LIPID FORMULATIONS FOR GENE EDITING

The present disclosure relates to PEG-lipids, cationic and/or ionizable lipids and nucleic acid-lipid particle compositions comprising the same. The present disclosure also relates to methods of making, using and delivering the described lipids and lipid-containing particles.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 14, 2023, is named 53989-703_301_US_SL.xml and is 1,069,846 bytes in size.

BACKGROUND

Lipid-containing particles have been used to encapsulate, and as transport vehicles for therapeutic agents such as nucleic acids, small molecules compounds, and proteins into cells and other intracellular compartments. There remains an ongoing need to develop new lipids to encapsulate therapeutic agents and improve the safety, efficacy, and specificity of such nanoparticle-based transport vehicles.

SUMMARY

Described herein are novel lipids and lipid nanoparticles comprised thereof. In one aspect, novel amino lipids are described. In another aspect novel PEG-lipids are described. In yet another aspects, novel lipid nanoparticles are described comprising one or more of the novel amino lipids and/or novel PEG-lipids. The nanoparticles, as described herein, in one aspect, are comprised of one or more of the following: an amino lipid, a neutral lipid, a PEG-lipid, a sterol or a derivative thereof, and optionally one or more of a nucleic acid molecular entity, a nucleic acid stabilizer, a surfactant, and an antioxidant. Further described herein are methods of using and making the same.

In one aspect, disclosed herein is an amino lipid having a structure of Formula (I), or a pharmaceutically acceptable salt or solvate thereof,

In one aspect, disclosed herein is an amino lipid having a structure of Formula (I*), or a pharmaceutically acceptable salt or solvate thereof,

In some embodiments, the amino lipid of Formula (I) or (I*) has a structure of Formula (Ia), or a pharmaceutically acceptable salt or solvate thereof,

In another aspect, disclosed herein is an amino lipid having structure of Formula (Ib), or a pharmaceutically acceptable salt thereof,

In one aspect, disclosed herein is an amino lipid having a structure of Formula (II), or a pharmaceutically acceptable salt or solvate thereof,

In one aspect, disclosed herein is an amino lipid having a structure of Formula (II*), or a pharmaceutically acceptable salt or solvate thereof,

In one aspect, disclosed herein is a nanoparticle composition that comprises an amino lipid described herein or a pharmaceutically acceptable salt or solvate thereof. In one aspect, disclosed herein is an amino lipid having a structure selected from Table 1A, or a pharmaceutically acceptable salt or solvate thereof. In one aspect, disclosed herein is a nanoparticle composition that comprises an amino lipid having a structure selected from Table 1A, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, said amino lipid or a pharmaceutically acceptable salt or solvate thereof, comprises from 20 mol % to 80 mol % of a total lipid content present in said nanoparticle composition. In some embodiments, said amino lipid or a pharmaceutically acceptable salt or solvate thereof, comprises from 40 mol % to 60 mol % of a total lipid content present in said nanoparticle composition. In some embodiments, said amino lipid or a pharmaceutically acceptable salt or solvate thereof, comprises from 50 mol % to 60 mol % of a total lipid content present in said nanoparticle composition. In some embodiments, said amino lipid or a salt or solvate thereof, comprises one or more ionizable nitrogen atoms. In some embodiments, the nanoparticle composition comprises one or more ionizable nitrogen atoms that are from the one or more amino lipids. In some embodiments, said nanoparticle composition comprises only one amino lipid or a salt or solvate thereof. In some embodiments, said nanoparticle composition comprises one or more nucleic acid molecular entities. In some embodiments, a molar ratio of said ionizable nitrogen atoms to phosphate groups present in said nucleic acid molecular entities (the N to P or N/P ratio) is from about 2 to about 20. In some embodiments, said N/P ratio is from about 2 to about 15, from about 2 to about 10, from about 2 to about 8, from about 2 to about 6, from about 3 to about 15, from about 3 to about 10, from about 3 to about 8, from about 3 to about 6, from about 4 to about 15, from about 4 to about 10, from about 4 to about 8, or from about 4 to about 6. In some embodiments, said N/P ratio is from about 3.5 to about 10. In some embodiments, said nanoparticle composition comprises a neutral lipid. In some embodiments, said neutral lipid comprises from about 1 mol % to about 20 mol % of a total lipid content present in said nanoparticle composition. In some embodiments, said neutral lipid comprises from about 2 mol % to about 25 mol % of a total lipid content present in said nanoparticle composition. In some embodiments, said neutral lipid comprises from about 5 mol % to about 10 mol % of a total lipid content present in said nanoparticle composition. In some embodiments, said neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and sphingomyelin. In some embodiments, said neutral lipid is DSPC. In some embodiments, said nanoparticle composition comprises a structural lipid. In some embodiments, said structural lipid is sterol or a derivative thereof. In some embodiments, said sterol or said derivative thereof is cholesterol or cholesterol derivative. In some embodiments, said structural lipid comprises from about 15 mol % to about 65 mol % of a total lipid content present in the nanoparticle composition. In some embodiments, said structural lipid comprises from about 30 mol % to about 60 mol % of a total lipid content present in the nanoparticle composition. In some embodiments, said structural lipid comprises from about 30 mol % to about 40 mol % of a total lipid content present in the nanoparticle composition. In some embodiments, said nanoparticle composition comprises a PEG-lipid. In some embodiments, said PEG-lipid is a PEG-lipid of Table 2. In some embodiments, said PEG-lipid comprises from about 0.1 mol % to about 6 mol % of a total lipid content present in said nanoparticle composition. In some embodiments, said PEG-lipid comprises about 2.0 mol % to about 2.5 mol % of a total lipid content present in said nanoparticle composition. In some embodiments, a number average molecular weight of said PEG-lipid is from about 200 Da to about 5000 Da. In some embodiments, said one or more nucleic acid molecular entities comprise a guide RNA (gRNA) targeting a disease causing gene of interest. In some embodiments, the guide RNA is a single guide RNA (sgRNA). In some embodiments, the disease causing gene of interest is produced in hepatocytes. In some embodiments, said one or more nucleic acid molecular entities comprise an mRNA encoding SpCas9, CBE, and/or ABE proteins. In some embodiments, the composition comprises a nucleic acid stabilizer.

In one aspect, disclosed herein is a nanoparticle composition comprising: (a) one or more nucleic acid molecular entities; (b) an amino lipid described herein, or a salt or solvate thereof, wherein said amino lipid or a salt thereof, comprises from 20 mol % to 80 mol % of a total lipid content present in said nanoparticle composition, wherein said amino lipid or a salt thereof, comprises one or more ionizable nitrogen atoms, and wherein a molar ratio of said ionizable nitrogen atoms to phosphate groups present in said nucleic acid molecular entity is from 2 to 12; (c) a neutral lipid, comprising from 2 mol % to 25 mol % of said total lipid content present in said nanoparticle composition; (d) a structural lipid, comprising from 30 mol % to 60 mol % of said total lipid content present in said nanoparticle composition; and (e) a PEG-lipid, comprising from 0.1 mol % to 6 mol % of said total lipid content present in said nanoparticle composition. In some embodiments, the composition further comprises a nucleic acid stabilizer. In some embodiments, the nucleic acid stabilizer comprises polyethylene glycol, cetrimonium bromide, or chitosan. In some embodiments, the nucleic acid stabilizer comprises polyethylene glycol that has a number average molecular weight of about 120 to about 2000 Da.

In one aspect, disclosed herein is a nanoparticle composition comprising: (a) one or more nucleic acid molecular entities; (b) an amino lipid or a salt thereof, wherein said amino lipid or a salt thereof, comprises from 20 mol % to 80 mol % of a total lipid content present in said nanoparticle composition, wherein said amino lipid or a salt thereof, comprises one or more ionizable nitrogen atoms, and wherein a molar ratio of said ionizable nitrogen atoms to phosphate groups present in said nucleic acid molecular entity is from 2 to 12; (c) a neutral lipid, comprising from 2 mol % to 25 mol % of said total lipid content present in said nanoparticle composition; (d) a structural lipid, comprising from 30 mol % to 60 mol % of said total lipid content present in said nanoparticle composition; (e) a PEG-lipid, comprising from 0.1 mol % to 6 mol % of said total lipid content present in said nanoparticle composition; and (f) a nucleic acid stabilizer, wherein the nucleic acid stabilizer comprises chitosan, cetrimonium bromide, polyethylene glycol that has a number average molecular weight of about 120 to about 2000 Da, or a combination thereof. In some embodiments, the nucleic acid stabilizer comprises PEG 200, PEG 400, or PEG 600. In some embodiments, the nucleic acid stabilizer is PEG 400. In some embodiments, the nucleic acid stabilizer is present in the nanoparticle composition in an amount of about 0.01% to about 20% by total weight. In some embodiments, the nucleic acid stabilizer is present in the nanoparticle composition in an amount of about 0.5% to about 5% by total weight. In some embodiments, said one or more nucleic acid molecular entities comprise a PCSK9 gRNA. In some embodiments, said one or more nucleic acid molecular entities comprise an mRNA encoding a Cas nuclease. In some embodiments, said one or more nucleic acid molecular entities comprise an mRNA, a gRNA, a siRNA, an antisense oligonucleotide, a microRNA, an anti-microRNA, an RNA activator, an aptamer, or a combination thereof. In some embodiments, said nanoparticle composition comprises an antioxidant. In some embodiments, said antioxidant comprise EDTA. In some embodiments, the composition comprises a surfactant. In some embodiments, the surfactant is a fatty acid or a fatty alcohol. In some embodiments, the surfactant is a C12-C24 fatty alcohol. In some embodiments, the C12-C24 fatty alcohol is lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, palmitoleyl alcohol, heptadecyl alcohol, stearyl alcohol, oleyl alcohol, nonadecyl alcohol, arachidyl alcohol, or a combination thereof. In some embodiments, the C12-C24 fatty alcohol is oleyl alcohol, stearyl alcohol, or a mixture thereof. In some embodiments, the surfactant comprises about 1.0 mol % to about 10 mol % of a total lipid content present in said nanoparticle composition. In some embodiments, a median diameter of the nanoparticle is from about 50 nm to about 150 nm. In some embodiments, a polydispersity index of the nanoparticle is from 0 to 0.15. In some embodiments, a polydispersity index of the nanoparticle is from 0 to 0.05. In some embodiments, a nucleic acid entrapment efficiency of the nanoparticle composition is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In one aspect, disclosed herein is a PEG-lipid having the structure of Formula (III), or a pharmaceutically acceptable salt or solvate thereof,

In one aspect, disclosed herein is a PEG-lipid having the structure of Formula (III*), or a pharmaceutically acceptable salt or solvate thereof,

In some embodiments, the PEG-lipid of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, wherein

In one aspect, disclosed herein is a PEG-lipid having the structure selected from Table 2, or a pharmaceutically acceptable salt or solvate thereof. In one aspect, disclosed herein is a pharmaceutical composition comprising a described nanoparticle composition, and an excipient or carrier. In some embodiments, said pharmaceutical composition comprises an mRNA encoding a gene editor nuclease. In some embodiments, said pharmaceutical composition comprises one or more guide RNA molecules. In some embodiments, said pharmaceutical composition comprises a PCSK9 guide RNA. In some embodiments, said pharmaceutical composition comprises two or more guide RNA molecules. In some embodiments, said two or more guide RNA molecules target two or more genes of interest. In some embodiments, said mRNA encodes Cas9 nuclease. In some embodiments, said mRNA encodes a base editor nuclease. In some embodiments, said mRNA and said one or more guide RNA molecules are present in a same nanoparticle composition. In some embodiments, said mRNA and said one or more guide RNA molecules are present in different nanoparticle compositions. In some embodiments, a ratio of said gRNA molecules to said mRNA in said pharmaceutical composition is from about 0.01 to about 100 by weight or by mole. In some embodiments, a ratio of said gRNA molecules to said mRNA in said pharmaceutical composition is about 50:1, about 40:1, about 30:1, about 20:1, about 18:1, about 16:1, about 14:1, about 12:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight or by mole. In one aspect, disclosed herein is a pharmaceutical composition comprising: a first nanoparticle composition as described herein, and a second nanoparticle composition as described herein. In some embodiments, said first nanoparticle composition comprises a gene editor mRNA, and said second nanoparticle composition comprises one or more guide RNA molecules. In some embodiments, a ratio of guide RNA molecules to mRNA in said pharmaceutical composition is about 50:1, about 40:1, about 30:1, about 20:1, about 18:1, about 16:1, about 14:1, about 12:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight or by mole. In some embodiments, a ratio of guide RNA to mRNA in said pharmaceutical composition is about 1:1 by weight or by mole.

In one aspect, disclosed herein is a method of delivering a nucleic acid molecular entity to a cell, the method comprising contacting said cell with a nanoparticle composition or a pharmaceutical composition as described herein, whereby said nucleic acid molecular entity is delivered to said cell. In some embodiments, said cell is contacted in vivo, ex vivo, or in intro. In one aspect, disclosed herein is a method of producing a polypeptide of interest in a cell, the method comprising contacting said cell with a nanoparticle composition or a pharmaceutical composition described herein, wherein said nanoparticle composition or said pharmaceutical composition comprises a nucleic acid molecular entity, and wherein said nucleic acid molecular entity is translated in the cell thereby producing the polypeptide. In one aspect, disclosed herein is a method of making a pharmaceutical composition, comprising combining a first nanoparticle composition and a second nanoparticle composition described herein. In one aspect, disclosed herein is a method of treating a disease or condition in a mammal, the method comprising administering to a mammal a therapeutically effective amount of a pharmaceutical composition described herein. In one aspect, disclosed herein is a method of editing PCSK9 gene in a cell comprising contacting said cell with a nanoparticle composition or a pharmaceutical composition described herein, wherein said nanoparticle composition or said pharmaceutical composition comprises a PCSK9 guide RNA. In one aspect, disclosed herein is a method of producing a stabilized nanoparticle composition described herein, comprising combining a nucleic acid stabilizer with a nanoparticle composition that lacks the nucleic acid stabilizer. In some embodiments, the nucleic acid stabilizer is combined with the nanoparticle composition before freezing or storage. In some embodiments, the nucleic acid stabilizer is combined with the nanoparticle composition before, concurrently, or after the addition of the one or more nucleic acid entities. In some embodiments, the combining comprises mixing the nucleic acid stabilizer with the one or more nucleic acid entities in an aqueous buffer.

In one aspect, disclosed herein is a method of producing a stabilized nanoparticle composition, comprising (a) combining a nucleic acid stabilizer with one or more nucleic acid molecular entities thereby producing a solution comprising the stabilized one or more nucleic acid molecular entities; and (b) combining the solution of (a) with a nanoparticle composition that comprises one or more of an amino lipid, a neutral lipid, a structural lipid, and a PEG-lipid. In some embodiments, the combining in (a) comprises mixing the nucleic acid stabilizer with the one or more nucleic acid entities in an aqueous buffer. In some embodiments, the method comprising collecting and dialyzing the nanoparticle composition against a buffer with a pH of about 6.5 to about 8.0. In some embodiments, the nucleic acid stabilizer is polyethylene glycol that has a number average molecular weight of about 120 to about 1000.

In one aspect, disclosed herein is a method of preparing a formulation comprising lipid nanoparticles, wherein the nanoparticles comprise (i) one or more nucleic acid molecular entities, (ii) an amino lipid, and (iii) one or more lipids selected from a structural lipid, a neutral lipid, and a PEG-lipid, the method comprising, (a) combining a first faction of the amino lipid with the one or more nucleic acid molecular entities in a first solution, wherein the first fraction comprises 0.1 mol % to 99 mol % of the total amino lipid; (b) combining the remaining of the amino lipid with the one or more lipids selected from a structural lipid, a neutral lipid, and a PEG-lipid in a second solution; (c) mixing the first solution and the second solution, thereby producing the lipid nanoparticles. In some embodiments, the first fraction of the amino lipid is configured to neutralize between 0.1-99% of the phosphate (on an N:P basis) in the one or more nucleic acid molecular entities. In some embodiments, the first fraction of the amino lipid is configured to neutralize between 0.5-90% of the phosphate (on an N:P basis) in the one or more nucleic acid molecular entities. In some embodiments, the first fraction of the amino lipid is configured to neutralize about 10%, 15%, 25%, 50%, or 75% of the phosphate (on an N:P basis) in the one or more nucleic acid molecular entities. In some embodiments, the first solution is an aqueous buffer solution. In some embodiments, the first solution further comprises a nucleic acid stabilizer. In some embodiments, the first solution and the second solution are mixed in an inline mixer.

