Patent Publication Number: US-2022226255-A1

Title: Encapsulation of messenger rna

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation application of U.S. patent application Ser. No. 15/495,157 filed Apr. 24, 2017, now abandoned, which is a continuation application of U.S. application Ser. No. 14/790,562, filed on Jul. 2, 2015, now issued as U.S. Pat. No. 9,668,980, which claims priority to U.S. provisional patent application Ser. No. 62/020,163, filed Jul. 2, 2014, each of which are hereby incorporated by reference in their entireties. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 12, 2015, is named 2006685-1078_SL.txt and is 20,862 bytes in size. The entire contents of the Sequence Listing are herein incorporated by reference. 
     BACKGROUND 
     Messenger RNA therapy (MRT) is becoming an increasingly important approach for the treatment of a variety of diseases. MRT involves administration of messenger RNA (mRNA) into a patient in need of the therapy for production of the protein encoded by the mRNA within the patient body. Lipid nanoparticles are commonly used to encapsulate mRNA for efficient in vivo delivery of mRNA. However, current methods for producing mRNA-loaded lipid nanoparticles suffer poor encapsulation efficiency, low mRNA recovery and/or heterogeneous particle sizes. 
     SUMMARY OF INVENTION 
     The present invention provides, among other things, an improved process for lipid nanoparticle formulation and mRNA encapsulation. In particular, the present invention is based on the surprising discovery that pre-heating a mRNA solution and/or a lipid solution prior to mixing resulted in significantly improved encapsulation efficiency, mRNA recovery rate, and more homogeneous and smaller particle sizes (e.g., less than 100 nm). 
     Thus, in some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a mRNA solution and a lipid solution, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature. In some embodiments, a pre-determined temperature suitable for the present invention is or is greater than about 30° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C. In some embodiments, a pre-determined temperature suitable for the present invention ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In particular embodiments, a pre-determined temperature suitable for the present invention is about 65° C. 
     In some embodiments, the mRNA solution and the lipid solution are heated to the pre-determined temperature separately prior to the mixing. In some embodiments, the mRNA solution is heated to the pre-determined temperature and the lipid solution is at ambient temperature prior to the mixing. In some embodiments, the mRNA solution is heated to the pre-determined temperature by adding a mRNA stock solution at ambient temperature to a heated buffering solution to the pre-determined temperature. In some embodiments, the buffering solution has a pH no greater than about 4.5 (e.g., no greater than about 4.4, 4.2, 4.0 or 3.8). 
     In some embodiments, the mRNA solution and the lipid solution are mixed by a pulse-less flow pump. In some embodiments, a suitable pump is a gear pump. In some embodiments, a suitable pump is a peristaltic pump. In some embodiments, a suitable pump is a centrifugal pump. 
     In some embodiments, the mRNA solution is mixed at a flow rate ranging from about 150-250 ml/minute, 250-500 ml/minute, 500-1000 ml/minute, 1000-2000 ml/minute, 2000-3000 ml/minute, 3000-4000 ml/minute, or 4000-5000 ml/minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 200 ml/minute, about 500 ml/minute, about 1000 ml/minute, about 2000 ml/minute, about 3000 ml/minute, about 4000 ml/minute, or about 5000 ml/minute. 
     In some embodiments, the lipid solution is mixed at a flow rate ranging from about 25-75 ml/minute, about 75-200 ml/minute, about 200-350 ml/minute, about 350-500 ml/minute, about 500-650 ml/minute, about 650-850 ml/minute, or about 850-1000 ml/minute. In some embodiments, the lipid solution is mixed at a flow rate of about 50 ml/minute, about 100 ml/minute, about 150 ml/minute, about 200 ml/minute, about 250 ml/minute, about 300 ml/minute, about 350 ml/minute, about 400 ml/minute, about 450 ml/minute, about 500 ml/minute, about 550 ml/minute, about 600 ml/minute, about 650 ml/minute, about 700 ml/minute, about 750 ml/minute, about 800 ml/minute, about 850 ml/minute, about 900 ml/minute, about 950 ml/minute, or about 1000 ml/minute. 
     In some embodiments, a process according to the present invention includes a step of first generating the mRNA solution by mixing a citrate buffer with a mRNA stock solution. In certain embodiments, a suitable citrate buffer contains about 10 mM citrate, about 150 mM NaCl, pH of about 4.5. In some embodiments, a suitable mRNA stock solution contains the mRNA at a concentration at or greater than about 0.10 mg/mL, 1 mg/ml, about 10 mg/ml, about 50 mg/ml, or about 100 mg/ml. 
     In some embodiments, the citrate buffer is mixed at a flow rate ranging between about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, or 4800-6000 ml/minute. In some embodiments, the citrate buffer is mixed at a flow rate of about 220 ml/minute, about 600 ml/minute, about 1200 ml/minute, about 2400 ml/minute, about 3600 ml/minute, about 4800 ml/minute, or about 6000 ml/minute. 
     In some embodiments, the mRNA stock solution is mixed at a flow rate ranging between about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute. In some embodiments, the mRNA stock solution is mixed at a flow rate of about 20 ml/minute, about 40 ml/minute, about 60 ml/minute, about 80 ml/minute, about 100 ml/minute, about 200 ml/minute, about 300 ml/minute, about 400 ml/minute, about 500 ml/minute, or about 600 ml/minute. 
     In some embodiments, the lipid solution contains one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids in ethanol. In some embodiments, the mRNA solution and the lipid solution are mixed into a 20% ethanol, resulting in a suspension of lipid nanoparticles. In some embodiments, the lipid nanoparticles are further purified by Tangential Flow Filtration. 
     In some embodiments, greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size less than about 100 nm (e.g., less than about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, substantially all of the purified nanoparticles have a size less than 100 nm (e.g., less than about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). 
     In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size ranging from about 40-90 nm (e.g., about 40-85 nm, about 40-80 nm, about 40-75 nm, about 40-70 nm, about 40-65 nm, or about 40-60 nm). In some embodiments, substantially all of the purified nanoparticles have a size ranging from about 40-90 nm (e.g., about 40-85 nm, about 40-80 nm, about 40-75 nm, about 40-70 nm, about 40-65 nm, or about 40-60 nm). 
     In some embodiments, the purified nanoparticles have an encapsulation efficiency of greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, a process according to the present invention results in greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA. 
     In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising (a) separately heating a mRNA solution and/or a lipid solution to a pre-determined temperature greater than ambient temperature; (b) mixing the heated mRNA solution and/or the heated lipid solution to generate a suspension of lipid nanoparticles; and (c) purifying the lipid nanoparticles. 
     In another aspect, the present invention provides a composition of lipid nanoparticles generated by a process described herein. In some embodiments, the present invention provides a composition comprising purified lipid nanoparticles, wherein greater than about 90% of the purified lipid nanoparticles have an individual particle size of less than about 100 nm (e.g., less than about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm) and greater than about 70% of the purified lipid nanoparticles encapsulate a mRNA within each individual particle. In some embodiments, greater than about 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles have an individual particle size of less than about 100 nm (e.g., less than about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, substantially all of the purified lipid nanoparticles have an individual particle size of less than about 100 nm (e.g., less than about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles encapsulate a mRNA within each individual particle. In some embodiments, substantially all of the purified lipid nanoparticles encapsulate a mRNA within each individual particle. In some embodiments, a composition according to the present invention contains at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. 
     In some embodiments, each individual lipid nanoparticle comprises one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids. In some embodiments, the one or more cationic lipids are selected from the group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (Imidazol-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof. 
     In some embodiments, the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)). 
     In some embodiments, the one or more cholesterol-based lipids is cholesterol or PEGylated cholesterol. In some embodiments, the one or more PEG-modified lipids contain a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20  length. 
     In some embodiments, the present invention is used to encapsulate mRNA containing one or more modified nucleotides. In some embodiments, the present invention is used to encapsulate mRNA that is unmodified. 
     Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The drawings are for illustration purposes only and not for limitation. 
         FIG. 1 : shows a schematic of an exemplary scaled-up lipid nanoparticle encapsulated mRNA formulation process with homogenous flow pumps. 
         FIG. 2 : depicts an exemplary purification and buffer exchange system for lipid nanoparticles. 
         FIG. 3 : depicts a schematic of an exemplary scaled-up lipid nanoparticle encapsulated mRNA formulation process with peristaltic pumps. 
         FIG. 4 : depicts an alternative exemplary tangential flow filtration system for purification and buffer exchange. 
         FIG. 5 : depicts an alternative schematic of an exemplary scaled-up lipid nanoparticle encapsulated mRNA formulation process with peristaltic pumps. 
     
