug)
(mg/kg)















PBS loaded pump;
PBS
1.00
50
50
2.5


SC Luciferase


Luciferase loaded
PBS
1.00
—
200
10.0


pump


PBS loaded pump
PBS
—
—
—
—


SC Luciferase
PBS
1.00
50
50
2.5









Example 94. Fibrin Sealant Study
Fibrin sealant, such as Tisseel (Baxter Healthcare Corp., Deerfield, Ill.), is composed of fibrinogen and thrombin in a dual-barreled syringe. Upon mixing, fibrinogen is converted to fibrin to form a fibrin clot in about 10 to 30 seconds. This clot can mimic the natural clotting mechanism of the body. Additionally a fibrin hydrogel is a three dimensional structure that can potentially be used in sustained release delivery. Currently, fibrin sealant is approved for application in hemostasis and sealing to replace conventional surgical techniques such as suture, ligature and cautery.
The thrombin and fibrinogen components were loaded separately into a dual barreled syringe. Balb-C mice (n=3) were injected subcutaneously with 50 ul of fibrinogen, 50 ul of thrombin and they were also injected at the same site with modified luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) (Tisseel+Luciferase), 50 ul of fibrinogen and 50 ul thrombin (Tisseel) or modified luciferase mRNA (Luciferase). The injection of fibrinogen and thrombin was done simultaneously using the dual-barreled syringe. The SC injection of luciferase was done 15 minutes after the fibrinogen/thrombin injection to allow the fibrin hydrogel to polymerize (Tisseel+Luciferase group). A control group of untreated mice were also evaluated. The mice were imaged at 5 hours and 24 hours. Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The luciferase formulations are outlined in Table 140 and the average total flux (photons/second) is shown in Table 141. The fibrin sealant was found to not interfere with imaging and the injection of luciferase and Tisseel showed expression of luciferase.







TABLE 140







Luciferase Formulations














Conc
Inj. Vol.
Amt
Dose


Group
Vehicle
(mg/ml)
(ul)
(ug)
(mg/kg)





Tisseel +
PBS
1.00
50
50
2.5


Luciferase


Tisseel
—
—
—
—
—


Luciferase
PBS
1.00
50
50
2.5


Untreated
—
—
—
—
—
















TABLE 141







Total Flux












5 Hours
24 Hours



Group
Flux (p/s)
Flux (p/s)







Tisseel + Luciferase
4.59E+05
3.39E+05



Tisseel
1.99E+06
1.06E+06



Luciferase
9.94E+05
7.44E+05



Untreated
3.90E+05
3.79E+05










Example 95. Fibrin Containing mRNA Sealant Study
A. Modified mRNA and Calcium Chloride
Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridine is added to calcium chloride. The calcium chloride is then used to reconstitute thrombin. Fibrinogen is reconstituted with fibrinolysis inhibitor solution per the manufacturer's instructions. The reconstituted thrombin containing modified mRNA and fibrinogen is loaded into a dual barreled syringe. Mice are injected subcutaneously with 50 ul of fibrinogen and 50 ul of thrombin containing modified mRNA or they were injected with 50 ul of PBS containing an equivalent dose of modified luciferase mRNA. A control group of untreated mice is also evaluated. The mice are imaged at predetermined intervals to determine the average total flux (photons/second).
B. Lipid Nanoparticle Formulated Modified mRNA and Calcium Chloride
Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridine is formulated in a lipid nanoparticle is added to calcium chloride. The calcium chloride is then used to reconstitute thrombin. Fibrinogen is reconstituted with fibrinolysis inhibitor solution per the manufacturer's instructions. The reconstituted thrombin containing modified mRNA and fibrinogen is loaded into a dual barreled syringe. Mice are injected subcutaneously with 50 ul of fibrinogen and 50 ul of thrombin containing modified mRNA or they were injected with 50 ul of PBS containing an equivalent dose of modified luciferase mRNA. A control group of untreated mice is also evaluated. The mice are imaged at predetermined intervals to determine the average total flux (photons/second).
C. Modified mRNA and Fibrinogen
Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridine is added to the fibrinolysis inhibitor solution. The fibrinolysis inhibitor solution is then used to reconstitute fibrinogen. Thrombin is reconstituted with the calcium chloride solution per the manufacturer's instructions. The reconstituted fibrinogen containing modified mRNA and thrombin is loaded into a dual barreled syringe. Mice are injected subcutaneously with 50 ul of thrombin and 50 ul of fibrinogen containing modified mRNA or they were injected with 50 ul of PBS containing an equivalent dose of modified luciferase mRNA. A control group of untreated mice is also evaluated. The mice are imaged at predetermined intervals to determine the average total flux (photons/second).
D. Lipid Nanoparticle Formulated Modified mRNA and Fibrinogen
Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridine is formulated in a lipid nanoparticle is added to the fibrinolysis inhibitor solution. The fibrinolysis inhibitor solution is then used to reconstitute fibrinogen. Thrombin is reconstituted with the calcium chloride solution per the manufacturer's instructions. The reconstituted fibrinogen containing modified mRNA and thrombin is loaded into a dual barreled syringe. Mice are injected subcutaneously with 50 ul of thrombin and 50 ul of fibrinogen containing modified mRNA or they were injected with 50 ul of PBS containing an equivalent dose of modified luciferase mRNA. A control group of untreated mice is also evaluated. The mice are imaged at predetermined intervals to determine the average total flux (photons/second).
E. Modified mRNA and Thrombin
Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridine is added to the reconstituted thrombin after it is reconstituted with the calcium chloride per the manufacturer's instructions. The fibrinolysis inhibitor solution is then used to reconstitute fibrinogen per the manufacturer's instructions. The reconstituted fibrinogen and thrombin containing modified mRNA is loaded into a dual barreled syringe. Mice are injected subcutaneously with 50 ul of thrombin containing modified mRNA and 50 ul of fibrinogen or they were injected with 50 ul of PBS containing an equivalent dose of modified luciferase mRNA. A control group of untreated mice is also evaluated. The mice are imaged at predetermined intervals to determine the average total flux (photons/second).
F. Lipid Nanoparticle Formulated Modified mRNA and Thrombin
Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridine is formulated in a lipid nanoparticle is added to the reconstituted thrombin after it is reconstituted with the calcium chloride per the manufacturer's instructions. The fibrinolysis inhibitor solution is then used to reconstitute fibrinogen per the manufacturer's instructions. The reconstituted fibrinogen and thrombin containing modified mRNA is loaded into a dual barreled syringe. Mice are injected subcutaneously with 50 ul of thrombin containing modified mRNA and 50 ul of fibrinogen or they were injected with 50 ul of PBS containing an equivalent dose of modified luciferase mRNA. A control group of untreated mice is also evaluated. The mice are imaged at predetermined intervals to determine the average total flux (photons/second).
Example 96. Cationic Lipid Formulation of 5-Methylcytosine and N1-Methyl-Pseudouridine Modified mRNA
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine was formulated in the cationic lipids described in Table 142. The formulations were administered intravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.







TABLE 142







Cationic Lipid Formulations












Formulation
NPA-126-1
NPA-127-1
NPA-128-1
NPA-129-1
111612-B





Lipid
DLin-MC3-
DLin-KC2-
C12-200
DLinDMA
DODMA



DMA
DMA


Lipid/mRNA
20:1
20:1
20:1
20:1
20:1


ratio (wt/wt)


Mean Size
122 nm
114 nm
153 nm
137 nm
223.2 nm



PDI: 0.13
PDI: 0.10
PDI: 0.17
PDI: 0.09
PDI: 0.142


Zeta at pH 7.4
−1.4 mV
−0.5 mV
−1.4 mV
2.0 mV
−3.09 mV


Encaps.
95%
77%
69%
80%
64%


(RiboGr)









Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hours after dosing and the average total flux (photons/second) was measured for each route of administration and cationic lipid formulation. The background flux was about 4.17E+05 p/s. The results of the imaging are shown in Table 143. In Table 143, “NT” means not tested.