INCORPORATION BY REFERENCE

DETAILED DESCRIPTION

The present disclosure relates to cationic and/or ionizable lipids and lipid-containing particles comprising the same. The disclosure also relates to methods of delivering a therapeutic agent (such as a nucleic acid) to a mammalian cell, methods of producing a polypeptide of interest in a mammalian cell, and methods of treating a disease or disorder in a mammal in need thereof. For example, a method of producing a polypeptide of interest in a cell can comprise a step of contacting a herein described lipid-containing particle with the cell, thereby delivering an mRNA that encodes the polypeptide of interest into the cell, and thereby the mRNA can be translated to produce the polypeptide of interest. For another example, a method of delivering a therapeutic agent to a mammalian cell or organ may involve administration of a herein described lipid-containing particle comprising the therapeutic agent to a subject, in which the administration comprises contacting the cell or organ with the lipid-containing particle, whereby the therapeutic agent is delivered to the cell or organ.

The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this present disclosure, which are encompassed within its scope.

Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.

All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

In this application, the use of “or” means “and/or” unless stated otherwise. The terms “and/or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

The term “derivative” as used herein indicates a chemical or biological substance that is related structurally to a second substance and derivable from the second substance through a modification of the second substance. In particular, if a first compound is a derivative of a second compound and the second compound is associated with a chemical and/or biological activity, the first compound differs from the second compound for at least one structural feature, while retaining (at least to a certain extent) the chemical and/or biological activity of the second compound and at least one structural feature (e.g. a sequence, a fragment, a functional group and others) associated thereto. A skilled person will be able to identify, on a case by case basis and upon reading of the present disclosure, structural features of the second compound that have to be maintained in the first compound to retain the second compound chemical and/or biological activity as well as assays that can be used to prove retention of the chemical and/or biological activity. Exemplary “derivatives” can include a prodrug, a metabolite, an enantiomer, a diastereomer, esters (e.g. acyloxyalkyl esters, alkoxycarbonyloxyalkyl esters, alkyl esters, aryl esters, phosphate esters, sulfonate esters, sulfate esters and disulfide containing esters), ethers, amides, carbonates, thiocarbonates, N-acyl derivatives, N-acyloxyalkyl derivatives, quaternary derivatives of tertiary amines, N-Mannich bases, Schiff bases, amino acid conjugates, phosphate esters, metal salts, sulfonate esters, and the like. In some cases, a derivative may include trivial substitutions (i.e. additional alkyl/akylene groups) to a parent compound that retains the chemical and/or biological activity of the parent compound.

The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

A “pharmaceutically acceptable excipient, carrier or diluent” refers to an excipient, carrier or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

A “pharmaceutically acceptable salt” may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2-hydroxyethyl sulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC—(CH2)n-COOH where n is 0-4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize from this disclosure and the knowledge in the art that further pharmaceutically acceptable salts include those listed by Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.

As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, payload, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

The term “subject” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.

The terms “treat,” “treated,” “treating,” “treatment,” and the like are meant to refer to reducing or ameliorating a disorder and/or symptoms associated therewith (e.g., a neoplasia or tumor). “Treating” may refer to administration of the LNP composition to a subject after the onset, or suspected onset, of a disease or condition. “Treating” includes the concepts of “alleviating”, which refers to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to a disease or condition and/or the side effects associated with the disease or condition. The term “treating” also encompasses the concept of “managing” which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. The term “treating” further encompasses the concept of “prevent,” “preventing,” and “prevention,” as previously stated. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

As used herein, the term “substituent” means positional variables on the atoms of a core molecule that are substituted at a designated atom position, replacing one or more hydrogens on the designated atom, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. A person of ordinary skill in the art should note that any carbon as well as heteroatom with valences that appear to be unsatisfied as described or shown herein is assumed to have a sufficient number of hydrogen atom(s) to satisfy the valences described or shown. In certain instances one or more substituents having a double bond (e.g., “oxo” or “=0”) as the point of attachment may be described, shown or listed herein within a substituent group, wherein the structure may only show a single bond as the point of attachment to the core structure of Formula (I), Formula (I*), Formula (Ia), Formula (II), Formula (II*), Formula (III) or Formula (III*). A person of ordinary skill in the art would understand that, while only a single bond is shown, a double bond is intended for those substituents.

The term “alkyl” refers to a straight or branched hydrocarbon chain radical, having from one to twenty carbon atoms, and which is attached to the rest of the molecule by a single bond. An alkyl comprising up to 10 carbon atoms is referred to as a C1-C10 alkyl, likewise, for example, an alkyl comprising up to 6 carbon atoms is a C1-C6 alkyl. Alkyls (and other moieties defined herein) comprising other numbers of carbon atoms are represented similarly. Alkyl groups include, but are not limited to, C1-C10 alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl and C4-C8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl, and the like. In some embodiments, the alkyl is methyl or ethyl. In some embodiments, the alkyl is —CH(CH3)2 or —C(CH3)3. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted as described below. “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group. In some embodiments, the alkylene is —CH2—, —CH2CH2—, or —CH2CH2CH2—. In some embodiments, the alkylene is —CH2—. In some embodiments, the alkylene is —CH2CH2—. In some embodiments, the alkylene is —CH2CH2CH2—.

The term “aryl” refers to a radical derived from a hydrocarbon ring system comprising at least one aromatic ring. In some embodiments, an aryl comprises hydrogens and 6 to 30 carbon atoms. The aryl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused (when fused with a cycloalkyl or heterocycloalkyl ring, the aryl is bonded through an aromatic ring atom) or bridged ring systems. In some embodiments, the aryl is a 6- to 10-membered aryl. In some embodiments, the aryl is a 6-membered aryl. Aryl radicals include, but are not limited to, aryl radicals derived from the hydrocarbon ring systems of anthrylene, naphthylene, phenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. In some embodiments, the aryl is phenyl. Unless stated otherwise specifically in the specification, an aryl can be optionally substituted, for example, with halogen, amino, alkylamino, aminoalkyl, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, —S(O)2NH—C1-C6alkyl, and the like. In some embodiments, an aryl is optionally substituted with halogen, methyl, ethyl, —CN, —CF3, —OH, —OMe, —NH2, —NO2, —S(O)2NH2, —S(O)2NHCH3, —S(O)2NHCH2CH3, —S(O)2NHCH(CH3)2, —S(O)2N(CH3)2, or —S(O)2NHC(CH3)3. In some embodiments, an aryl is optionally substituted with halogen, methyl, ethyl, —CN, —CF3, —OH, or —OMe. In some embodiments, the aryl is optionally substituted with halogen. In some embodiments, the aryl is substituted with alkyl, alkenyl, alkynyl, haloalkyl, or heteroalkyl, wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl is independently unsubstituted, or substituted with halogen, methyl, ethyl, —CN, —CF3, —OH, —OMe, —NH2, or —NO2.

The term “alkenyl” refers to a type of alkyl group in which at least one carbon-carbon double bond is present. In one embodiment, an alkenyl group has the formula —C(Ra)=CRa2, wherein Ra refers to the remaining portions of the alkenyl group, which may be the same or different. In some embodiments, Ra is H or an alkyl. In some embodiments, an alkenyl is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, and the like. Non-limiting examples of an alkenyl group include —CH═CH2, —C(CH3)=CH2, —CH═CHCH3, —C(CH3)=CHCH3, and —CH2CH═CH2. “Alkenylene” or “alkenylene chain” refers to a alkylene group in which at least one carbon-carbon double bond is present. In some embodiments, the alkenylene is —CH═CH—, —CH2CH2CH═CH—, or —CH═CHCH2CH2—. In some embodiments, the alkenylene is —CH═CH—. In some embodiments, the alkenylene is —CH2CH2CH═CH—. In some embodiments, the alkenylene is —CH═CHCH2CH2—.

The term “alkynyl” refers to a type of alkyl group in which at least one carbon-carbon triple bond is present. In one embodiment, an alkynyl group has the formula —C≡CRa, wherein Ra refers to the remaining portions of the alkynyl group. In some embodiments, Ra is H or an alkyl. In some embodiments, an alkynyl is selected from ethynyl (i.e., acetylenyl), propynyl (i.e., propargyl), butynyl, pentynyl, and the like. Non-limiting examples of an alkynyl group include —C≡CH, —C≡CCH3, and —CH2C≡CH. “Alkynylene” or “alkynylene chain” refers to a alkylene group in which at least one carbon-carbon triple bond is present. In some embodiments, the alkynylene is —C≡C—, —CH2CH2C≡C—, or —C≡CCH2CH2—. In some embodiments, the alkynylene is —C═C—. In some embodiments, the alkynylene is —CH2CH2C≡C—. In some embodiments, the alkynylene is —C≡CCH2CH2—.

The term “cycloalkyl” refers to a monocyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In some embodiments, cycloalkyls are saturated or partially unsaturated. In some embodiments, cycloalkyls are spirocyclic or bridged compounds. In some embodiments, cycloalkyls are fused with an aromatic ring (in which case the cycloalkyl is bonded through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having from 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having from three to ten carbon atoms, from three to eight carbon atoms, from three to six carbon atoms, or from three to five carbon atoms. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl. Polycyclic radicals include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetrainyl, decalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl, norbornyl and bicycle[1.1.1]pentyl. Unless otherwise stated specifically in the specification, a cycloalkyl group may be optionally substituted. Depending on the structure, a cycloalkyl group can be monovalent or divalent (i.e., a cycloalkylene group).

The term “heterocycloalkyl” refers to a cycloalkyl group that includes at least one ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless stated otherwise specifically in the specification, the heterocycloalkyl radical may be a monocyclic, or bicyclic ring system, which may include fused (when fused with an aryl or a heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized. The nitrogen atom may be optionally quaternized. The heterocycloalkyl radical is partially or fully saturated. Examples of heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 1 or 2 N atoms. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 3 or 4 N atoms. In some embodiments, heterocycloalkyls have from 2 to 12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring. In some embodiments, heterocycloalkyls have from 2 to 12 carbons, 1-3 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring). Unless stated otherwise specifically in the specification, a heterocycloalkyl group may be optionally substituted. As used herein, the term “heterocycloalkylene” can refer to a divalent heterocycloalkyl group.

The term “heteroalkyl” refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g. —NH—, —N(alkyl)-, or —N(aryl)-), sulfur (e.g. —S—, —S(═O)—, or —S(═O)2—), or combinations thereof. In some embodiments, a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In some embodiments, a heteroalkyl is attached to the rest of the molecule at a heteroatom of the heteroalkyl. In some embodiments, a heteroalkyl is a C1-C6 heteroalkyl. Representative heteroalkyl groups include, but are not limited to —OCH2OMe, —OCH2CH2OH, —OCH2CH2OMe, or —OCH2CH2OCH2CH2NH2. “Heteroalkylene” or “heteroalkylene chain” refers to a straight or branched divalent heteroalkyl chain linking the rest of the molecule to a radical group. Unless stated otherwise specifically in the specification, the heteroalkyl or heteroalkylene group may be optionally substituted. Representative heteroalkylene groups include, but are not limited to —OCH2CH2O—, —OCH2CH2OCH2CH2O—, or —OCH2CH2OCH2CH2OCH2CH2O—.

The term “heteroalkenyl” refers to an alkenyl group in which one or more skeletal atoms of the alkenyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g. —NH—, —N(alkyl)-, or —N(aryl)-), sulfur (e.g. —S—, —S(═O)—, or —S(═O)2—), or combinations thereof. In some embodiments, a heteroalkenyl is attached to the rest of the molecule at a carbon atom of the heteroalkenyl. In some embodiments, a heteroalkenyl is attached to the rest of the molecule at a heteroatom of the heteroalkenyl. In some embodiments, a heteroalkyl is a C1-C6 heteroalkenyl.

The term “heteroalkynyl” refers to an alkynyl group in which one or more skeletal atoms of the alkynyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g. —NH—, —N(alkyl)-, or —N(aryl)-), sulfur (e.g. —S—, —S(═O)—, or —S(═O)2—), or combinations thereof. In some embodiments, a heteroalkynyl is attached to the rest of the molecule at a carbon atom of the heteroalkynyl. In some embodiments, a heteroalkynyl is attached to the rest of the molecule at a heteroatom of the heteroalkynyl. In some embodiments, a heteroalkyl is a C1-C6 heteroalkynyl.

As used herein, the “N/P ratio” is the molar ratio of ionizable (e.g., within a pH range close to the pKa of the lipid nanoparticle) nitrogen atoms in an amino lipid (or lipids) to phosphate groups in a nucleic acid molecular entity (or nucleic acid molecular entities), e.g., in a nanoparticle composition comprising a lipid component and an RNA. Ionizable nitrogen atoms can include, for example, nitrogen atoms that can be protonated at about pH 1, about pH 2, about pH 3, about pH 4, about pH5, about pH 6, about pH 7, about pH 7.5, or about pH 8 or higher. The physiological pH range can include, for example, the pH range of different cellular compartments (such as organs, tissues, and cells) and bodily fluids (such as blood, CSF, gastric juice, milk, bile, saliva, tears, and urine). In certain specific embodiments, the physiological pH range refers to the pH range of blood in a mammal, for example, from about 7.35 to about 7.45. In some embodiments, ionizable nitrogen atoms refer to those nitrogen atoms that are ionizable within a pH range between 5 and 14.

For the payload that does not contain a phosphate group, the N/P ratio can refer to a molar ratio of ionizable nitrogen atoms in a lipid to the total negative charge in the payload. For example, the N/P ratio of an LNP composition can refer to a molar ratio of the total ionizable nitrogen atoms in the LNP composition to the total negative charge in the payload that is present in the composition.

As used herein, amino lipids can contain at least one primary, secondary or tertiary amine moiety that is protonatable (or ionizable) between pH range 4 and 14. In some embodiments, and the amine moiety/moieties function as the hydrophilic headgroup of the amino lipids described in Tables 1A and 1B. When most of the amine moiety(ies) of an amino lipid (or amino lipids) in a nucleic acid-lipid nanoparticle formulation is protonated at physiological pH, then the nanoparticles can be termed as cationic lipid nanoparticle (cLNP). When most of the amine moiety(ies) of an amino lipid (or amino lipids) in a nucleic acid-lipid nanoparticle formulation is not protonated at physiological pH but can be protonated at acidic pH, endosomal pH for example, can be termed as ionizable lipid nanoparticle (iLNP). The amino lipids that constitute cLNPs can be generally called cationic amino lipids (cLipids). The amino lipids that constitute iLNPs can be called ionizable amino lipids (iLipids). The amino lipids described in Tables 1A and 1B can be an iLipid or a cLipid at physiological pH.

As used herein, a “lipid nanoparticle (LNP) composition” or a “nanoparticle composition” is a composition comprising one or more described lipids. LNP compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or less. The LNPs described herein can have a mean diameter of from about 1 nm to about 2500 nm, from about 10 nm to about 1500 nm, from about 20 nm to about 1000 nm, from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, or from about 70 nm to about 80 nm. The LNPs described herein can have a mean diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, or greater. The LNPs described herein can be substantially non-toxic.

As used herein, a “PEG-lipid” or “PEG-lipid” refers to a lipid comprising a polyethylene glycol component.

As used herein, a “phospholipid” can refer to a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds. In some embodiments, a phospholipid may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of an LNP to pass through the membrane, i.e., delivery of the one or more elements to a cell.

The term “therapeutic agent” can refer to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. Therapeutic agents can also be referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

The term “nucleic acid molecular entity” is used interchangeably with “nucleic acid.” The term “nucleic acid” as used herein generally refers to one or more nucleobases, nucleosides, or nucleotides, and the term includes polynucleobases, polynucleosides, and polynucleotides. A nucleic acid can include polynucleotides, mononucleotides, and oligonucleoitdes. A nucleic acid can include DNA, RNA, or a mixture thereof, and can be single stranded, double stranded, or partially single or double stranded, and can form secondary structures. In some embodiments, a nucleic acid has multiple double-stranded segments and single stranded segments. For example, a nucleic acid may comprise a polynucleotide, e.g. a mRNA, with multiple double stranded segments within it. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA., a PCR product, vectors, expression cassettes, chimeric sequences, chromosomalDNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), CRISPR RNA, base editor RNA and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2′, 3′, 4′ and 5′ substituted ribonucleotide, 2′, 3′, 4′ and 5′ substituted 2′-ribonucleotide, substituted and unsubstituted carbocyclic nucleotides, substituted and unsubstituted acyclic nucleotides and peptide-nucleic acids (PNAs). Examples of nucleic acids also include acyclic and carbocyclic nucleotide, such as Glycol nucleic acid. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mal. Cell. Probes, 8:91-98 (1994)). “Nucleotides” contain a substituted and/or unsubstituted sugar deoxyribose (DNA), or a substituted and/or unsubstituted sugar ribose (RNA), or a substituted and/or unsubstituted carbocyclic, or a substituted and/or unsubstituted acyclic moiety (e.g., glycol nucleic), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

The present disclosure encompasses isolated or substantially purified nucleic acid molecules and compositions containing those molecules. As used herein, an “isolated” or “purified” DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment. An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” nucleic acid molecule or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in some embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

Amino Lipid

Described herein are LNP compositions comprising an amino lipid, a phospholipid, a PEG-lipid, a cholesterol or a derivative thereof, a payload, or any combination thereof. In some embodiments, the LNP composition comprises an amino lipid. Exemplary amino lipids include, but are not limited to, the lipids in Table 1A. In some embodiments, the LNP composition comprises an amino lipid having the structure of Formula (I), Formula (I*), Formula (Ia), Formula (II), Formula (II*), or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the LNP composition comprises an amino lipid of having the structure of Formula (Ia), or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the LNP comprises a plurality of amino lipids. For example, the LNP composition can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino lipids. For another example, the LNP composition can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, or at least 20 amino lipids. For yet another example, the LNP composition can comprise at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 9, at most 10, at most 20, or at most 30 amino lipids.