    
    
     DEFINITIONS 
     In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. 
     Approximately or about: 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). 
     Encapsulation: As used herein, the term “encapsulation,” or grammatical equivalent, refers to the process of confining an individual mRNA molecule within a nanoparticle. 
     Improve, increase, or reduce: As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated. 
     Impurities: As used herein, the term “impurities” refers to substances inside a confined amount of liquid, gas, or solid, which differ from the chemical composition of the target material or compound. Impurities are also referred to as contaminants. 
     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, etc., rather than within a multi-cellular organism. 
     In Vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems). 
     Isolated: As used herein, the term “isolated” refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, 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% of the other components with which they were initially associated. In some embodiments, isolated agents are 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. As used herein, calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.). 
     messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. 
     Nucleic acid: As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA and/or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and/or encode the same amino acid sequence. Nucleotide sequences that encode proteins and/or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). In some embodiments, the present invention is specifically directed to “unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and/or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery. 
     Salt: As used herein the term “salt” refers to an ionic compound that does or may result from a neutralization reaction between an acid and a base. 
     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. 
     Yield: As used herein, the term “yield” refers to the percentage of mRNA recovered after encapsulation as compared to the total mRNA as starting material. In some embodiments, the term “recovery” is used interchangeably with the term “yield”. 
     DETAILED DESCRIPTION 
     The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a mRNA solution and a lipid solution, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature. 
     Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and/or” unless stated otherwise. 
     mRNA 
     The present invention may be used to encapsulate any mRNA. mRNA is typically thought of as the type of RNA that carries information from DNA to the ribosome. The existence of mRNA is typically very brief and includes processing and translation, followed by degradation. Typically, in eukaryotic organisms, mRNA processing comprises the addition of a “cap” on the N-terminal (5′) end, and a “tail” on the C-terminal (3′) end. A typical cap is a 7-methylguanosine cap, which is a guanosine that is linked through a 5′-5′-triphosphate bond to the first transcribed nucleotide. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The tail is typically a polyadenylation event whereby a polyadenylyl moiety is added to the 3′ end of the mRNA molecule. The presence of this “tail” serves to protect the mRNA from exonuclease degradation. Messenger RNA is translated by the ribosomes into a series of amino acids that make up a protein. 
     mRNAs may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application. 
     In some embodiments, in vitro synthesized mRNA may be purified before formulation and encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis. 
     The present invention may be used to formulate and encapsulate mRNAs of a variety of lengths. In some embodiments, the present invention may be used to formulate and encapsulate in vitro synthesized mRNA of or greater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb in length. In some embodiments, the present invention may be used to formulate and encapsulate in vitro synthesized mRNA ranging from about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length. 
     The present invention may be used to formulate and encapsulate mRNA that is unmodified or mRNA containing one or more modifications that typically enhance stability. In some embodiments, modifications are selected from modified nucleotide, modified sugar phosphate backbones, 5′ and/or 3′ untranslated region. 
     In some embodiments, modifications of mRNA may include modifications of the nucleotides of the RNA. An modified mRNA according to the invention can include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, .beta.-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosure of which is included here in its full scope by reference. 
     Typically, mRNA synthesis includes the addition of a “cap” on the N-terminal (5′) end, and a “tail” on the C-terminal (3′) end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation. 
     Thus, in some embodiments, mRNAs include a 5′ cap structure. A 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. 2′-O-methylation may also occur at the first base and/or second base following the 7-methyl guanosine triphosphate residues. Examples of cap structures include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA and m7GpppNmpNmp-RNA (where m indicates 2′-Omethyl residues). 
     In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region. In some embodiments, a 5′ untranslated region includes one or more elements that affect a mRNA&#39;s stability or translation, for example, an iron responsive element. In some embodiments, a 5′ untranslated region may be between about 50 and 500 nucleotides in length. 
     In some embodiments, a 3′ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect a mRNA&#39;s stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3′ untranslated region may be between 50 and 500 nucleotides in length or longer. 
     While mRNA provided from in vitro transcription reactions may be desirable in some embodiments, other sources of mRNA are contemplated as within the scope of the invention including mRNA produced from bacteria, fungi, plants, and/or animals. 
     The present invention may be used to formulate and encapsulate mRNAs encoding a variety of proteins. Non-limiting examples of mRNAs suitable for the present invention include mRNAs encoding spinal motor neuron 1 (SMN), alpha-galactosidase (GLA), argininosuccinate synthetase (ASS1), firefly luciferase, Factor IX (FIX), phenylalanine hydroxylase (PAH), and cystic fibrosis transmembrane conductance receptor (CFTR). Exemplary mRNA sequences are described in detail in the Examples section. 
     mRNA Solution 
     mRNA may be provided in a solution to be mixed with a lipid solution such that the mRNA may be encapsulated in lipid nanoparticles. A suitable mRNA solution may be any aqueous solution containing mRNA to be encapsulated at various concentrations. For example, a suitable mRNA solution may contain a mRNA at a concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml. In some embodiments, a suitable mRNA solution may contain a mRNA at a concentration ranging from about 0.01-1.0 mg/ml, 0.01-0.9 mg/ml, 0.01-0.8 mg/ml, 0.01-0.7 mg/ml, 0.01-0.6 mg/ml, 0.01-0.5 mg/ml, 0.01-0.4 mg/ml, 0.01-0.3 mg/ml, 0.01-0.2 mg/ml, 0.01-0.1 mg/ml, 0.05-1.0 mg/ml, 0.05-0.9 mg/ml, 0.05-0.8 mg/ml, 0.05-0.7 mg/ml, 0.05-0.6 mg/ml, 0.05-0.5 mg/ml, 0.05-0.4 mg/ml, 0.05-0.3 mg/ml, 0.05-0.2 mg/ml, 0.05-0.1 mg/ml, 0.1-1.0 mg/ml, 0.2-0.9 mg/ml, 0.3-0.8 mg/ml, 0.4-0.7 mg/ml, or 0.5-0.6 mg/ml. In some embodiments, a suitable mRNA solution may contain a mRNA at a concentration up to about 5.0 mg/ml, 4.0 mg/ml, 3.0 mg/ml, 2.0 mg/ml, 1.0 mg/ml, 0.09 mg/ml, 0.08 mg/ml, 0.07 mg/ml, 0.06 mg/ml, or 0.05 mg/ml. 
     Typically, a suitable mRNA solution may also contain a buffering agent and/or salt. Generally, buffering agents can include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate. In some embodiments, suitable concentration of the buffering agent may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. In some embodiments, suitable concentration of the buffering agent is or greater than about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. 
     Exemplary salts can include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable concentration of salts in a mRNA solution may range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM. Salt concentration in a suitable mRNA solution is or greater than about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. 
     In some embodiments, a suitable mRNA solution may have a pH ranging from about 3.5-6.5, 3.5-6.0, 3.5-5.5., 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5. In some embodiments, a suitable mRNA solution may have a pH of or no greater than about 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6.5. 
     Various methods may be used to prepare a mRNA solution suitable for the present invention. In some embodiments, mRNA may be directly dissolved in a buffering solution described herein. In some embodiments, a mRNA solution may be generated by mixing a mRNA stock solution with a buffering solution prior to mixing with a lipid solution for encapsulation. In some embodiments, a mRNA solution may be generated by mixing a mRNA stock solution with a buffering solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration at or greater than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, or 1.6 mg/ml, 2.0 mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, or 5.0 mg/ml. 
     In some embodiments, a mRNA stock solution is mixed with a buffering solution using a pump. Exemplary pumps include but are not limited to gear pumps, peristaltic pumps and centrifugal pumps. 
     Typically, the buffering solution is mixed at a rate greater than that of the mRNA stock solution. For example, the buffering solution may be mixed at a rate at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, or 20× greater than the rate of the mRNA stock solution. In some embodiments, a buffering solution is mixed at a flow rate ranging between about 100-6000 ml/minute (e.g., about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute). In some embodiments, a buffering solution is mixed at a flow rate of or greater than about 60 ml/minute, 100 ml/minute, 140 ml/minute, 180 ml/minute, 220 ml/minute, 260 ml/minute, 300 ml/minute, 340 ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540 ml/minute, 600 ml/minute, 1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000 ml/minute. 
     In some embodiments, a mRNA stock solution is mixed at a flow rate ranging between about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute). In some embodiments, a mRNA stock solution is mixed at a flow rate of or greater than about 5 ml/minute, 10 ml/minute, 15 ml/minute, 20 ml/minute, 25 ml/minute, 30 ml/minute, 35 ml/minute, 40 ml/minute, 45 ml/minute, 50 ml/minute, 60 ml/minute, 80 ml/minute, 100 ml/minute, 200 ml/minute, 300 ml/minute, 400 ml/minute, 500 ml/minute, or 600 ml/minute. 
     Lipid Solution 
     According to the present invention, a lipid solution contains a mixture of lipids suitable to form lipid nanoparticles for encapsulation of mRNA. In some embodiments, a suitable lipid solution is ethanol based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol based. In another embodiment, a suitable lipid solution is dimethylsulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide. 
     A suitable lipid solution may contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of or greater than about 0.1 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0 mg/ml, 3.0 mg/ml, 4.0 mg/ml, 5.0 mg/ml, 6.0 mg/ml, 7.0 mg/ml, 8.0 mg/ml, 9.0 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, or 100 mg/ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration ranging from about 0.1-100 mg/ml, 0.5-90 mg/ml, 1.0-80 mg/ml, 1.0-70 mg/ml, 1.0-60 mg/ml, 1.0-50 mg/ml, 1.0-40 mg/ml, 1.0-30 mg/ml, 1.0-20 mg/ml, 1.0-15 mg/ml, 1.0-10 mg/ml, 1.0-9 mg/ml, 1.0-8 mg/ml, 1.0-7 mg/ml, 1.0-6 mg/ml, or 1.0-5 mg/ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration up to about 100 mg/ml, 90 mg/ml, 80 mg/ml, 70 mg/ml, 60 mg/ml, 50 mg/ml, 40 mg/ml, 30 mg/ml, 20 mg/ml, or 10 mg/ml. 
     Any desired lipids may be mixed at any ratios suitable for encapsulating mRNAs. In some embodiments, a suitable lipid solution contain a mixture of desired lipids including cationic lipids, helper lipids (e.g. non cationic lipids and/or cholesterol lipids) and/or PEGylated lipids. In some embodiments, a suitable lipid solution contain a mixture of desired lipids including one or more cationic lipids, one or more helper lipids (e.g. non cationic lipids and/or cholesterol lipids) and one or more PEGylated lipids. 
     Cationic Lipids 
     As used herein, the phrase “cationic lipids” refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. Particularly suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publications WO 2010/053572 (and particularly, C12-200 described at paragraph [00225]) and WO 2012/170930, both of which are incorporated herein by reference. In certain embodiments, cationic lipids suitable for the compositions and methods of the invention include an ionizable cationic lipid described in U.S. provisional patent application 61/617,468, filed Mar. 29, 2012 (incorporated herein by reference), such as, e.g, (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002). 
     In some embodiments, cationic lipids suitable for the compositions and methods of the invention include a cationic lipid described in WO 2013063468 and in U.S. provisional application entitled “Lipid Formulations for Delivery of Messenger RNA” both of which are incorporated by reference herein. In some embodiments, a cationic lipid comprises a compound of formula I-c1-a: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof, wherein:
 
each R 2  independently is hydrogen or C 1-3  alkyl;
 
each q independently is 2 to 6;
 
each R′ independently is hydrogen or C 1-3  alkyl;
 
and each R L  independently is C 8-12  alkyl.
 