TABLE 143







Flux















DLin-
DLin-







MC3-
KC2-






Time
DMA
DMA
C12-200
DLinDMA
DODMA


Route
Point
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s) 
Flux (p/s)





I.V.
 2 hrs
1.92E+08
2.91E+08
1.08E+08
2.53E+07
8.40E+06


I.V.
 8 hrs
1.47E+08
2.13E+08
3.72E+07
3.82E+07
5.62E+06


I.V.
24 hrs
1.32E+07
2.41E+07
5.35E+06
4.20E+06
8.97E+05


I.M.
 2 hrs
8.29E+06
2.37E+07
1.80E+07
1.51E+06
NT


I.M.
 8 hrs
5.83E+07
2.12E+08
2.60E+07
1.99E+07
NT


I.M.
24 hrs
4.30E+06
2.64E+07
3.01E+06
9.46E+05
NT


S.C.
 2 hrs
1.90E+07
5.16E+07
8.91E+07
4.66E+06
9.61E+06


S.C.
 8 hrs
7.74E+07
2.00E+08
4.58E+07
9.67E+07
1.90E+07


S.C.
24 hrs
7.49E+07
2.47E+07
6.96E+06
6.50E+06
1.28E+06









Example 97. Lipid Nanoparticle Intravenous Study
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) was formulated in a lipid nanoparticle containing 50% DLin-MC3-DMA OR DLin-KC2-DMA as described in Table 144, 38.5% cholesterol, 10% DSPC and 1.5% PEG. The formulation was administered intravenously (I.V.) to Balb-C mice at a dose of 0.5 mg/kg, 0.05 mg/kg, 0.005 mg/kg or 0.0005 mg/kg. Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse.







TABLE 144







Formulations









Formulation
NPA-098-1
NPA-100-1





Lipid
DLin-KC2-DMA
DLin-MC3-DMA


Lipid/mRNA ratio (wt/wt)
20:1
20:1


Mean Size
135 nm
152 nm



PDI: 0.08
PDI: 0.08


Zeta at pH 7.4
−0.6 mV
−1.2 mV


Encaps. (RiboGr)
91%
94%









For DLin-KC2-DMA the mice were imaged at 2 hours, 8 hours, 24 hours, 72 hours, 96 hours and 168 hours after dosing and the average total flux (photons/second) was measured for each route of administration and cationic lipid formulation. The background flux was about 3.66E+05 p/s. The results of the imaging are shown in Table 145. Organs were imaged at 8 hours and the average total flux (photons/second) was measured for the liver, spleen, lung and kidney. A control for each organ was also analyzed. The results are shown in Table 146. The peak signal for all dose levels was at 8 hours after administration. Also, distribution to the various organs (liver, spleen, lung, and kidney) may be able to be controlled by increasing or decreasing the LNP dose.







TABLE 145







Flux












0.5 mg/kg
0.05 mg/kg
0.005 mg/kg
0.0005 mg/kg











Time Point
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)















2
hrs
3.54E+08
1.75E+07
2.30E+06
4.09E+05


8
hrs
1.67E+09
1.71E+08
9.81E+06
7.84E+05


24
hrs
2.05E+08
2.67E+07
2.49E+06
5.51E+05


72
hrs
8.17E+07
1.43E+07
1.01E+06
3.75E+05


96
hrs
4.10E+07
9.15E+06
9.58E+05
4.29E+05


168
hrs
3.42E+07
9.15E+06
1.47E+06
5.29E+05
















TABLE 146







Organ Flux












Liver
Spleen
Lung
Kidney



Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)
















0.5
mg/kg
1.42E+08
4.86E+07
1.90E+05
3.20E+05


0.05
mg/kg
7.45E+06
4.62E+05
6.86E+04
9.11E+04


0.005
mg/kg
3.32E+05
2.97E+04
1.42E+04
1.15E+04


0.0005
mg/kg
2.34E+04
1.08E+04
1.87E+04
9.78E+03











Untreated
1.88E+04
1.02E+04
1.41E+04
9.20E+03









For DLin-MC3-DMA the mice were imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours and 144 hours after dosing and the average total flux (photons/second) was measured for each route of administration and cationic lipid formulation. The background flux was about 4.51E+05 p/s. The results of the imaging are shown in Table 147. Organs were imaged at 8 hours and the average total flux (photons/second) was measured for the liver, spleen, lung and kidney. A control for each organ was also analyzed. The results are shown in Table 148. The peak signal for all dose levels was at 8 hours after administration. Also, distribution to the various organs (liver, spleen, lung, and kidney) may be able to be controlled by increasing or decreasing the LNP dose.







TABLE 147







Flux












0.5 mg/kg
0.05 mg/kg
0.005 mg/kg
0.0005 mg/kg











Time Point
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)















2
hrs
1.23E+08
7.76E+06
7.66E+05
4.88E+05


8
hrs
1.05E+09
6.79E+07
2.75E+06
5.61E+05


24
hrs
4.44E+07
1.00E+07
1.06E+06
5.71E+05


48
hrs
2.12E+07
4.27E+06
7.42E+05
4.84E+05


72
hrs
1.34E+07
5.84E+06
6.90E+05
4.38E+05


144
hrs
4.26E+06
2.25E+06
4.58E+05
3.99E+05
















TABLE 148







Organ Flux












Liver
Spleen
Lung
Kidney



Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)
















0.5
mg/kg
1.19E+08
9.66E+07
1.19E+06
1.85E+05


0.05
mg/kg
1.10E+07
1.79E+06
7.23E+04
5.82E+04


0.005
mg/kg
3.58E+05
6.04E+04
1.33E+04
1.33E+04


0.0005
mg/kg
2.25E+04
1.88E+04
2.05E+04
1.65E+04











Untreated
1.91E+04
1.66E+04
2.63E+04
2.14E+04









Example 98. Lipid Nanoparticle Subcutaneous Study
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) was formulated in a lipid nanoparticle containing 50% DLin-KC2-DMA as described in Table 149, 385% cholesterol, 10% DSPC and 1.5% PEG. The formulation was administered subcutaneously (S.C.) to Balb-C mice at a dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg.







TABLE 149







DLin-KC2-DMA Formulation










Formulation
NPA-098-1







Lipid
DLin-KC2-DMA



Lipid/mRNA ratio (wt/wt)
20:1



Mean Size
135 nm




PDI: 0.08



Zeta at pH 7.4
−0.6 mV



Encaps. (RiboGr)
91%










Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours and 144 hours after dosing and the average total flux (photons/second) was measured for each route of administration and cationic lipid formulation. The lower limit of detection was about 3E+05 p/s. The results of the imaging are shown in Table 150. Organs were imaged at 8 hours and the average total flux (photons/second) was measured for the liver, spleen, lung and kidney. A control for each organ was also analyzed. The results are shown in Table 151. The peak signal for all dose levels was at 8 hours after administration. Also, distribution to the various organs (liver, spleen, lung, and kidney) may be able to be controlled by increasing or decreasing the LNP dose. At high doses, the LNP formulations migrates outside of the subcutaneous injection site, as high levels of luciferase expression are detected in the liver, spleen, lung, and kidney.







TABLE 150







Flux











0.5 mg/kg
0.05 mg/kg
0.005 mg/kg












Time Point
Flux (p/s)
Flux (p/s)
Flux (p/s)















2
hrs
3.18E+07
7.46E+06
8.94E+05


8
hrs
5.15E+08
2.18E+08
1.34E+07


24
hrs
1.56E+08
5.30E+07
7.16E+06


48
hrs
5.22E+07
8.75E+06
9.06E+05


72
hrs
8.87E+06
1.50E+06
2.98E+05


144
hrs
4.55E+05
3.51E+05
2.87E+05
















TABLE 151







Organ Flux












Liver
Spleen
Lung
Kidney



Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)
















0.5
mg/kg
1.01E+07
7.43E+05
9.75E+04
1.75E+05


0.05
mg/kg
1.61E+05
3.94E+04
4.04E+04
3.29E+04


0.005
mg/kg
2.84E+04
2.94E+04
2.42E+04
9.79E+04











Untreated
1.88E+04
1.02E+04
1.41E+04
9.20E+03









Example 99. Cationic Lipid Nanoparticle Subcutaneous Study
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) is formulated in a lipid nanoparticle containing 50% DLin-MC3-DMA, 38.5% cholesterol, 10% DSPC and 1.5% PEG. The formulation is administered subcutaneously (S.C.) to Balb-C mice at a dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg.
The mice are imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours and 144 hours after dosing and the average total flux (photons/second) was measured for each route of administration and cationic lipid formulation. Organs are imaged at 8 hours and the average total flux (photons/second) is measured for the liver, spleen, lung and kidney. A control for each organ is also analyzed.
Example 100. Luciferase Lipoplex Study
Lipoplexed luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine (5mC/pU), fully modified with 5-methylcytosine and N1-methyl-pseudouridine (5mC/N1mpU) or modified where 25% of the cytosines replaced with 5-methylcytosine and 25% of the uridines replaced with 2-thiouridine (5mC/s2U). The formulation was administered intravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.10 mg/kg.
Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 8 hours, 24 hours and 48 hours after dosing and the average total flux (photons/second) was measured for each route of administration and chemical modification. The background signal was about 3.91E+05 p/s. The results of the imaging are shown in Table 152. Organs were imaged at 6 hours and the average total flux (photons/second) was measured for the liver, spleen, lung and kidney. A control for each organ was also analyzed. The results are shown in Table 153.