In some embodiments, the LNP composition comprises one or more amino lipids. In some embodiments, the one or more amino lipids comprise from about 40 mol % to about 65 mol % of the total lipid present in the particle. In some embodiments, the one or more amino lipids comprise about 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %, about 45 mol %, about 46 mol %, about 47 mol %, about 48 mol %, about 49 mol %, about 50 mol %, about 51 mol %, about 52 mol %, about 53 mol %, about 54 mol %, about 55 mol %, about 56 mol %, about 57 mol %, about 58 mol %, about 59 mol %, about 60 mol %, about 61 mol %, about 62 mol %, about 63 mol %, about 64 mol %, or about 65 mol % of the total lipid present in the particle.

In some embodiments, the amino lipid is an ionizable lipid. An ionizable lipid can comprise one or more ionizable nitrogen atoms. In some embodiments, at least one of the one or more ionizable nitrogen atoms is positively charged. In some embodiments, at least 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, 90 mol %, 95 mol %, or 99 mol % of the ionizable nitrogen atoms in the LNP composition are positively charged. In some embodiments, the amino lipid comprises a primary amine, a secondary amine, a tertiary amine, an imine, an amide, a guanidine moiety, a histidine residue, a lysine residue, an arginine residue, or any combination thereof. In some embodiments, the amino lipid comprises a primary amine, a secondary amine, a tertiary amine, a guanidine moiety, or any combination thereof. In some embodiments, the amino lipid comprises a tertiary amine.

In some embodiments, the amino lipid is symmetric, which may result in fewer metabolites and make the molecule achiral. In some embodiments, the amino lipid contains more number of hydrolysable bonds, which results in a faster metabolic clearance of the nanoparticles comprising the lipid. In some embodiments the symmetric achiral amino lipid helps yield tighter and/or smaller lipid nanoparticle than unsymmetric amino lipid after LNP formation wherein small particles translate efficient delivery to livers of mammalian subject compared to LNP particle of larger size

In one aspect, disclosed herein is an amino lipid having the structure of Formula (I), or a pharmaceutically acceptable salt or solvate thereof,

In some embodiments of Formula (I),

In one aspect, disclosed herein is an amino lipid having a structure of Formula (I*), or a pharmaceutically acceptable salt or solvate thereof,

In some embodiments, R5 of Formula (I*) is hydrogen or substituted or unsubstituted C1-C16 alkyl. In some embodiments, R5 of Formula (I*) is substituted or unsubstituted C1-C16 alkyl.

In some embodiments of Formula (I) or (I*), if the structure carries more than one asymmetric C-atom, each asymmetric C-atom independently represents racemic, chirally pure R and/or chirally pure S isomer, or a combination thereof.

In some embodiments, each of n, m, and q in Formula (I) or (I*) is independently 0, 1, 2, or 3. In some embodiments, each of n and m in Formula (I) or (I*) is 1. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, q is 0. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

In some embodiments, the compound of Formula (I) or (I*) has a structure of Formula (Ia), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof:

In some embodiments of Formula (Ia), if the structure carries more than one asymmetric C-atom, each asymmetric C-atom independently represents racemic, chirally pure R and/or chirally pure S isomer, or a combination thereof.

In some embodiments, a compound of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) comprises an unsymmetrical heteroatom on R1 and/or R2.

In some embodiments of a compound of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R1 and R2 are each substituted or unsubstituted Cy-C3-30 alkyl. In some embodiments, Cy is a bicyclic. In some embodiments, Cy is a monocylic. In some embodiments, Cy is bicyclic heteroaryl with 0-2 Nitrogen and 0-1 oxygen. In some embodiments, Cy is bicyclic heteroalkyl with 0-2 Nitrogen and 0-1 oxygen.

In some embodiments, R1 and/or R2 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently

In some embodiments, R1 and/or R2 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, R1 and/or R2 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, R1 and/or R2 in Formula (I), Formula (I*) Formula (Ia) or Formula (Ib) is

In some embodiments, R1 and/or R2 in Formula (I), Formula (*), Formula (Ia), or Formula (Ib) is

In some embodiments, R1 and/or R2 in Formula (I), Formula (I*) Formula (Ia) or Formula (Ib) is

In some embodiments, R1 and/or R2 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, R1 and/or R2 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, R1 and/or R2 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —O—, —S—, —C1-C10 alkylene-O—, —C1-C10 alkylene-C(═O)O—, —C1-C10 alkylene-OC(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)NR4—, —NR4C(═O)—, or a bond, wherein the alkylene is substituted or unsubstituted. In some embodiments, at least one Lin Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —O—, —S—, —C1-C3 alkylene-O—, —C1-C3 alkylene-C(═O)O—, —C1-C3 alkylene-OC(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)NR4—, —NR4C(═O)—, or a bond, wherein the alkylene is substituted or unsubstituted. In some embodiments, at least one Lin Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —O—, —S—, —C1-C3 alkylene-O—, —C1-C3 alkylene-C(═O)O—, —C1-C3 alkylene-OC(═O)—, or a bond, wherein the alkylene is linear or branched unsubstituted alkylene. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)O—, —OC(═O)—, —C(═O)NR4—, —NR4C(═O)—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)O—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —OC(═O)—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)NR4—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)NH—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)N(CH3)—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —NR4C(═O)—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —NHC(═O)—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —N(CH3)C(═O)—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —O—N═CR4— or —CR4=N—O—. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R1 and R2 are the same. In some embodiments, at least one L in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R1 and R2 are different.

In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R1 is —C0-C10 alkylene-L-R6. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R2 is —C0-C10 alkylene-L-R6. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), X is —C(═O)O—. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), X is —OC(═O)—. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), X is —OC(═O)NR4—. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), Y is —C(═O)O—. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), Y is —OC(═O)—. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), Y is —OC(═O)NR4—. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), L is —OC(═O)—. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), L is —C(═O)—. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R1 is —C4-C8 alkylene-L-R6. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R2 is —C4-C8 alkylene-L-R6.

In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R1 is optionally substituted by oxo. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R1 or R2 does not contain any hydrylozable bonds. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R1 or R2 does not contain any C═C bonds. In some embodiments of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), R1 or R2 does not contain any carbon-carbon triple bonds.

In some embodiments, p in Formula (Ib) is 1 to 6. In some embodiments, p in Formula (Ib) is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, or 5 to 6. In some embodiments, p in Formula (Ib) is 1, 2, 3, 4, 5, or 6. In some embodiments, p in Formula (Ib) is at least 1, 2, 3, 4, or 5. In some embodiments, p in Formula (Ib) is at most 2, 3, 4, 5, or 6. In some embodiments, p in Formula (Ib) is 1-3. In some embodiments, p in Formula (Ib) is 1. In some embodiments, p in Formula (Ib) is 2. In some embodiments, p in Formula (Ib) is 3. In some embodiments, p in Formula (Ib) is 4. In some embodiments, p in Formula (Ib) is 5. In some embodiments, p in Formula (Ib) is 6.

In some embodiments, R6 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently

In some embodiments, at least one R6 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, at least one R6 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, at least one R6 in Formula (T) Formula (T*) Formula (Ta) or Formula (Th) is

In some embodiments, at least one R6 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, at least one R6 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, at least one R6 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, R4 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently H or substituted or unsubstituted C1-C4 alkyl. In some embodiments, at least one R4 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently substituted or unsubstituted linear or branched C1-C4 alkyl. In some embodiments, at least one R4 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is H. In some embodiments, at least one R4 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently H, —CH3, —CH2CH3, —CH2CH2CH3, or —CH(CH3)2. In some embodiments, at least one R4 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently H or —CH3. In some embodiments, at least one R4 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —CH3.

In some embodiments, W, X, and Y in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —C(═O)O— or —OC(═O)—. In some embodiments, at least one W, X, and Y in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)O—. In some embodiments, at least one W, X, and Y in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —OC(═O)—. In some embodiments, at least one W, X, and Y in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —C(═O)NR4— or —NR4C(═O)—. In some embodiments, at least one W, X, and Y in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —C(═O)N(CH3)—, —N(CH3)C(═O)—, —C(═O)NH—, or —NHC(═O)—. In some embodiments, at least one W, X, and Y in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —C(═O)NH—, —C(═O)N(CH3)—. —OC(═O)—, —NHC(═O)—, —N(CH3)C(═O)—, —C(═O)O—, —OC(═O)O—, —NHC(═O)O—, —N(CH3)C(═O)O—, —OC(═O)NH—, —OC(═O)N(CH3)—, —NHC(═O)NH—, —N(CH3)C(═O)NH—, —NHC(═O)N(CH3)—, —N(CH3)C(═O)N(CH3)—, NHC(═NH)NH—, —N(CH3)C(═NH)NH—, —NHC(═NH)N(CH3)—, —N(CH3)C(═NH)N(CH3)—, NHC(═NMe)NH—, —N(CH3)C(═NMe)NH—, —NHC(═NMe)N(CH3)—, or —N(CH3)C(═NMe)N(CH3)—. In some embodiments, at least one W, X, and Y in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —OC(═O)O—, —NR4C(═O)O—, —OC(═O)NR4—, or —NR4C(═O)NR4—. In some embodiments, at least one W, X, and Y in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, —NHC(═O)NH—, —N(CH3)C(═O)O—, —OC(═O)N(CH3)—, —N(CH3)C(═O)N(CH3)— or —N(CH3)C(═O)NH—. In some embodiments, at least one W, X, and Y in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, or —NHC(═O)NH—.

In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C0-C10 alkylene-NR7R8 or —C0-C10 alkylene-heterocycloalkyl, wherein the alkylene and heterocycloalkyl is independently substituted or unsubstituted. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C0-C10 alkylene-NR7R8. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C1-C6 alkylene-NR7R8. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C1-C4 alkylene-NR7R8. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C1-alkylene-NR7R8. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C2-alkylene-NR7R8. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C3-alkylene-NR7R8. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C4-alkylene-NR7R8. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C5— alkylene-NR7R8. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C0-C10 alkylene-heterocycloalkyl. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C1-C6 alkylene-heterocycloalkyl, wherein the heterocycloalkyl comprises 1 to 3 nitrogen and 0-2 oxygen. In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is quaternized.

In some embodiments, R7 and R8 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently hydrogen or substituted or unsubstituted C1-C6 alkyl. In some embodiments, R7 and R8 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, R7 and R8 is independently hydrogen or substituted or unsubstituted C1-C3 alkyl. In some embodiments, at least one R7 and R8 is independently substituted or unsubstituted C1-C3 alkyl. In some embodiments, at least one R7 and R8 is independently —CH3, —CH2CH3, —CH2CH2CH3, or —CH(CH3)2. In some embodiments, at least one R7 and R8 is CH3. In some embodiments, at least one R7 and R8 is —CH2CH3.

In some embodiments, R7 and R8 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) Formula (I), Formula (I*), Formula (Ia), or Formula (Ib), taken together with the nitrogen to which they are attached, form a substituted or unsubstituted C2-C6 heterocyclyl. In some embodiments, R7 and R8 taken together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocycloalkyl. In some embodiments, R7 and R8 taken together with the nitrogen to which they are attached form a substituted or unsubstituted 3-7 membered heterocycloalkyl.

In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments R3 in Formula (I), Formula (I*) Formula (Ia), or Formula (Ib) is

In some embodiments R3 in Formula (I) Formula (I*), Formula (Ia) or Formula (Ib is

In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, R3 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)O— or —OC(═O)—. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)O—. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —OC(═O)—. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)NR4— or —NR4C(═O)—. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)NR4—. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —NR4C(═O)—. In some embodiments, R4 in Z is hydrogen or C1-C4 alkyl. In some embodiments, R4 in Z is hydrogen. In some embodiments, R4 in Z is C1-C4 alkyl. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —C(═O)N(CH3)—, —N(CH3)C(═O)—, —C(═O)NH—, or —NHC(═O)—. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —OC(═O)O—, —NR4C(═O)O—, —OC(═O)NR4—, or —NR4C(═O)NR4—. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, —NHC(═O)NH—, —N(CH3)C(═O)O—, —OC(═O)N(CH3)—, —N(CH3)C(═O)N(CH3)—, —NHC(═O)N(CH3)— or —N(CH3)C(═O)NH—. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, or —NHC(═O)NH—. In some embodiments, Z in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is a bond.

In some embodiments, R5 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is hydrogen or substituted or unsubstituted C1-C3 alkyl. In some embodiments, R5 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is H, —CH3, —CH2CH3, —CH2CH2CH3, or —CH(CH3)2. In some embodiments, R5 in Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is H. In some embodiments, R5 is substituted or unsubstituted —C0-C10 alkylene-L-R4. In some embodiments, R5 is substituted or unsubstituted C1-C16 alkyl.

In another aspect, disclosed herein is an amino lipid having structure of Formula (Ib), or a pharmaceutically acceptable salt thereof,

R12 is hydrogen, C1-C10 alkyl, or C1-C10 heteroalkyl, wherein the alkyl and heteroalkyl are each independently substituted or unsubstituted; and

In some embodiments, the amino lipid of Formula (I), Formula (I*), Formula (Ia), or Formula (Ib) is

In another aspect, disclosed herein is an amino lipid having the structure of Formula (II), or a pharmaceutically acceptable salt or solvate thereof,

In one aspect, disclosed herein is an amino lipid having a structure of Formula (II*), or a pharmaceutically acceptable salt or solvate thereof,

In some embodiments of Formula (II) or (II*), if the structure carries more than one asymmetric C-atom, each asymmetric C-atom independently represents racemic, chirally pure R and/or chirally pure S isomer, or a combination thereof.

In some embodiments, each of a and b in Formula (II) or (II*) is independently 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5.

In some embodiments, m in Formula (II) or (II*) is 1, 2, 3, 4, or 5. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5.

In some embodiments, each R1 and R2 in Formula (II) or (II*) is independently C5-C28 alkyl, C5-C28 alkenyl, C5-C28 alkynyl, C3-C10 cycloalkyl, —C1-C10 alkylene-L-R6, or —C1-C10 alkenylene-L-R6, wherein each of the alkyl, alkylene, alkenyl, alkenylene, alkynyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, each R1 and R2 in Formula (II) or (II*) is independently C7-C25 alkyl, C7-C25 alkenyl, C7-C25 alkynyl, C3-C10 cycloalkyl, —C1-C10 alkylene-L-R6, or —C1-C10 alkenylene-L-R6, wherein each of the alkyl, alkylene, alkenyl, alkenylene, alkynyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, each R1 and R2 in Formula (II) or (II*) is independently C7-C22 alkyl, C7-C22 alkenyl, C7-C22 alkynyl, C3-C10 cycloalkyl, —C1-C10 alkylene-L-R6, or —C1-C10 alkenylene-L-R6, wherein each of the alkyl, alkylene, alkenyl, alkenylene, alkynyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, each R1 and R2 in Formula (II) or (II*) is independently C10-C20 alkyl, C10-C20 alkenyl, —C4-C8 alkylene-L-R6, or —C4-C8 alkylene-L-R6, wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, each R1 and R2 in Formula (II) or (II*) is —C1-C10 alkylene-L-R6. In some embodiments, each R1 and R2 in Formula (II) or (II*) is C7-C30 alkenyl. In some embodiments, each R1 and R2 in Formula (II) or (II*) is C7-C28 alkenyl. In some embodiments, each R1 and R2 in Formula (II) or (II*) is C7-C25 alkenyl. In some embodiments, each R1 and R2 in Formula (II) or (II*) is C7-C22 alkenyl.

In some embodiments, R1 and/or R2 in Formula (II) or (II*) is independently

In some embodiments, R1 and/or R2 in Formula (II) or (II*) is

In some embodiments, R1 and/or R2 in Formula (II) or (II*) is

In some embodiments, R1 and/or R2 in Formula (II) or (II*) is

In some embodiments, R1 and/or R2 in Formula (II) or (II*) is

In some embodiments, R1 in Formula (II) or (II*) is

In some embodiments, R2 in Formula (II) or (II*) is

In some embodiments, R1 in Formula (II) or (II*) is

In some embodiments, R2 in Formula (II) or (II*) is

In some embodiments, each of L in Formula (II) or (II*) is independently —O—, —S—, —C1-C10 alkylene-O—, —C1-C10 alkylene-C(═O)O—, —C1-C10 alkylene-OC(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)NR4—, —NR4C(═O)—, or a bond, wherein the alkylene is substituted or unsubstituted. In some embodiments, at least one Lin Formula (II) or (II*) is independently —O—, —S—, —C1-C3 alkylene-O—, —C1-C3 alkylene-C(═O)O—, —C1-C3 alkylene-OC(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)NR4—, —NR4C(═O)—, or a bond, wherein the alkylene is substituted or unsubstituted. In some embodiments, at least one L in Formula (II) or (II*) is independently —O—, —S—, —C1-C3 alkylene-O—, —C1-C3 alkylene-C(═O)O—, —C1-C3 alkylene-OC(═O)—, or a bond, wherein the alkylene is linear or branched unsubstituted alkylene. In some embodiments, at least one L in Formula (II) or (II*) is —C(═O)O—, —OC(═O)—, —C(═O)NR4—, —NR4C(═O)—. In some embodiments, at least one Lin Formula (II) or (II*) is —C(═O)O—. In some embodiments, at least one Lin Formula (II) or (II*) is —OC(═O)—. In some embodiments, at least one L in Formula (II) or (II*) is —C(═O)NR4—. In some embodiments, at least one L in Formula (II) or (II*) is —C(═O)NH—. In some embodiments, at least one L in Formula (II) or (II*) is —C(═O)N(CH3)—. In some embodiments, at least one Lin Formula (II) or (II*) is —NR4C(═O)—. In some embodiments, at least one L in Formula (II) or (II*) is —NHC(═O)—. In some embodiments, at least one L in Formula (II) or (II*) is —N(CH3)C(═O)—.

In some embodiments, each R6 in Formula (II) or (II*) is independently

In some embodiments, each R6 in Formula (II) or (II*) is

In some embodiments, each R6 in Formula (II) or (II*) is

In some embodiments, each R6 in Formula (II) or (II*) is

In some embodiments, R4 in Formula (II) or (II*) is independently H or substituted or unsubstituted C1-C4 alkyl. In some embodiments, at least one R4 in Formula (II) or (II*) is independently substituted or unsubstituted linear C1-C4 alkyl. In some embodiments, at least one R4 in Formula (II) or (II*) is H. In some embodiments, at least one R4 in Formula (II) or (II*) is independently H, —CH3, —CH2CH3, —CH2CH2CH3, or —CH(CH3)2. In some embodiments, at least one R4 in Formula (II) or (II*) is independently H or —CH3. In some embodiments, at least one R4 in Formula (II) or (II*) is —CH3.

In some embodiments, each X and Y in Formula (II) or (II*) is independently —C(═O)O— or —OC(═O)—. In some embodiments, each X and Y in Formula (II) or (II*) is —C(═O)O—. In some embodiments, each X and Y in Formula (II) or (II*) is —OC(═O)—. In some embodiments, Y in Formula (II) or (II*) is —C(═O)O—. In some embodiments, Y in Formula (II) or (II*) is —OC(═O)—. In some embodiments, each X and Y in Formula (II) or (II*) is independently —C(═O)NR4— or —NR4C(═O)—. In some embodiments, X in Formula (II) or (II*) is —C(═O)NR4— or —NR4C(═O)—. In some embodiments, X in Formula (II) or (II*) is —C(═O)NR4—. In some embodiments, X in Formula (II) or (II*) is —NR4C(═O)—. In some embodiments, each X and Y in Formula (II) or (II*) is independently —C(═O)N(CH3)—, —N(CH3)C(═O)—, —C(═O)NH—, or —NHC(═O)—. In some embodiments, each X and Y in Formula (II) or (II*) is independently —C(═O)NH—, —C(═O)N(CH3)—. —OC(═O)—, —NHC(═O)—, —N(CH3)C(═O)—, —C(═O)O—, —OC(═O)O—, —NHC(═O)O—, —N(CH3)C(═O)O—, —OC(═O)NH—, —OC(═O)N(CH3)—, —NHC(═O)NH—, —N(CH3)C(═O)NH—, —NHC(═O)N(CH3)—, —N(CH3)C(═O)N(CH3)—, NHC(═NH)NH—, —N(CH3)C(═NH)NH—, —NHC(═NH)N(CH3)—, —N(CH3)C(═NH)N(CH3)—, NHC(═NMe)NH—, —N(CH3)C(═NMe)NH—, —NHC(═NMe)N(CH3)—, or —N(CH3)C(═NMe)N(CH3)—. In some embodiments, each X and Y in Formula (II) or (II*) is independently —OC(═O)O—, —NR4C(═O)O—, —OC(═O)NR4—, or —NR4C(═O)NR4—. In some embodiments, each X and Y in Formula (II) or (II*) is independently —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, —NHC(═O)NH—, —N(CH3)C(═O)O—, —OC(═O)N(CH3)—, —N(CH3)C(═O)N(CH3)— or —N(CH3)C(═O)NH—. In some embodiments, each X and Y in Formula (II) or (II*) is independently —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, or —NHC(═O)NH—.

In some embodiments, R3 in Formula (II) or (II*) is —C0-C10 alkylene-NR7R8 or —C0-C10 alkylene-heterocycloalkyl, wherein the alkylene and heterocycloalkyl is independently substituted or unsubstituted. In some embodiments, R3 in Formula (II) or (II*) is —C0-C10 alkylene-NR7R8. In some embodiments, R3 in Formula (II) or (II*) is —C1-C6 alkylene-NR7R8. In some embodiments, R3 in Formula (II) or (II*) is —C1-C4 alkylene-NR7R8. In some embodiments, R3 in Formula (II) or (II*) is —C1-alkylene-NR7R8. In some embodiments, R3 in Formula (II) or (II*) is —C2-alkylene-NR7R8. In some embodiments, R3 in Formula (II) or (II*) is —C3-alkylene-NR7R8. In some embodiments, R3 in Formula (II) or (II*) is —C4-alkylene-NR7R8. In some embodiments, R3 in Formula (II) or (II*) is —C5-alkylene-NR7R8. In some embodiments, R3 in Formula (II) or (II*) is —C0-C10 alkylene-heterocycloalkyl. In some embodiments, R3 in Formula (II) or (II*) is —C1-C6 alkylene-heterocycloalkyl, wherein the heterocycloalkyl comprises 1 to 3 nitrogen and 0-2 oxygen. In some embodiments, R3 in Formula (II) or (II*) is quaternized.

In some embodiments, R7 and R8 in Formula (II) or (II*) is independently hydrogen or substituted or unsubstituted C1-C6 alkyl. In some embodiments, at least one R7 and R8 in Formula (II) or (II*) is independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, at least one R7 and R8 is independently hydrogen or substituted or unsubstituted C1-C3 alkyl. In some embodiments, at least one R7 and R8 is independently substituted or unsubstituted C1-C3 alkyl. In some embodiments, at least one R7 and R8 is independently —CH3, —CH2CH3, —CH2CH2CH3, or —CH(CH3)2. In some embodiments, at least one R7 and R8 is CH3. In some embodiments, at least one R7 and R8 is —CH2CH3.

In some embodiments, R7 and R8 in Formula (II) or (II*), taken together with the nitrogen to which they are attached, form a substituted or unsubstituted C2-C6 heterocyclyl. In some embodiments, R7 and R8 taken together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocycloalkyl. In some embodiments, R7 and R8 taken together with the nitrogen to which they are attached form a substituted or unsubstituted 3-7 membered heterocycloalkyl.

In some embodiments, R3 in Formula (II) or (II*) is

In some embodiments, R3 in Formula (II) or (II*) is

In some embodiments, R3 in Formula (II) or (II*) is

In some embodiments, R3 in Formula (II) or (II*) is

In some embodiments, R3 in Formula (II) or (II*) is

In some embodiments, R3 in Formula (II) or (II*) is

In some embodiments, R3 in Formula (II) or (II*) is

In some embodiments, Z in Formula (II) or (II*) is —C(═O)O— or —OC(═O)—. In some embodiments, Z in Formula (II) or (II*) is —C(═O)O—. In some embodiments, Z in Formula (II) or (II*) is —OC(═O)—. In some embodiments, Z in Formula (II) or (II*) is —C(═O)NR4— or —NR4C(═O)—. In some embodiments, Z in Formula (II) or (II*) is —C(═O)NR4—. In some embodiments, Z in Formula (II) or (II*) is —NR4C(═O)—. In some embodiments, R4 in Z is hydrogen or C1-C4 alkyl. In some embodiments, R4 in Z is hydrogen. In some embodiments, R4 in Z is C1-C4 alkyl. In some embodiments, Z in Formula (II) or (II*) is —C(═O)N(CH3)—, —N(CH3)C(═O)—, —C(═O)NH—, or —NHC(═O)—. In some embodiments, Z in Formula (II) or (II*) is —OC(═O)O—, —NR4C(═O)O—, —OC(═O)NR4—, or —NR4C(═O)NR4—. In some embodiments, Z in Formula (II) or (II*) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, —NHC(═O)NH—, —N(CH3)C(═O)O—, —OC(═O)N(CH3)—, —N(CH3)C(═O)N(CH3)—, —NHC(═O)N(CH3)— or —N(CH3)C(═O)NH—. In some embodiments, Z in Formula (II) or (II*) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, or —NHC(═O)NH—.

In one aspect, disclosed herein is an amino lipid of Table 1A, or a salt or solvate thereof.

Amino lipids for constituting lipid nanoparticles. The chiral carbon atom(s) in any of the

In some embodiments of a compound of Table 1A, m is 1-10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10.

In some embodiments, an asymmetric carbon atom in Table 1A represents racemic, chirally pure R or chirally S. In some embodiments, if a compound of Table 1A contains two or more chiral centers, all the combinations of the stereochemistries for each of the chiral centers are encompassed by the disclosure.

In some embodiments, the disclosed amino lipids can be converted to N-oxides. In some embodiments, N-oxides are formed by a treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid and/or hydrogen peroxides). Accordingly, disclosed herein are N-oxide compounds of the described amino lipids, when allowed by valency and structure, which can be designated as N→0 or N+-0−. In some embodiments, the nitrogen in the compounds of the disclosure can be converted to N-hydroxy or N-alkoxy. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as ra-CPBA. All shown and claimed nitrogen-containing compounds are also considered. Accordingly, also disclosed herein are N-hydroxy and N-alkoxy (e.g., N—OR, wherein R is substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-14-membered carbocycle or 3-14-membered heterocycle) derivatives of the described amino lipids.

In some embodiments, an amino lipid described herein can take the form of a salt, such as a pharmaceutically acceptable salt. All pharmaceutically acceptable salts of the amino lipid are encompassed by this disclosure. As used herein, the term “amino lipid” also includes its pharmaceutically acceptable salts, and its diastereomeric, enantiomeric, and epimeric forms.

In some embodiments, an amino lipid described herein, possesses one or more stereocenters and each stereocenter exists independently in either the R or S configuration. The lipids presented herein include all diastereomeric, enantiomeric, and epimeric forms as well as the appropriate mixtures thereof. The lipids provided herein include all cis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well as the appropriate mixtures thereof. In certain embodiments, lipids described herein are prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds/salts, separating the diastereomers and recovering the optically pure enantiomers. In some embodiments, resolution of enantiomers is carried out using covalent diastereomeric derivatives of the compounds described herein. In another embodiment, diastereomers are separated by separation/resolution techniques based upon differences in solubility. In other embodiments, separation of stereoisomers is performed by chromatography or by the forming diastereomeric salts and separation by recrystallization, or chromatography, or any combination thereof. Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John Wiley And Sons, Inc., 1981. In one aspect, stereoisomers are obtained by stereoselective synthesis.

In some embodiments, the lipids such as the amino lipids are substituted based on the structures disclosed herein. In some embodiments, the amino lipid is a lipid of Table 1A with one or more substituents. In some embodiments, the lipids such as the amino lipids are unsubstituted. In another embodiment, the lipids described herein are labeled isotopically (e.g. with a radioisotope) or by another other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

Lipids described herein include isotopically-labeled compounds, which are identical to those recited in the various formulae and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the present lipids include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as, for example, 2H, 3h, 13C, 14C, 15N, 18O, 17O, 35S, 18F, 36 cl. In one aspect, isotopically-labeled lipids described herein, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. In one aspect, substitution with isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements.

In some embodiments, the asymmetric carbon atom of the amino lipid is present in enantiomerically enriched form. In certain embodiments, the asymmetric carbon atom of the amino lipid has at least 5000 enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (S)- or (R)-configuration.

Also disclosed herein is a lipid of Table 11B, or a salt or solvate thereof.

Amino lipids and excipients for constituting lipid nanoparticles

In some embodiments, an amino lipid (or other lipids) provided herein can be designated by more than one compound TD number in different parts of the disclosure.

In some embodiments, the described LNP composition comprises a PEG-lipid. In some embodiments, the described LNP composition comprises two or more PEG-lipids. Exemplary PEG-lipids include, but are not limited to, the lipids in Table 2. Exemplary PEG-lipids also include, but are not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the one or more PEG-lipids can comprise PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, a PEG-DSPE lipid, or a combination thereof. In some embodiments, PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG moiety is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, the PEG moiety includes PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., j. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)). In some embodiments, the PEG moiety does not include PEG copolymers, e.g., it may be a PEG monopolymer. Exemplary PEG-lipids include, but are not limited to, PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylgiycerol (PEG-DSPE), PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol, and PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]).

In one aspect, disclosed herein is a PEG-lipid having the structure of Formula (III), or a pharmaceutically acceptable salt or solvate thereof,

In one aspect, disclosed herein is a PEG-lipid having the structure of Formula (III*), or a pharmaceutically acceptable salt or solvate thereof,

In some embodiments, the PEG-lipid of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, wherein

In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, each of R21 and R22 is independently unsubstituted or substituted, linear or branched C12-C30 alkyl. In some embodiments, each of R21 and R22 is independently unsubstituted, linear C12-C25 alkyl. In some embodiments of a compound of Formula (III), or a pharmaceutically acceptable salt or solvate thereof, each of R21 and R22 is independently unsubstituted or substituted, linear or branched C12-C30 alkenyl. In some embodiments, each of R21 and R22 is independently unsubstituted, linear C12-C25 alkenyl.

In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, each of R26 and R27 is —O—, —C(═O)O—, or —OC(═O)—. In some embodiments, each of R26 and R27 is —O—. In some embodiments, each of R26 and R27 is a bond.

In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, R28 is —O—, —C(═O)O—, —OC(═O)—, or —OC(═O)NR4. In some embodiments of a compound of Formula (III*), R28 is —OC(═O)NR4. In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, R28 is —C(═O)O— or —OC(═O)—.

In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, k1 is 0, 1, or 2. In some embodiment, k1 is 0. In some embodiment, k1 is 1. In some embodiment, k1 is 2.

In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, k2 is 0, 1, or 2. In some embodiment, k2 is 0. In some embodiment, k2 is 1. In some embodiment, k2 is 2.

In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, k3 is 0, 1, or 2. In some embodiment, k3 is 0. In some embodiment, k3 is 1. In some embodiment, k3 is 2.

In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, R23 is —C0-C3 alkylene-(CH2—CH2—O)k4—R24, wherein R24 is H, ethyl or methyl. In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, R23 is —C1-C5 heteroalkylene-(CH2—CH2—O)k4—R24, wherein R24 is H or methyl. In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, R23 is —C2 alkylene-(CH2—CH2—O)k4—R24; wherein R24 is —O—CH3. In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, R23 is —C3 alkylene-(CH2—CH2-0)k4—R24; wherein R24 is —O—CH3.

In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, k4 is 1 to 75. In some embodiments, k4 is 1 to 50, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 20 to 50, or 5 to 50. In some embodiments, k4 is 30 to 50. In some embodiments, k4 is 35 to 45. In some embodiments, k4 is 40 to 50. In some embodiments, k4 is 36 to 48.

In some embodiments of a compound of Formula (III) or (III*), or a pharmaceutically acceptable salt or solvate thereof, R25 is hydrogen.

In some embodiments of a compound of Formula (III*), R22 is the the unsubstituted C28 alkenyl:

In some embodiments, disclosed herein is a PEG-lipid comprising a structure of —O(CH2)2C(O)O— or —O(CH2)2C(O)NH—.

In one aspect, disclosed herein is a PEG-lipid of Table 2.

Exemplary PEG-Lipids for constituting LNP

A PEG-lipid can comprise one or more ethylene glycol units, for example, at least 1, at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, or at least 150 ethylene glycol units. In some embodiments, a number average molecular weight of the PEG-lipids is from about 200 Da to about 5000 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 500 Da to about 3000 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 750 Da to about 2500 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 750 Da to about 2500 Da. In some embodiments, a number average molecular weight of the PEG-lipids is about 500 Da, about 750 Da, about 1000 Da, about 1250 Da, about 1500 Da, about 1750 Da, or about 2000 Da. In some embodiments, a polydispersity index (PDI) of the one or more PEG-lipids is smaller than 2. In some embodiments, a PDI of the one or more PEG-lipids is at most 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In some embodiments, a PDI of the one or more PEG-lipids is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0.

In some embodiments, the PEG-lipid comprises from about 0.1 mol % to about 10 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises from about 0.1 mol % to about 6 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises from about 0.5 mol % to about 5 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises from about 1 mol % to about 3 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises about 2.0 mol % to about 2.5 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises about 1 mol %, about 1.1 mol %, about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %, about 1.6 mol %, about 1.7 mol %, about 1.8 mol %, about 1.9 mol %, about 2.0 mol %, about 2.1 mol %, about 2.2 mol %, about 2.3 mol %, about 2.4 mol %, about 2.5 mol %, about 2.6 mol %, about 2.7 mol %, about 2.8 mol %, about 2.9 mol %, or about 3.0 mol % of the total lipid present in the particle.

In some embodiments, the LNP composition comprises a plurality of PEG-lipids, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct PEG-lipids. In some embodiments, at least one of the plurality of PEG-lipids is selected from Table 2.

In some embodiments, the described LNP composition comprises a phospholipid. In some embodiments, the phospholipid comprises a lipid selected from the group consisting of: phosphatidylcholine (PC), phosphatidylethanolamine amine, glycerophospholipid, sphingophospholipids, Guriserohosuhono, sphingolipids phosphono lipids, natural lecithins, and hydrogenated phospholipid. In some embodiments, the phospholipid comprises a phosphatidylcholine. Exemplary phosphatidylcholines include, but are not limited to, soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoyl phosphatidylcholine, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), dimyristoyl phosphatidylcholine (DMPC), and dioleoyl phosphatidylcholine (DOPC). In certain specific embodiments, the phospholipid is DSPC.

In some embodiments, the phospholipid comprises a phosphatidylethanolamine amine. In some embodiments, the phosphatidylethanolamine amine is distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphoethanolamine (DMPE), 16-O-Monome Le PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyl oleoyl-phosphatidylethanolamine (POPE), or 1-stearoyl-2-oleoyl-phosphatidyl ethanolamine (SOPE). In some embodiments, the phospholipid comprises a glycerophospholipid. In some embodiments, the glycerophospholipid is plasmalogen, phosphatidate, or phosphatidylcholine. In some embodiments, the glycerophospholipid is phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyl oleoyl phosphatidylglycerol (POPG), or lysophosphatidylcholine. In some embodiments, the phospholipid comprises a sphingophospholipid. In some embodiments, the sphingophospholipid is sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, or ceramide phosphoglycerophosphoric acid. In some embodiments, the phospholipid comprises a natural lecithin. In some embodiments, the natural lecithin is egg yolk lecithin or soybean lecithin. In some embodiments, the phospholipid comprises a hydrogenated phospholipid. In some embodiments, the hydrogenated phospholipid is hydrogenated soybean phosphatidylcholine. In some embodiments, the phospholipid is selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.

In some embodiments, the LNP composition comprises a plurality of phospholipids, for example, at least 2, 3, 4, 5, or more distinct phospholipids. In some embodiments, the phospholipid comprises from 1 mol % to 20 mol % of the total lipid present in the particle. In some embodiments, the phospholipid comprises from about 5 mol % to about 15 mol % of the total lipid present in the particle. In some embodiments, the phospholipid comprises from about 8 mol % to about 12 mol % of the total lipid present in the particle. In some embodiments, the phospholipid comprises from about 9 mol %, 10 mol %, or 11 mol % of the total lipid present in the particle.

Cholesterol

In some embodiments, the LNP composition comprises a cholesterol or a derivative thereof. In some embodiments, the LNP composition comprises a structural lipid. The structural lipid can be selected from steroid, sterol, alkyl resoreinol, cholesterol or derivative thereof, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and a combination thereof. In some embodiments, the structural lipid is a corticosteroid such as prednisolone, dexamethasone, prednisone, and hydrocortisone. In some embodiments, the cholesterol or derivative thereof is cholesterol, 5-heptadecylresorcinol, or cholesterol hemisuccinate. In some embodiments, the cholesterol or derivative thereof is cholesterol.

In some embodiments, the cholesterol or derivative thereof is a cholesterol derivative. In some embodiments, the cholesterol derivative is a polar cholesterol analogue. In some embodiments, the polar cholesterol analogue is 5α-cholestanol, 5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butyl ether, or 6-ketocholestanol. In some embodiments, the polar cholesterol analogue is cholesteryl-(4′-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is a non-polar cholesterol analogue. In some embodiments, the non-polar cholesterol analogue is 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, or cholesteryl decanoate.

In some embodiments, the cholesterol or the derivative thereof comprises from 20 mol % to 50 mol % of the total lipid present in the particle. In some embodiments, the cholesterol or the derivative thereof comprises about 20 mol %, about 21 mol %, about 22 mol %, about 23 mol %, about 24 mol %, about 25 mol %, about 26 mol %, about 27 mol %, about 28 mol %, about 29 mol %, about 30 mol %, about 31 mol %, about 32 mol %, about 33 mol %, about 34 mol %, about 35 mol %, about 36 mol %, about 37 mol %, about 38 mol %, about 39 mol %, about 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %, about 45 mol %, about 46 mol %, about 47 mol %, about 48 mol %, or about 50 mol % of the total lipid present in the particle.

Antioxidants

In some embodiments, the LNP described herein comprises one or more antioxidants. In some embodiments, the one or more antioxidants function to reduce a degradation of the cationic lipids, the payload, or both. In some embodiments, the one or more antioxidants comprise a hydrophilic antioxidant. In some embodiments, the one or more antioxidants is a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and citrate. In some embodiments, the one or more antioxidants is EDTA. In some embodiments, the one or more antioxidants comprise a lipophilic antioxidant. In some embodiments, the lipophilic antioxidant comprises a vitamin E isomer or a polyphenol. In some embodiments, the one or more antioxidants are present in the LNP composition at a concentration of at least 1 mM, at least 10 mM, at least 20 mM, at least 50 mM, or at least 100 mM. In some embodiments, the one or more antioxidants are present in the particle at a concentration of about 20 mM.

Payload

The LNPs described herein can be designed to deliver a payload, such as a therapeutic agent, or a target of interest. Exemplary therapeutic agents include, but are not limited to, antibodies (e.g., monoclonal, chimeric, humanized, nanobodies, and fragments thereof etc.), cholesterol, hormones, peptides, proteins, chemotherapeutics and other types of antineoplastic agents, low molecular weight drugs, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys and analogs thereof, plasmids and other types of expression vectors, and small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siRNA), messenger ribonucleic acid (messenger RNA, mRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), siRNA (small internally segmented interfering RNA), aiRNA (asymmetric interfering RNA), and siRNA with 1, 2 or more mismatches between the sense and anti-sense strand to relevant cells and/or tissues, such as in a cell culture, subject or organism. Therapeutic agents can be purified or partially purified, and can be naturally occurring or synthetic, or chemically modified. In some embodiments, the therapeutic agent is an RNAi agent, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or a short hairpin RNA (shRNA) molecule. In some embodiments, the therapeutic agent is an mRNA.

In some embodiments, the payload comprises one or more nucleic acid(s) (i.e., one or more nucleic acid molecular entities). In some embodiments, the nucleic acid is a single-stranded nucleic acid. In some embodiments, single-stranded nucleic acid is a DNA. In some embodiments, single-stranded nucleic acid is an RNA. In some embodiments, the nucleic acid is a double-stranded nucleic acid. In some embodiments, the double-stranded nucleic acid is a DNA. In some embodiments, the double-stranded nucleic acid is an RNA. In some embodiments, the double-stranded nucleic acid is a DNA-RNA hybrid. In some embodiments, the nucleic acid is a messenger RNA (mRNA), a microRNA, an asymmetrical interfering RNA (aiRNA), a small hairpin RNA (shRNA), or a Dicer-Substrate dsRNA.

In some embodiments, the payload comprises an mRNA. In some embodiments, the payload comprises an mRNA molecule encoding a Cas nuclease, i.e., a Cas nuclease mRNA. In some embodiments, the payload comprises one or more guide RNAs or nucleic acids encoding guide RNAs. In some embodiments, the payload comprises a template nucleic acid for repair or recombination. In some embodiments, the payload comprises an mRNA encoding a gene editor nuclease. In some embodiments, the payload comprises an mRNA encoding a base editor nuclease. In some embodiments, the payload comprises an mRNA encoding a restriction enzyme. In some embodiments, the payload comprises zinc-finger nuclease or TALEN nuclease.

In some embodiments, the mRNA payload, such as a Cas nuclease mRNA, can be modified for improved stability and/or immunogenicity properties. The modifications may be made to one or more nucleosides within the mRNA. Examples of chemical modifications to mRNA nucleobases include pseudouridine, 1-methyl-pseudouridine, and 5-methyl-cytidine. Additional modifications to improve stability, expression, and immunogenicity can also be made. The mRNA encoding a Cas nuclease can be codon optimized for expression in a particular cell type, such as a eukaryotic cell, a mammalian cell, or more specifically, a human cell. In some embodiments, the mRNA encodes a human codon optimized Cas9 nuclease or human codon optimized Cpf nuclease as the Cas nuclease. In some embodiments, the mRNA encodes a gene editor (i.e., genome editor) nuclease and is called a gene editor mRNA. In some embodiments, the gene editor is a Cas protein, such as the ones described herein. In some embodiments, the gene editor is an engineered nuclease. In some embodiments, the gene editor introduces a double stranded break in a gene of interest. In some embodiments, the gene editor introduces a double stranded break at a targeted point within a gene of interest. In some embodiments, the gene editor introduces a single stranded break in a gene of interest. In some embodiments, the gene editor is a base editor. In some embodiments, the gene editor inserts a nucleic acid sequence into a gene of interest. In some embodiments, the gene editor deletes a targeted sequence from a gene of interest. In some embodiments, the gene editor mRNA encodes Cas9 nuclease. In some embodiments, the gene editor mRNA encodes base editor nuclease. In some embodiments, the gene editor mRNA encodes a restriction enzyme. In some embodiments, the gene editor mRNA encodes zinc-finger nuclease. In some embodiments, the gene editor mRNA encodes transcription activator-like effector-based nucleases (TALEN). In some embodiments, the gene editor mRNA encodes a meganuclease. In some embodiments, the gene editor mRNA encodes an Argonaute protein. In some embodiments, the mRNA is purified. In some embodiments, the mRNA is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or an equivalent method, e.g., as described herein) or a chromatography-based method (e.g., an HPLC-based method or an equivalent method).

In some embodiments, the Cas nuclease mRNA comprises a 3′ or 5′ untranslated region (UTR). In some embodiments, the 3′ or 5′ UTR can be derived from a human gene sequence. Exemplary 3′ and 5′ UTRs include α- and β-globin, albumin, HSD17B4, and eukaryotic elongation factor 1a. In addition, viral-derived 5′ and 3′ UTRs can also be used and include orthopoxvirus and cytomegalovirus UTR sequences. In certain embodiments, the mRNA includes a 5′ cap, such as m7G(5′)ppp(5′)N. In certain embodiments, this cap can be a cap-0 where nucleotide N does not contain 2′OMe, or cap-1 where nucleotide N contains 2′OMe, or cap-2 where nucleotides N and N+1 contain 2′OMe. In some embodiments, the 5′ cap can regulate nuclear export; prevent degradation by exonucleases; promote translation; and promote 5′ proximal intron excision. In addition, caps can also contain a non-nucleic acid entity that acts as the binding element for eukaryotic translation initiation factor 4E, eIF4E. In certain embodiments, the mRNA includes a poly(A) tail. This tail can be about 40 to about 300 nucleotides in length. In some embodiments, the tail is about 40 to about 100 nucleotides in length. In some embodiments, the tail is about 100 to about 300 nucleotides in length. In some embodiments, the tail is about 100 to about 300 nucleotides in length. In some embodiments, the tail is about 50 to about 200 nucleotides in length. In some embodiments, the tail is about 50 to about 250 nucleotides in length. In certain embodiments, the tail is about 100, 150, or 200 nucleotides in length. The poly(A) tail can contain modifications to prevent exonuclease degradation including phosphorotioate linkages and modifications to the nucleobase. In some embodiments, the poly(A) tail contains a 3′ “cap” which could include modified or non-natural nucleobases or other synthetic moieties. In some embodiments, the mRNA comprises at least one element that is capable of modifying the intracellular half-life of the RNA. The half-life of the RNA can be increased or decreased. In some embodiments, the element is capable of increasing or decreasing the stability of the RNA. In some embodiments the element may promote RNA decay. In some embodiments, the element can activate translation. In some embodiments, the element may be within the 3′ UTR of the RNA. For example, the element may be an mRNA decay signal or may include a polyadenylation signal (PA).

In some embodiments, the Cas nuclease mRNA encodes a Cas protein from a CRISPR/Cas system. In some embodiments, the Cas protein comprises at least one domain that interacts with a guide RNA (“gRNA”). In some embodiments, the Cas protein is directed to a target sequence by a guide RNA. The guide RNA can interact with the Cas protein as well as the target sequence such that, it can direct binding to the target sequence. In some embodiments, the guide RNA provides the specificity for the targeted cleavage, and the Cas protein may be universal and paired with different guide RNAs to cleave different target sequences. In certain embodiments, the Cas protein may cleave single or double-stranded DNA. In certain embodiments, the Cas protein may cleave RNA. In certain embodiments, the Cas protein may nick RNA. In some embodiments, the Cas protein comprises at least one DNA binding domain and at least one nuclease domain. In some embodiments, the nuclease domain may be heterologous to the DNA binding domain. In certain embodiments, the Cas protein may be modified to reduce or eliminate nuclease activity. The Cas protein may be used to bind to and modulate the expression or activity of a DNA sequence.

In some embodiments, the CRISPR/Cas system comprises Class 1 or Class 2 system components, including ribonucleic acid protein complexes. The Class 2 Cas nuclease families of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as described further herein. A Class 2 CRISPR/Cas system component may be from a Type-IIA, Type-JIB, Type-IIC, Type V, or Type VI system. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. In some embodiments, the Cas protein is from a Type-II CRISPR/Cas system, i.e., a Cas9 protein from a CRISPR/Cas9 system, or a Type-V CRISPR/Cas system, e.g., a Cpf1 protein. In some embodiments, the Cas protein is from a Class 2 CRISPR/Cas system, i.e., a single-protein Cas nuclease such as a Cas9 protein or a Cpf1 protein.

In some embodiments, the payload comprises at least one guide RNA. The guide RNA may guide the Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule, where the guide RNA hybridizes with and the Cas nuclease cleaves or modulates the target sequence. In some embodiments, a guide RNA binds with and provides specificity of cleavage by a Class 2 nuclease. In some embodiments, the guide RNA and the Cas protein may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex. In some embodiments, the CRISPR complex may be a Type-II CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex may be a Type-V CRISPR/Cas complex, such as a Cpf1/guide RNA complex. In some embodiments, the Cas nuclease may be a single-protein Cas nuclease, e.g. a Cas9 protein or a Cpf 1 protein. In some embodiments, the guide RNA targets cleavage by a Cas9 protein. In some embodiments, the payload comprises two or more guide RNA molecules. In some embodiments, the two or more guide RNA molecules target the same disease-causing gene. In some embodiments, the two or more guide RNA molecules target different genes. In some specific embodiments, the two guide RNA molecules target two separate disease-causing genes of interest.

A guide RNA for a CRISPR/Cas9 nuclease system comprises a CRISPR RNA (crRNA) and a tracr RNA (tracr). In some embodiments, the crRNA may comprise a targeting sequence that is complementary to and hybridizes with the target sequence on the target nucleic acid molecule. The crRNA may also comprise a flagpole that is complementary to and hybridizes with a portion of the tracrRNA. In some embodiments, the crRNA may parallel the structure of a naturally occurring crRNA transcribed from a CRISPR locus of a bacteria, where the targeting sequence acts as the spacer of the CRISPR/Cas9 system, and the flagpole corresponds to a portion of a repeat sequence flanking the spacers on the CRISPR locus. The guide RNA may target any sequence of interest via the targeting sequence of the crRNA. In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may be 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1-6 mismatches.

In some embodiments, the length of the targeting sequence depends on the CRISPR/Cas system and components used. For example, different Cas proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence comprised 18-24 nucleotides in length. In some embodiments, the targeting sequence comprises 19-21 nucleotides in length. In some embodiments, the targeting sequence comprises 20 nucleotides in length.

In some embodiments, the guide RNA is a “dual guide RNA” or “dgRNA”. In some embodiments, the dgRNA comprises a first RNA molecule comprising a crRNA, and a second RNA molecule comprising a tracr RNA. The first and second RNA molecules may form a RNA duplex via the base pairing between the flagpole on the crRNA and the tracr RNA. In some embodiments, the guide RNA is a “single guide RNA” or “sgRNA”. In some embodiments, the sgRNA may comprise a crRNA covalently linked to a tracr RNA. In some embodiments, the crRNA and the tracr RNA may be covalently linked via a linker. In some embodiments, the single-molecule guide RNA may comprise a stem-loop structure via the base pairing between the flagpole on the crRNA and the tracr RNA. In some embodiments, the sgRNA is a “Cas9 sgRNA” capable of mediating RNA-guided DNA cleavage by a Cas9 protein. In certain embodiments, the guide RNA comprises a crRNA and tracr RNA sufficient for forming an active complex with a Cas9 protein and mediating RNA-guided DNA cleavage. In some embodiments, the payload comprises more than one guide RNAs; each guide RNA contains a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target sequence. In some embodiments, one or more guide RNAs may have the same or differing properties such as activity or stability within a CRISPR/Cas complex. Where more than one guide RNA is used, each guide RNA can be encoded on the same or on different expression cassettes. The promoters used to drive expression of the more than one guide RNA may be the same or different.

In some embodiments, the nucleic acid payload, such as RNAs, is modified. Modified nucleosides or nucleotides can be present in a guide RNA or mRNA. A guide RNA or Cas nuclease encoding mRNA comprising one or more modified nucleosides or nucleotides is called a “modified” RNA to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified RNA is synthesized with a non-canonical nucleoside or nucleotide. Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3′ or 5′ cap modifications may comprise a sugar and/or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification).

In some embodiments, the payload can include a template nucleic acid. The template can be used to alter or insert a nucleic acid sequence at or near a target site for a Cas nuclease. In some embodiments, the template is used in homologous recombination. In some embodiments, the homologous recombination may result in the integration of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In some embodiments, a single template is provided. In other embodiments, two or more templates are provided such that homologous recombination may occur at two or more target sites.

In some embodiments, the payload, such as one or more RNAs, are fully encapsulated within the lipid portion of the particle, thereby protecting the RNAs from nuclease degradation. Fully encapsulated can indicate that the RNA in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free DNA or RNA. In some embodiments, the nucleic acid-lipid particle composition comprises a RNA molecule that is fully encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or range therein) of the particles have the RNA encapsulated therein.

In some embodiments, the payload comprises an mRNA and one or more guide RNA. In some embodiments, the mRNA encodes a gene editor nuclease and is called a gene editor mRNA. In some embodiments, the gene editor mRNA encodes Cas9 nuclease. In some embodiments, the mRNA encodes base editor nuclease. In some embodiments, the gene editor mRNA encodes zinc-finger nuclease. In some embodiments, the gene editor mRNA encodes TALEN nuclease.

Surfactants and Other Components

In some embodiments, a nanoparticle described herein comprises one or more non-ionic surfactants. In some embodiments, the one or more non-ionic surfactants comprise a fatty alcohol, a fatty acid, or both. In some embodiments, the fatty alcohol is a C12-C24 fatty alcohol. In some embodiments, the fatty alcohol is lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, palmitoleyl alcohol, heptadecyl alcohol, stearyl alcohol, oleyl alcohol, nonadecyl alcohol, arachidyl alcohol, or a combination thereof. In some embodiments, the C12-C24 fatty alcohol is oleyl alcohol, stearyl alcohol, or a mixture thereof. In some embodiments, the nanoparticle comprises oleyl alcohol. In some embodiments, the nanoparticle comprises stearyl alcohol.

In some embodiments, the one or more surfactants (such as fatty alcohol) are present in a herein described nanoparticle composition in an amount of about 0.5 mol % to about 20 mol % of a total lipid content present in the nanoparticle composition, or any ranges therebetween. In some embodiments, the one or more surfactants (such as fatty alcohol) are present in the nanoparticle composition in an amount of about 1 mol % to about 10 mol % of a total lipid content present in the nanoparticle composition. In some embodiments, the one or more surfactants (such as fatty alcohol) are present in the nanoparticle composition in an amount of about 3 mol % to about 8 mol % of a total lipid content present in the nanoparticle composition. In some embodiments, the one or more surfactants (such as fatty alcohol) are present in the nanoparticle composition at about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, or about 10 mol % of a total lipid content present in the nanoparticle composition. In some embodiments, the one or more surfactants (such as fatty alcohol) are present in the nanoparticle composition in a range from about 0.5 mol %, 1 mol %, 1.5 mol %, 2 mol %, 2.5 mol %, 3 mol %, or 3.5 mol % to about 4 mol %, 4.5 mol %, 5 mol %, 5.5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % of a total lipid content present in the nanoparticle composition. In some embodiments, the one or more surfactants (such as fatty alcohol) are present in the nanoparticle composition in a range from about 1 mol %, 2 mol %, 3 mol %, 4 mol %, 5 mol %, 6 mol %, or 7 mol % to about 8 mol %, 9 mol %, 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol %, 15 mol %, or 20 mol % of a total lipid content present in the nanoparticle composition.

Nanoparticle compositions described herein can comprise one or more permeability enhancer molecules, carbohydrates, polymers, or other components. A permeability enhancer molecule can be, e.g., a molecule described by U.S. patent application publication No. 2005/0222064. The carbohydrates can be simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

Nucleic Acid Stabilizer

In some embodiments, nanoparticle compositions described herein comprise excipients that stabilize lipid nanoparticles encapsulating nucleic acid payload (e.g., nucleic acid therapeutics). For example, the nucleic acid therapeutics can be mRNA to modulate disease causing proteins; mRNA and guide RNA to edit therapeutic genes of interest; RNA-editing nucleic acid payloads; siRNA; antisense oligonucleotides such as those to elicit RNase H-mediated gene silencing; microRNA and anti-microRNA; RNA activators; aptamers and the like.

Lipid nanoparticles (LNPs) represent an efficacious non-viral delivery system for nucleic acid therapeutics. Efficacy of an LNP treatment can be determined by numerous variables, including the stability and functionality of the encapsulated RNA cargo. If the RNA cargo experiences nicking, degradation, or a structural change during long-term storage or freeze/thaw, a partial or complete loss in therapeutic potency may occur. As such, the herein disclosed nanoparticle compositions are advantageous in the delivering of nucleic acid payloads, at least partially due to the enhanced stability of the nanoparticle compositions and/or encapsulated compositions including e.g. nucleic acid therapeutics. In some embodiments, the enhanced stability can prolong the shelf-storage life and improve the freeze-thaw resistance of a pharmaceutical composition comprising the nanoparticle. In some embodiments, the enhanced stability is achieved, at least partially, by the selection of the type, the pH, the osmolarity, and the counter ions of the buffer used to solubilize the nucleic acid payload. In some embodiments, the enhanced stability is achieved, at least partially, by the addition of a nucleic acid stabilizer. The nucleic acid stabilizer can be selected from excipients that are designated by the FDA as generally recognized as safe (GRAS). Accordingly, in one aspect, the present disclosure describes the innovative use of commonly used GRAS excipients to improve RNA stability and delivery by LNPs

Nanoparticle compositions described herein can comprise a nucleic acid stabilizer. In some embodiments, the nucleic acid stabilizer is polyethylene glycol. In some embodiments, the nucleic acid stabilizer is a cationic surfactant such as cetrimonium bromide and cetrimonium chloride. In some embodiments, the nucleic acid stabilizer is cetrimonium bromide (i.e., cetyltrimethylammonium bromide or CTAB). In some embodiments, the nucleic acid stabilizer is polysaccharide or oligosaccharide such as low molecular weight chitosan. Exemplary low molecular weight chitosan can have a molecular weight of 50,000 to 190,000 Da, or lower than 50,000 Da. In some embodiments, the nucleic acid stabilizer is a cryoprotectant. Exemplary cryoprotectants include, but are not limited to, a polyol (e.g., a diol or a triol such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, 2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a nondetergent sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propane sulfonate), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a water soluble polymer (e.g., polyethylene glycol, polyethylene glycol monomethyl ether (mPEG) such as mPEG 550, mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, and mPEG 5000, polyvinylpyrrolidone, pentaerythritol propoxylate, and a block polymer of polyethylene glycol and polypropylene glycol), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof), or any combination thereof. In some embodiments, the nanoparticle composition comprises two or more nucleic acid stabilizers.

In some embodiments, the nucleic acid stabilizer is polyethylene glycol (PEG). In some embodiments, the polyethylene glycol is PEG 200, PEG 400, PEG 600, PEG 1000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, or a combination thereof. In some embodiments, the polyethylene glycol is PEG 200, PEG 400, PEG 600, or a combination thereof. In some embodiments, the polyethylene glycol has a number average molecular weight (Mn) of from about 120 to about 5000 Da, or any numbers or ranges therebetween. In some embodiments, the polyethylene glycol has a Mn of about 120 to about 200 Da, about 200 to about 800 Da, about 200 to about 600 Da, about 120 to about 1000 Da, or about 40 to about 1200 Da. In some embodiments, the polyethylene glycol has a number average molecular weight of about 120, about 160, or about 200 to about 300, about 400, about 500, about 600, about 700, or about 800 Da. In some embodiments, the nucleic acid stabilizer comprise PEG 400. In some embodiments, a herein described nanoparticle composition comprises a polyethylene glycol (such as PEG400) and a nucleic acid payload (such as mRNA, guide RNA, and/or siRNA).

In one aspect, disclosed herein is a method of making a nanoparticle composition that comprises a nucleic acid stabilizer. The nucleic acid stabilizer (e.g., polyethylene glycol) can be added into the nanoparticle composition before, concurrently, or after with addition of nucleic acid payload. In some embodiments, the nucleic acid stabilizer is added to the nanoparticle composition concurrently with the nucleic acid. In some embodiments, the nucleic acid stabilizer is pre-mixed with the nucleic acid payload. In some embodiments, the nucleic acid stabilizer is combined with the nucleic acid payload in a buffer solution. In some embodiments, the nucleic acid stabilizer (such as PEG400) is combined with a nucleic acid payload (such as mRNA, guide RNA, and/or siRNA) in a buffer solution. In some embodiments, the nucleic acid stabilizer (such as PEG400) is mixed with a nucleic acid payload in a buffer solution. In some embodiments, the nucleic acid stabilizer (such as PEG400) is added into a buffer that comprises the nucleic acid payload. In some embodiments, adding the nucleic acid stabilizer such as PEG-400 along with the RNA cargo to the drug substance buffer before lipid mixing can allow it to incorporate into the core of the LNP, where it is expected to confer several advantages: 1) increase long-term stability both at 2-8° C. and at freezing temperature such as −20, −40 and −80° C. by providing cushion to the RNA core, 2) improve RNA encapsulation (also known to those skilled in the art as “entrapment”) efficiency, and 3) preserve RNA potency and prevent nicking/degradation. In some embodiments, the nucleic acid stabilizer (such as PEG400) is added into the nanoparticle composition after the addition of the lipids and the nucleic acid payload. In some embodiments, the nucleic acid stabilizer (such as PEG400) is added into the nanoparticle composition after formulation and before freezing. In some embodiments, the method of making a nanoparticle composition comprises collecting or buffer exchanging LNPs into a drug product or freezing buffer solution containing the target amount of a nucleic acid stabilizer such as PEG400. In some embodiments, adding a nucleic acid stabilizer such as PEG400 in the freezing buffer solution as well as in the drug substance buffer can increase long-term stability both at 2-8° C. and at freezing temperature such as −20, −40 and −80° C. by providing cushion to the RNA core and preserve RNA potency and prevent nicking/degradation.

In some embodiments, the nucleic acid stabilizer (such as PEG400) is present in the described nanoparticle composition in an amount of about 0.001% to about 50% by total weight. In some embodiments, the nucleic acid stabilizer (such as PEG400) is present in the nanoparticle composition in an amount of about 0.01% to about 20% by total weight. In some embodiments, the nucleic acid stabilizer (such as PEG400) is present in the nanoparticle composition in an amount of about 0.01% to about 5% by total weight. In some embodiments, the nucleic acid stabilizer (such as PEG400) is present in the nanoparticle composition in an amount up to about 15% by total weight. In some embodiments, the nucleic acid stabilizer is present in the nanoparticle composition at about 0.01%, about 0.05%, about 0.1%, about 0.15%, or about 0.2% to about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0% or about 2.0% by total weight. In some embodiments, the nucleic acid stabilizer is present in the nanoparticle composition within a range of about 0.01%, 0.25%, about 0.5%, about 0.75%, about 1%, or about 5% to about 7.5%, about 10%, about 15%, or about 20% by total weight. In some embodiments, the nucleic acid stabilizer is present in the nanoparticle composition in an amount of about 0.01% to about 0.5%, 0.01% to about 2%, 0.01% to about 3%, about 0.2% to about 0.8%, about 0.4% to about 0.6%, about 0.6% to about 0.8%, about 0.5% to about 1.0%, about 0.5% to about 2.0%, about 1% to about 2%, about 1% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 2.5% to about 7.5%, about 7.5% to about 12.5%, about 12.5% to about 17.5%, or about 15% to about 25% by total weight. In some embodiments, the nucleic acid stabilizer is present in the nanoparticle composition in an amount of at least 0.1%, at least 0.2%, at least 0.25%, at least 0.5%, at least 0.75%, at least 1%, at least 2%, at least 5%, at least 10%, or at least 15% by total weight. In some embodiments, the nucleic acid stabilizer is present in the nanoparticle composition in an amount of at most 0.25%, at most 0.5%, at most 0.75%, at most 1%, at most 2%, at most 5%, at most 7.5%, at most 10%, at most 15%, at most 20%, at most 25%, or at most 30% by total weight. In some embodiments, the nucleic acid stabilizer is present in the nanoparticle composition in an amount of about 0.25%, about 0.5%, about 0.75%, about 5%, about 10%, or about 15% by total weight.

In some embodiments, adding the nucleic acid stabilizer (such as PEG-400) to the drug substance buffer prior to or co-mixed with nucleic acid payload, the w/w % in the non-RNA containing drug substance buffer is higher than the target w/w % in the final RNA containing drug substance buffer, such that eventual dilution with RNA dilutes the nucleic acid stabilizer % to the target w/w %. In some embodiments, the nucleic acid stabilizer (e.g., PEG-400) is trapped within the core of the particle at an amount proportional to its weight percentage in the drug substance buffer.

Other Lipids

In some embodiments, the disclosed LNP compositions comprise a helper lipid. In some embodiments, the disclosed LNP compositions comprise a neutral lipid. In some embodiments, the disclosed LNP compositions comprise a stealth lipid. In some embodiments, the disclosed LNP compositions comprises additional lipids.

“Helper lipids” can refer to lipids that enhance transfection (e.g. transfection of the nanoparticle including the biologically active agent). The mechanism by which the helper lipid enhances transfection includes enhancing particle stability. In some embodiments, the helper lipid enhances membrane fusogenicity. In some embodiments, the helper lipid is a neutral lipid.

“Stealth lipids” can refer to lipids that alter the length of time the nanoparticles can exist in vivo {e.g., in the blood). Stealth lipids can assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the LNP. Stealth lipids suitable for use in a lipid composition of the disclosure can include, but are not limited to, stealth lipids having a hydrophilic head group linked to a lipid moiety. Stealth lipids suitable for use in a lipid composition of the present disclosure and information about the biochemistry of such lipids can be found in Romberg et al, Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and Hoekstra et al, Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG-lipids are disclosed, e.g., in WO 2006/007712.

In some embodiments, the stealth lipid is a PEG-lipid. In one embodiment, the hydrophilic head group of stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyaminoacids and poly N-(2-hydroxypropyl)methacrylamide]. Stealth lipids can comprise a lipid moiety. In some embodiments, the lipid moiety of the stealth lipid may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups.

LNP Formulations

The LNPs described herein can be designed for one or more specific applications or targets. The elements of a nanoparticle composition can be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, and availability. Similarly, the particular formulation of a nanoparticle composition may be selected for the particular application or target. Exemplary LNP formulations include, but are not limited to, the formulations described in Tables 4-6, 8-15 and 17. Exemplary LNP formulations also include compositions comprising an amino lipid of Formula (I), (I*), (Ia) (Ib), (II), (II*) and a PEG lipid of Formula (III) and (III*).

The described LNP formulations can be designed for one or more specific applications or targets. For example, a nanoparticle composition may be designed to deliver a therapeutic agent such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal's body. Physiochemical properties of nanoparticle compositions may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs. The therapeutic agent included in a nanoparticle composition may also be selected based on the desired delivery target or targets. For example, a therapeutic agent may be selected for a particular indication, condition, disease, or disorder and/or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery). In certain embodiments, a nanoparticle composition may include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest. Such a composition may be designed to be specifically delivered to a particular organ.

The amount of a therapeutic agent in an LNP composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition. For example, the amount of an RNA comprised in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to a therapeutic agent in a nanoparticle composition may be from about 5:1 to about 60:1, such as about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt/wt ratio of the lipid component to a therapeutic agent may be from about 10:1 to about 40:1. In certain embodiments, the wt/wt ratio is about 20:1. The amount of a therapeutic agent in a nanoparticle composition can be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

In some embodiments, an LNP composition comprises one or more nucleic acids such as RNAs. In some embodiments, the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N/P ratio. The N/P ratio can be selected from about 1 to about 30. The N/P ratio can be selected from about 2 to about 10. In some embodiments, the N/P ratio is from about 0.1 to about 50. In some embodiments, the N/P ratio is from about 2 to about 8. In some embodiments, the N/P ratio is from about 2 to about 15, from about 2 to about 10, from about 2 to about 8, from about 2 to about 6, from about 3 to about 15, from about 3 to about 10, from about 3 to about 8, from about 3 to about 6, from about 4 to about 15, from about 4 to about 10, from about 4 to about 8, or from about 4 to about 6. In some embodiments, the N/P ratio is from about 2 to about 8. In some embodiments, the N/P ratio is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, or about 6.5. In some embodiments, the N/P ratio is from about 4 to about 6. In some embodiments, the N/P ratio is about 4, about 4.5, about 5, about 5.5, or about 6.

In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 70%, from about 70% to about 90%, or from about 90% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 95%. In some embodiments, a nucleic acid (e.g., RNA) entrapment efficiency of a nanoparticle described herein is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, a nucleic acid entrapment efficiency of a nanoparticle described herein is from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 95% to about 100%, from about 90% to about 99%, or from about 95% to about 99%. In some embodiments, a nucleic acid entrapment efficiency of a nanoparticle described herein is from about 90% to about 99%,

In some embodiments, nanoparticles described herein have a median diameter of about 10 nm to about 500 nm. In some embodiments, the median diameter of the nanoparticles described herein is from about 50 nm to about 150 nm, from about 60 nm to about 140 nm, from about 70 nm to about 130 nm, from about 80 nm to about 120 nm, or from about 90 nm to about 110 nm. In some embodiments, the median diameter of the nanoparticles described herein is about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, or about 120 nm. Particle size and particle size distribution of the nanoparticles can be measured by light scattering using, for example, a Zetasizer Ultra ZSU 5700 (Malvern, USA). In some embodiments, the particle size distribution is unimodal.

Preparation of LNP Formulation

In one aspect, described in the present disclosure are processes for making LNP compositions.

A process for making lipid nanoparticles can comprise several general steps: (i) providing a first solution, such as citrate or phosphate buffer, comprising one or more nucleic acid molecular entities in a first reservoir; (ii) providing a second solution comprising one or more lipids and an organic solvent, such as an alcohol (e.g., ethanol) in a second reservoir; and (iii) mixing the first solution with the second solution. The first reservoir is optionally in fluid communication with the second reservoir.

In some embodiments, disclosed herein is a method of preparing a formulation comprising lipid nanoparticles, wherein the nanoparticles comprise (i) one or more nucleic acid molecular entities, (ii) an amino lipid, and (iii) one or more lipids selected from a structural lipid, a neutral lipid, and a PEG-lipid. In some embodiments, the method comprises (a) combining a first faction of the amino lipid with the one or more nucleic acid molecular entities in a first solution, wherein the first fraction comprises 0.1 mol % to 99 mol % of the total amino lipid; (a) combining the remaining of the amino lipid with the one or more lipids selected from a structural lipid, a neutral lipid, and a PEG-lipid in a second solution; (c) mixing the first solution and the second solution, thereby producing the lipid nanoparticles. In some embodiments, the first fraction of the amino lipid is configured to neutralize between 0.1-99% of the phosphate (on an N:P basis) in the one or more nucleic acid molecular entities, or any numbers or ranges therebetween. In some embodiments, the first fraction of the amino lipid is configured to neutralize between 0.5-90% of the phosphate (on an N:P basis) in the one or more nucleic acid molecular entities. In some embodiments, the first fraction of the amino lipid is configured to neutralize about 5 to 90%, 10 to 75%, 25 to 50%, 25 to 75%, 50 to 75%, or 50 to 90% of the phosphate (on an N:P basis) in the one or more nucleic acid molecular entities. In some embodiments, the first fraction of the amino lipid is configured to neutralize about 10%, 15%, 25%, 50%, or 75% of the phosphate (on an N:P basis) in the one or more nucleic acid molecular entities. In some embodiments, the first solution is an aqueous buffer solution, e.g., a citrate buffer. In some embodiments, the first solution further comprises a nucleic acid stabilizer, e.g., PEG 400. In some embodiments, the first solution and the second solution are mixed in an inline mixer.

The process can optionally comprise one or more dilution steps, one or more incubation steps, one or more buffer exchange steps, one or more concentration steps, and/or one or more filtrations steps. In some embodiments, the dilution step involves dilution by adding a dilution buffer. In some embodiments, the dilution step involves dilution with aqueous buffer (e.g. citrate buffer or pure water) e.g., using a pumping apparatus (e.g. a peristaltic pump). In some embodiments, the dilution buffer is an organic solution such as alcohol. The dilution step can comprise a dilution that is 1 to 20 times of the initial volume, or any numbers or ranges therebetween. In some embodiments, the dilution step comprises a dilution that is 1 to 10 times of the initial volume. In some embodiments, the dilution step is followed by the buffer exchange step or the incubation step.

The incubation step comprises allowing a solution from the mixing step to stand in a vessel for about 0 to about 100 hours at about room temperature and optionally protected from light. In some embodiments, the incubation step runs from 0 to 24 hours, 1 minute to 2 hours, or 1 minute to 60 minutes. In some embodiments, the incubation step runs from 1 minutes to 120 minutes. In some embodiments, the incubation step is followed by the buffer exchange step. In some embodiments, the incubation step follows the buffer exchange step.

In some embodiments, the buffer exchange step comprises a solvent exchange that results in a higher concentration of phosphate buffered saline (PBS) buffer. In some embodiments, the buffer exchange step comprises removing all or a portion of organic solvent. In some embodiments, the buffer exchange step comprises dialysis through a suitable membrane (e.g. 10,000 mwc snakeskin membrane). In some embodiments, the buffer exchange step comprises filtration such as tangential flow filtration (TFF)). In some embodiments, the buffer exchange step comprises chromatography such as using a desalting column, e.g., PD10 column. In some embodiments, the buffer exchange step comprises ultrafiltration. Ultrafiltration comprises concentration of the diluted solution followed by diafiltration, e.g., using a suitable pumping system (e.g. pumping apparatus such as a peristaltic pump or equivalent thereof) in conjunction with a suitable ultrafiltration membrane (e.g. GE Hollow fiber cartridges or equivalent).

In some embodiments, the mixing step provides a clear single phase. In some embodiments, after the mixing step, the organic solvent is removed to provide a suspension of particles, wherein the one or more nucleic acid molecular entities are encapsulated by the lipid(s). The selection of an organic solvent can involve consideration of solvent polarity and the ease with which the solvent can be removed at the later stages of particle formation. The organic solvent, which can serve as a solubilizing agent, can be in an amount sufficient to provide a clear single phase mixture of the one or more nucleic acid molecular entities and lipid(s). The organic solvent may be selected from one or more (e.g., two) of chloroform, dichloromethane, diethylether, cyclohexane, cyclopentane, benzene, toluene, methanol, and other aliphatic alcohols (e.g. C1 to C8) such as ethanol, propanol, isopropanol, butanol, tert-butanol, iso-butanol, pentanol and hexanol. The methods used to remove the organic solvent can involve diafiltration or dialysis or evaporation at reduced pressures or blowing a stream of inert gas (e.g. nitrogen or argon) across the mixture.

In some embodiments, the method further comprises adding nonlipid polycations which are useful to effect the transformation of cells using the present compositions. Examples of suitable nonlipid polycations include, but are limited to, hexadimethrine bromide (sold under the brand name POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts of hexadimethrine. Other suitable polycations include, e.g., salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine and polyethyleneimine. In certain embodiments, the formation of the lipid nanoparticles can be carried out either in a mono-phase system (e.g. a Bligh and Dyer monophase or similar mixture of aqueous and organic solvents) or in a two-phase system with suitable mixing.

The lipid nanoparticles can be formed in a mono- or a bi-phase system. In some embodiments, in a mono-phase system, the amino lipid(s) and one or more nucleic acid molecular entities are each dissolved in a volume of the mono-phase mixture. Combining the two solutions provides a single mixture in which the complexes form. In some embodiments, in a bi-phase system, the amino lipids bind to the one or more nucleic acid molecular entities (which is present in the aqueous phase), and thus increasing the solubility in organic phase.

In some embodiments, the lipid nanoparticles are prepared in an apparatus comprising a first reservoir for holding an aqueous solution and a second reservoir for holding an organic lipid solution. In some embodiments, the apparatus comprises additional reservoirs for holding an aqueous solution (such as for a portion of the one or more nucleic acid molecular entities) and/or an organic solution. The apparatus can include a pump mechanism configured to pump the aqueous and the organic lipid solutions into a mixing region or mixing chamber at substantially equal flow rates. In some embodiments, the mixing region or mixing chamber comprises a T coupling or equivalent thereof, which allows the aqueous and organic fluid streams to combine as input into the T connector and the resulting combined aqueous and organic solutions to exit out of the T connector into a collection reservoir or equivalent thereof.

In some embodiments, the first solution comprises an aqueous buffer. In some embodiments, the first solution comprises a mixture of an aqueous buffer mixed with an organic solvent. In some embodiments, the organic solvent present in the aqueous buffer is ethanol. In some embodiments, the second solution comprises a mixture of an aqueous buffer mixed with an organic solvent. In some embodiments, the second solution comprises ethanol. In some embodiments, the second solution comprises ethanol and water. In some embodiments, the ethanol percentage in the aqueous buffer ranges from 0.1% to 50%, or any numbers or ranges therebetween. In some embodiments, the dilution buffer comprises an aqueous buffer. In some embodiments, the dilution buffer comprises an organic solvent. In some embodiments, the dilution buffer comprises ethanol and water. In some embodiments, the dilution buffer comprises 10% to 20% of ethanol in PBS buffer.

In some embodiments, the mixing comprises laminar mixing, vortex mixing, turbulent mixing, or a combination thereof. In some embodiments, the mixing comprises cross-mixing. In some embodiments, the mixing comprises inline mixing. In some embodiments, the mixing comprises introducing at least a portion of the first solution through a first inlet channel and at least a portion of the second solution through a second inlet channel, and wherein an angle between the first inlet channel and the second inlet channel is from about 0 to 180 degrees. In some embodiments, the angle between the first inlet channel and the second inlet channel is from about 15 to 180 degrees, from about 30 to 180 degrees, from about 45 to 180 degrees, from about 60 to 180 degrees, from about 90 to 180 degrees, or any numbers or ranges therebetween. In some embodiments, the mixing comprises introducing a portion of the first solution through a third inlet channel. The mixing step can take place by any number of methods, e.g., by mechanical means such as a vortex mixer. In some embodiments, the mixing step comprises inline mixing.

In some embodiments, a method of making a formulation comprising the herein-described nanoparticles comprises a filtration step. In some embodiments, a method of making a formulation comprising the herein-described nanoparticles comprises buffer exchange. In some embodiments, the buffer exchange comprises dialysis, chromatography, or tangential flow filtration (TFF).

In one aspect, disclosed herein are pharmaceutical compositions comprising one or more described particle compositions. For example, a pharmaceutical composition can include one or more LNP compositions including one or more different payloads. Pharmaceutical compositions can further include one or more pharmaceutically acceptable excipients, carrier, or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Excipients or carriers can include any ingredient other than the compound(s) of the disclosure, the other lipid component(s) and the payload. An excipient may impart either a functional (e.g. drug release rate controlling) and/or a nonfunctional (e.g. processing aid or diluent) characteristic to the formulations. The choice of excipient and carrier can depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. Parenteral formulations are typically aqueous or oily solutions or suspensions. Excipients or carrier such as sugars (including but not restricted to glucose, mannitol, sorbitol, etc.), salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9) can be used. In some embodiments, the LNP compositions can be formulated with a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water (WFI).

In some embodiments, the excipient or carrier can make up greater than 50% of the total mass or volume of a pharmaceutical composition comprising a nanoparticle composition. For example, the excipient or carrier can make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical composition. In some embodiments, a pharmaceutically acceptable excipient or carrier is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, a pharmaceutical composition can comprise between 0.1% and 100% (wt/wt) of one or more nanoparticle compositions. In certain embodiments, the nanoparticle compositions and/or pharmaceutical compositions are refrigerated or frozen for storage and/or shipment (e.g., being stored at a temperature of 4° C. or lower, such as a temperature between about −150° C. and about 0° C. or between about −80° C. and about −20° C. In some embodiments, the nanoparticle compositions and/or pharmaceutical compositions are refrigerated or frozen at about −5° C., −10° C., −15° C., −20° C., −25° C., −30° C., −40° C., −50° C., −60° C., −70° C., −80° C., −90° C., −130° C., or −150° C.

The described LNP compositions and/or pharmaceutical compositions can be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery of the payload to one or more particular cells, tissues, organs, or systems or groups thereof. In some embodiments, the subject is a mammal such as human. In some embodiments, the subject is non-human primates or mammals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and/or rats.

A pharmaceutical composition including one or more nanoparticle compositions may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and/or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., nanoparticle composition). The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. Pharmaceutical compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and/or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

In some embodiments, the pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more payloads. In some embodiments, the pharmaceutical composition comprises two distinct payloads, such guide RNA and mRNA. The guide RNA and mRNA can be located in the same LNP composition, or they can be located at separate LNP compositions. For example, a pharmaceutical composition can comprise two distinct LNP compositions, one comprising a guide RNA payload and the other comprising an mRNA payload. For another example, a pharmaceutical composition can comprise two distinct LNP compositions, one comprising a guide RNA (or mRNA) payload and the other comprising both an mRNA payload and a guide RNA payload. For yet another example, a pharmaceutical composition can comprise one LNP composition, which comprising an mRNA payload and a guide RNA payload. In some embodiments, the pharmaceutical composition comprises two or more distinct LNP compositions. In some embodiments, the two or more distinct LNP compositions are present in the pharmaceutical composition such that the mRNA molecule(s) and the guide RNA molecule(s) are at a mole or weight ratio described herein.

The gRNA and mRNA payloads can be present in the pharmaceutical composition at various molar or weight ratios. For example, the gRNA to mRNA ratio in the pharmaceutical composition can be from 0.01 to 100 by weight, and/or any value therebetween. For example, the gRNA to mRNA ratio in the pharmaceutical composition can be from 0.01 to 100 by mole, and/or any value therebetween. In some embodiments, the ratio of gRNA to mRNA in the pharmaceutical composition is from about 1 to about 50 by weight or by mole, and/or any value therebetween. In some embodiments, the ratio of gRNA to mRNA in the pharmaceutical composition is from about 0.1 to about 10 by weight or by mole, and/or any value therebetween. In some embodiments, the ratio of gRNA to mRNA in the pharmaceutical composition is from about 0.2 to about 5, from about 0.25 to about 4, from about 0.3 to about 3, or from about 0.5 to about 2 by weight. In some embodiments, the ratio of gRNA to mRNA in the pharmaceutical composition is from about 0.2 to about 5, from about 0.25 to about 4, from about 0.3 to about 3, or from about 0.5 to about 2 by mole. In some embodiments, the gRNA to mRNA ratio in the pharmaceutical composition is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight. In some embodiments, the gRNA to mRNA ratio in the pharmaceutical composition is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by mole. In some embodiments, the mRNA to gRNA ratio in the pharmaceutical composition is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by weight. In some embodiments, the mRNA to gRNA ratio in the pharmaceutical composition is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 by mole. In some embodiments, the gRNA to mRNA ratio in the pharmaceutical composition is about 1:1 by weight. In some embodiments, the gRNA to mRNA ratio in the pharmaceutical composition is about 1:1 by mole.

In some embodiments, the gRNA in the pharmaceutical composition targets a disease-causing gene that is produced in the hepatocytes. In some embodiments, the target cells of the disclosed compositions are selected from hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells. In some embodiments, the pharmaceutical composition comprises more than one guide RNA. For example, the pharmaceutical composition can comprise 2, 3, 4, 5, or more distinct guide RNAs. In some embodiments, the pharmaceutical composition comprises two guide RNA molecules. In some embodiments, the pharmaceutical composition comprises one mRNA and two or more guide RNA molecules. In some embodiments, the two or more guide RNA molecules target the same disease-causing gene. In some embodiments, the two or more guide RNA molecules target different genes. In some specific embodiments, the two or more guide RNA molecules target two separate disease-causing genes of interest produced in the hepatocytes. In some embodiments, the gRNA is a sgRNA. In some embodiments, the gRNA is a dgRNA.

IV. Method of Use

The LNP compositions and pharmaceutical compositions disclosed herein can be used in methods for gene editing, both in vivo and in vitro. In some embodiments, the methods comprise contacting a cell with an LNP composition or a pharmaceutical composition described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a liver cell. In certain embodiments, the cell is a human liver cell. In some embodiments, the liver cell is a hepatocyte. In some embodiments, the hepatocyte is a human hepatocyte. In some embodiments, the liver cell is a stem cell. In some embodiments, the human liver cell is a liver sinusoidal endothelial cell (LSEC). In some embodiments, the human liver cell is a Kupffer cell. In some embodiments, the human liver cell is a hepatic stellate cell. In some embodiments, the human liver cell is a tumor cell. In some embodiments, the human liver cell is a liver stem cell. In some embodiments, the cell comprises ApoE-binding receptors. In some embodiments, engineered cells are provided; for example an engineered cell can be derived from any one of the cell types as described herein. Such engineered cells can be produced according to the methods described herein. In some embodiments, the engineered cell resides within a tissue or organ, e.g., a liver within a subject.

In some embodiments, the cell comprises a modification, for example an insertion or deletion (“indel”) or substitution of nucleotides in a target sequence. In some embodiments, the modification comprises an insertion of 1, 2, 3, 4 or 5 or more nucleotides in a target sequence. In some embodiments, the modification comprises an insertion of either 1 or 2 nucleotides in a target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a deletion of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification comprises an indel which results in a frameshift mutation in a target sequence. In some embodiments, the modification comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a substitution of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from the incorporation of a template nucleic acid, for example any of the template nucleic acids described herein.

In some embodiments, the method comprises contacting a population of cells, such as a population of engineered cells. In some embodiments, the population of cells comprises engineered cells cultured in vitro. In some embodiments, the population resides within a tissue or organ, e.g., a liver within a subject. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% or more of the cells within the population is engineered. In certain embodiments, a method disclosed herein results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% editing efficiency (or “percent editing”), defined by detection of indels. In other embodiments, a method disclosed herein, results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% DNA modification efficiency, defined by detecting a change in sequence, whether by insertion, deletion, substitution or otherwise. In certain embodiments, a method disclosed herein results in an editing efficiency level or a DNA modification efficiency level of between about 5% to about 100%, about 10% to about 50%, about 20 to about 100%, about 20 to about 80%, about 40 to about 100%, or about 40 to about 80%.

In some embodiments, cells within the population comprise a modification. In some embodiments, the modification comprises an insertion of 1, 2, 3, 4 or 5 or more nucleotides in a target sequence. In some embodiments, the modification comprises an insertion of either 1 or 2 nucleotides in a target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a deletion of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification comprises an indel which results in a frameshift mutation in a target sequence. In some embodiments, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the engineered cells in the population comprise a frameshift mutation.

In some embodiments, the LNP compositions can be used to edit a gene resulting in a gene knockout. In some embodiments, the LNP compositions can be used to edit a gene resulting in a gene correction. In some embodiments, the LNP compositions can be used to edit a cell resulting in gene insertion. In some embodiments, disclosed are methods for silencing expression of a target gene in a cell. In some embodiments, the method comprises contacting a cell comprising an expressed target gene with an LNP composition or a pharmaceutical composition described herein under conditions whereby the gRNA enters the cell and silences the expression of the target gene within the cell. In certain embodiments, the cell is in a mammal, such as a human. In some embodiments, the method for silencing expression of a target gene comprises administering to the mammal a therapeutically effective amount of an LNP composition or pharmaceutical composition comprising one or more gRNAs described herein. In some embodiments, the administration of the LNP composition or pharmaceutical composition comprising one or more gRNAs described herein reduces target RNA levels by at least about 5%, 100%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range therein) relative to RNA levels detected in the absence of the gRNA (e.g., buffer control or irrelevant gRNA control). In some embodiments, the administration of the LNP composition or pharmaceutical composition comprising one or more gRNAs reduces target RNA levels for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days or more (or any range therein) relative to a negative control such as, e.g., a buffer control or an irrelevant non-targeting gRNA control.

In some embodiments, administration of the LNP composition or pharmaceutical composition may result in gene editing which results in persistent response. For example, administration can result in a duration of response of a day, a month, a year, or longer. As used herein, “duration of response” means that, after cells have been edited using an LNP composition or pharmaceutical composition disclosed herein, the resulting modification is still present for a certain period of time after the administration. The modification can be detected by measuring target protein levels. The modification can be detected by detecting the target DNA. In some embodiments, the duration of response can be at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, or at least 1 year. In certain embodiments, the duration of response may be about 26 weeks. In some embodiments, the duration of response can be at least 5 years or at least 10 years. A persistent response is detectable after at least 6 months, either by measuring target protein levels or by detection of the target DNA.

In one aspect, disclosed herein are methods for treating a disease or condition, including raising an immune response to an immunogen, in a subject. In one embodiment, the disease or condition is treatable by administering the payload. In some embodiments, the disease or condition is characterized by missing or aberrant protein or polypeptide activity. For example, an LNP composition comprising an mRNA encoding a missing or aberrant polypeptide may be administered or delivered to a cell. Subsequent translation of the mRNA may produce the polypeptide, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide. A payload included in an LNP composition may also be capable of altering the rate of transcription of a given species, thereby affecting gene expression.

Diseases and/or conditions characterized by dysfunctional or aberrant protein or polypeptide activity can include, but are not limited to, rare diseases, infectious diseases (as both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases. Multiple diseases and/or conditions may be characterized by missing (or substantially diminished such that proper protein function does not occur) protein activity. Such proteins may not be present, or they may be essentially non-functional. A specific example of a dysfunctional protein is the missense mutation variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some embodiments, the present disclosure provides a method for treating such diseases and/or conditions in a subject by administering an LNP composition or pharmaceutical composition comprising an RNA payload, wherein the RNA can be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes an aberrant protein activity present in the cell of the subject.

Further embodiments of the present disclosure, for example, the LNP compositions (including the various components of the LNP composition), the pharmaceutical compositions, the methods of preparing the compositions, the methods of gene editing, the methods of treatment, and other methods of using the compositions, can be found in various WO and US patent/patent application publications, including U.S. Pat. No. 9,868,692B2, US20180353434A1, U.S. Pat. No. 8,492,359B2, U.S. Pat. No. 9,878,042B2, US20180148719, U.S. Pat. No. 9,687,448B2, U.S. Pat. No. 9,415,109B2, U.S. Pat. No. 7,858,117B2, U.S. Pat. No. 9,404,127B2, U.S. Pat. No. 9,504,651, US20070087045A1, US20180092848A1, US20170273907A1, US20180147298A1, WO2019067992A1, WO2019067999A1, WO2018185241, WO 2018170306A1, WO 2019046809A1, WO2017173054A1, WO2015095340, WO2016197133A1, and WO2018191750, all of which are hereby incorporated by reference in their entirety. Further description about the LNP compositions, the pharmaceutical compositions, and the methods are described in WO2016153012 A1, WO2018062413 A1, WO2019027055 A1, WO2017/173054A1, WO2015/095340A1, WO2013063468A1, WO2010054401A1, and WO 2018/170306, all of which are hereby incorporated by reference in their entirety.

The present invention is further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

EXAMPLES

Example 1. Synthesis of VL401 and VL469

To a stirred solution of 8 (700 mg, 1.06 mmol) in THE (4 mL) was added TBAF (0.46 mL, 1.593 mmol) at ice temperature and the reaction mixture was stirred at RT for 4 h. The reaction mixture was diluted with water (10 mL) and the product was extracted with CH2Cl2 (2×50 mL); the organic layer was separated and evaporated to get the crude. Crude material was purified by silica gel column eluting with 50% EtOAc in pet-ether yielded 9 (300 mg, 52%) as a colourless gummy liquid.

Compound VL469. Compound 5A is reacted with 4-nitropehnyl chloroformate in dichloromethane in the presence of pyridine and DMAP. The 4-nitropehnyl carbonate thus formed is then reacted with compound 7 to obtain compound VL469.

Example 2. Synthesis of VL403

Example 3. Synthesis of VL404

Example 4. Synthesis of VL406 and VL422

Compound 21A. To a solution of compound 21 (1.0 g, 1.99 mmol) in CH2Cl2 (10 mL) were added compound 23 (517.4 mg, 1.19 mmol) followed by TEA (2.8 mL, 19.98 mmol) and DMAP (244 mg, 1.98 mmol) and the reaction mixture was stirred at RT for 18 h. After completion, the reaction mixture was diluted with water (20 mL) and the product was extracted with CH2Cl2 (2×50 mL), washed with brine (20 mL) and dried over Na2SO4. The crude product was purified on silica column by eluting with 50% EtOAc in pet-ether to obtain 21A (220 mg, 14%) as a gummy liquid.

Synthesis of VL421. To a stirred solution of compound 21 (1.0 g, 1.83 mmol) in CH2Cl2 (10 mL) were added compound 24 (1.09 g, 1.83 mmol) followed by pyridine (0.37 mL, 3.54 mmol) and DMAP (67 mg, 0.55 mmol) and the reaction was stirred at RT for 1 h. Reaction mixture was diluted with water (20 mL) and extracted with DCM (2×50 mL), washed with brine solution (20 mL) and dried on Na2SO4. The crude was purified on silica column by eluting with 30% EtOAc in pet-ether to get 25 (480 mg, 30%) as a gummy liquid.

Compound 102 is prepared from desired mPEG-amine 101 by Michael addition of the PEG-amine to tert-butyl acrylate. The mono adduct 102 and diadduct 103 are separated by column chromatography. Acid treatment of 102 affords the acid 105. Acetylation of 102 with acetic anhydride followed by acid treatment affords the carboxylic acid 104. Treatment of 103 with acid affords the dicarboxylic acid 106.

Compound 108 is prepared from desired mPEG-OH 107 by Michael addition of the mPEG-OH to tert-butyl acrylate. Treatment of compound 108 with acid affords the carboxylic acid 109.

Activation of the carboxylic acid 109 using peptide coupling agent HBTU in the presence of DIEAN and DAMP followed by addition of the alcohol 110 affords the PEG-lipid VP101. Similarly, addition of the alcohols 111-118 to the activated carboxylic acid afford PEG-lipids VP101-VP110.

Activation of the carboxylic acid 104 using peptide coupling agent HBTU in the presence of DIEAN and DAMP followed by addition of the alcohol 110 affords the PEG-lipid VP111. Similarly, addition of the alcohols 111-118 to the activated carboxylic acid afford PEG-lipids VP112-VP119.

Michael addition of the alcohol 110 to tert-butyl acrylate affords the ester 119. Treatment of the ester 119 with acid affords 123. Activation of the carboxylic acid 123 with peptide coupling agent followed by addition of mPEG2000 affords VP120, the racemic compounds. Similarly, starting with chirally pure 110 affords the enantiomers VP121 and VP122. The PEG-lipids VP123-VP131 are similarly prepared starting from corresponding racemic or chirally pure alcohols 111-113.

Activation of the carboxylic acid 123 with peptide coupling agent followed by addition of mPEG2000-NH2 affords VP132, the racemic compounds. Similarly, starting with chirally pure 110 affords the enantiomers VP133 and VP134. The PEG-lipids VP135-VP143 are similarly prepared starting from corresponding racemic or chirally pure alcohols 111-113.

Michael addition of the alcohol 114 to tert-butyl acrylate affords the ester 127. Treatment of the ester 127 with acid affords the carboxylic acid 132. Activation of the carboxylic acid 132 with peptide coupling agent followed by addition of mPEG2000 affords VP144. The PEG-lipids VP145-VP148 are similarly prepared starting from corresponding alcohols 115-118.

Activation of the carboxylic acid 132 with peptide coupling agent followed by addition of mPEG2000-NH2 affords VP149. The PEG-lipids VP150-VP153 are similarly prepared starting from corresponding racemic or chirally pure alcohols 115-118.

To a stirred solution of compound 139 (1.6 g, 7.0 mmol) in THE (16 mL) were added N-hydroxysuccinimide (1.12 g, 9.80 mmol) followed by DCC (1.7 g, 8.40 mmol) at 0° C. and the reaction mixture was stirred at RT for overnight. Reaction mixture filtered and the filtrate was evaporated under reduced pressure to get the crude material. The crude was taken in isopropyl alcohol (10 mL) and heated at 80° C. for 45 min and then cooled to 0° C. and the precipitated solid was filtered and dried to get compound 142 (1.6 g, 74% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 2.83 (s, 4H), 2.61 (t, J=7.2 Hz, 2H), 1.76-1.72 (m, 2H), 1.40-1.25 (m, 20H), 0.89 (t, J=6.4 Hz, 3H).

Example 8. Guide RNA (gRNA) and mRNA for LNP Evaluation

The guide RNAs (gRNA) shown in Table 3 were synthesized under solid phase oligonucleotide synthesis and deprotection conditions using controlled pore glass support and commercially available phosphoramidite monomers and oligonucleotide synthesis reagents (Methods in Molecular Biology, 1993, 20, 81-114; ACS Chem. Biol. 2015, 10, 1181-1187, incorporated herein by reference in its entirety). The deprotected guide RNAs were purified by HPLC and the integrity of each guide RNA was confirmed by mass spectrometric analysis. The observed mass of each guide RNA was conformed to calculated mass.

Single guide RNA (gRNA) used in the studies described in Examples 9-23

G
usu

*The gRNAs were designed to target mouse, rat, monkey and human PCSK9 and ANGPTL3 genes.

#uppercase and lowercase letters in the guide RNA sequence indicate nucleotides carrying 2′-ribo (2′-OH) and 2′-O-methyl (2′-OMe) ribosugar moiety, respectively, and the letter ‘s’ indicates phosphorothioate (PS) linkage.

The term “protospacer,” or “target sequence” and their grammatical equivalents as used herein can refer to a DNA sequence of a target gene. In the native state, a protospacer is adjacent to a PAM (protospacer adjacent motif). The term “spacer” can be the RNA version of the protospacer that binds to the complementary strand of the protospacer. A spacer can be within a guide RNA (gRNA). The site of cleavage by an RNA-guided nuclease is within a protospacer sequence. Please see FIG. 1A for an illustration.

mRNA Encoding SpCas9, CBE, and ABE Proteins

mRNA for SpCas9, CBE, and ABE were produced by different methods well known in the art. One of such methods used herein was in vitro transcription (IVT) using T7 polymerase or additional RNA polymerase variants. Typically, IVT of mRNA uses a linearized DNA template that comprises a T7 polymerase promoter, mRNA coding sequence (CDS), 3′ and 5′ untranslated regions (UTRs), poly A tail, and additional replication and transcription regulatory elements. Prior to IVT, the DNA template was in the form of a plasmid, PCR product, or additional double-stranded DNA construct. A typical IVT reaction includes T7 polymerase, DNA template, RNase inhibitor, cap analog, inorganic pyrophosphatase, and naturally occurring ribonucleotides (rNTPs) such as GTP, ATP, CTP, UTP, or substitutions of natural rNTPs with modified rNTPs such as pseudouridine, N1-methylpseudouridine, 5-methyl cytidine, 5-methoxyuridine, N6-methyl adenosine, and N4-acetylcytidine. The cap analog was a dinucleotide or trinucleotide cap structure with the first initiating nucleotide containing standard 2′-hydroxyl group, 2′—O-methyl group, or additional 2′chemical modification. Cap analog also was added after the IVT reaction using a vaccinia capping enzyme. After IVT, in some cases DNase is added to the transcription mixture to remove DNA template; alternatively, residual DNA was removed by ion exchange column chromatography. Purification and concentration of mRNA were performed with ion exchange chromatography, affinity chromatography, precipitation, ion-pairing reverse-phase chromatography, enzymatic reactions, size exclusion chromatography, and/or tangential flow filtration. Similar IVT and purification process were used to produce mRNA encoding SpCas9, CBE, and ABE; in all cases the DNA template, reaction conditions, and purification parameters were optimized for the specific gene of interest. In some examples, capped and polyadenylated SpCas9 mRNA MS002 was obtained from commercially sources (TriLink, for e.g.). The SpCas9 mRNA MS002 and adenosine base editor (ABE) mRNAs MA002 and MA004 prepared in Verve's laboratory.

The adenosine nucleobase editor mRNA MA002 comprises a fusion protein comprising a polypeptide encoded by the polynucleotide sequence provided below