     In some embodiments, each R 2  independently is hydrogen, methyl or ethyl. In some embodiments, each R 2  independently is hydrogen or methyl. In some embodiments, each R 2  is hydrogen. 
     In some embodiments, each q independently is 3 to 6. In some embodiments, each q independently is 3 to 5. In some embodiments, each q is 4. 
     In some embodiments, each R′ independently is hydrogen, methyl or ethyl. In some embodiments, each R′ independently is hydrogen or methyl. In some embodiments, each R′ independently is hydrogen. 
     In some embodiments, each R L  independently is C 8-12  alkyl. In some embodiments, each R L  independently is n-C 8-12  alkyl. In some embodiments, each R L  independently is C 9-11  alkyl. In some embodiments, each R L  independently is n-C 9-11  alkyl. In some embodiments, each R L  independently is C 10  alkyl. In some embodiments, each R L  independently is n-C 10  alkyl. 
     In some embodiments, each R 2  independently is hydrogen or methyl; each q independently is 3 to 5; each R′ independently is hydrogen or methyl; and each R L  independently is C 8-12  alkyl. 
     In some embodiments, each R 2  is hydrogen; each q independently is 3 to 5; each R′ is hydrogen; and each R L  independently is C 8-12  alkyl. 
     In some embodiments, each R 2  is hydrogen; each q is 4; each R′ is hydrogen; and each R L  independently is C 8-12  alkyl. 
     In some embodiments, a cationic lipid comprises a compound of formula I-g: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof, wherein each R L  independently is C 8-12  alkyl. In some embodiments, each R L  independently is n-C 8-12  alkyl. In some embodiments, each R L  independently is C 9-11  alkyl. In some embodiments, each R L  independently is n-C 9-11  alkyl. In some embodiments, each R L  independently is C 10  alkyl. In some embodiments, each R L  is n-C 10  alkyl. 
     In particular embodiments, a suitable cationic lipid is cKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione). Structure of cKK-E12 is shown below: 
     
       
         
         
             
             
         
       
     