TABLE 152







Flux











5mC/pU
5mC/N1mpU
5mC/s2U











Route
Time Point
Flux (p/s)
Flux (p/s)
Flux (p/s)















I.V.
8
hrs
5.76E+06
1.78E+06
1.88E+06


I.V.
24
hrs
1.02E+06
7.13E+05
5.28E+05


I.V.
48
hrs
4.53E+05
3.76E+05
4.14E+05


I.M.
8
hrs
1.90E+06
2.53E+06
1.29E+06


I.M.
24
hrs
9.33E+05
7.84E+05
6.48E+05


I.M.
48
hrs
8.51E+05
6.59E+05
5.49E+05


S.C.
8
hrs
2.85E+06
6.48E+06
1.14E+06


S.C.
24
hrs
6.66E+05
7.15E+06
3.93E+05


S.C.
48
hrs
3.24E+05
3.20E+06
5.45E+05
















TABLE 153







Organ Flux















Liver
Spleen
Lung
Kidney
Inj. Site


Route
Chemistry
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)





I.V.
5 mC/pU
5.26E+05
2.04E+07
4.28E+06
1.77E+04
n/a


I.V.
5 mC/N1mpU
1.48E+05
5.00E+06
1.93E+06
1.77E+04
n/a


I.V.
5 mC/s2U
2.14E+04
3.29E+06
5.48E+05
2.16E+04
n/a


I.M.
5 mC/pU
2.46E+04
1.38E+04
1.50E+04
1.44E+04
1.15E+06


I.M.
5 mC/N1mpU
1.72E+04
1.76E+04
1.99E+04
1.56E+04
1.20E+06


I.M.
5 mC/s2U
1.28E+04
1.36E+04
1.33E+04
1.07E+04
7.60E+05


S.C.
5 mC/pU
1.55E+04
1.67E+04
1.45E+04
1.69E+04
4.46E+04


S.C.
5 mC/N1mpU
1.20E+04
1.46E+04
1.38E+04
1.14E+04
8.29E+04


S.C.
5 mC/s2U
1.22E+04
1.31E+04
1.45E+04
1.08E+04
5.62E+04



Untreated
2.59E+04
1.34E+04
1.26E+04
1.22E+04
n/a









Example 101. Cationic Lipid Formulation of Modified mRNA
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) modified where 25% of the cytosines replaced with 5-methylcytosine and 25% of the uridines replaced with 2-thiouridine (5mC/s2U) was formulated in the cationic lipids described in Table 154. The formulations were administered intravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.







TABLE 154







Cationic Lipid Formulations












Formulation
NPA-130-1
NPA-131-1
NPA-132-1
NPA-133-1
111612-C





Lipid
DLin-MC3-
DLin-KC2-
C12-200
DLinDMA
DODMA



DMA
DMA


Lipid/mRNA
20:1
20:1
20:1
20:1
20:1


ratio (wt/wt)


Mean Size
120 nm
105 nm
122 nm
105 nm
221.3 nm



PDI: 0.10
PDI: 0.11
PDI: 0.13
PDI: 0.14
PDI: 0.063


Zeta at pH 7.4
0.2 mV
−0.6 mV
−0.5 mV
−0.3 mV
−3.10 mV


Encaps.
100%
100%
93%
93%
60%


(RiboGr)









Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hours after dosing and the average total flux (photons/second) was measured for each route of administration and cationic lipid formulation. The background flux was about 3.31E+05 p/s. The results of the imaging are shown in Table 155. In Table 155, “NT” means not tested. Untreated mice showed an average flux of 3.14E+05 at 2 hours, 3.33E+05 at 8 hours and 3.46E+05 at 24 hours. Peak expression was seen for all three routes tested at 8 hours. DLin-KC2-DMA has better expression than DLin-MC3-DMA and DODMA showed expression for all routes evaluated.







TABLE 155







Flux















DLin-
DLin-







MC3-
KC2-






Time
DMA
DMA
C12-200
DLinDMA
DODMA


Route
Point
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)





I.V.
 2 hrs
9.88E+06
6.98E+07
9.18E+06
3.98E+06
5.79E+06


I.V.
 8 hrs
1.21E+07
1.23E+08
1.02E+07
5.98E+06
6.14E+06


I.V.
24 hrs 
2.02E+06
1.05E+07
1.25E+06
1.35E+06
5.72E+05


I.M.
 2 hrs
6.72E+05
3.66E+06
3.25E+06
7.34E+05
4.42E+05


I.M.
 8 hrs
7.78E+06
2.85E+07
4.29E+06
2.22E+06
1.38E+05


I.M.
24 hrs
4.22E+05
8.79E+05
5.95E+05
8.48E+05
4.80E+05


S.C.
 2 hrs
2.37E+06
4.77E+06
4.44E+06
1.07E+06
1.05E+06


S.C.
 8 hrs
3.65E+07
1.17E+08
3.71E+06
9.33E+06
2.57E+06


S.C.
24 hrs 
4.47E+06
1.28E+07
6.39E+05
8.89E+05
4.27E+05









Example 102. Formulation of 5-Methylcytosine and N1-Methyl-Pseudouridine Modified mRNA
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and N1-methyl-pseudouridine was formulated in PBS (pH of 7.4). The formulations were administered intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 2.5 mg/kg.
Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 5 minutes, 30 minutes, 60 minutes and 120 minutes after dosing and the average total flux (photons/second) was measured for each route of administration and cationic lipid formulation. The background flux was about 3.78E+05 p/s. The results of the imaging are shown in Table 156. Expression of luciferase was already seen at 30 minutes with both routes of delivery. Peak expression from subcutaneous administration appears between 30 to 60 minutes. Intramuscular expression was still increasing at 120 minutes.







TABLE 156







Flux









PBS (pH 7.4)












Route

Time Point
Flux (p/s)
















I.M.
5
min
4.38E+05



I.M.
30
min
1.09E+06



I.M.
60
min
1.18E+06



I.M.
120
min
2.86E+06



S.C.
5
min
4.19E+05



S.C.
30
min
6.38E+06



S.C.
60
min
5.61E+06



S.C.
120
min
2.66E+06










Example 103. Intramuscular and Subcutaneous Administration of Chemically Modified mRNA
Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with N4-acetylcytidine, fully modified with 5-methoxyuridine, fully modified with N4-acetylcytidine and N1-methyl-pseudouridine or fully modified 5-methylcytosine and 5-methoxyuridine formulated in PBS (pH 7.4) was administered to Balb-C mice intramuscularly or subcutaneously at a dose of 2.5 mg/kg. Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hours. The average total flux (photons/second) for intramuscular administration is shown in Table 157 and the average total flux (photons/second) for subcutaneous administration is shown in Table 158. The background signal was 3.84E+05 (p/s). The peak expression for intramuscular administration was seen between 24 and 48 hours for all chemistry and expression was still detected at 120 hours. For subcutaneous delivery the peak expression was seen at 2-8 hours and expression was detected at 72 hours.







TABLE 157







Intramuscular Administration











2 hours
8 hours
24 hours



Flux (p/s)
Flux (p/s)
Flux (p/s)














N4-acetylcytidine
1.32E+07
2.15E+07
4.01E+07


5-methoxyuridine
4.93E+06
1.80E+07
4.53E+07


N4-acetylcytidine/
2.02E+07
1.93E+07
1.63E+08


N1-methyl-pseudouridine


5-methylcytosine/5-
6.79E+06
4.55E+07
3.44E+07


methoxyuridine
















TABLE 158







Subcutaneous Administration











2 hours
8 hours
24 hours



Flux (p/s)
Flux (p/s)
Flux (p/s)














N4-acetylcytidine
3.07E+07
1.23E+07
1.28E+07


5-methoxyuridine
7.10E+06
9.38E+06
1.32E+07


N4-acetylcytidine/
7.12E+06
3.07E+06
1.03E+07


N1-methyl-pseudouridine


5-methylcytosine/5-
7.15E+06
1.25E+07
1.11E+07


methoxyuridine









Example 104. In Vivo Study
Luciferase modified mRNA containing at least one chemical modification is formulated as a lipid nanoparticle (LNP) using the syringe pump method and characterized by particle size, zeta potential, and encapsulation.
As outlined in Table 159, the luciferase LNP formulation is administered to Balb-C mice intramuscularly (I.M.), intravenously (I.V.) and subcutaneously (S.C.). As a control luciferase modified RNA formulated in PBS is administered intravenously to mice.







TABLE 159







Luciferase Formulations
















Concen-

Amount 






tration
Injection
of



Formu-


Volume
modified
RNA
Dose


lation
Vehicle
Route
(mg/ml)
(ul)
(ug)
(mg/kg)
















Luc-LNP
PBS
S.C.
0.2000
50
10
0.5000


Luc-LNP
PBS
S.C.
0.0200
50
1
0.0500


Luc-LNP
PBS
S.C.
0.0020
50
0.1
0.0050


Luc-LNP
PBS
S.C.
0.0002
50
0.01
0.0005


Luc-LNP
PBS
I.V.
0.2000
50
10
0.5000


Luc-LNP
PBS
I.V.
0.0200
50
1
0.0500


Luc-LNP
PBS
I.V.
0.0020
50
0.1
0.0050


Luc-LNP
PBS
I.V.
0.0002
50
0.01
0.0005


Luc-LNP
PBS
I.M.
0.2000
50
10
0.5000


Luc-LNP
PBS
I.M.
0.0200
50
1
0.0500


Luc-LNP
PBS
I.M.
0.0020
50
0.1
0.0050


Luc-LNP
PBS
I.M.
0.0002
50
0.01
0.0005


Luc-PBS
PBS
I.V.
0.20
50
10
0.50









The mice are imaged at 2, 8, 24, 48, 120 and 192 hours to determine the bioluminescence (measured as total flux (photons/second) of the entire mouse). At 8 hours or 192 hours the liver, spleen, kidney and injection site for subcutaneous and intramuscular administration are imaged to determine the bioluminescence.
Example 105. Cationic Lipid Formulation Studies of Chemically Modified mRNA
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine (5mC/pU), pseudouridine (pU) or N1-methyl-pseudouridine (N1mpU) was formulated in the cationic lipids described in Table 160. The formulations were administered intravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.







TABLE 160







Cationic Lipid Formulations












Formulation
NPA-137-1
NPA-134-1
NPA-135-1
NPA-136-1
111612-A





Lipid
DLin-MC3-
DLin-MC3-
DLin-KC2-
C12-200
DODMA



DMA
DMA
DMA


Lipid/mRNA
20:1
20:1
20:1
20:1
20:1


ratio (wt/wt)


Mean Size
111 nm
104 nm
95 nm
143 nm
223.2 nm



PDI: 0.15
PDI: 0.13
PDI: 0.11
PDI: 0.12
PDI: 0.142


Zeta at pH 7.4
−4.1 mV
−1.9 mV
−1.0 mV
0.2 mV
−3.09 mV


Encaps.
97%
100%
100%
78%
64%


(RiboGr)


Chemistry
pU
N1mpU
N1mpU
N1mpU
5mC/pU









Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hours after dosing and the average total flux (photons/second) was measured for each route of administration and cationic lipid formulation. The background flux was about 4.11E+05 p/s. The results of the imaging are shown in Table 161. Peak expression was seen for all three routes tested at 8 hours.







TABLE 161







Flux















DLin-
DLin-
DLin-






MC3-
MC3-
KC2-






DMA
DMA
DMA
C12-200
DODMA



Time
(pU)
(N1mpU)
(N1mpU)
(N1mpU)
(5 mC/pU)


Route
Point
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)





I.V.
 2 hrs
3.21E+08
1.24E+09
1.01E+09
9.00E+08
3.90E+07


I.V.
 8 hrs
1.60E+09
3.22E+09
2.38E+09
1.11E+09
1.17E+07


I.V.
24 hrs
1.41E+08
3.68E+08
3.93E+08
8.06E+07
1.11E+07


I.M.
 2 hrs
2.09E+07
3.29E+07
8.32E+07
9.43E+07
4.66E+06


I.M.
 8 hrs
2.16E+08
6.14E+08
1.00E+09
8.77E+07
7.05E+06


I.M.
24 hrs
1.23E+07
1.40E+08
5.09E+08
1.36E+07
1.14E+06


S.C.
 2 hrs
2.32E+07
3.60E+07
2.14E+08
1.01E+08
3.11E+07


S.C.
 8 hrs
5.55E+08
9.80E+08
4.93E+09
1.01E+09
8.04E+07


S.C.
24 hrs
1.81E+08
2.74E+08
2.12E+09
4.74E+07
1.34E+07









Example 106. Studies of Chemical Modified mRNA
Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with N4-acetylcytidine (N4-acetyl), fully modified with 5-methoxyuridine (5meth), fully modified with N4-acetylcytidine and N1-methyl-pseudouridine (N4-acetyl/N1mpU) or fully modified with 5-methylcytosine and 5-methoxyuridine (5mC/5-meth) was formulated in DLin-MC3-DMA as described in Table 162. The formulations were administered intravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.







TABLE 162







Cationic Lipid Formulations











Formulation
NPA-141-1
NPA-142-1
NPA-143-1
NPA-144-1





Lipid
DLin-MC3-
DLin-MC3-
DLin-MC3-
DLin-MC3-



DMA
DMA
DMA
DMA


Lipid/mRNA
20:1
20:1
20:1
20:1


ratio (wt/wt)


Mean Size
138 nm
116 nm
144 nm
131 nm



PDI: 0.16
PDI: 0.15
PDI: 0.15
PDI: 0.15


Zeta at pH 7.4
−2.8 mV
−2.8 mV
−4.3 mV
−5.0 mV


Encaps.
97%
100%
75%
72%


(RiboGr)


Chemistry
N4-acetyl
5meth
N4-acetyl/
5mC/5-meth





N1mpU









Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 2 hours, 6 hours and 24 hours after dosing and the average total flux (photons/second) was measured for each route of administration and cationic lipid formulation. The background flux was about 2.70E+05 p/s. The results of the imaging are shown in Table 163.







TABLE 163







Flux














N4-acetyl/
5mC/5-



N4-acetyl
5meth
N1mpU
meth












Route
Time Point
Flux (p/s)
Flux (p/s)
Flux (p/s)
Flux (p/s)
















I.V.
2
hrs
9.17E+07
3.19E+06
4.21E+07
1.88E+06


I.V.
6
hrs
7.70E+08
9.28E+06
2.34E+08
7.75E+06


I.V.
24
hrs
6.84E+07
1.04E+06
3.55E+07
3.21E+06


I.M.
2
hrs
8.59E+06
7.86E+05
5.30E+06
5.11E+05


I.M.
6
hrs
1.27E+08
8.88E+06
3.82E+07
3.17E+06


I.M.
24
hrs
4.46E+07
1.38E+06
2.00E+07
1.39E+06


S.C.
2
hrs
1.83E+07
9.67E+05
4.45E+06
1.01E+06


S.C.
6
hrs
2.89E+08
1.78E+07
8.91E+07
1.29E+07


S.C.
24
hrs
6.09E+07
6.40E+06
2.08E+08
6.63E+06









Example 107. Lipid Nanoparticle Containing a Plurality of Modified mRNAs
EPO mRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine), G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) and Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 5659; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine), is formulated in DLin-MC3-DMA as described in Table 164. The formulations are administered intravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg. Control LNP formulations containing only one mRNA are also administered at an equivalent dose.







TABLE 164







DLin-MC3-DMA Formulation










Formulation
NPA-157-1







Lipid
DLin-MC3-DMA



Lipid/mRNA
20:1



ratio (wt/wt)



Mean Size
89 nm




PDI: 0.08



Zeta at pH 7.4
1.1 mV



Encaps.
97%



(RiboGr)










Serum is collected from the mice at 8 hours, 24 hours, 72 hours and/or 7 days after administration of the formulation. The serum is analyzed by ELISA to determine the protein expression of EPO, G-CSF, and Factor IX.
Example 108. Cationic Lipid Formulation Studies of 5-Methylcytosine and N1-Methyl-Pseudouridine Modified mRNA
EPO mRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) or G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5655; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) is formulated in DLin-MC3-DMA and DLin-KC2-DMA as described in Table 165. The formulations are administered intravenously (I.V), intramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.







TABLE 165







DLin-MC3-DMA and DLin-KC2-DMA Formulations











Formulation
NPA-147-1
NPA-148-1
NPA-150-1
NPA-151-1





mRNA
EPO
EPO
G-CSF
G-CSF


Lipid
DLin-MC3-
DLin-KC2-
DLin-MC3-
DLin-KC2-



DMA
DMA
DMA
DMA


Lipid/mRNA
20:1
20:1
20:1
20:1


ratio (wt/wt)


Mean Size
117 nm
82 nm
119 nm
88 nm



PDI: 0.14
PDI: 0.08
PDI: 0.13
PDI: 0.08


Zeta at pH 7.4
−1.7 mV
0.6 mV
3.6 mV
2.2 mV


Encaps.
100%
96%
100%
100%


(RiboGr)









Serum is collected from the mice at 8 hours, 24 hours, 72 hours and/or 7 days after administration of the formulation. The serum is analyzed by ELISA to determine the protein expression of EPO and G-CSF.
Example 109. In Vitro VEGF PBMC Study
500 ng of VEGF mRNA (mRNA sequence shown in SEQ ID NO: 5668 polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine (VEGF 5mC/pU), fully modified with 5-methylcytosine and N1-methyl-pseudouridine (VEGF 5mC/N1mpU) or unmodified (VEGF unmod) was transfected with 0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells (PBMC) from three normal blood donors (D1, D2, and D3). Cells were also untreated for each donor as a control. The supernatant was harvested and run by ELISA 22 hours after transfection to determine the protein expression and cytokine induction. The expression of VEGF and IFN-alpha induction is shown in Table 166.







TABLE 166







Protein and Cytokine levels










VEGF Expression
IFN-alpha Induction



(pg/ml)
(pg/ml)














D1
D2
D3
D1
D2
D3
















VEGF unmod
2
0
0
5400
3537
4946


VEGF 5 mC/pU
424
871
429
145
294
106


VEGF
5088 
10331
6183
205
165
6


5 mC/N1mpU















Example 110. In Vitro Expression of Modified mRNA
HEK293 cells were transfected with VEGF-A modified mRNA (mRNA sequence shown in SEQ ID NO: 5668; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) or EPO modified mRNA (mRNA sequence shown in SEQ ID NO: 5658; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine), HeLa cells were forward transfected with Transforming growth factor beta (TGF-beta) modified mRNA (mRNA sequence shown in SEQ ID NO 5669; poly A tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) and HepG2 cells were transfected with bactericidal/permeability-increasing protein (rBPI-21) modified mRNA (mRNA sequence shown in SEQ ID NO 5670; poly A tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) which had been complexed with Lipofectamine2000 from Invitrogen (Carlsbad, Calif.) at the concentrations shown in Table 167, 168, and 169 using the procedures described herein. The protein expression was detected by ELISA and the protein (pg/ml) is also shown in Table 167, 168, and 169. In Table 167, “>” means greater than. For TGF-beta a control of untreated cells and a mock transfection of Lipofectamine2000 was also tested.







TABLE 167







EPO Protein Expression















Amount
5
1
200
40
8
1.6
320
64


Transfected
ng
ng
pg
pg
pg
pg
fg
fg





Protein (pg/ml)
>2000
609.486
114.676
0
0
0
0
0
















TABLE 168







TGF-beta Protein Expression












Amount Transfected
750 ng
250 ng
83 ng
Mock
Untreated





Protein (pg/ml)
5058
4325
3210
2
0
















TABLE 169







rBPI-21 Protein Expression















Amount
2
400
80
16
3.2
640
128
26


Transfected
ug
ng
ng
ng
ng
pg
pg
pg





Protein (pg/ml)
20.683
9.269 
4.768
0
0
0
0
0









Example 111. Bicistronic Modified mRNA
Human embryonic kidney epithelial (HEK293) were seeded on 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany) HEK293 were seeded at a density of 30,000 in 100 μl cell culture medium (DMEM, 10% FCS, adding 2 mM L-Glutamine, 1 mM Sodiumpyruvate and 1× non-essential amino acids (Biochrom AG, Berlin, Germany) and 1.2 mg/ml Sodiumbicarbonate (Sigma-Aldrich, Munich, Germany)) 75 ng of the bi-cistronic modified mRNA (mCherry-2A-GFP) (mRNA sequence shown in SEQ ID NO 5671; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine), mCherry modified mRNA (mRNA SEQ ID NO: 5657; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) or green fluorescent protein (GFP) modified mRNA (mRNA sequence shown in SEQ ID NO: 5667; polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) were added after seeding the cells and incubated. A control of untreated cells was also evaluated. mCherry-2A-GFP refers to a modified mRNA sequence comprising the coding region of mCherry, the 2A peptide and the coding region of GFP.
Cells were harvested by transferring the culture media supernatants to a 96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg, Germany). Cells were trypsinized with 1⁄2 volume Trypsin/EDTA (Biochrom AG, Berlin, Germany), pooled with respective supernatants and fixed by adding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck, Darmstadt, Germany). Samples then were submitted to a flow cytometer measurement with a 532 nm excitation laser and the 610/20 filter for PE-Texas Red in a LSRII cytometer (Beckton Dickinson GmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI) of all events is shown in Table 170. Cells transfected with the bi-cistronic modified mRNA were able to express both mCherry and GFP.







TABLE 170







MFI of Modified mRNA











Modified mRNA
mCherry MFI
GFP MFI















mCherry
17746
427



GFP
427
20019



mCherry-2A-GFP
5742
6783



Untreated
427
219










Example 112. Cationic Lipid Formulation Studies of 5-Methylcytosine and N1-Methyl-Pseudouridine Modified mRNA to Produce an Antibody
Herceptin heavy chain (HC) mRNA (mRNA sequence shown in SEQ ID NO 5672; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) and Herceptin light chain (LC) modified mRNA (mRNA sequence shown in SEQ ID NO 5673; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and N1-methyl-pseudouridine) are formulated in DLin-MC3-DMA and DLin-KC2-DMA as described in Table 171. The formulations are administered intravenously (I.V) to Balb-C mice at a dose of 0.500, 0.050, and 0.005 mg/kg.







TABLE 171







DLin-MC3-DMA and DLin-KC2-DMA Formulations











Formulation
NPA-158-1
NPA-159-1







Herceptin HC:LC
 2:1
 2:1



Ratio (wt/wt)



Lipid
DLin-MC3-DMA
DLin-KC2-DMA



Lipid/Total mRNA
20:1
20:1



ratio (wt/wt)



Mean Size
129 nm
100 nm




PDI: 0.14
PDI: 0.10



Zeta at pH 7.4
0.9 mV
1.9 mV



Encaps. (RiboGr)
100%
100%










Serum was collected from the mice at 8 hours, 24 hours, 72 hours and/or 7 days after administration of the formulation. The serum was analyzed by ELISA to determine the protein expression of Herceptin.
Example 113. Directed SAR of Pseudouridine and N1-Methyl PseudoUridine
With the recent focus on the pyrimidine nucleoside pseudouridine, a series of structure-activity studies were designed to investigate mRNA containing modifications to pseudouridine or N1-methyl-pseudourdine.
The study was designed to explore the effect of chain length, increased lipophilicity, presence of ring structures, and alteration of hydrophobic or hydrophilic interactions when modifications were made at the N1 position, C6 position, the 2-position, the 4-position and on the phosphate backbone. Stability is also investigated.
To this end, modifications involving alkylation, cycloalkylation, alkyl-cycloalkylation, arylation, alkyl-arylation, alkylation moieties with amino groups, alkylation moieties with carboxylic acid groups, and alkylation moieties containing amino acid charged moieties are investigated. The degree of alkylation is generally C1-C6. Examples of the chemistry modifications include those listed in Table 172 and Table 173.







TABLE 172







Pseudouridine and N1-methyl Pseudo Uridine SAR










Compound
Naturally


Chemistry Modification
#
occuring












N1-Modifications




N1-Ethyl-pseudo-UTP
1
N


N1-Propyl-pseudo-UTP
2
N


N1-iso-propyl-pseudo-UTP
3
N


N1-(2,2,2-Trifluoroethyl)-pseudo-UTP
4
N


N1-Cyclopropyl-pseudo-UTP
5
N


N1-Cyclopropylmethyl-pseudo-UTP
6
N


N1-Phenyl-pseudo-UTP
7
N


N1-Benzyl-pseudo-UTP
8
N


N1-Aminomethyl-pseudo-UTP
9
N


Pseudo-UTP-N1-2-ethanoic acid
10
N


N1-(3-Amino-3-carboxypropyl)pseudo-UTP
11
N


N1-Methyl-3-(3-amino-3-carboxy-
12
Y


propyl)pseudo-UTP


C-6 Modifications


6-Methyl-pseudo-UTP
13
N


6-Trifluoromethyl-pseudo-UTP
14
N


6-Methoxy-pseudo-UTP
15
N


6-Phenyl-pseudo-UTP
16
N


6-Iodo-pseudo-UTP
17
N


6-Bromo-pseudo-UTP
18
N


6-Chloro-pseudo-UTP
19
N


6-Fluoro-pseudo-UTP
20
N


2- or 4-position Modifications


4-Thio-pseudo-UTP
21
N


2-Thio-pseudo-UTP
22
N


Phosphate backbone Modifications


Alpha-thio-pseudo-UTP
23
N


N1-Me-alpha-thio-pseudo-UTP
24
N
















TABLE 173







Pseudouridine and N1-methyl Pseudo Uridine SAR










Compound
Naturally


Chemistry Modification
#
occurring





N1-Methyl-pseudo-UTP
 1
Y


N1-Butyl-pseudo-UTP
 2
N


N1-tert-Butyl-pseudo-UTP
 3
N


N1-Pentyl-pseudo-UTP
 4
N


N1-Hexyl-pseudo-UTP
 5
N


N1-Trifluoromethyl-pseudo-UTP
 6
Y


N1-Cyclobutyl-pseudo-UTP
 7
N


N1-Cyclopentyl-pseudo-UTP
 8
N


N1-Cyclohexyl-pseudo-UTP
 9
N


N1-Cycloheptyl-pseudo-UTP
10
N


N1-Cyclooctyl-pseudo-UTP
11
N


N1-Cyclobutylmethyl-pseudo-UTP
12
N


N1-Cyclopentylmethyl-pseudo-UTP
13
N


N1-Cyclohexylmethyl-pseudo-UTP
14
N


N1-Cycloheptylmethyl-pseudo-UTP
15
N


N1-Cyclooctylmethyl-pseudo-UTP
16
N


N1-p-tolyl-pseudo-UTP
17
N


N1-(2,4,6-Trimethyl-phenyl)pseudo-UTP
18
N


N1-(4-Methoxy-phenyl)pseudo-UTP
19
N


N1-(4-Amino-phenyl)pseudo-UTP
20
N


N1(4-Nitro-phenyl)pseudo-UTP
21
N


Pseudo-UTP-N1-p-benzoic acid
22
N


N1-(4-Methyl-benzyl)pseudo-UTP
24
N


N1-(2,4,6-Trimethyl-benzyl)pseudo-UTP
23
N


N1-(4-Methoxy-benzyl)pseudo-UTP
25
N


N1-(4-Amino-benzyl)pseudo-UTP
26
N


N1-(4-Nitro-benzyl)pseudo-UTP
27
N


Pseudo-UTP-N1-methyl-p-benzoic acid
28
N


N1-(2-Amino-ethyl)pseudo-UTP
29
N


N1-(3-Amino-propyl)pseudo-UTP
30
N


N1-(4-Amino-butyl)pseudo-UTP
31
N


N1-(5-Amino-pentyl)pseudo-UTP
32
N


N1-(6-Amino-hexyl)pseudo-UTP
33
N


Pseudo-UTP-N1-3-propionic acid
34
N


Pseudo-UTP-N1-4-butanoic acid
35
N


Pseudo-UTP-N1-5-pentanoic acid
36
N


Pseudo-UTP-N1-6-hexanoic acid
37
N


Pseudo-UTP-N1-7-heptanoic acid
38
N


N1-(2-Amino-2-carboxyethyl)pseudo-UTP
39
N


N1-(4-Amino-4-carboxybutyl)pseudo-UTP
40
N


N3-Alkyl-pseudo-UTP
41
N


6-Ethyl-pseudo-UTP
42
N


6-Propyl-pseudo-UTP
43
N


6-iso-Propyl-pseudo-UTP
44
N


6-Butyl-pseudo-UTP
45
N


6-tert-Butyl-pseudo-UTP
46
N


6-(2,2,2-Trifluoroethyl)-pseudo-UTP
47
N


6-Ethoxy-pseudo-UTP
48
N


6-Trifluoromethoxy-pseudo-UTP
49
N


6-Phenyl-pseudo-UTP
50
N


6-(Substituted-Phenyl)-pseudo-UTP
51
N


6-Cyano-pseudo-UTP
52
N


6-Azido-pseudo-UTP
53
N


6-Amino-pseudo-UTP
54
N


6-Ethylcarboxylate-pseudo-UTP
 54b
N


6-Hydroxy-pseudo-UTP
55
N


6-Methylamino-pseudo-UTP
 55b
N


6-Dimethylamino-pseudo-UTP
57
N


6-Hydroxyamino-pseudo-UTP
59
N


6-Formyl-pseudo-UTP
60
N


6-(4-Morpholino)-pseudo-UTP
61
N


6-(4-Thiomorpholino)-pseudo-UTP
62
N


N1-Me-4-thio-pseudo-UTP
63
N


N1-Me-2-thio-pseudo-UTP
64
N


1,6-Dimethyl-pseudo-UTP
65
N


1-Methyl-6-trifluoromethyl-pseudo-UTP
66
N


1-Methyl-6-ethyl-pseudo-UTP
67
N


1-Methyl-6-propyl-pseudo-UTP
68
N


1-Methyl-6-iso-propyl-pseudo-UTP
69
N


1-Methyl-6-butyl-pseudo-UTP
70
N


1-Methyl-6-tert-butyl-pseudo-UTP
71
N


1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP
72
N


1-Methyl-6-iodo-pseudo-UTP
73
N


1-Methyl-6-bromo-pseudo-UTP
74
N


1-Methyl-6-chloro-pseudo-UTP
75
N


1-Methyl-6-fluoro-pseudo-UTP
76
N


1-Methyl-6-methoxy-pseudo-UTP
77
N


1-Methyl-6-ethoxy-pseudo-UTP
78
N


1-Methyl-6-trifluoromethoxy-pseudo-UTP
79
N


1-Methyl-6-phenyl-pseudo-UTP
80
N


1-Methyl-6-(substituted phenyl)pseudo-UTP
81
N


1-Methyl-6-cyano-pseudo-UTP
82
N


1-Methyl-6-azido-pseudo-UTP
83
N


1-Methyl-6-amino-pseudo-UTP
84
N


1-Methyl-6-ethylcarboxylate-pseudo-UTP
85
N


1-Methyl-6-hydroxy-pseudo-UTP
86
N


1-Methyl-6-methylamino-pseudo-UTP
87
N


1-Methyl-6-dimethylamino-pseudo-UTP
88
N


1-Methyl-6-hydroxyamino-pseudo-UTP
89
N


1-Methyl-6-formyl-pseudo-UTP
90
N


1-Methyl-6-(4-morpholino)-pseudo-UTP
91
N


1-Methyl-6-(4-thiomorpholino)-pseudo-UTP
92
N


1-Alkyl-6-vinyl-pseudo-UTP
93
N


1-Alkyl-6-allyl-pseudo-UTP
94
N


1-Alkyl-6-homoallyl-pseudo-UTP
95
N


1-Alkyl-6-ethynyl-pseudo-UTP
96
N


1-Alkyl-6-(2-propynyl)-pseudo-UTP
97
N


1-Alkyl-6-(1-propynyl)-pseudo-UTP
98
N









Example 114. Incorporation of Naturally and Non-Naturally Occurring Nucleosides
Naturally and non-naturally occurring nucleosides are incorporated into mRNA encoding a polypeptide of interest. Examples of these are given in Tables 174 and 175. Certain commercially available nucleoside triphosphates (NTPs) are investigated in the polynucleotides of the invention. A selection of these are given in Table 175. The resultant mRNA are then examined for their ability to produce protein, induce cytokines, and/or produce a therapeutic outcome.