     In some embodiments, one or more cationic lipids suitable for the present invention may be N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA”. (Feigner et al. (Proc. Nat&#39;l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or “DOGS,” 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium or “DOSPA” (Behr et al. Proc. Nat.&#39;l Acad. Sci. 86, 6982 (1989); U.S. Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propane or “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”. 
     Additional exemplary cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”, 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”, 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”, 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”, N-dioleyl-N,N-dimethylammonium chloride or “DODAC”, N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide or “DMRIE”, 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane or “CLinDMA”, 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy 1-1-(cis,cis-9′,1-2′-octadecadienoxy)propane or “CpLinDMA”, N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”, 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”, 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”, 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-DMA”, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or “DLin-K-XTC2-DMA”, and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (see, WO 2010/042877; Semple et al., Nature Biotech. 28: 172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). In some embodiments, one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety. 
     In some embodiments, one or more cationic lipids may be chosen from XTC (2,2-Dilinoley1-4-dimethylaminoethy1-[1,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide), DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011/068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61/617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001 (cis or trans) (Provisional Patent Application No. 61/617,468), aminoalcohol lipidoids such as those disclosed in WO2010/053572, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids” J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al. “Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech. 2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materials for low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869). 
     In some embodiments, cationic lipids constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipids in a suitable lipid solution by weight or by molar. In some embodiments, cationic lipid(s) constitute(s) about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid mixture by weight or by molar. 
     Non-Cationic/Helper Lipids 
     As used herein, the phrase “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected H, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof. 
     In some embodiments, non-cationic lipids may constitute at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% of the total lipids in a suitable lipid solution by weight or by molar. In some embodiments, non-cationic lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution by weight or by molar. 
     Cholesterol-Based Lipids 
     In some embodiments, a suitable lipid solution include one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE. In some embodiments, cholesterol-based lipid(s) constitute(s) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in a suitable lipid solution by weight or by molar. In some embodiments, cholesterol-based lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution by weight or by molar. 
     PEGylated Lipids 
     In some embodiments, a suitable lipid solution includes one or more PEGylated lipids. For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention. Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20  length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C 14  or C 18 ). 
     PEG-modified phospholipid and derivatized lipids may constitute at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in a suitable lipid solution by weight or by molar. In some embodiments, PEGylated lipid lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution by weight or by molar. 
     Exemplary combinations of cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids are described in the Examples section. For example, a suitable lipid solution may contain cKK-E12, DOPE, chol, and DMG-PEG2K; C12-200, DOPE, cholesterol, and DMG-PEG2K; HGT5000, DOPE, chol, and DMG-PEG2K; HGT5001, DOPE, chol, and DMG-PEG2K; cKK-E12, DPPC, chol, and DMG-PEG2K; C12-200, DPPC, cholesterol, and DMG-PEG2K; HGT5000, DPPC, chol, and DMG-PEG2K; or HGT5001, DPPC, chol, and DMG-PEG2K. The selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid mixture as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s) and the nature of the and the characteristics of the mRNA to be encapsulated. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly. 
     Mixing Process 
     The present invention is based on the discovery of unexpected effect of temperature on the mRNA encapsulation efficiency and recovery rate. Thus, in some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles by mixing a mRNA solution and a lipid solution, described herein, wherein the mRNA solution and/or the lipid solution are heated to a pre-determined temperature greater than ambient temperature. As used herein, the term “ambient temperature” refers to the temperature in a room, or the temperature which surrounds an object of interest (e.g., a mRNA solution or lipid solution) without heating or cooling. In some embodiments, the ambient temperature refers to temperature ranging from about 20-25° C. 
     Therefore, a pre-determined temperature greater than ambient temperature is typically greater than about 25° C. In some embodiments, a pre-determined temperature suitable for the present invention is or is greater than about 30° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C. In some embodiments, a pre-determined temperature suitable for the present invention ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In particular embodiments, a pre-determined temperature suitable for the present invention is about 65° C. 
     The mRNA solution, or the lipid solution, or both, may be heated to a pre-determined temperature above the ambient temperature prior to mixing. In some embodiments, the mRNA solution and the lipid solution are heated to the pre-determined temperature separately prior to the mixing. In some embodiments, the mRNA solution and the lipid solution are mixed at the ambient temperature but then heated to the pre-determined temperature after the mixing. In some embodiments, the lipid solution is heated to the pre-determined temperature and mixed with a mRNA solution at the ambient temperature. In some embodiments, the mRNA solution is heated to the pre-determined temperature and mixed with a lipid solution at ambient temperature. 
     In some embodiments, the mRNA solution is heated to the pre-determined temperature by adding a mRNA stock solution that is at ambient temperature to a heated buffering solution to achieve the desired pre-determined temperature. 
     A mRNA solution and a lipid solution may be mixed using a pump. As the encapsulation procedure can occur on a wide range of scales, different types of pumps may be used to accommodate desired scale. It is however generally desired to use a pulse-less flow pumps. As used herein, a pulse-less flow pump refers to any pump that can establish a continuous flow with a stable flow rate. Types of suitable pumps may include, but are not limited to, gear pumps and centrifugal pumps. Exemplary gear pumps include, but are not limited to, Cole-Parmer or Diener gear pumps. Exemplary centrifugal pumps include, but are not limited to, those manufactured by Grainger or Cole-Parmer. 
     A mRNA solution and a lipid solution may be mixed at various flow rates. Typically, the mRNA solution may be mixed at a rate greater than that of the lipid solution. For example, the mRNA solution may be mixed at a rate at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, or 20× greater than the rate of the lipid solution. 
     Suitable flow rates for mixing may be determined based on the scales. In some embodiments, a mRNA solution is mixed at a flow rate ranging from about 40-400 ml/minute, 60-500 ml/minute, 70-600 ml/minute, 80-700 ml/minute, 90-800 ml/minute, 100-900 ml/minute, 110-1000 ml/minute, 120-1100 ml/minute, 130-1200 ml/minute, 140-1300 ml/minute, 150-1400 ml/minute, 160-1500 ml/minute, 170-1600 ml/minute, 180-1700 ml/minute, 150-250 ml/minute, 250-500 ml/minute, 500-1000 ml/minute, 1000-2000 ml/minute, 2000-3000 ml/minute, 3000-4000 ml/minute, or 4000-5000 ml/minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 200 ml/minute, about 500 ml/minute, about 1000 ml/minute, about 2000 ml/minute, about 3000 ml/minute, about 4000 ml/minute, or about 5000 ml/minute. 
     In some embodiments, a lipid solution is mixed at a flow rate ranging from about 25-75 ml/minute, 20-50 ml/minute, 25-75 ml/minute, 30-90 ml/minute, 40-100 ml/minute, 50-110 ml/minute, 75-200 ml/minute, 200-350 ml/minute, 350-500 ml/minute, 500-650 ml/minute, 650-850 ml/minute, or 850-1000 ml/minute. In some embodiments, the lipid solution is mixed at a flow rate of about 50 ml/minute, about 100 ml/minute, about 150 ml/minute, about 200 ml/minute, about 250 ml/minute, about 300 ml/minute, about 350 ml/minute, about 400 ml/minute, about 450 ml/minute, about 500 ml/minute, about 550 ml/minute, about 600 ml/minute, about 650 ml/minute, about 700 ml/minute, about 750 ml/minute, about 800 ml/minute, about 850 ml/minute, about 900 ml/minute, about 950 ml/minute, or about 1000 ml/minute. 
     Typically, a mRNA solution and a lipid solution are mixed into a solution such that the lipids can form nanoparticles encapsulating mRNA. Such a solution is also referred to as a formulation or encapsulation solution. A suitable formulation or encapsulation solution may be based on a solvent such as ethanol. For example, a suitable formulation or encapsulation solution may be based on about 10% ethanol, about 15% ethanol, about 20% ethanol, about 25% ethanol, about 30% ethanol, about 35% ethanol, or about 40% ethanol. 
     A suitable formulation or encapsulation solution may be based on a solvent such as isopropyl alcohol. For example, a suitable formulation or encapsulation solution may be based on about 10% isopropyl alcohol, about 15% isopropyl alcohol, about 20% isopropyl alcohol, about 25% isopropyl alcohol, about 30% isopropyl alcohol, about 35% isopropyl alcohol, or about 40% isopropyl alcohol. 
     A suitable formulation or encapsulation solution may be based on a solvent such as dimethyl sulfoxide. For example, a suitable formulation or encapsulation solution may be based on about 10% dimethyl sulfoxide, about 15% dimethyl sulfoxide, about 20% dimethyl sulfoxide, about 25% dimethyl sulfoxide, about 30% dimethyl sulfoxide, about 35% dimethyl sulfoxide, or about 40% dimethyl sulfoxide. 
     A suitable formulation or encapsulation solution may also contain a buffering agent or salt. Exemplary buffering agent may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate. Exemplary salt may include sodium chloride, magnesium chloride, and potassium chloride. 
     Purification 
     Typically, subsequent to formulation and encapsulation, lipid nanoparticles are purified and/or concentrated. Various purification methods may be used. In some embodiments, lipid nanoparticles are purified using Tangential Flow Filtration. Tangential flow filtration (TFF), also referred to as cross-flow filtration, is a type of filtration wherein the material to be filtered is passed tangentially across a filter rather than through it. In TFF, undesired permeate passes through the filter, while the desired retentate passes along the filter and is collected downstream. It is important to note that the desired material is typically contained in the retentate in TFF, which is the opposite of what one normally encounters in traditional-dead end filtration. 
     Depending upon the material to be filtered, TFF is usually used for either microfiltration or ultrafiltration. Microfiltration is typically defined as instances where the filter has a pore size of between 0.05 μm and 1.0 μm, inclusive, while ultrafiltration typically involves filters with a pore size of less than 0.05 μm. Pore size also determines the nominal molecular weight limits (NMWL), also referred to as the molecular weight cut off (MWCO) for a particular filter, with microfiltration membranes typically having NMWLs of greater than 1,000 kilodaltons (kDa) and ultrafiltration filters having NMWLs of between 1 kDa and 1,000 kDa. 
     A principal advantage of tangential flow filtration is that non-permeable particles that may aggregate in and block the filter (sometimes referred to as “filter cake”) during traditional “dead-end” filtration, are instead carried along the surface of the filter. This advantage allows tangential flow filtration to be widely used in industrial processes requiring continuous operation since down time is significantly reduced because filters do not generally need to be removed and cleaned. 
     Tangential flow filtration can be used for several purposes including concentration and diafiltration, among others. Concentration is a process whereby solvent is removed from a solution while solute molecules are retained. In order to effectively concentrate a sample, a membrane having a NMWL or MWCO that is substantially lower than the molecular weight of the solute molecules to be retained is used. Generally, one of skill may select a filter having a NMWL or MWCO of three to six times below the molecular weight of the target molecule(s). 
     Diafiltration is a fractionation process whereby small undesired particles are passed through a filter while larger desired nanoparticles are maintained in the retentate without changing the concentration of those nanoparticles in solution. Diafiltration is often used to remove salts or reaction buffers from a solution. Diafiltration may be either continuous or discontinuous. In continuous diafiltration, a diafiltration solution is added to the sample feed at the same rate that filtrate is generated. In discontinuous diafiltration, the solution is first diluted and then concentrated back to the starting concentration. Discontinuous diafiltration may be repeated until a desired concentration of nanoparticles is reached. 
     Purified and/or concentrated lipid nanoparticles may be formulated in a desired buffer such as, for example, PBS. 
     Provided Nanoparticles Encapsulating mRNA 
     A process according to the present invention results in more homogeneous and smaller particle sizes (e.g., less than 100 nm), as well as significantly improved encapsulation efficiency and/or mRNA recovery rate as compared to a prior art process. 
     Thus, the present invention provides a composition comprising purified nanoparticles described herein. In some embodiments, majority of purified nanoparticles in a composition, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles, have a size less than about 100 nm (e.g., less than about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments, substantially all of the purified nanoparticles have a size less than 100 nm (e.g., less than about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm). 
     In addition, more homogeneous nanoparticles with narrow particle size range are achieved by a process of the present invention. For example, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles in a composition provided by the present invention have a size ranging from about 40-90 nm (e.g., about 40-85 nm, about 40-80 nm, about 40-75 nm, about 40-70 nm, about 40-65 nm, or about 40-60 nm). In some embodiments, substantially all of the purified nanoparticles have a size ranging from about 40-90 nm (e.g., about 40-85 nm, about 40-80 nm, about 40-75 nm, about 40-70 nm, about 40-65 nm, or about 40-60 nm). 
     In some embodiments, the dispersity, or measure of heterogeneity in size of molecules (PDI), of nanoparticles in a composition provided by the present invention is less than about 0.16 (e.g., less than about 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, or 0.08). 
     In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles in a composition provided by the present invention encapsulate a mRNA within each individual particle. In some embodiments, substantially all of the purified lipid nanoparticles in a composition encapsulate a mRNA within each individual particle. 
     In some embodiments, a composition according to the present invention contains at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. In some embodiments, a process according to the present invention results in greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA. 
     EXAMPLES 
     While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same. 
     Example 1. Effect of Temperature on Nanoparticle Encapsulation Process 
     This example demonstrates that an increase in temperature during nanoparticle encapsulation process results in increased yield and/or encapsulation efficiency. 
     Lipid Materials 
     The formulations described in the following Examples, unless otherwise specified, contain a multi-component lipid mixture of varying ratios employing one or more cationic lipids, helper lipids (e.g., non-cationic lipids and/or cholesterol lipids) and PEGylated lipids designed to encapsulate various nucleic acid materials. Cationic lipids for the process can include but are not limited to DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP (1,2-dioleyl-3-dimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids” J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al. “Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech. 2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materials for low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869), cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), HGT5000, HGT5001, HGT4003, ICE, dialkylamino-based, imidazole-based, guanidinium-based, etc. Helper lipids can include but are not limited to DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), cholesterol, etc. The PEGylated lipids can include but are not limited to a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. 
     Messenger RNA Material 
     Codon-optimized human spinal motor neuron 1 (SMN) messenger RNA, argininosuccinate synthetase (ASS1) messenger RNA, modified cystic fibrosis transmembrane conductance regulator (SNIM® CFTR, 25% pseudouridine, 25% 5-methyl-cytidine) messenger RNA, firefly luciferase (FFL) messenger RNA, Factor IX (FIX) messenger RNA, phelyalanine hydroxylase (PAH) messenger RNA and alpha-galactosidase (GLA) messenger RNA was synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5′ cap structure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNA cap structures by viral and cellular proteins” J. Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail of approximately 250 nucleotides in length (SEQ ID NO: 1) as determined by gel electrophoresis. 5′ and 3′ untranslated regions present in each mRNA product are represented as X and Y, respectively and defined as stated (vide infra). 
     
       
         
           
               
               
            
               
                 Codon-Optimized Human Spinal Motor Neuron 1(SMN) mRNA: 
                   
               
               
                 (SEQ ID NO: 2) 
                   
               
               
                   X AUGGCCAUGAGCAGCGGAGGCAGCGGCGGAGGAGUGCCCGAGCAGGAGGACAG 
                   
               
               
                   
               
               
                 CGUGCUGUUCAGGAGAGGCACCGGCCAGAGCGAUGACAGCGAUAUCUGGGACGA 
               
               
                   
               
               
                 UACCGCUCUGAUCAAGGCCUACGACAAGGCCGUGGCCAGCUUCAAGCACGCCCUG 
               
               
                   
               
               
                 AAAAACGGCGACAUCUGCGAGACCAGCGGCAAGCCCAAGACAACCCCCAAGAGAA 
               
               
                   
               
               
                 AGCCCGCCAAGAAGAAUAAGAGCCAGAAAAAGAACACCGCCGCCAGCCUGCAGCA 
               
               
                   
               
               
                 GUGGAAGGUGGGCGACAAGUGCAGCGCCAUCUGGAGCGAGGACGGCUGCAUCUA 
               
               
                   
               
               
                 CCCCGCCACCAUCGCCAGCAUCGACUUCAAGAGAGAGACCUGCGUGGUCGUGUAC 
               
               
                   
               
               
                 ACCGGCUACGGCAACAGAGAGGAGCAGAACCUGAGCGACCUGCUGAGCCCCAUUU 
               
               
                   
               
               
                 GUGAGGUGGCCAAUAACAUCGAACAGAACGCCCAGGAGAACGAGAAUGAAAGCC 
               
               
                   
               
               
                 AGGUGAGCACCGACGAGAGCGAGAACAGCAGAUCUCCUGGCAACAAGAGCGACAA 
               
               
                   
               
               
                 CAUCAAGCCUAAGUCUGCCCCUUGGAACAGCUUCCUGCCCCCUCCUCCACCCAUG 
               
               
                   
               
               
                 CCCGGACCCAGACUGGGACCCGGAAAACCUGGCCUGAAGUUCAACGGACCACCUC 
               
               
                   
               
               
                 CCCCUCCACCUCCUCCCCCACCUCAUCUCCUGAGCUGCUGGCUGCCACCCUUCCCC 
               
               
                   
               
               
                 AGCGGACCCCCUAUCAUCCCACCACCCCCUCCCAUCUGCCCCGACAGCCUGGACGA 
               
               
                   
               
               
                 CGCCGAUGCCCUGGGCAGCAUGCUGAUCAGCUGGUACAUGAGCGGCUACCACACA 
               
               
                   
               
               
                 GGAUACUACAUGGGCUUCAGACAGAACCAGAAGGAGGGCAGAUGCUCCCACUCCC 
               
               
                   
               
               
                 UGAACUGA Y   
               
               
                   
               
               
                 Human alpha-galactosidase (GLA) mRNA: 
               
               
                 (SEQ ID NO: 3) 
                   
               
               
                   X AUGCAGCUGAGGAACCCAGAACUACAUCUGGGCUGCGCGCUUGCGCUUCGCUUC 
                   
               
               
                   
               
               
                 CUGGCCCUCGUUUCCUGGGACAUCCCUGGGGCUAGAGCACUGGACAAUGGAUUGG 
               
               
                   
               
               
                 CAAGGACGCCUACCAUGGGCUGGCUGCACUGGGAGCGCUUCAUGUGCAACCUUGA 
               
               
                   
               
               
                 CUGCCAGGAAGAGCCAGAUUCCUGCAUCAGUGAGAAGCUCUUCAUGGAGAUGGC 
               
               
                   
               
               
                 AGAGCUCAUGGUCUCAGAAGGCUGGAAGGAUGCAGGUUAUGAGUACCUCUGCAU 
               
               
                   
               
               
                 UGAUGACUGUUGGAUGGCUCCCCAAAGAGAUUCAGAAGGCAGACUUCAGGCAGA 
               
               
                   
               
               
                 CCCUCAGCGCUUUCCUCAUGGGAUUCGCCAGCUAGCUAAUUAUGUUCACAGCAAA 
               
               
                   
               
               
                 GGACUGAAGCUAGGGAUUUAUGCAGAUGUUGGAAAUAAAACCUGCGCAGGCUUC 
               
               
                   
               
               
                 CCUGGGAGUUUUGGAUACUACGACAUUGAUGCCCAGACCUUUGCUGACUGGGGA 
               
               
                   
               
               
                 GUAGAUCUGCUAAAAUUUGAUGGUUGUUACUGUGACAGUUUGGAAAAUUUGGCA 
               
               
                   
               
               
                 GAUGGUUAUAAGCACAUGUCCUUGGCCCUGAAUAGGACUGGCAGAAGCAUUGUG 
               
               
                   
               
               
                 UACUCCUGUGAGUGGCCUCUUUAUAUGUGGCCCUUUCAAAAGCCCAAUUAUACAG 
               
               
                   
               
               
                 AAAUCCGACAGUACUGCAAUCACUGGCGAAAUUUUGCUGACAUUGAUGAUUCCU 
               
               
                   
               
               
                 GGAAAAGUAUAAAGAGUAUCUUGGACUGGACAUCUUUUAACCAGGAGAGAAUUG 
               
               
                   
               
               
                 UUGAUGUUGCUGGACCAGGGGGUUGGAAUGACCCAGAUAUGUUAGUGAUUGGCA 
               
               
                   
               
               
                 ACUUUGGCCUCAGCUGGAAUCAGCAAGUAACUCAGAUGGCCCUCUGGGCUAUCAU 
               
               
                   
               
               
                 GGCUGCUCCUUUAUUCAUGUCUAAUGACCUCCGACACAUCAGCCCUCAAGCCAAA 
               
               
                   
               
               
                 GCUCUCCUUCAGGAUAAGGACGUAAUUGCCAUCAAUCAGGACCCCUUGGGCAAGC 
               
               
                   
               
               
                 AAGGGUACCAGCUUAGACAGGGAGACAACUUUGAAGUGUGGGAACGACCUCUCU 
               
               
                   
               
               
                 CAGGCUUAGCCUGGGCUGUAGCUAUGAUAAACCGGCAGGAGAUUGGUGGACCUC 
               
               
                   
               
               
                 GCUCUUAUACCAUCGCAGUUGCUUCCCUGGGUAAAGGAGUGGCCUGUAAUCCUGC 
               
               
                   
               
               
                 CUGCUUCAUCACACAGCUCCUCCCUGUGAAAAGGAAGCUAGGGUUCUAUGAAUGG 
               
               
                   
               
               
                 ACUUCAAGGUUAAGAAGUCACAUAAAUCCCACAGGCACUGUUUUGCUUCAGCUA 
               
               
                   
               
               
                 GAAAAUACAAUGCAGAUGUCAUUAAAAGACUUACUUUAA Y    
               
               
                   
               
               
                 Codon-Optimized Human Argininosuccinate Synthetase (ASS1) mRNA: 
               
               
                 (SEQ ID NO: 4) 
                   
               
               
                   X AUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACCAGC 
                   
               
               
                   
               
               
                 UGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCCUACCUGGCCA 
               
               
                   
               
               
                 ACAUCGGCCAGAAGGAGGACUUCGAGGAGGCCCGCAAGAAGGCCCUGAAGCUGGG 
               
               
                   
               
               
                 CGCCAAGAAGGUGUUCAUCGAGGACGUGAGCCGCGAGUUCGUGGAGGAGUUCAU 
               
               
                   
               
               
                 CUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGACCGCUACCUGCUGGGCACC 
               
               
                   
               
               
                 AGCCUGGCCCGCCCCUGCAUCGCCCGCAAGCAGGUGGAGAUCGCCCAGCGCGAGG 
               
               
                   
               
               
                 GCGCCAAGUACGUGAGCCACGGCGCCACCGGCAAGGGCAACGACCAGGUGCGCUU 
               
               
                   
               
               
                 CGAGCUGAGCUGCUACAGCCUGGCCCCCCAGAUCAAGGUGAUCGCCCCCUGGCGC 
               
               
                   
               
               
                 AUGCCCGAGUUCUACAACCGCUUCAAGGGCCGCAACGACCUGAUGGAGUACGCCA 
               
               
                   
               
               
                 AGCAGCACGGCAUCCCCAUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGA 
               
               
                   
               
               
                 GAACCUGAUGCACAUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAACCAG 
               
               
                   
               
               
                 GCCCCCCCCGGCCUGUACACCAAGACCCAGGACCCCGCCAAGGCCCCCAACACCCC 
               
               
                   
               
               
                 CGACAUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUGAAGGUGACCAACGU 
               
               
                   
               
               
                 GAAGGACGGCACCACCCACCAGACCAGCCUGGAGCUGUUCAUGUACCUGAACGAG 
               
               
                   
               
               
                 GUGGCCGGCAAGCACGGCGUGGGCCGCAUCGACAUCGUGGAGAACCGCUUCAUCG 
               
               
                   
               
               
                 GCAUGAAGAGCCGCGGCAUCUACGAGACCCCCGCCGGCACCAUCCUGUACCACGC 
               
               
                   
               
               
                 CCACCUGGACAUCGAGGCCUUCACCAUGGACCGCGAGGUGCGCAAGAUCAAGCAG 
               
               
                   
               
               
                 GGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACCGGCUUCUGGCACAGCCCCG 
               
               
                   
               
               
                 AGUGCGAGUUCGUGCGCCACUGCAUCGCCAAGAGCCAGGAGCGCGUGGAGGGCAA 
               
               
                   
               
               
                 GGUGCAGGUGAGCGUGCUGAAGGGCCAGGUGUACAUCCUGGGCCGCGAGAGCCCC 
               
               
                   
               
               
                 CUGAGCCUGUACAACGAGGAGCUGGUGAGCAUGAACGUGCAGGGCGACUACGAG 
               
               
                   
               
               
                 CCCACCGACGCCACCGGCUUCAUCAACAUCAACAGCCUGCGCCUGAAGGAGUACC 
               
               
                   
               
               
                 ACCGCCUGCAGAGCAAGGUGACCGCCAAGUGA Y   
               
               
                   
               
               
                 Codon-Optimized Firefly Luciferase mRNA: 
               
               
                 (SEQ ID NO: 5) 
                   
               
               