TABLE 174







Naturally and non-naturally occurring nucleosides










Compound
Naturally


Chemistry Modification
#
occuring












N4-Methyl-Cytosine
1
Y


N4,N4-Dimethyl-2′-OMe-Cytosine
2
Y


5-Oxyacetic acid-methyl ester-Uridine
3
Y


N3-Methyl-pseudo-Uridine
4
Y


5-Hydroxymethyl-Cytosine
5
Y


5-Trifluoromethyl-Cytosine
6
N


5-Trifluoromethyl-Uridine
7
N


5-Methyl-amino-methyl-Uridine
8
Y


5-Carboxy-methyl-amino-methyl-Uridine
9
Y


5-Carboxymethylaminomethyl-2′-OMe-Uridine
10
Y


5-Carboxymethylaminomethyl-2-thio-Uridine
11
Y


5-Methylaminomethyl-2-thio-Uridine
12
Y


5-Methoxy-carbonyl-methyl-Uridine
13
Y


5-Methoxy-carbonyl-methyl-2′-OMe-Uridine
14
Y


5-Oxyacetic acid-Uridine
15
Y


3-(3-Amino-3-carboxypropyl)-Uridine
16
Y


5-(carboxyhydroxymethyl)uridine methyl ester
17
Y


5-(carboxyhydroxymethyl)uridine
18
Y
















TABLE 175







Non-naturally occurring nucleoside triphosphates












Compound
Naturally



Chemistry Modification
#
occuring















N1-Me-GTP
1
N



2′-OMe-2-Amino-ATP
2
N



2′-OMe-pseudo-UTP
3
Y



2′-OMe-6-Me-UTP
4
N



2′-Azido-2′-deoxy-ATP
5
N



2′-Azido-2′-deoxy-GTP
6
N



2′-Azido-2′-deoxy-UTP
7
N



2′-Azido-2′-deoxy-CTP
8
N



2′-Amino-2′-deoxy-ATP
9
N



2′-Amino-2′-deoxy-GTP
10
N



2′-Amino-2′-deoxy-UTP
11
N



2′-Amino-2′-deoxy-CTP
12
N



2-Amino-ATP
13
N



8-Aza-ATP
14
N



Xanthosine-5′-TP
15
N



5-Bromo-CTP
16
N



2′-F-5-Methyl-2′-deoxy-UTP
17
N



5-Aminoallyl-CTP
18
N



2-Amino-riboside-TP
19
N










Example 115. Incorporation of Modifications to the Nucleobase and Carbohydrate (Sugar)
Naturally and non-naturally occurring nucleosides are incorporated into mRNA encoding a polypeptide of interest. Commercially available nucleosides and NTPs having modifications to both the nucleobase and carbohydrate (sugar) are examined for their ability to be incorporated into mRNA and to produce protein, induce cytokines, and/or produce a therapeutic outcome. Examples of these nucleosides are given in Tables 176 and 177.







TABLE 176







Combination modifications










Chemistry Modification
Compound #














5-iodo-2′-fluoro-deoxyuridine
1



5-iodo-cytidine
6



2′-bromo-deoxyuridine
7



8-bromo-adenosine
8



8-bromo-guanosine
9



2,2′-anhydro-cytidine hydrochloride
10



2,2′-anhydro-uridine
11



2′-Azido-deoxyuridine
12



2-amino-adenosine
13



N4-Benzoyl-cytidine
14



N4-Amino-cytidine
15



2′-O-Methyl-N4-Acetyl-cytidine
16



2′Fluoro-N4-Acetyl-cytidine
17



2′Fluor-N4-Bz-cytidine
18



2′O-methyl-N4-Bz-cytidine
19



2′O-methyl-N6-Bz-deoxyadenosine
20



2′Fluoro-N6-Bz-deoxyadenosine
21



N2-isobutyl-guanosine
22



2′Fluro-N2-isobutyl-guanosine
23



2′O-methyl-N2-isobutyl-guanosine
24

















TABLE 177







Naturally occuring combinations










Compound
Naturally


Name
#
occurring












5-Methoxycarbonylmethyl-2-thiouridine TP
1
Y


5-Methylaminomethyl-2-thiouridine TP
2
Y


5-Crbamoylmethyluridine TP
3
Y


5-Carbamoylmethyl-2′-O-methyluridine TP
4
Y


1-Methyl-3-(3-amino-3-carboxypropyl)
5
Y


pseudouridine TP


5-Methylaminomethyl-2-selenouridine TP
6
Y


5-Carboxymethyluridine TP
7
Y


5-Methyldihydrouridine TP
8
Y


lysidine TP
9
Y


5-Taurinomethyluridine TP
10
Y


5-Taurinomethyl-2-thiouridine TP
11
Y


5-(iso-Pentenylaminomethyl)uridine TP
12
Y


5-(iso-Pentenylaminomethyl)-2-thiouridine TP
13
Y


5-(iso-Pentenylaminomethyl)-2′-O-
14
Y


methyluridine TP


N4-Acetyl-2′-O-methylcytidine TP
15
Y


N4,2′-O-Dimethylcytidine TP
16
Y


5-Formyl-2′-O-methylcytidine TP
17
Y


2′-O-Methylpseudouridine TP
18
Y


2-Thio-2′-O-methyluridine TP
19
Y


3,2′-O-Dimethyluridine TP
20
Y









In the tables “UTP” stands for uridine triphosphate, “GTP” stands for guanosine triphosphate, “ATP” stands for adenosine triphosphate, “CTP” stands for cytosine triphosphate, “TP” stands for triphosphate and “Bz” stands for benzyl.
Example 116. Additional Cosmetic Polypeptides of Interest
The cosmetic polypeptide of interest may be a toxin such as but not limited to botulinum toxin and/or tetanus toxin. Shown in Table 178, in addition to the name and description of the gene encoding the cosmetic polypeptide of interest is the NCBI Accession ID for the nucleotide (NCBI Nucleotide) or protein sequence (NCBI Protein). For any particular gene there may exist one or more variants or isoforms. Where these exist, they are shown in the tables as well. It will be appreciated by those of skill in the art that disclosed in the Tables are potential flanking regions. These are encoded in each ENST transcript or NCBI nucleotide sequence either to the 5′ (upstream) or 3′ (downstream) of the ORF or coding region. The coding region is definitively and specifically disclosed by teaching the ENSP protein or NCBI protein sequence. Consequently, the sequences taught flanking that encoding the protein are considered flanking regions. It is also possible to further characterize the 5′ and 3′ flanking regions by utilizing one or more available databases or algorithms. Databases have annotated the features contained in the flanking regions of the ENST transcripts, NCBI nucleotide sequences and these are available in the art.







TABLE 178







Cosmetic Targets












Target
Target Description
NCBI Nucleotide
SEQ ID NO
NCBI Protein
SEQ ID NO





729
botulinum toxin
GQ168376.1
5674
ACS52168.1
5691



type A


730
botulinum toxin
N/A
5675
N/A
5692



type A light chain


731
botulinum toxin
AB665558.1
5676
YP_001693307.1
5693



type B


732
botulinum toxin
N/A
5677
N/A
5694



type B light chain


733
botulinum toxin
AB200359.1
5678
P18640.2
5695



type C1


734
botulinum toxin
N/A
5679
N/A
5696



type C1 light chain


735
botulinum toxin
AB465554.1
5680
BAH29485.1
5697



type C2 component I


736
botulinum toxin
AB465554.1
5681
BAH29486.1
5698



type C2 component II


737
botulinum toxin
S49407.1
5682
P19321.1
5699



type D


738
botulinum toxin
N/A
5683
N/A
5700



type D light chain


739
botulinum toxin
AM695761.1
5684
CAQ03495.1
5701



type E


740
botulinum toxin
N/A
5685
N/A
5702



type E light chain


741
botulinum toxin
M92906.1
5686
P30996.1
5703



type F


742
botulinum toxin
N/A
5687
N/A
5704



type F light chain


743
botulinum toxin
X74162.1
5688
Q60393.2
5705



type G


744
botulinum toxin
N/A
5689
N/A
5706



type G light chain


745
tetanus toxin
X06214.1
5690
P04958.2
5707









A. Tetanus Toxin
The cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of neurodegenerative and other muscle disorders. The cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA of the present invention can encode a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 5707.
B. Botulinum Toxin
The cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA may be used in the treatment, amelioration or prophylaxis of cosmetic diseases, disorders and/or conditions and other muscle disorders. The cosmetic polynucleotides, cosmetic primary constructs and/or cosmetic mmRNA can encode a cosmetic polypeptide of interest, a light chain of a cosmetic polypeptide of interest such as, but not limited to, SEQ ID NOs: 5691-5706. The light chain of the polypeptide of interest may be produced by a method known in the art.
Example 117. Protein Complexes Associated with Neurotoxins
The cosmetic polynucleotides, cosmetic primary construct and/or cosmetic mmRNA can encode and/or form toxin complexes from the noncovalent association of the neurotoxin with up to seven other proteins which are known as neurotoxin-associated proteins (NAPs). Table 179 describes a non-limiting listing of NAPs for the seven botulinum toxin serotypes.







TABLE 179







Neurotoxin-Associated Proteins









Botulinum Toxin
Neurotoxin-associated



Serotype
protein (NAP)
SEQ ID NO





A
NTNH
5708


A
NTNH
5709


A
NTNH
5710


A
NTNH
5711


A
NTNH
5712


A
NTNH
5713


A
NTNH
5714


A
NTNH
5715


A
NTNH
5716


A
HA-17/15
5717


A
HA-33/35
5718


A
HA-33/35
5719


A
HA-33/35
5720


A
HA-19/20


A
HA-70
5721


A
HA-70
5722


B
NTNH
5723


B
NTNH
5724


B
NTNH
5725


B
NTNH
5726


B
NTNH
5727


B
HA-17
5728


B
HA-33/35
5729


B
HA-33/35
5730


B
HA-33/35
5731


B
HA-70
5732


B
HA-70
5733


C
NTNH
5734


C
NTNH
5735


C
HA-17
5736


C
HA-17
5737


C
HA-26/21


C
HA-33
5738


C
HA-33
5739


C
HA-33
5740


C
HA-70
5741


C
HA-70
5742


D
NTNH
5743


D
NTNH
5744


D
HA-17
5745


D
HA-33
5746


D
HA-33
5747


D
HA-70
5748


E
NTNH
5749


F
NTNH
5750


F
NTNH
5751


F
NTNH
5752


G
NTNH
5753


G
HA-70 (partial sequence)
5754


G
HA-17









Example 118. Dose Response and Injection Site Selection and Timing
To determine the dose response trends, impact of injection site and impact of injection timing, studies are performed following the protocol outlined in Example 35. In these studies, varied doses of 1 ug, 5 ug, 10 ug, 25 ug, 50 ug, and values in between are used to determine dose response outcomes. Split dosing for a 100 ug total dose includes three or six doses of 1.6 ug, 4.2 ug, 8.3 ug, 16.6 ug, or values and total doses equal to administration of the total dose selected.
Injection sites are chosen from the limbs or any body surface presenting enough area suitable for injection. This may also include a selection of injection depth to target the dermis (Intradermal), epidermis (Epidermal), subcutaneous tissue (SC) or muscle (IM). Injection angle will vary based on targeted delivery site with injections targeting the intradermal site to be 10-15 degree angles from the plane of the surface of the skin, between 20-45 degrees from the plane of the surface of the skin for subcutaneous injections and angles of between 60-90 degrees for injections substantially into the muscle.
Example 119. Intranasal Lung Delivery of 1-Methylpseudouridine or 5-Methylcytosine and 1-Methylpseudouridine Modified Luciferase mRNA Formulated in Cationic Lipid Nanoparticle
Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 1-methylpseudouridine (Luc-G5-LNP-KC2) or fully modified with 1-methylpseudouridine and 5-methylcytosine (Luc-G2-LNP-KC2) were formulated in the cationic Lipid Nanoparticle (LNP-KC2) as described in Table 180.







TABLE 180







Formulation











Formulation
NPA-127-1
NPA-135-1







Lipid
DLin-KC2-DMA
DLin-KC2-DMA



Lipid/mRNA
20:1
20:1



ratio (wt/wt)



Mean Size
114 nm
95 nm




PDI: 0.10
PDI: 0.11



Zeta at pH 7.4
−0.5 mV
−1.0 mV



Encaps. (RiboGr)
77%
100%










The formulations were administered intranasally (I.N.) to Balb-C mice at a dose of 0.3 mg/kg. Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 2 hours, 8 hours, 48, and 72 hours after dosing and the average total flux (photons/second) was measured. The background flux was about 6.3+05 p/s. The results of the imaging of Luc-G5-LNP-KC2 or Luc-G5-LNP-KC2 vs. Vehicle are shown in the Table 181, “NT” means not tested.







TABLE 181







Luciferase expression after intranasal dosing















Luc-G5-
Luc-G2-
Vehicle




Time
LNP-KC2
LNP-KC2
(PBS)



Route
Point
Flux (p/s)
Flux (p/s)
Flux (p/s)







I.N.
 2 hrs
1.53E+06
5.81E+05
3.87E+05



I.N.
 8 hrs
1.09E+07
9.35E+05
5.57E+05



I.N.
24 hrs
5.93E+06
5.33E+05
4.89E+05



I.N.
28 hrs
1.22E+06
7.63E+05
8.81E+05



I.N.
72 hrs
8.43E+05
NT
8.17E+05










Example 120. Intranasal Lung Delivery of 1-Methylpseudouridine or 5-Methylcytosine and 1-Methylpseudouridine Modified Luciferase mRNA Lipoplexed in Lipofectamine 2000
Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 1-methylpseudouridine (Luc-G5-Lipoplex) or fully modified with 1-methylpseudouridine and 5-methylcytosine (Luc-G2-Lipoplex) were lipoplexed in two steps, first step was the dilution of 16.6 ul from 3 mg/ml of 1-methylpseudouridine or 1-methylpseudouridine and 5-methylcytosine modified luciferase mRNA in 108.5 ul DMEM and the second step was the dilution of 100 ul LF2000 in 25 ul DMEM then both mixed together gently to make 250 ul total mixer which incubated 5-10 min at RT to form a Lipid-mRNA complex before injection. The lipoplex from both Luciferase mRNA fully modified with 1-methylpseudouridine or 1-methylpseudouridine and 5-methylcytosine were administered intranasally (I.N.) to Balb-C mice at a dose of 0.5 mg/kg.
Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hours after dosing and the average total flux (photons/second) was measured. The background signal was about 4.8+05 p/s. The background flux was about 6.3+05 p/s. The results of the imaging of Luc-G5-Lipoplex or Luc-G2-Lipoplex vs. Vehicle are shown in the Table 182.







TABLE 182







Luciferase expression after intranasal dosing















Luc-G5-
Luc-G2-
Vehicle




Time
Lipoplex
Lipoplex
(PBS)



Route
Point
Flux (p/s)
Flux (p/s)
Flux (p/s)







I.N.
2 hrs
8.37E+05
9.58E+05
3.87E+05



I.N.
8 hrs
8.42E+05
7.11E+05
5.57E+05



I.N.
24 hrs 
5.74E+05
5.53E+05
4.89E+05










Example 121. Intranasal Lung Delivery of 1-Methylpseudouridine or 5-Methylcytosine and 1-Methylpseudouridine Modified Luciferase mRNA Formulated in PBS
Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 5665; polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 1-methylpseudouridine (Luc-G5-Buffer(PBS)) or fully modified with 1-methylpseudouridine and 5-methylcytosine (Luc-G2-Buffer(PBS)) formulated in PBS (pH 7.4) were administered intranasally (I.N.) to Balb-C mice at a dose of 7.5 mg/kg.
Twenty minutes prior to imaging, mice were injected intraperitoneally with a D-luciferin solution at 150 mg/kg. Animals were then anesthetized and images were acquired with an IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence was measured as total flux (photons/second) of the entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hours after dosing and the average total flux (photons/second) was measured. The background flux was about 4.8+05 p/s. The background flux was about 6.3+05 p/s. The results of the imaging of Luc-G5-in Buffer vs. Vehicle are shown in the Table 183.







TABLE 183







Luciferase expression after intranasal dosing















Luc-G5-in
Luc-G2-in
Vehicle




Time
Buffer (PBS)
Buffer (PBS)
(PBS)



Route
Point
Flux [p/s]
Flux [p/s]
Flux (p/s)







I.N.
2 hrs
4.50E+05
9.58E+05
3.87E+05



I.N.
8 hrs
7.12E+05
7.11E+05
5.57E+05



I.N.
24 hrs 
4.47E+05
5.53E+05
4.89E+05










It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.
While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

## Claims

1. A cosmetic mRNA encoding SEQ ID NO: 1245, wherein said mRNA comprises a coding region, said coding region having at least 80% identity to SEQ ID NO: 1973.

2. The cosmetic mRNA of claim 1, wherein the cosmetic mRNA comprises a poly-A tail.

3. The cosmetic mRNA of claim 1, wherein the cosmetic mRNA comprises at least one 5′ terminal cap.

4. The cosmetic mRNA of claim 1 where the cosmetic mRNA is substantially purified.

5. The cosmetic mRNA of claim 1, wherein the coding region is codon optimized.

6. The cosmetic mRNA of claim 1, wherein the cosmetic mRNA comprises a 5′ untranslated region (UTR) at the 5′ terminus of the coding region and a 3′ UTR at the 3′ terminus of the coding region.

7. The cosmetic mRNA of claim 6, wherein the 5′UTR and the 3′UTR are not derived from the same species.

8. The cosmetic mRNA of claim 6, wherein at least one of the 5′UTR or the 3′UTR is not derived from beta-globin.

9. The cosmetic mRNA of claim 1, wherein the coding region is selected from the group consisting of SEQ ID NO: 1973, 1974, 1987, 1976, 1975, 1986, 1978, 1981, 1977, 1985, 3423, 3437, 3428, 3436, 3424, 3431, 3425, 3427, and 4148.