                   X AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAA 
                   
               
               
                   
               
               
                 GACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGC 
               
               
                   
               
               
                 CCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGA 
               
               
                   
               
               
                 GUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAA 
               
               
                   
               
               
                 UACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCC 
               
               
                   
               
               
                 GUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACA 
               
               
                   
               
               
                 ACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGU 
               
               
                   
               
               
                 GAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAU 
               
               
                   
               
               
                 ACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUG 
               
               
                   
               
               
                 UACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGC 
               
               
                   
               
               
                 CCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAG 
               
               
                   
               
               
                 UACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUC 
               
               
                   
               
               
                 AGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCC 
               
               
                   
               
               
                 UCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUU 
               
               
                   
               
               
                 GAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUU 
               
               
                   
               
               
                 GCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUU 
               
               
                   
               
               
                 AGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACG 
               
               
                   
               
               
                 AGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAA 
               
               
                   
               
               
                 ACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGC 
               
               
                   
               
               
                 GCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGG 
               
               
                   
               
               
                 UGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUG 
               
               
                   
               
               
                 UGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGU 
               
               
                   
               
               
                 UAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGC 
               
               
                   
               
               
                 GGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGA 
               
               
                   
               
               
                 AGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAU 
               
               
                   
               
               
                 CCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGAC 
               
               
                   
               
               
                 GAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGA 
               
               
                   
               
               
                 CCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCU 
               
               
                   
               
               
                 GCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUU 
               
               
                   
               
               
                 GGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUC 
               
               
                   
               
               
                 GCCGUGUAA Y   
               
               
                   
               
               
                 Human Factor IX (FIX) mRNA: 
               
               
                 (SEQ ID NO: 6) 
                   
               
               
                   X AUGCAGCGCGUGAACAUGAUCAUGGCAGAAUCACCAGGCCUCAUCACCAUCUGC 
                   
               
               
                   
               
               
                 CUUUUAGGAUAUCUACUCAGUGCUGAAUGUACAGUUUUUCUUGAUCAUGAAAAC 
               
               
                   
               
               
                 GCCAACAAAAUUCUGAGGCGGAGAAGGAGGUAUAAUUCAGGUAAAUUGGAAGAG 
               
               
                   
               
               
                 UUUGUUCAAGGGAACCUUGAGAGAGAAUGUAUGGAAGAAAAGUGUAGUUUUGAA 
               
               
                   
               
               
                 GAAGCACGAGAAGUUUUUGAAAACACUGAAAGAACAACUGAAUUUUGGAAGCAG 
               
               
                   
               
               
                 UAUGUUGAUGGAGAUCAGUGUGAGUCCAAUCCAUGUUUAAAUGGCGGCAGUUGC 
               
               
                   
               
               
                 AAGGAUGACAUUAAUUCCUAUGAAUGUUGGUGUCCCUUUGGAUUUGAAGGAAAG 
               
               
                   
               
               
                 AACUGUGAAUUAGAUGUAACAUGUAACAUUAAGAAUGGCAGAUGCGAGCAGUUU 
               
               
                   
               
               
                 UGUAAAAAUAGUGCUGAUAACAAGGUGGUUUGCUCCUGUACUGAGGGAUAUCGA 
               
               
                   
               
               
                 CUUGCAGAAAACCAGAAGUCCUGUGAACCAGCAGUGCCAUUUCCAUGUGGAAGA 
               
               
                   
               
               
                 GUUUCUGUUUCACAAACUUCUAAGCUCACCCGUGCUGAGGCUGUUUUUCCUGAUG 
               
               
                   
               
               
                 UGGACUAUGUAAAUUCUACUGAAGCUGAAACCAUUUUGGAUAACAUCACUCAAA 
               
               
                   
               
               
                 GCACCCAAUCAUUUAAUGACUUCACUCGGGUUGUUGGUGGAGAAGAUGCCAAAC 
               
               
                   
               
               
                 CAGGUCAAUUCCCUUGGCAGGUUGUUUUGAAUGGUAAAGUUGAUGCAUUCUGUG 
               
               
                   
               
               
                 GAGGCUCUAUCGUUAAUGAAAAAUGGAUUGUAACUGCUGCCCACUGUGUUGAAA 
               
               
                   
               
               
                 CUGGUGUUAAAAUUACAGUUGUCGCAGGUGAACAUAAUAUUGAGGAGACAGAAC 
               
               
                   
               
               
                 AUACAGAGCAAAAGCGAAAUGUGAUUCGAAUUAUUCCUCACCACAACUACAAUG 
               
               
                   
               
               
                 CAGCUAUUAAUAAGUACAACCAUGACAUUGCCCUUCUGGAACUGGACGAACCCUU 
               
               
                   
               
               
                 AGUGCUAAACAGCUACGUUACACCUAUUUGCAUUGCUGACAAGGAAUACACGAA 
               
               
                   
               
               
                 CAUCUUCCUCAAAUUUGGAUCUGGCUAUGUAAGUGGCUGGGGAAGAGUCUUCCA 
               
               
                   
               
               
                 CAAAGGGAGAUCAGCUUUAGUUCUUCAGUACCUUAGAGUUCCACUUGUUGACCG 
               
               
                   
               
               
                 AGCCACAUGUCUUCGAUCUACAAAGUUCACCAUCUAUAACAACAUGUUCUGUGCU 
               
               
                   
               
               
                 GGCUUCCAUGAAGGAGGUAGAGAUUCAUGUCAAGGAGAUAGUGGGGGACCCCAU 
               
               
                   
               
               
                 GUUACUGAAGUGGAAGGGACCAGUUUCUUAACUGGAAUUAUUAGCUGGGGUGAA 
               
               
                   
               
               
                 GAGUGUGCAAUGAAAGGCAAAUAUGGAAUAUAUACCAAGGUAUCCCGGUAUGUC 
               
               
                   
               
               
                 AACUGGAUUAAGGAAAAAACAAAGCUCACUUAA Y   
               
               
                   
               
               
                 Codon-Optimized Human Phenylalanine Hydroxylase (PAH) mRNA: 
               
               
                 (SEQ ID NO: 7) 
                   
               
               
                   X AUGAGCACCGCCGUGCUGGAGAACCCCGGCCUGGGCCGCAAGCUGAGCGACUUC 
                   
               
               
                   
               
               
                 GGCCAGGAGACCAGCUACAUCGAGGACAACUGCAACCAGAACGGCGCCAUCAGCC 
               
               
                   
               
               
                 UGAUCUUCAGCCUGAAGGAGGAGGUGGGCGCCCUGGCCAAGGUGCUGCGCCUGUU 
               
               
                   
               
               
                 CGAGGAGAACGACGUGAACCUGACCCACAUCGAGAGCCGCCCCAGCCGCCUGAAG 
               
               
                   
               
               
                 AAGGACGAGUACGAGUUCUUCACCCACCUGGACAAGCGCAGCCUGCCCGCCCUGA 
               
               
                   
               
               
                 CCAACAUCAUCAAGAUCCUGCGCCACGACAUCGGCGCCACCGUGCACGAGCUGAG 
               
               
                   
               
               
                 CCGCGACAAGAAGAAGGACACCGUGCCCUGGUUCCCCCGCACCAUCCAGGAGCUG 
               
               
                   
               
               
                 GACCGCUUCGCCAACCAGAUCCUGAGCUACGGCGCCGAGCUGGACGCCGACCACC 
               
               
                   
               
               
                 CCGGCUUCAAGGACCCCGUGUACCGCGCCCGCCGCAAGCAGUUCGCCGACAUCGC 
               
               
                   
               
               
                 CUACAACUACCGCCACGGCCAGCCCAUCCCCCGCGUGGAGUACAUGGAGGAGGAG 
               
               
                   
               
               
                 AAGAAGACCUGGGGCACCGUGUUCAAGACCCUGAAGAGCCUGUACAAGACCCACG 
               
               
                   
               
               
                 CCUGCUACGAGUACAACCACAUCUUCCCCCUGCUGGAGAAGUACUGCGGCUUCCA 
               
               
                   
               
               
                 CGAGGACAACAUCCCCCAGCUGGAGGACGUGAGCCAGUUCCUGCAGACCUGCACC 
               
               
                   
               
               
                 GGCUUCCGCCUGCGCCCCGUGGCCGGCCUGCUGAGCAGCCGCGACUUCCUGGGCG 
               
               
                   
               
               
                 GCCUGGCCUUCCGCGUGUUCCACUGCACCCAGUACAUCCGCCACGGCAGCAAGCC 
               
               
                   
               
               
                 CAUGUACACCCCCGAGCCCGACAUCUGCCACGAGCUGCUGGGCCACGUGCCCCUG 
               
               
                   
               
               
                 UUCAGCGACCGCAGCUUCGCCCAGUUCAGCCAGGAGAUCGGCCUGGCCAGCCUGG 
               
               
                   
               
               
                 GCGCCCCCGACGAGUACAUCGAGAAGCUGGCCACCAUCUACUGGUUCACCGUGGA 
               
               
                   
               
               
                 GUUCGGCCUGUGCAAGCAGGGCGACAGCAUCAAGGCCUACGGCGCCGGCCUGCUG 
               
               
                   
               
               
                 AGCAGCUUCGGCGAGCUGCAGUACUGCCUGAGCGAGAAGCCCAAGCUGCUGCCCC 
               
               
                   
               
               
                 UGGAGCUGGAGAAGACCGCCAUCCAGAACUACACCGUGACCGAGUUCCAGCCCCU 
               
               
                   
               
               
                 GUACUACGUGGCCGAGAGCUUCAACGACGCCAAGGAGAAGGUGCGCAACUUCGCC 
               
               
                   
               
               
                 GCCACCAUCCCCCGCCCCUUCAGCGUGCGCUACGACCCCUACACCCAGCGCAUCGA 
               
               
                   
               
               
                 GGUGCUGGACAACACCCAGCAGCUGAAGAUCCUGGCCGACAGCAUCAACAGCGAG 
               
               
                   
               
               
                 AUCGGCAUCCUGUGCAGCGCCCUGCAGAAGAUCAAGUAA Y   
               
               
                   
               
               
                 Codon-Optimized Cystic Fibrosis Transmembrane Conductance  
               
               
                 Regulator (CFTR) mRNA: 
               
               
                 (SEQ ID NO: 8) 
                   
               
               
                 AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAU 
                   
               
               
                   
               
               
                 GGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACA 
               
               
                   
               
               
                 UCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACG 
               
               
                   
               
               
                 GGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUG 
               
               
                   
               
               
                 AGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGG 
               
               
                   
               
               
                 GAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACG 
               
               
                   
               
               
                 ACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUG 
               
               
                   
               
               
                 UUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAU 
               
               
                   
               
               
                 CACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACA 
               
               
                   
               
               
                 CUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCC 
               
               
                   
               
               
                 CUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUC 
               
               
                   
               
               
                 GUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUG 
               
               
                   
               
               
                 UUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUU 
               
               
                   
               
               
                 CAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAA 
               
               
                   
               
               
                 AUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCA 
               
               
                   
               
               
                 AAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAA 
               
               
                   
               
               
                 CUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGC 
               
               
                   
               
               
                 GUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUG 
               
               
                   
               
               
                 AUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUA 
               
               
                   
               
               
                 UUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGAC 
               
               
                   
               
               
                 UCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAG 
               
               
                   
               
               
                 ACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCU 
               
               
                   
               
               
                 UUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAAC 
               
               
                   
               
               
                 AACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGC 
               
               
                   
               
               
                 UCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUC 
               
               
                   
               
               
                 UCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUA 
               
               
                   
               
               
                 UGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAU 
               
               
                   
               
               
                 UCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUU 
               
               
                   
               
               
                 UCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGU 
               
               
                   
               
               
                 UGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAG 
               
               
                   
               
               
                 GGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGG 
               
               
                   
               
               
                 UAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACG 
               
               
                   
               
               
                 UAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUA 
               
               
                   
               
               
                 AGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGA 
               
               
                   
               
               
                 UCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCA 
               
               
                   
               
               
                 AAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAG 
               
               
                   
               
               
                 UUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGC 
               
               
                   
               
               
                 UUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGC 
               
               
                   
               
               
                 AGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACU 
               
               
                   
               
               
                 CAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUG 
               
               
                   
               
               
                 AAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGA 
               
               
                   
               
               
                 GCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUU 
               
               
                   
               
               
                 CAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGG 
               
               
                   
               
               
                 GGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACC 
               
               
                   
               
               
                 CCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACC 
               
               
                   
               
               
                 GGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUU 
               
               
                   
               
               
                 GAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUAC 
               
               
                   
               
               
                 AUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUC 
               
               
                   
               
               
                 UCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUU 
               
               
                   
               
               
                 GCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUC 
               
               
                   
               
               
                 ACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUC 
               
               
                   
               
               
                 UGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGU 
               
               
                   
               
               
                 CUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCC 
               
               
                   
               
               
                 ACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUU 
               
               
                   
               
               
                 GCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGC 
               
               
                   
               
               
                 UGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUG 
               
               
                   
               
               
                 UCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCA 
               
               
                   
               
               
                 GACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUAC 
               
               
                   
               
               
                 GCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCA 
               
               
                   
               
               
                 GCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGG 
               
               
                   
               
               
                 UUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUU 
               
               
                   
               
               
                 GUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGC 
               
               
                   
               
               
                 GAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUG 
               
               
                   
               
               
                 CAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCG 
               
               
                   
               
               
                 AGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACG 
               
               
                   
               
               
                 AAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCAC 
               
               
                   
               
               
                 GUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUG 
               
               
                   
               
               
                 ACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCA 
               
               
                   
               
               
                 UUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGA 
               
               
                   
               
               
                 CGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCG 
               
               
                   
               
               
                 ACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAG 
               
               
                   
               
               
                 UAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCC 
               
               
                   
               
               
                 UUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCU 
               
               
                   
               
               
                 UCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGG 
               
               
                   
               
               
                 GGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUC 
               
               
                   
               
               
                 CUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGG 
               
               
                   
               
               
                 UAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGG 
               
               
                   
               
               
                 UGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUG 
               
               
                   
               
               
                 UCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGA 
               
               
                   
               
               
                 GAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCC 
               
               
                   
               
               
                 ACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAA 
               
               
                   
               
               
                 GAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA 
               
               
                   
               
               
                 5′ and 3′ UTR Sequences 
               
               
                 X = 
               
               
                 (SEQ ID NO: 9) 
                   
               
               
                 GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACC 
                   
               
               
                   
               
               
                 GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG 
               
               
                   
               
               
                 UGCCAAGAGUGACUCACCGUCCUUGACACG 
               
               
                   
               
               
                 Y = 
               
               
                 (SEQ ID NO: 10) 
                   
               
               
                 CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCC 
                   
               
               
                   
               
               
                 ACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU 
               
            
           
         
       
     
     Lipid Nanoparticle Formulations 
     Ethanolic solution of mixture of lipids (cationic lipid, helper lipids, zwitterionic lipids, PEG lipids etc.) was prepared to the reported volume and heated to the selected temperature. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of mRNA was prepared from a 1 mg/mL stock and heated to the selected temperature for 5-10 minutes. 
     For small scale formulations, the lipid solution was injected rapidly into the aqueous mRNA solution using a syringe pump (3.71 mL/sec) and the resulting suspension was shaken to yield the lipid nanoparticles in 20% ethanol. The resulting nanoparticle suspension was dia-filtrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. 
     Representative Example at 25° C. 
     Aliquots of 50 mg/mL ethanolic solutions of cKK-E12, DOPE, Chol and DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of FFL mRNA was prepared from a 1 mg/mL stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. Final concentration=0.20 mg/mL FFL mRNA (encapsulated). Z ave =91 nm PDI (0.16). 
     Formulation at 37° C. 
     Aliquots of 50 mg/mL ethanolic solutions of cKK-E12, DOPE, Chol and DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of FIX mRNA was prepared from a 1 mg/mL stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. Final concentration=0.20 mg/mL FIX mRNA (encapsulated). Z ave =64 nm; PDI (0.12). 
     Formulation at 65° C. 
     Aliquots of 50 mg/mL ethanolic solutions of cKK-E12, DOPE, Chol and DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of FIX mRNA was prepared from a 1 mg/mL stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. Final concentration=0.20 mg/mL FIX mRNA (encapsulated). Z ave =73 nm; PDI (0.13). 
     Effect of Temperature on the Nanoparticle Encapsulation Process 
     Both the ethanol lipid solution and the aqueous buffered solution of mRNA (10 mM citrate/150 mM NaCl, pH 4.5) were heated at different selected temperatures before the formulation process to determine the effect of temperature on the final yield and the encapsulation efficiency of the formulation. 
     The effect of temperature on the nanoparticle formulation process was evaluated for size, size dispersity, encapsulation efficiency and yield (or recovery). Exemplary data are shown in Table 1. As can be seen, an increase in temperature (e.g., above the ambient temperature) results in increased encapsulation efficiency and/or yield/recover, as well as reduced particle size and/or size dispersity. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Effect of Temperature on Nanoparticle Formation and mRNA Encapsulation 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Formulation # 
                 mRNA 
                 Temperature 
                 Size 
                 PDI 
                 Encapsulation 
                 Recovery 
               
               
                   
               
            
           
           
               
            
               
                 Formulation process at ambient temperature (25° C.): 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 FFL 
                 25 
                 91 
                 0.16 
                 71% 
                 30% 
               
               
                 2 
                 FFL 
                 25 
                 88 
                 0.14 
                 76% 
                 33% 
               
            
           
           
               
            
               
                 Formulation process at 37° C.: 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 3 
                 FIX 
                 37 
                 77 
                 0.13 
                 57% 
                 29% 
               
               
                 4 
                 FIX 
                 37 
                 80 
                 0.12 
                 68% 
                 36% 
               
               
                 5 
                 FIX 
                 37 
                 64 
                 0.12 
                 69% 
                 37% 
               
               
                 6 
                 FIX 
                 37 
                 63 
                 0.12 
                 65% 
                 51% 
               
            
           
           
               
            
               
                 Formulation process at 65° C.: 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 7 
                 ASS1 
                 65 
                 86 
                 0.12 
                 85% 
                 64% 
               
               
                 8 
                 ASS1 
                 65 
                 84 
                 0.11 
                 96% 
                 98% 
               
               
                 9 
                 ASS1 
                 65 
                 81 
                 0.16 
                 86% 
                 64% 
               
               
                 10 
                 ASS1 
                 65 
                 84 
                 0.11 
                 96% 
                 98% 
               
               
                 11 
                 PAH 
                 65 
                 79 
                 0.12 
                 82% 
                 77% 
               
               
                 12 
                 FIX 
                 65 
                 73 
                 0.13 
                 81% 
                 77% 
               
               
                 13 
                 FIX 
                 65 
                 79 
                 0.14 
                 92% 
                 82% 
               
               
                 14 
                 FIX 
                 65 
                 85 
                 0.13 
                 95% 
                 70% 
               
               
                 15 
                 FFL 
                 65 
                 68 
                 0.10 
                 92% 
                 78% 
               
               
                 16 
                 FFL 
                 65 
                 83 
                 0.12 
                 91% 
                 77% 
               
               
                 17 
                 FFL 
                 65 
                 80 
                 0.11 
                 91% 
                 75% 
               
               
                 18 
                 FFL 
                 65 
                 83 
                 0.11 
                 88% 
                 72% 
               
               
                 19 
                 FFL 
                 65 
                 80 
                 0.16 
                 90% 
                 75% 
               
               
                 20 
                 FFL 
                 65 
                 78 
                 0.11 
                 81% 
                 77% 
               
               
                 21 
                 FFL 
                 65 
                 86 
                 0.12 
                 82% 
                 75% 
               
               
                   
               
            
           
         
       
     
     Example 2. Scaled-Up Formulation Process 
     This example illustrates an exemplary scaled-up formulation process for encapsulating mRNA at an increased temperature. 
     An exemplary scaled-up formulation process is shown in  FIG. 1 . Ismatec programmable digital drive pumps (Cole Parmer Model #CP 78008-10) were used. Micropump A-mount Suction Shoe Pump Head 316 SS body/graphite gears/PTFE seals, 0.084 mL/rev, w/out internal bypass (Cole Parmer Model #07002-27) and Pharma Pure Tubing Size 14, 0.06″ ID, 1/16″ (Spectrum labs Part #ACTU-P14-25N) were used. 
     Nanoparticle formulation and encapsulation of mRNA is prepared by mixing an ethanol lipid solution with mRNA in citrate buffer (10 mM citrate buffer, 150 mM NaCl, pH 4.5) using a ‘T’ junction (or “Y” junction). Exemplary flow rates for the mRNA in citrate buffer and lipids in ethanol solution are 200 mL/minute and 50 mL/minute respectively. During this process, both pumps are started simultaneously. Both the starting and the end fractions of the formulations are discarded, only the intermediate formulation is collected. Accurate flow rates and pulse less flow are two important parameters of this process. 
     Purification and Buffer Exchange 
     Purification and buffer exchange of the formulation from the above step is performed with KrosFlo® Research IIi Tangential Flow Filtration system from Spectrum labs using the modified polyethersulfone hollow fiber filter modules. Buffer exchange is performed with 6× volumes of sterile PBS (pH 7.4) in a continuous diafiltration form. See  FIG. 2 . Formulation is analyzed for Size (PDI) and encapsulation (yield). Exemplary data is presented in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Examples of scaled-up formulation 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Formulation 
                 Batch Size (mg) 
                 N/P 
                 mRNA 
                 Size (nm) 
                 PDI 
                 Encapsulation 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 22 
                 5 
                 4 
                 ASS1 
                 59 
                 0.09 
                 88% 
               
               
                 23 
                 5 
                 4 
                 ASS1 
                 60 
                 0.12 
                 81% 
               
               
                 24 
                 5 
                 4 
                 ASS1 
                 59 
                 0.11 
                 92% 
               
               
                 25 
                 5 
                 4 
                 ASS1 
                 62 
                 0.12 
                 91% 
               
               
                 26 
                 5 
                 4 
                 ASS1 
                 59 
                 0.11 
                 89% 
               
               
                 27 
                 5 
                 4 
                 ASS1 
                 62 
                 0.07 
                 97% 
               
               
                 28 
                 5 
                 4 
                 ASS1 
                 57 
                 0.12 
                 91% 
               
               
                 29 
                 5 
                 4 
                 ASS1 
                 62 
                 0.07 
                 97% 
               
               
                 30 
                 5 
                 4 
                 ASS1 
                 67 
                 0.12 
                 88% 
               
               
                 31 
                 5 
                 4 
                 ASS1 
                 60 
                 0.15 
                 82% 
               
               
                 32 
                 5 
                 4 
                 ASS1 
                 75 
                 0.09 
                 92% 
               
               
                 33 
                 5 
                 4 
                 ASS1 
                 67 
                 0.12 
                 91% 
               
               
                 34 
                 5 
                 4 
                 ASS1 
                 71 
                 0.13 
                 92% 
               
               
                 35 
                 5 
                 4 
                 ASS1 
                 69 
                 0.11 
                 92% 
               
               
                 36 
                 5 
                 4 
                 ASS1 
                 66 
                 0.13 
                 94% 
               
               
                 37 
                 5 
                 4 
                 ASS1 
                 72 
                 0.11 
                 94% 
               
               
                 38 
                 5 
                 4 
                 ASS1 
                 82 
                 0.13 
                 96% 
               
               
                 39 
                 5 
                 4 
                 ASS1 
                 62 
                 0.12 
                 90% 
               
               
                 40 
                 5 
                 4 
                 ASS1 
                 60 
                 0.11 
                 86% 
               
               
                 41 
                 5 
                 4 
                 ASS1 
                 67 
                 0.15 
                 91% 
               
               
                 42 
                 5 
                 4 
                 ASS1 
                 69 
                 0.14 
                 94% 
               
               
                 43 
                 5 
                 4 
                 ASS1 
                 65 
                 0.16 
                 90% 
               
               
                 44 
                 5 
                 4 
                 ASS1 
                 63 
                 0.12 
                 89% 
               
               
                 45 
                 5 
                 4 
                 ASS1 
                 65 
                 0.08 
                 86% 
               
               
                 46 
                 5 
                 4 
                 GLA 
                 62 
                 0.11 
                 95% 
               
               
                 47 
                 5 
                 4 
                 GLA 
                 57 
                 0.16 
                 89% 
               
               
                 48 
                 5 
                 4 
                 GLA 
                 54 
                 0.08 
                 95% 
               
               
                 49 
                 5 
                 4 
                 GLA 
                 62 
                 0.12 
                 88% 
               
               
                 50 
                 5 
                 2 
                 SMN 
                 61 
                 0.14 
                 81% 
               
               
                 51 
                 5 
                 4 
                 25% s2U, 
                 60 
                 0.13 
                 96% 
               
               
                   
                   
                   
                 25% 5mC CFTR 
                   
                   
                   
               
               
                 52 
                 20 
                 2 
                 ASS1 
                 72 
                 0.10 
                 90% 
               
               
                 53 
                 20 
                 4 
                 ASS1 
                 75 
                 0.12 
                 92% 
               
               
                 54 
                 20 
                 4 
                 ASS1 
                 81 
                 0.11 
                 82% 
               
               
                 55 
                 20 
                 6 
                 FFluc 
                 82 
                 0.11 
                 94% 
               
               
                 56 
                 20 
                 4 
                 FFLuc 
                 78 
                 0.11 
                 94% 
               
               
                 57 
                 20 
                 4 
                 CFTR 
                 80 
                 0.12 
                 98% 
               
               
                 58 
                 30 
                 4 
                 CFTR 
                 75 
                 0.12 
                 85% 
               
               
                 59 
                 50 
                 4 
                 CFTR 
                 69 
                 0.17 
                 92% 
               
               
                 60 
                 50 
                 2 
                 ASS1 
                 73 
                 0.15 
                 82% 
               
               
                 61 
                 60 
                 4 
                 ASS1 
                 71 
                 0.13 
                 95% 
               
               
                 62 
                 300 
                 2 
                 ASS1 
                 59 
                 0.18 
                 95% 
               
               
                 63 
                 300 
                 4 
                 ASS1 
                 64 
                 0.11 
                 96% 
               
               
                 64 
                 1000 
                 2 
                 ASS1 
                 51 
                 0.18 
                 89% 
               
               
                 65 
                 1000 
                 4 
                 ASS1 
                 61 
                 0.19 
                 91% 
               
               
                 66 
                 1000 
                 2 
                 ASS1 
                 56 
                 0.18 
                 81% 
               
               
                 67 
                 1000 
                 4 
                 ASS1 
                 71 
                 0.08 
                 92% 
               
               
                 68 
                 1000 
                 2 
                 ASS1 
                 51 
                 0.12 
                 90% 
               
               
                 69 
                 1000 
                 4 
                 ASS1 
                 73 
                 0.13 
                 89% 
               
               
                 AVERAGE 
                   
                   
                   
                 65.8 
                 0.12 
                 91% 
               
               
                   
               
            
           
         
       
     
     Using this process, very narrow particle size range is achieved as well as high encapsulation efficiency (e.g., &gt;90% average). 
     To test the importance of pulse-less homogeneous flow, peristaltic pumps that have some degree of pulsating flow were used for the formulation process. See  FIG. 3 . mRNA in citrate buffer and lipids in pure ethanol were mixed at flow rate of 200 mL/minute and 50 mL/minute respectively. Exemplary results were shown in Table 3. As can be seen, the use of peristaltic pumps within this process results in the formulation of nanoparticles with larger size. This is likely due to non-homogeneous mixing due to pulsating flow. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Examples of formulations with peristaltic pumps 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Formulation # 
                 mRNA 
                 Size(nm) 
                 PDI 
               
               
                   
                   
               
               
                   
                 70 
                 CFTR 
                 112 
                 0.19 
               
               
                   
                 71 
                 CFTR 
                 116 
                 0.17 
               
               
                   
                 72 
                 FFL 
                 128 
                 0.14 
               
               
                   
                 73 
                 FFL 
                 134 
                 0.16 
               
               
                   
                   
               
            
           
         
       
     
     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 of 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 following claims: