Compounds and compositions for delivery of therapeutic agents

Abstract

The disclosure features novel lipids and compositions involving the same. Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) include a novel cationic lipid as well as additional lipids such as ionizable lipids, phospholipids, structural lipids, and PEG lipids. Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) further including therapeutic and / or prophylactic agents such as RNA are useful in the delivery of therapeutic and / or prophylactic agents to mammalian cells or organs to, for example, regulate polypeptide, protein, or gene expression.

Description

COMPOUNDS AND COMPOSITIONS FOR DELIVERY OF THERAPEUTICAGENTSRELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Application No. 63 / 508, 196, filed June 14, 2023, the entire content of which is incorporated herein by reference.FIELD OF DISCLOSURE

[0002] The present disclosure provides novel cationic lipids, compositions comprising such lipids, and methods involving lipid nanoparticle compositions to deliver one or more therapeutic and / or prophylactic agents to and / or produce polypeptides in mammalian cells or organs. In addition to a novel cationic lipid, lipid nanoparticle compositions of the disclosure may include one or more ionizable amino lipids, phospholipids including polyunsaturated lipids, PEG lipids, structural lipids, and / or therapeutic and / or prophylactic agents in specific fractions.BACKGROUND

[0003] The effective targeted delivery of biologically active substances such as small molecule drugs, proteins, and nucleic acids represents a continuing medical challenge. In particular, the delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of such species. Thus, there exists a need to develop methods and compositions to facilitate the delivery of therapeutic and / or prophylactic agents such as nucleic acids to cells.

[0004] Lipid-containing nanoparticle compositions, liposomes, and lipoplexes have proven effective as transport vehicles into cells and / or intracellular compartments for biologically active substances such as small molecule drugs, proteins, and nucleic acids. Such compositions generally include one or more ionizable lipids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), and / or lipids containing polyethylene glycol (PEG lipids). Ionizable lipids include, for example, amine-containing lipids that can be readily protonated. Though a variety of such lipid-containing nanoparticle compositions have been demonstrated, improvements in safety, efficacy, and specificity are still lacking.SUMMARY

[0005] In an aspect, the present disclosure provides a compound of Formula (I):or an ionic form or a salt thereof, wherein: q is 1, 2, 3, 4, 5, or 6;Q is -ORqor -NHRq;Rqis H, or C3-6cycloalkyl substituted one or more oxo, -NH2, -NH(C1-6alkyl), or -N(C1-6alkyl)2;T1is (i) C1 -24alkyl optionally substituted with one or more -OH, -C(=O)OH or 3- to 10- membered heterocyclyl, or (ii) -M1-L1-R1;M1is C1-12alkylene optionally substituted with one or more Rm1; each Rm1independently is oxo, halogen, -OH, -O(C1-6alkyl), -NH2, - NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl; or two Rm1, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6-membered heterocycloalkyl;cycloalkylene)-**, wherein * denotes attachment to T1and ** denotes attachment to R1;R1is W1or -U1-V1-W1;U1is C1-6alkylene optionally substituted with one or more oxo, halogen, -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl;V1is *-C(=O)-O-**, *-O-C(=O)-**, *-S-S-**, *-(C6-10arylene)-**, *- 0-(C6-10arylene)-**, *-(C6-10arylene)-O-**, *-NH-(C6-10arylene)-**, *-O-**, or *-(C6-10arylene)-NH-**, wherein * denotes attachment to U1and ** denotes attachment to W1;W1is C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl, wherein the C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl is optionally substituted with one or more Rwl;each Rwlindependently is oxo, halogen, -OH, -O(C1-6alkyl), - NH2-NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl; or two Rwl, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6-membered heterocycloalkyl; b is 1, 2, 3, 4, 5, or 6;L2is -O-C(=O)-** or -C(=O)-O-** , wherein ** denotes attachment to R2;R2ais H, C 1-12 alkyl, C2-12alkenyl, or C2-12alkynyl;R2bis C5 -12alkyl, C5 -12alkenyl, or C 5- 12 alkynyl; c is 1, 2, 3, 4, 5, or 6;L3is -O-C(=O)-** or -C(=O)-O-** , wherein ** denotes attachment to R3;r3 is 0, 1, 2, 3, 4, 5, or 6;R3ais H, V3a, or -U3a-V3a;U3ais -O-C(=O)-** or -C(=O)-O-**, wherein ** denotes attachment to V3a;V3ais C1-12 alkyl, C2-12alkenyl, or C2-12alkynyl;R3bis V3b, or -U3b-V3b;U3bis -O-C(=O)-** or -C(=O)-O-** , wherein ** denotes attachment to V3b; andV3bis C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl.

[0006] In an aspect, the present disclosure provides a method of preparing a lipid nanoparticle formulation (LNP formulation) comprising: i) a nanoprecipitation step, comprising: i-a) mixing a lipid solution comprising a cationic lipid, e.g., a cationic lipid of the present disclosure, an ionizable lipid, a structural lipid, and a phospholipid, with an aqueous buffer solution comprising a buffering agent, thereby forming an intermediate lipid nanoparticle solution (intermediate LNP solution); i-b) adding a diluting solution to the intermediate LNP solution;i-c) holding the intermediate LNP solution for a residence time; i-d) adding a pH-adjusting solution to the intermediate LNP solution, thereby forming a lipid nanoparticle solution (LNP solution); and ii) processing the LNP solution, thereby forming an LNP formulation, wherein the processing comprises: ii-a) adding a PEG lipid solution to the LNP solution.

[0007] In some aspects, the present disclosure provides a pharmaceutical composition comprising a LNP of the present disclosure, e.g., a LNP prepared by a method of the present disclosure.

[0008] In some aspects, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a cell, comprising administering to the cell a LNP of the present disclosure. In some embodiments, the LNP comprises a compound of the present disclosure, e.g., a compound of Formula (I).

[0009] In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a LNP comprising a compound of the present disclosure, e.g., a compound of Formula (I).

[0010] In some aspects, the present disclosure provides a LNP of the present disclosure for use in delivering a therapeutic or prophylactic agent to a cell.

[0011] In some aspects, the present disclosure provides the use of a LNP of the present disclosure in the manufacture of a medicament for delivering a therapeutic and / or prophylactic agent to a cell.

[0012] In some aspects, the present disclosure provides a LNP comprising a compound of the present disclosure, e.g., a compound of Formula (I), for us in delivering a therapeutic and / or prophylactic agent to a cell.

[0013] In some aspects, the present disclosure provides the use of a compound of the present disclosure in the manufacture of a medicament for delivering a therapeutic and / or prophylactic agent to a cell.

[0014] In some aspects, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject, comprising administering to the subject the LNP of the present disclosure.

[0015] In some aspects, the present disclosure provides a LNP of the present disclosure for use in delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject.

[0016] In some aspects, the present disclosure provides the use of a LNP of the present disclosure in the manufacture of a medicament for delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject.

[0017] In some aspects, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject, comprising administering to the subject a LNP comprising a compound of the present disclosure, e.g., a compound of Formula (I).

[0018] In some aspects, the present disclosure provides a LNP comprising a compound of the present disclosure for use in delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject.

[0019] In some aspects, the present disclosure provides the use of a compound of the present disclosure, e.g., a compound of Formula (I), in the manufacture of a medicament for delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject.

[0020] In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder in a subject, comprising administering to the LNP the present disclosure.

[0021] In some aspects, the present disclosure provides a LNP of the present disclosure for use in treating or preventing a disease or disorder in a subject.

[0022] In some aspects, the present disclosure provides the use of a LNP of the present disclosure in the manufacture of a medicament for treating or preventing a disease or disorder in a subject.

[0023] In some aspects, the present disclosure provides a LNP comprising a compound of the present disclosure, e.g., a compound of Formula (I), for use in treating or preventing a disease or disorder in a subject.

[0024] In some aspects, the present disclosure provides the use of a compound of the present disclosure, e.g., a compound of Formula (I), in the manufacture of a medicament for treating or preventing a disease or disorder in a subject.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the presentspecification, including definitions, will control. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting. In the case of conflict between the chemical structures and names of the compounds disclosed herein, the chemical structures will control.

[0026] Other features and advantages of the disclosure will be apparent from the following detailed description and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a flow-chart of an exemplary process used to manufacture LNPs comprising Compound Ref-1, wherein the process comprises the steps of adding PEG lipid following pH adjustment and following nanoprecipitation.

[0028] FIG. 2 is a flow-chart of an exemplary process used to manufacture LNPs comprising Compound Ref-l / DMG, wherein the process comprises the steps of adding PEG lipid following pH adjustment.

[0029] FIG. 3 is a flow-chart of an exemplary process previously used to manufacture LNPs comprising Compound Ref-1.

[0030] FIG. 4A is a chart showing the bioluminescence in rat lung cells as a result of luciferase expression effectuated by LNPs made by the process shown in FIG. 1, wherein various buffers are used in the diluting solution. LNPs manufactured using citrate buffered saline or citrate were efficacious than LNPs manufactured using sodium chloride or water. The t-test analysis compares each buffer against the citrate buffer saline (CBS) group.

[0031] FIG. 4B is a chart showing the expression of mOX40 ligand in rat lung cells effectuated by LNPs made in the process wherein various buffers are used in the diluting fluid. The leftmost dataset (“0”) is for LNPs made in a process shown in FIG. 1, and middle dataset (“1”) and rightmost dataset (“2”) are for LNPs made in a similar process via forming an empty LNP composition first, and then adding mRNA to the empty LNP composition.

[0032] FIG. 5A is a flow-chart showing a process of the present disclosure featuring the step of storing the resultant LNP in a storage buffer comprising 20 mM tris, 50 mM tris, or 100 mM tris.

[0033] FIG. 5B is a diagram showing the change in purity of a sample of mRNA in an LNP produced by a process of the present disclosure, wherein the LNP was stored in a storage buffer comprising 20 mM tris, 50 mM tris, or 100 mM tris. The change in purity of the mRNA was monitored by HPLC. A decrease in the proportion of the sample that appeared in the main peak was indicated of mRNA degradation. Increasing concentrations of tris were correlated withslower purity loss (e.g., slower mRNA degradation as observed by slower disappearance of the main peak).DETAILED DESCRIPTION

[0034] The present disclosure provides novel cationic lipids, e.g., including a central amine moiety and at least one biodegradable group. Without wishing to be bound by theory, the cationic lipids described herein may be advantageously used in lipid nanoparticles (e.g., empty LNPs or loaded LNPs) for the delivery of therapeutic and / or prophylactic agents to mammalian cells or organs. For example, the cationic lipids described herein may be advantageously used in lipid nanoparticles (e.g., empty LNPs or loaded LNPs) for the delivery of therapeutic and / or prophylactic agents to specific mammalian cells or organs. In some embodiments, the cationic lipids described herein may be advantageously used in lipid nanoparticles (e.g., empty LNPs or loaded LNPs) for the delivery of therapeutic and / or prophylactic agents to endothelial cells or the lung.Definitions

[0035] As used herein, the term “alkyl” or “alkyl group” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, or more carbon atoms), which is optionally substituted. The notation “C1-14 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms. The term “alkylene” refers to a divalent alkyl group. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.

[0036] As used herein, the term “alkenyl” or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. The notation “C2-14 alkenyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. In some embodiments, C18alkenyl may include one or more double bonds. A C18alkenyl group including two double bonds may be a linoleyl group. The term “alkenylene” refers to a divalent alkenyl group.Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.

[0037] As used herein, the term “alkynyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, “alkynyl” includes straight chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. In certain embodiments, a straight chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6for straight chain, C3-C6for branched chain). The term “C2- C6” includes alkynyl groups containing two to six carbon atoms. The term “C3- C6” includes alkynyl groups containing three to six carbon atoms. As used herein, “C2-C6alkenylene linker” or “C2-C6alkynylene linker” is intended to include C2, C3, C4, C5or C6chain (linear or branched) divalent unsaturated aliphatic hydrocarbon groups. For example, C2- C6alkenylene linker is intended to include C2, C3, C4, C5and C6alkenylene linker groups. The term “alknylene” refers to a divalent alkynyl group. Unless otherwise specified, an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.

[0038] As used herein, the term “carbocycle” or “carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings. The notation “C3-6carbocycle” means a carbocycle including a single ring having 3-6 carbon atoms. Carbocycles may include one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups.

[0039] As used herein, the term “cycloalkyl” refers to a saturated or partially unsaturated hydrocarbon monocyclic or polycyclic (e.g., fused, bridged, or spiro rings) system having 3 to 30 carbon atoms (e.g., C3-C12, C3-C10, or C3-C8). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,2,3,4-tetrahydronaphthalenyl, and adamantyl. In the case of polycyclic cycloalkyl, only one of the rings in the cycloalkyl needs to be non- aromatic.

[0040] As used herein, the term “heterocycle” or “heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom. Heteroatoms may be, for example, nitrogen, oxygen, or sulfuratoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen membered rings. Heterocycles may include one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocycloalkyl groups include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1, 2,3,6- tetrahydropyridinyl, tetrahydropyranyl, dihydropyranyl, pyranyl, morpholinyl, tetrahydrothiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2. l]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl, l,4-dioxa-8-azaspiro[4.5]decanyl, l,4-dioxaspiro[4.5]decanyl, l-oxaspiro[4.5]decanyl, 1- azaspiro[4.5]decanyl, 3'H-spiro[cyclohexane-l, l'-isobenzofuran]-yl, 7'H-spiro[cyclohexane- l,5'-furo[3,4-b]pyridin]-yl, 3'H-spiro[cyclohexane-l,l'-furo[3,4-c]pyridin]-yl, 3- azabicyclo[3.1.0]hexanyl, 3-azabicyclo[3.1.0]hexan-3-yl, l,4,5,6-tetrahydropyrrolo[3,4- c]pyrazolyl, 3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidinyl, 4,5,6,7-tetrahydro-lH- pyrazolo[3,4-c]pyridinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, 2- azaspiro[3.3]heptanyl, 2-methyl-2-azaspiro[3.3]heptanyl, 2-azaspiro[3.5]nonanyl, 2-methyl-2- azaspiro[3.5]nonanyl, 2-azaspiro[4.5]decanyl, 2-methyl-2-azaspiro[4.5]decanyl, 2-oxa- azaspiro[3 ,4]octanyl, 2-oxa-azaspiro[3 ,4]octan-6-yl, 5,6-dihydro-4H- cyclopenta[b]thiophenyl, and the like. In the case of multicyclic heterocycloalkyl, only one of the rings in the heterocycloalkyl needs to be non-aromatic (e.g., 4, 5,6,7- tetrahydrobenzo[c]isoxazolyl). The term “heterocycloalkyl” as used herein means a non- aromatic heterocycle and may or may not include any double or triple bond. Unless otherwise specified, heterocycles described herein refers to both unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles.

[0041] As used herein, the term “hydroxy” or “hydroxyl” includes groups with an -OH or -O'

[0042] As used herein, the term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

[0043] The term “haloalkyl” or “haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or more halogen atoms, respectively.

[0044] As used herein, the term “alkoxy” or “alkoxyl” includes substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with groups such as alkenyl,alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy and tri chi or om ethoxy .

[0045] As used herein, a “biodegradable group” is a group that may facilitate faster metabolism of a lipid in a mammalian entity. A biodegradable group may be selected from the group consisting of, but is not limited to, -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, - C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group.

[0046] As used herein, an “aryl group” is an optionally substituted carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. The term “arylene” refers to a divalent aryl group. As used herein, a “heteroaryl group” is an optionally substituted heterocyclic group including one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted. Unless otherwise specified, aryl or heteroaryl groups described herein refers to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0047] Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified. Optional substituents may be selected from the group consisting of, but are not limited to, a halogen atom (e.g., a chloride, bromide, fluoride, or iodide group), a carboxylic acid (e.g., -C(O)OH), an alcohol (e.g., a hydroxyl, -OH), an ester (e.g., -C(O)OR or -OC(O)R), an aldehyde (e.g.,-C(O)H), a carbonyl (e.g., -C(O)R, alternatively represented by C=O), an acyl halide (e.g.,-C(O)X, in which X is a halide selected from bromide, fluoride, chloride, and iodide), a carbonate (e.g., -OC(O)OR), an alkoxy (e.g., -OR), an acetal (c.g,-C(OR)2R””, in which each OR are alkoxy groups that can be the same or different and R”” is an alkyl or alkenyl group), a phosphate (e.g., P(O)43-), a thiol (e.g., -SH), a sulfoxide (e.g., -S(O)R), a sulfinic acid (e.g., -S(O)OH), a sulfonic acid (e.g., -S(O)2OH), athial (e.g., -C(S)H), a sulfate (e.g., S(O)42'), a sulfonyl (e.g., -S(O)2-), an amide (e.g., -C(0)NR2, or -N(R)C(O)R), an azido (e.g., -N3), a nitro (e.g., -NO2), a cyano (e.g., -CN), an isocyano (e.g., -NC), an acyloxy (e.g.,-OC(O)R), an amino (e.g, -NR2, -NRH, or -NH2), a carbamoyl (e.g, - OC(O)NR2, -OC(O)NRH, or -OC(O)NH2), a sulfonamide (e.g., -S(O)2NR2, -S(O)2NRH, - S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H, or -N(H)S(O)2H), an alkyl group, an alkenyl group, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any of the preceding, R is an alkyl or alkenyl group, as defined herein. In some embodiments, the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. In some embodiments, a C1-6alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.

[0048] As used herein, the terms “approximately” and “about,” as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). In some embodiments, when used in the context of an amount of a given compound in a lipid component of a LNP, “about” may mean + / - 10% of the recited value. For instance, a LNP including a lipid component having about 40% of a given compound may include 30-50% of the compound.

[0049] As used herein, the term “compound,” is meant to include all isomers and isotopes of the structure depicted. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. In some embodiments, isotopes of hydrogen include tritium and deuterium. Further, a compound, salt, or complex of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.

[0050] As used herein, the term “upon” intends to refer to the time point being after an action happens. For example, “upon administration” refers to the time point being after the action of administration.

[0051] As used herein, the term “contacting” means establishing a physical connection between two or more entities. In some embodiments, contacting a mammalian cell with a LNP means that the mammalian cell and a nanoparticle are made to share a physical connection. Methods of contacting cells with external entities both in vivo and ex vivo are well known in the biological arts. In some embodiments, contacting a LNP and a mammalian cell disposedwithin a mammal may be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve varied amounts of lipid nanoparticles. Moreover, more than one mammalian cell may be contacted by a LNP.

[0052] As used herein, the term “comparable method” refers to a method with comparable parameters or steps, as of the method being compared (e.g., the producing the LNP formulation of the present disclosure). In some embodiments, the “comparable method” is a method with one or more of steps i), i-a), i-b), i-c), i-d), ii), and ii-a) of the method being compared. In some embodiments, the “comparable method” is a method without one or more of steps i), i-a), i-b), i-c), i-d), ii), and ii-a) of the method being compared. In some embodiments, the “comparable method” is a method without one or more of steps ia) and ib) of the method being compared. In some embodiments, the “comparable method” is a method employing a water-soluble salt of a nucleic acid. In some embodiments, the “comparable method” is a method employing an organic solution that does not comprise an organic solvent-soluble nucleic acid. In some embodiments, the “comparable method” is a method comprising processing the lipid nanoparticle prior to administering the lipid nanoparticle formulation.

[0053] As used herein, the term “delivering” means providing an entity to a destination. In some embodiments, delivering a therapeutic and / or prophylactic agent to a subject may involve administering a LNP including the therapeutic and / or prophylactic agent to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of a LNP to a mammal or mammalian cell may involve contacting one or more cells with the lipid nanoparticle.

[0054] As used herein, the term “enhanced delivery” means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10- fold more) of a therapeutic and / or prophylactic agent by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to the level of delivery of a therapeutic and / or prophylactic agent by a control nanoparticle to a target tissue of interest (e.g., MC3, KC2, or DLinDMA). The level of delivery of a nanoparticle to a particular tissue may be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of therapeutic and / or prophylactic agent in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of therapeutic and / or prophylactic agent in a tissue to the amount of total therapeutic and / or prophylactic agent in said tissue. It will be understood thatthe enhanced delivery of a nanoparticle to a target tissue need not be determined in a subject being treated, it may be determined in a surrogate such as an animal model (e.g., a rat model).

[0055] As used herein, the term “specific delivery,” “specifically deliver,” or “specifically delivering” means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3- fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a therapeutic and / or prophylactic agent by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to an off-target tissue (e.g., mammalian spleen). The level of delivery of a nanoparticle to a particular tissue may be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of therapeutic and / or prophylactic agent in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of therapeutic and / or prophylactic agent in a tissue to the amount of total therapeutic and / or prophylactic agent in said tissue. In some embodiments, for renovascular targeting, a therapeutic and / or prophylactic agent is specifically provided to a mammalian kidney as compared to the liver and spleen if 1.5, 2-fold, 3-fold, 5-fold, 10-fold, 15 fold, or 20 fold more therapeutic and / or prophylactic agent per 1 g of tissue is delivered to a kidney compared to that delivered to the liver or spleen following systemic administration of the therapeutic and / or prophylactic agent. It will be understood that the ability of a nanoparticle to specifically deliver to a target tissue need not be determined in a subject being treated, it may be determined in a surrogate such as an animal model (e.g., a rat model).

[0056] As used herein, “encapsulation efficiency” refers to the amount of a therapeutic and / or prophylactic agent that becomes part of a LNP, relative to the initial total amount of therapeutic and / or prophylactic agent used in the preparation of a LNP. In some embodiments, if 97 mg of therapeutic and / or prophylactic agent are encapsulated in a LNP out of a total 100 mg of therapeutic and / or prophylactic agent initially provided to the composition, the encapsulation efficiency may be given as 97%.

[0057] As used herein, “encapsulation”, “encapsulated”, “loaded”, and “associated” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement. As used herein, “encapsulation” or “association” may refer to the process of confining an individual nucleic acid molecule within a nanoparticle and / or establishing a physiochemical relationship between an individual nucleic acid molecule and a nanoparticle. As used herein, an “empty nanoparticle” may refer to a nanoparticle that is substantially free of a therapeutic or prophylactic agent. As used herein, an “empty nanoparticle” may refer to a nanoparticlethat is substantially free of a nucleic acid. As used herein, an “empty nanoparticle” may refer to a nanoparticle that consists substantially of only lipid components.

[0058] As used herein, “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide or protein and / or post-translational modification of a polypeptide or protein.

[0059] As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0060] As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0061] As used herein, the term “ex vivo” refers to events that occur outside of an organism (e.g., animal, plant, or microbe or cell or tissue thereof). Ex vivo events may take place in an environment minimally altered from a natural (e.g., in vivo) environment.

[0062] As used herein, the term “isomer” means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. Compounds may include one or more chiral centers and / or double bonds and may thus exist as stereoisomers, such as doublebond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cisltrans isomers). The present disclosure encompasses any and all isomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known.

[0063] As used herein, a “lipid component” is that component of a lipid nanoparticle that includes one or more lipids. In some embodiments, the lipid component may include one or more cationic / ionizable, PEGylated, structural, or other lipids, such as phospholipids.

[0064] As used herein, a “linker” is a moiety connecting two moieties, for example, the connection between two nucleosides of a cap species. A linker may include one or more groups including but not limited to phosphate groups (e.g., phosphates, boranophosphates, thiophosphates, selenophosphates, and phosphonates), alkyl groups, amidates, or glycerols. In some embodiments, two nucleosides of a cap analog may be linked at their 5’ positions by a triphosphate group or by a chain including two phosphate moieties and a boranophosphate moiety.

[0065] As used herein, “methods of administration” may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. Amethod of administration may be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body.

[0066] As used herein, “modified” means non-natural. In some embodiments, an RNA may be a modified RNA. That is, an RNA may include one or more nucleobases, nucleosides, nucleotides, or linkers that are non-naturally occurring. A “modified” species may also be referred to herein as an “altered” species. Species may be modified or altered chemically, structurally, or functionally. In some embodiments, a modified nucleobase species may include one or more substitutions that are not naturally occurring.

[0067] As used herein, the “N:P ratio” is the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in a lipid to phosphate groups in an RNA, e.g., in a LNP including a lipid component and an RNA.

[0068] As used herein, a “lipid nanoparticle” is a composition comprising one or more lipids. Lipid nanoparticles are typically sized on the order of micrometers or smaller and may include a lipid bilayer. Lipid nanoparticles, as used herein, unless otherwise specified, encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. In some embodiments, a LNP may be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0069] As used herein, “naturally occurring” means existing in nature without artificial aid.

[0070] As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.

[0071] As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.

[0072] As used herein, a “polymeric lipid” refers to a lipid comprising repeating subunits in its chemical structure. In some embodiments, the polymeric lipid is a lipid comprising a polymer component. In some embodiments, the polymeric lipid is a PEG lipid. In some embodiments, the polymeric lipid is not a PEG lipid. In some embodiments, the polymeric lipid is Brij or OH-PEG- stearate .

[0073] The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, composition, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complication, commensurate with a reasonable benefit / risk ratio.

[0074] The phrase “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than the compounds described herein (for example, a vehicle capable ofsuspending, complexing, or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: anti -adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other species disclosed herein.

[0075] Compositions may also include salts of one or more compounds. Salts may be pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety to its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, di gluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. In some embodiments, the nonaqueous media are ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0076] It is understood that the compounds of the present disclosure include the compounds themselves, as well as their ionic forms, if applicable.

[0077] As used herein, the term “ionic form” refers to a form of the referenced structure, in which one or more neutral atomic center(s) is replaced with one or more cationic atomic center(s) and / or one or more anionic center(s). The ionic form may be a free cation, a free anion, or a zwitterion. For example, when a referenced structure is described herein as a cation, the ionic form may include the corresponding zwitterion of the structure.

[0078] As used herein, a “phospholipid” is 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 (e.g., one or more unsaturations). A phospholipid or an analog or derivative thereof may include choline. A phospholipid or an analog or derivative thereof may not include choline. Particular phospholipids may facilitate fusion to a membrane. In some embodiments, 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 a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell.

[0079] As used herein, the “poly dispersity index” is a ratio that describes the homogeneity of the particle size distribution of a system. A small value, e.g., less than 0.3, indicates a narrow particle size distribution.

[0080] As used herein, an amphiphilic “polymer” is an amphiphilic compound that comprises an oligomer or a polymer. In some embodiments, an amphiphilic polymer can comprise an oligomer fragment, such as two or more PEG monomer units. In some embodiments, an amphiphilic polymer described herein can be PS 20.

[0081] As used herein, the term “polypeptide” or “polypeptide of interest” refers to a polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g, isolated or purified) or synthetically.

[0082] As used herein, an “RNA” refers to a ribonucleic acid that may be naturally or non- naturally occurring. In some embodiments, an RNA may include modified and / or non- naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and / or a polyadenylation signal. An RNA may have a nucleotide sequence encoding a polypeptide of interest. In some embodiments, an RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, for example, in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide. RNAs may be selected from the non-liming group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, long non-coding RNA (IncRNA) and mixtures thereof.

[0083] As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event.

[0084] As used herein, a “split dose” is the division of a single unit dose or total daily dose into two or more doses.

[0085] As used herein, a “total daily dose” is an amount given or prescribed in a 24 hour period. It may be administered as a single unit dose.

[0086] As used herein, the term “subject” refers to any organism to which a composition or formulation in accordance with the disclosure may be administered, e.g, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0087] As used herein, “Tx” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP solution, lyophilized LNP composition, or LNP formulation to degrade to about X of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP solution, lyophilized LNP composition, or LNP formulation. For example, “T80%” refers to the amount of time lasted for the nucleic acidintegrity (e.g., mRNA integrity) of aLNP, LNP solution, lyophilized LNP composition, orLNP formulation to degrade to about 80% of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP solution, lyophilized LNP composition, or LNP formulation. For another example, “T1 / 2” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP solution, lyophilized LNP composition, or LNP formulation to degrade to about 1 / 2 of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP solution, lyophilized LNP composition, or LNP formulation.

[0088] As used herein, “targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ, or in the tissue or organ of an organism. The organism may be an animal. In some embodiments, the organism is a mammal. In some embodiments, the organism is a human. In some embodiments, the organism is a patient.

[0089] As used herein, “target tissue” refers to any one or more tissue types of interest in which the delivery of a therapeutic and / or prophylactic agent would result in a desired biological and / or pharmacological effect. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. In particular applications, a target tissue may be a kidney, a lung, a spleen, vascular endothelium in vessels (e.g., intra-coronary or intra-femoral), or tumor tissue (e.g., via intratumoral injection). An “off-target tissue” refers to any one or more tissue types in which the expression of the encoded protein does not result in a desired biological and / or pharmacological effect. In particular applications, off-target tissues may include the liver and the spleen.

[0090] The term “therapeutic agent” or “prophylactic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Therapeutic agents are also 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.

[0091] As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, 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.

[0092] As used herein, the term “transfection” refers to the introduction of a species (e.g., an RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.

[0093] As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.

[0094] As used herein, the term “preventing” or “prevent” refers to reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.

[0095] As used herein, the term “zeta potential” refers to the electrokinetic potential of a lipid, e.g., in a particle composition.

[0096] As used herein, the term “polydispersity”, “poly dispersity index”, or “PDF’ refers to a measurement of the distribution of molecular mass in a given sample. The poly dispersity is calculated as Mw / Mn, in which Mwis the mass-average molar mass (or molecular weight) and Mnis the number-average molar mass (or molecular weight).

[0097] The term, “empty lipid nanoparticle” or “empty LNP”, as used herein, refers to a lipid nanoparticle which is substantially free of therapeutic or prophylactic agent. In some embodiments, therapeutic or prophylactic agent is nucleic acid (e.g., mRNA). In some embodiments, the empty LNP is substantially free of nucleic acid (e.g., mRNA). In some embodiments, the empty LNP comprises an ionizable lipid, a phospholipid, a structural lipid, and a PEG lipid. In some embodiments, the empty LNP comprises substantially less nucleic acid (e.g., RNA) as compared to the loaded LNP. In some embodiments, the empty LNP comprises less than about 5% w / w, less than about 4% w / w, less than 3% w / w, less than 2% w / w, less than 1% w / w, less than 0.5% w / w, less than 0.4% w / w, less than 0.3% w / w, less than 0.2% w / w, or less than 0.1% w / w of nucleic acid (e.g., RNA). In some embodiments, the empty LNP is free of nucleic acid (e.g., mRNA). In some embodiments, the empty LNP is further substantially free of nucleic acid (e.g., mRNA) associated with the surface of the LNP or conjugated to the exterior of the LNP.

[0098] The term, “loaded lipid nanoparticle” or “loaded LNP”, as used herein, refers to a lipid nanoparticle comprising a substantial amount of therapeutic or prophylactic agent. In some embodiments, therapeutic or prophylactic agent is nucleic acid (e.g., mRNA). In some embodiments, the loaded LNP comprises a substantial amount of nucleic acid (e.g., mRNA). In some embodiments, the empty LNP comprises an ionizable lipid, a phospholipid, a structural lipid, and a PEG lipid. In some embodiments, the empty LNP comprises a substantial amount of nucleic acid (e.g., mRNA) that is at least partially in the interior of the LNP. In someembodiments, the empty LNP comprises a substantial amount of nucleic acid (e.g., mRNA) that is associated with the surface of the LNP or conjugated to the exterior of the LNP.

[0099] The term “spread”, as used herein, refers to the width at half height of a peak (e.g., a mobility peak).

[0100] The term “free of’, as used herein, means not comprising the referenced component. For example, when a population, solution, or formulation is described as being “free of PEG lipid”, the population, solution, or formulation does not comprise PEG lipid (e.g., does not comprise a PEG lipid described herein (e.g., does not comprise PEG-DMG)).

[0101] As used herein, the expressions “one or more of A, B, or C,” “one or more A, B, or C,” “one or more of A, B, and C,” “one or more A, B, and C,” “selected from the group consisting of A, B, and C”, “selected from A, B, and C”, and the like are used interchangeably and all refer to a selection from a group consisting of A, B, and / or C, i.e., one or more As, one or more Bs, one or more C8, or any combination thereof, unless indicated otherwise.

[0102] It is understood that, when more than two of a variable (e.g., more than two variable X) are present, the phrase “two X, together with the one or more intervening atoms they are attached to, form” is not limited to describe only two of the more than two variable X . For example, when four variable X are present, the phrase intend to describe (i) two of the four variable X form a ring, and the other two are substitutions, or (ii) each two of the four variable X form a ring.

[0103] All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure.

[0104] In the synthetic schemes described herein, compounds may be drawn with one particular configuration for simplicity. Such particular configurations are not to be construed as limiting the disclosure to one or another isomer, tautomer, regioisomer or stereoisomer, nor does it exclude mixtures of isomers, tautomers, regioisomers or stereoisomers.

[0105] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of writtendescription, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.Compounds of the Present Disclosure - Cationic Lipids

[0106] Without wishing to be bound by theory, it is understood that the compounds of the present disclosure may serve as cationic lipids in lipid nanoparticles (LNPs).

[0107] In some aspects, the present disclosure provides a compound of Formula (I):or an ionic form or a salt thereof, wherein: q is 1, 2, 3, 4, 5, or 6;Q is -ORqor -NHRq;Rqis H, or C3-6cycloalkyl substituted one or more oxo, -NH2, -NH(C1-6alkyl), or -N(C1-6alkyl)2;T1is (i) C1-24 alkyl optionally substituted with one or more -OH, -C(=O)OH or 3- to 10- membered heterocyclyl, or (ii) -M^L^R1;M1is C 1-12 alkylene optionally substituted with one or more Rm1; each Rm1independently is oxo, halogen, -OH, -O(C1-6alkyl), -NH2, - NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl; or two Rm1, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6-membered heterocycloalkyl;cycloalkylene)-**, wherein * denotes attachment to T1and ** denotes attachment to R1;R1is W1or -U1-L1-W1;U1is C1-6alkylene optionally substituted with one or more oxo, halogen, -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl;V1is *-C(=O)-O-**, *-O-C(=O)-**, *-S-S-**, *-(C6-10arylene)-**, *- 0-(C6-10arylene)-**, *-(C6-10arylene)-O-**, *-NH-(C6-10arylene)-**, *-O-**, or *-(C6-10arylene)-NH-**, wherein * denotes attachment to U1and ** denotes attachment to W1;W1is C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl, wherein the C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl is optionally substituted with one or more Rwl; each Rwlindependently is oxo, halogen, -OH, -O(C1-6alkyl), - NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl; or two Rw1, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6-membered heterocycloalkyl; b is 1, 2, 3, 4, 5, or 6;L2is -O-C(=O)-** or -C(=O)-O-**, wherein ** denotes attachment to R2;R2ais H, C 1-12 alkyl, C2-12alkenyl, or C2-12alkynyl;R2bis C5 -12alkyl, C5 -12alkenyl, or C 5- 12 alkynyl; c is 1, 2, 3, 4, 5, or 6;L3is -O-C(=O)-** or -C(=O)-O-**, wherein ** denotes attachment to R3;r3 is 0, 1, 2, 3, 4, 5, or 6;R3ais H, V3a, or -U3a-V3a;U3ais -O-C(=O)-** or -C(=O)-O-**, wherein ** denotes attachment to V3a;V3ais C1-12 alkyl, C2-12alkenyl, or C2-12alkynyl;R3bis V3b, or -U3b-V3b;U3bis -O-C(=O)-** or -C(=O)-O-** , wherein ** denotes attachment to V3b; andV3bis C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl.

[0108] In some embodiments, T1is -M1-L1-R1, wherein:(i) M1is C1 -12alkylene substituted with one or more Rm1;(ii) R1is -U1-V1-W1; or(iii) R1is W1, wherein W1is C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl, wherein the C1-12alkyl, C2-12alkenyl, or C2-12alkynyl is substituted with one or more Rwl, wherein each Rwlindependently is oxo, halogen, -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1- 3 alkyl, C5-6alkyl, C2-6alkenyl, or C2-6alkynyl.

[0109] In some embodiments, T1is - M1-L1-R1, wherein M1is C1-12alkylene substituted with one or more Rm1.

[0110] In some embodiments, T1is - M1-L1-R1, wherein R1is -U1-V1-W1.

[0111] In some embodiments, T1is -M1-L1-R1, wherein R1is W1, wherein W1is C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl, wherein the C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl is substituted with one or more Rwl, wherein each Rwlindependently is oxo, halogen, -OH, - O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1-3alkyl, C5-6 alkyl, C2-6alkenyl, or C2- 6 alkynyl.

[0112] In some embodiments, T1is -M1-L1-R1, wherein:(i) M1is C1-12alkylene substituted with one or more Rm1; and(ii) R1is -U1-V1-W1; or(iii) R1is W1, wherein W1is C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl, wherein the C1- 12 alkyl, C2-12alkenyl, or C2-12alkynyl is substituted with one or more Rwl, wherein each Rwlindependently is oxo, halogen, -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1- 3 alkyl, C5-6alkyl, C2-6alkenyl, or C2-6alkynyl.Variables q, Q, and Rq

[0113] In some embodiments, q is 1.

[0114] In some embodiments, q is 2, 3, 4, 5, or 6.

[0115] In some embodiments, q is 2, 3, or 4.

[0116] In some embodiments, q is 2 or 3.

[0117] In some embodiments, q is 2.

[0118] In some embodiments, q is 3.

[0119] In some embodiments, q is 4.

[0120] In some embodiments, q is 5.

[0121] In some embodiments, q is 6.

[0122] In some embodiments, Q is -ORq.

[0123] In some embodiments, Q is -OH.

[0124] In some embodiments, Q is -NHRq.

[0125] In some embodiments,

[0126] In some embodiments, Rqis H.

[0127] In some embodiments, Rqis C3-6cycloalkyl substituted one or more oxo, -NH2, -NH(Cn6 alkyl), or -N(C1-6alkyl)2.

[0128] In some embodiments, Rqis cyclobutenyl substituted one or more oxo, -NH2, -NH(Cn 6 alkyl), or -N(C1-6alkyl)2.

[0129] In some embodiments,.

[0130] In some embodiments, Q is -NHRq, wherein Rqis C3-6cycloalkyl substituted one or more oxo, -NH2, -NH(C1-6alkyl), or -N(C1-6alkyl)2.

[0131] In some embodiments, Q is -NHRq, wherein Rqis cyclobutenyl substituted one or more oxo, -NH2, -NH(C1-6alkyl), or -N(C1-6alkyl)2.

[0132] In some embodiments, Q is -NHRq, wherein.Variables T1, M1, Rm1, L1, R1, U1, V1, W1, and Rw1

[0133] In some embodiments, T1is C1-24alkyl optionally substituted with one or more - C(=O)OH.

[0134] In some embodiments, T1is C1-24alkyl substituted with one or more -C(=O)OH.

[0135] In some embodiments, T1is C1-24alkyl optionally substituted with one or more -OH.

[0136] In some embodiments, T1is C1-24alkyl substituted with one or more -OH.

[0137] In some embodiments, T1is C1-24alkyl optionally substituted with one or more 3- to 10-membered heterocyclyl.

[0138] In some embodiments, T1is C1-24alkyl substituted with one or more 3- to 10-membered heterocyclyl.

[0139] In some embodiments, T1is C1-24alkyl optionally substituted with one or more 3- to 10-membered heterocycloalkyl.

[0140] In some embodiments, T1is C1-24alkyl substituted with one or more 3- to 10-membered heterocycloalkyl.

[0141] In some embodiments, T1is C1-24alkyl optionally substituted with one or more azetidinyl or pyrrolidinyl.

[0142] In some embodiments, T1is C1-24alkyl substituted with one or more azetidinyl or pyrrolidinyl.

[0143] In some embodiments, T1is C1-24alkyl optionally substituted with one or more 3- to 10-membered heteroaryl.

[0144] In some embodiments, T1is C1-24alkyl substituted with one or more 3- to 10-membered heteroaryl.

[0145] In some embodiments, T1is C1-24alkyl optionally substituted with one or more 3- to 10-membered heteroaryl.

[0146] In some embodiments, T1is C1-24alkyl substituted with one or more midazolyl, triazolyl, or tetrazolyl.

[0147] In some embodiments, T1is C1-24alkyl.

[0148] In some embodiments, T1is C1-16alkyl.

[0149] In some embodiments, T1is C1-12alkyl.

[0150] In some embodiments, T1is C1-8alkyl.

[0151] In some embodiments, T1is C1-6alkyl.

[0152] In some embodiments, T1is C1-6alkyl optionally substituted with one or more - C(=O)OH.

[0153] In some embodiments, T1is C1-6alkyl substituted with one or more -C(=O)OH.

[0154] In some embodiments, T1is C1-6alkyl optionally substituted with one or more -OH.

[0155] In some embodiments, T1is C1-6alkyl substituted with one or more -OH.

[0156] In some embodiments, T1is C1-6alkyl optionally substituted with one or more 3- to 10- membered heterocyclyl.

[0157] In some embodiments, T1is C1-6alkyl substituted with one or more 3- to 10-membered heterocyclyl.

[0158] In some embodiments, T1is C1-6alkyl optionally substituted with one or more 3- to 10- membered heterocycloalkyl.

[0159] In some embodiments, T1is C1-6alkyl substituted with one or more 3- to 10-membered heterocycloalkyl.

[0160] In some embodiments, T1is C1-6alkyl optionally substituted with one or more azetidinyl or pyrrolidinyl.

[0161] In some embodiments, T1is C1-6alkyl substituted with one or more azetidinyl or pyrrolidinyl.

[0162] In some embodiments, T1is C1-6alkyl optionally substituted with one or more 3- to 10- membered heteroaryl.

[0163] In some embodiments, T1is C1-6alkyl substituted with one or more 3- to 10-membered heteroaryl.

[0164] In some embodiments, T1is C1-6alkyl optionally substituted with one or more 3- to 10- membered heteroaryl.

[0165] In some embodiments, T1is C1-6alkyl substituted with one or more imidazolyl, triazolyl, or tetrazolyl.

[0166] In some embodiments, T1is C1-3alkyl.

[0167] In some embodiments, T1is methyl.

[0168] In some embodiments, T1is ethyl.

[0169] In some embodiments, T1is propyl.

[0170] In some embodiments, T1is -M1-L1-R1.

[0171] In some embodiments, M1is C1-12alkylene optionally substituted with one or more Rm1.

[0172] In some embodiments, M1is C1-12alkylene substituted with one or more Rm1.

[0173] In some embodiments, M1is C3-8alkylene (e.g., propylene, butylene, pentylene, hexylene, heptylene, or octylene) optionally substituted with one or more Rm1.

[0174] In some embodiments, M1is C3-8alkylene (e.g., propylene, butylene, pentylene, hexylene, heptylene, or octylene) substituted with one or more Rm1.

[0175] In some embodiments, M1is propylene optionally substituted with one or more Rm1.

[0176] In some embodiments, M1is propylene substituted with one or more Rm1.

[0177] In some embodiments, M1is butylene optionally substituted with one or more Rm1.

[0178] In some embodiments, M1is butylene substituted with one or more Rm1.

[0179] In some embodiments, M1is pentylene optionally substituted with one or more Rm1.

[0180] In some embodiments, M1is pentylene substituted with one or more Rm1.

[0181] In some embodiments, M1is hexylene optionally substituted with one or more Rm1.

[0182] In some embodiments, M1is hexylene substituted with one or more Rm1.

[0183] In some embodiments, M1is heptylene optionally substituted with one or more Rm1.

[0184] In some embodiments, M1is heptylene substituted with one or more Rm1.

[0185] In some embodiments, M1is octylene optionally substituted with one or more Rm1.

[0186] In some embodiments, M1is octylene substituted with one or more Rm1.

[0187] In some embodiments, at least one (e.g., each) Rm1is oxo.

[0188] In some embodiments, at least one (e.g., each) Rm1is halogen (e.g., F, Cl, or Br).

[0189] In some embodiments, at least one (e.g., each) Rm1is -OH, -O(C1-6alkyl), -NH2, - NH(C1-6alkyl), or -N(C1-6alkyl)2.

[0190] In some embodiments, at least one (e.g., each) Rm1is -OH.

[0191] In some embodiments, at least one (e.g., each) Rm1is -O(C1-6alkyl).

[0192] In some embodiments, at least one (e.g., each) Rm1is -NH2.

[0193] In some embodiments, at least one (e.g., each) Rm1is -NH(C1-6alkyl).

[0194] In some embodiments, at least one (e.g., each) Rm1is -N(C1-6alkyl)2.

[0195] In some embodiments, at least one (e.g., each) Rm1is C1-6alkyl, C2-6alkenyl, or C2-6alkynyl.

[0196] In some embodiments, at least one (e.g., each) Rm1is C1-6alkyl.

[0197] In some embodiments, at least one (e.g., each) Rm1is C2-6alkenyl.

[0198] In some embodiments, at least one (e.g., each) Rm1is C2-6alkynyl.

[0199] In some embodiments, at least two Rm1, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6-membered heterocycloalkyl.

[0200] In some embodiments, at least two Rm1, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl.

[0201] In some embodiments, at least two Rm1, together with the one or more intervening atoms they are attached to, form cyclopropyl.

[0202] In some embodiments, at least two Rm1, together with the one or more intervening atoms they are attached to, form 3- to 6-membered heterocycloalkyl.

[0203] In some embodiments, at least two Rm1, together with the one or more intervening atoms they are attached to, form 3 -membered heterocycloalkyl.

[0204] In some embodiments, at least two Rm1, together with the one or more intervening atoms they are attached to, form oxiranyl.

[0205] In some embodiments, L1is *-O-C(=O)-** or *-C(=O)-O-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

[0206] In some embodiments, L1is *-O-C(=O)-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

[0207] In some embodiments, L1is *-C(=O)-O-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

[0208] In some embodiments, L1is *-C(=O)-NH-** or *-NH-C(=O)-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

[0209] In some embodiments, L1is *-C(=O)-NH-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

[0210] In some embodiments, L1is *-NH-C(=O)-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

[0211] In some embodiments, L1is *-(C3-6cycloalkylene)-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

[0212] In some embodiments, L1is *-(cyclopropylene)-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

[0213] In some embodiments, R1is W1.

[0214] In some embodiments, R1is -L1-V1-W1.

[0215] In some embodiments, U1is C1-6alkylene optionally substituted with one or more oxo, halogen, -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl.

[0216] In some embodiments, U1is C1-6alkylene optionally substituted with one or more oxo, halogen, -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), or -N(C1-6alkyl)2.

[0217] In some embodiments, U1is C1-6alkylene.

[0218] In some embodiments, U1is C1-6alkylene substituted with one or more oxo, halogen, - OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl.

[0219] In some embodiments, U1is C1-6alkylene substituted with one or more oxo, halogen, - OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), or -N(C1-6alkyl)2.

[0220] In some embodiments, U1is C1-6alkylene optionally substituted with one or more oxo.

[0221] In some embodiments, U1is C1-6alkylene optionally substituted with one or more halogen (e.g., F, Cl, or Br).

[0222] In some embodiments, U1is C1-6alkylene optionally substituted with one or more -OH or -O(C1-6alkyl).

[0223] In some embodiments, U1is C1-6alkylene optionally substituted with one or more - NH2, -NH(C1-6alkyl), or -N(C1-6alkyl)2.

[0224] In some embodiments, U1is C1-6alkylene optionally substituted with one or more C1-6alkyl, C2-6alkenyl, or C2-6alkynyl.

[0225] In some embodiments, V1is *-C(=O)-O-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

[0226] In some embodiments, V1is *-O-C(=O)-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

[0227] In some embodiments, V1is *-O-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

[0228] In some embodiments, V1is wherein * denotes attachment to U1and ** denotes attachment to W1.

[0229] In some embodiments, V1is *-(C6-10arylene)-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

[0230] In some embodiments, V1is *-0-(C6-10arylene)-** or *-(C6-10arylene)-O-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

[0231] In some embodiments, V1is *-NH-(C6-10arylene)-** or *-(C6-10arylene)-NH-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

[0232] In some embodiments, W1is hydrogen.

[0233] In some embodiments, W1is C1-12alkyl optionally substituted with one or more Rwl.

[0234] In some embodiments, W1is C5 -12alkyl optionally substituted with one or more Rwl.

[0235] In some embodiments, W1is C1-12alkyl.

[0236] In some embodiments, W1is C5 -12alkyl.

[0237] In some embodiments, W1is C1-12alkyl substituted with one or more Rwl.

[0238] In some embodiments, W1is C5 -12alkyl substituted with one or more Rwl.

[0239] In some embodiments, W1is C2-12alkenyl optionally substituted with one or more Rwl.

[0240] In some embodiments, W1is C5 -12alkenyl optionally substituted with one or more Rwl.

[0241] In some embodiments, W1is C2-12alkenyl.

[0242] In some embodiments, W1is C5 -12alkenyl.

[0243] In some embodiments, W1is C2-12alkenyl substituted with one or more Rwl.

[0244] In some embodiments, W1is C5 -12alkenyl substituted with one or more Rwl.

[0245] In some embodiments, W1is C2-12alkynyl optionally substituted with one or more Rwl.

[0246] In some embodiments, W1is C5 -12alkynyl optionally substituted with one or more Rwl.

[0247] In some embodiments, W1is C2-12alkynyl.

[0248] In some embodiments, W1is C5 -12alkynyl.

[0249] In some embodiments, W1is C2-12alkynyl substituted with one or more Rwl.

[0250] In some embodiments, W1is C5 -12alkynyl substituted with one or more Rwl.

[0251] In some embodiments, at least one (e.g., each) Rwlis oxo, halogen, -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl.

[0252] In some embodiments, at least one (e.g., each) Rwlis oxo, halogen, -OH, -O(C1-6alkyl),-NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2.

[0253] In some embodiments, at least one (e.g., each) Rwlis oxo.

[0254] In some embodiments, at least one (e.g., each) Rwlis halogen (e.g., F, Cl, or Br).

[0255] In some embodiments, at least one (e.g., each) Rwlis -OH or -O(C1-6alkyl).

[0256] In some embodiments, at least one (e.g., each) Rwlis -NH2, -NH(C1-6alkyl), or -N(C1- 6 alkyl)2.

[0257] In some embodiments, at least one (e.g., each) Rwlis C1-6alkyl, C2-6alkenyl, or C2-6alkynyl.

[0258] In some embodiments, at least two Rwl, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6-membered heterocycloalkyl.

[0259] In some embodiments, at least two Rwl, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl.

[0260] In some embodiments, at least two Rwl, together with the one or more intervening atoms they are attached to, form cyclopropyl.

[0261] In some embodiments, at least two Rwl, together with the one or more intervening atoms they are attached to, form 3- to 6-membered heterocycloalkyl.

[0262] In some embodiments, at least two Rwl, together with the one or more intervening atoms they are attached to, form 3 -membered heterocycloalkyl.

[0263] In some embodiments, at least two Rwl, together with the one or more intervening atoms they are attached to, form oxiranyl.Variables b, L2, R2, R2a, andR2b

[0264] In some embodiments, b is 1.

[0265] In some embodiments, b is 2.

[0266] In some embodiments, b is 3, 4, or 5.

[0267] In some embodiments, b is 3.

[0268] In some embodiments, b is 4.

[0269] In some embodiments, b is 5.

[0270] In some embodiments, b is 6.

[0271] In some embodiments, L2is -O-C(=O)-** , wherein ** denotes attachment to R2.

[0272] In some embodiments, L2is -C(=O)-O-** , wherein ** denotes attachment to R2.

[0273] In some embodiments,

[0274] In some embodiments,

[0275] In some embodiments, R2ais H.

[0276] In some embodiments, R2ais C1-12alkyl, C2-12alkenyl, or C2-12alkynyl.

[0277] In some embodiments, R2ais C1-12alkyl (e.g., propyl, butyl, pentyl, hexyl, heptyl, or octyl).

[0278] In some embodiments, R2ais C2-12alkenyl.

[0279] In some embodiments, R2ais C2-12alkynyl.

[0280] In some embodiments, R2bis C1-12alkyl (e.g., propyl, butyl, pentyl, hexyl, heptyl, or octyl).

[0281] In some embodiments, R2bis C5 -12alkyl (e.g., pentyl, hexyl, heptyl, or octyl).

[0282] In some embodiments, R2bis C5 -12alkenyl.

[0283] In some embodiments, R2bis C5 -12alkenyl.

[0284] In some embodiments, R2bis C5 -12alkynyl.

[0285] In some embodiments, R2bis C5 -12alkynyl.Variables c, L3, R3, r3, R3a, U3a, V3a, R3b, U3b, and V3b

[0286] In some embodiments, c is 1.

[0287] In some embodiments, c is 2.

[0288] In some embodiments, c is 3, 4, or 5.

[0289] In some embodiments, c is 3.

[0290] In some embodiments, c is 4.

[0291] In some embodiments, c is 5.

[0292] In some embodiments, c is 6.

[0293] In some embodiments, L3is -O-C(=O)-** , wherein ** denotes attachment to R3.

[0294] In some embodiments, L3is -C(=O)-O-** , wherein ** denotes attachment to R3.

[0295] In some embodiments,

[0296] In some embodiments,

[0297] In some embodiments,

[0298] In some embodiments,

[0299] In some embodiments, r3 is 0.

[0300] In some embodiments, r3 is 1, 2, 3, 4, 5, or 6.

[0301] In some embodiments, r3 is 1.

[0302] In some embodiments, r3 is 2.

[0303] In some embodiments, r3 is 3.

[0304] In some embodiments, r3 is 4.

[0305] In some embodiments, r3 is 5.

[0306] In some embodiments, r3 is 6.

[0307] In some embodiments, R3ais H.

[0308] In some embodiments, R3ais V3aor -U3a-V3a.

[0309] In some embodiments, R3ais V3a.

[0310] In some embodiments, R3ais -U3a-V3a.

[0311] In some embodiments, U3ais -O-C(=O)-**, wherein ** denotes attachment to V3a.

[0312] In some embodiments, U3ais -C(=O)-O-**, wherein ** denotes attachment to V3a.

[0313] In some embodiments, V3ais C1-12alkyl, C2-12alkenyl, or C2-12alkynyl.

[0314] In some embodiments, V3ais C1-12alkyl (e.g., propyl, butyl, pentyl, hexyl, heptyl, or octyl).

[0315] In some embodiments, V3ais C2-12alkenyl.

[0316] In some embodiments, V3ais C2-12alkynyl.

[0317] In some embodiments, R3bis V3b.

[0318] In some embodiments, R3bis -U3b-V3b.

[0319] In some embodiments, U3bis -O-C(=O)-** , wherein ** denotes attachment to V3b.

[0320] In some embodiments, U3bis -C(=O)-O-** , wherein ** denotes attachment to V3b.

[0321] In some embodiments, V3bis C1-12alkyl, C2-12alkenyl, or C2-12alkynyl.

[0322] In some embodiments, V3bis C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl.

[0323] In some embodiments, V3bis C1-12alkyl (e.g., propyl, butyl, pentyl, hexyl, heptyl, or octyl).

[0324] In some embodiments, V3bis C5 -12alkyl (e.g., butyl, pentyl, hexyl, heptyl, or octyl).

[0325] In some embodiments, V3bis C2-12alkenyl.

[0326] In some embodiments, V3bis C5 -12alkenyl.

[0327] In some embodiments, V3bis C2-12alkynyl.

[0328] In some embodiments, V3bis C5 -12alkynyl.Exemplary Embodiments of the Compounds

[0329] In some embodiments, the compound is of Formula (la):or a salt thereof.

[0330] In some embodiments, b is 3, 4, or 5.

[0331] In some embodiments, c is 3 or 5.

[0332] In some embodiments, b is 3, 4, or 5; and c is 3 or 5.

[0333] In some embodiments, the compound is of Formula (lb):or a salt thereof.

[0334] In some embodiments, b is 4 or 5.

[0335] In some embodiments, c is 5.

[0336] In some embodiments, b is 4 or 5; and c is 5.

[0337] In some embodiments, the compound is of Formula (Ic):or a salt thereof. In some embodiments, q is 2 or 3.

[0338] In some embodiments, b is 3, 4, or 5.

[0339] In some embodiments, c is 3 or 5.

[0340] In some embodiments, R2bis C8-10alkyl.

[0341] In some embodiments, q is 2 or 3; b is 3, 4, or 5; c is 3 or 5; and R2bis C8-10alkyl.

[0342] In some embodiments, the compound is of Formula (Id):or a salt thereof.

[0343] In some embodiments, q is 2 or 3.

[0344] In some embodiments, b is 3, 4, or 5.

[0345] In some embodiments, c is 3 or 5.

[0346] In some embodiments, R3ais C8alkyl.

[0347] In some embodiments, R3bis C8alkyl.

[0348] In some embodiments, q is 2 or 3; b is 3, 4, or 5; c is 3 or 5; R3ais C8alkyl; and R3bis C8alkyl.

[0349] In some embodiments, the compound is of Formula (le):or a salt thereof.

[0350] In some embodiments, M1is a C3-7alkylene optionally substituted with one Rm1, wherein Rm1is -OH, or with two Rm1, which together with the one or more intervening atoms they are attached to, form a 3 -membered heterocycloalkyl.

[0351] In some embodiments, W1is a C9-13alkyl optionally substituted with one Rwl, wherein Rwlis C2-4alkyl; or C11alkynyl.

[0352] In some embodiments, q is 2 or 3.

[0353] In some embodiments, b is 3, 4, or 5.

[0354] In some embodiments, c is 3 or 5.

[0355] In some embodiments, M1is a C3-7alkylene optionally substituted with one Rm1, wherein Rm1is -OH, or with two Rm1, which together with the one or more intervening atoms they are attached to, form a 3 -membered heterocycloalkyl; W1is a C9-13alkyl optionallysubstituted with one Rwl, wherein Rwlis C2-4alkyl; or C11alkynyl; q is 2 or 3; b is 3, 4, or 5; and c is 3 or 5.

[0356] In some embodiments, the compound is of Formula (If):or a salt thereof.

[0357] In some embodiments, M1is a C6-8alkylene.

[0358] In some embodiments, L1is *-C(=O)-O-** or *-(C3cycloalkylene)-**, wherein * denotes attachment to T1and ** denotes attachment to U1.

[0359] In some embodiments, U1is a C1-2alkylene.

[0360] In some embodiments, V1is *-S-S-**, *-C(=O)-O-**, or *-(C6-10arylene)-O-**; wherein * denotes attachment to U1and ** denotes attachment to W1.

[0361] In some embodiments, W1is a C3-8alkyl.

[0362] In some embodiments, M1is a C6-8 alkylene; L1is *-C(=O)-O-** or *-(C3cycloalkylene)-**, wherein * denotes attachment to T1and ** denotes attachment to U1; U1is a C1-2alkylene; V1is *-S-S-**, *-C(=O)-O-**, or *-(C6-10arylene)-O-**; wherein * denotes attachment to U1and ** denotes attachment to W1; and W1is a C3-8alkyl.

[0363] In some embodiments, the compound is of Formula (Ig):or a salt thereof. In some embodiments, U1is a C1-2alkylene.

[0364] In some embodiments, V1is *-S-S-**, *-C(=O)-O-**, or *-(C6-10arylene)-O-**; wherein * denotes attachment to U1and ** denotes attachment to W1.

[0365] In some embodiments, W1is a C3-8alkyl.

[0366] In some embodiments, U1is a C1-2alkylene; V1is *-S-S-**, *-C(=O)-O-**, or *-(C6-10arylene)-O-**; wherein * denotes attachment to U1and ** denotes attachment to W1; and W1is a C3-8alkyl.

[0367] In some embodiments, the compounds is selected from the compounds described inTable 1 and salts thereof.

[0368] In some embodiments, the compounds is selected from the compounds described inTable 1.Table 1Table 1RMethods of the Present Disclosure

[0369] In some aspects, the present disclosure provides a method of preparing a lipid nanoparticle formulation (LNP formulation), comprising: i) a nanoprecipitation step, comprising: i-a) mixing a lipid solution comprising a cationic lipid, an ionizable lipid, a structural lipid, and a phospholipid, with an aqueous buffer solution comprising a buffering agent, thereby forming an intermediate lipid nanoparticle solution (intermediate LNP solution); i-b) adding a diluting solution to the intermediate LNP solution; i-c) holding the intermediate LNP solution for a residence time; i-d) adding a pH-adjusting solution to the intermediate LNP solution, thereby forming a lipid nanoparticle solution (LNP solution); ii) processing the LNP solution, thereby forming an LNP formulation, wherein the processing comprises: ii-a) adding a PEG lipid solution to the LNP solution.

[0370] In some embodiments, the method further comprises: iii) storing the LNP formulation in a buffered storage solution.

[0371] In some embodiments, the buffered storage solution comprises tris.

[0372] In some embodiments, the buffered storage solution comprises acetate.

[0373] In some embodiments, the buffered storage solution comprises sodium chloride.

[0374] In some embodiments, the buffered storage solution comprises citrate.

[0375] In some embodiments, the buffered storage solution comprises a mixture of tris, acetate, citrate, and / or sodium chloride.

[0376] In some embodiments, the ionic strength of the buffered storage solution is from about 0.15 molar to about 0.2 molar.

[0377] In some embodiments, the ionic strength of the buffered storage solution is about 0.15 molar.

[0378] In some embodiments, the ionic strength of the buffered storage solution is 0.15 molar.

[0379] In some embodiments, the ionic strength of the buffered storage solution is about 0.2 molar.

[0380] In some embodiments, the ionic strength of the buffered storage solution is 0.2 molar.

[0381] In some embodiments, the ionic strength of the buffered storage solution is less than 0.2 molar.Lipid Solutions

[0382] In some embodiments, the cationic lipid is a compound of the present disclosure (e.g., a compound of Formula (I) or a salt thereof).

[0383] In some embodiments, the cationic lipid is Compound No. Ref-1.

[0384] In some embodiments, the cationic lipid is selected from the compounds shown in Table 1 and salts thereof.

[0385] In some embodiments, the cationic lipid is selected from the compounds shown in Table 1.

[0386] In some embodiments, the ionizable lipid is Compound No. 1-18, Compound No. VI-4, or Compound No. II-6.

[0387] In some embodiments, the structural lipid is cholesterol.

[0388] In some embodiments, the phospholipid is DOPE or DSPC.

[0389] In some embodiments, the lipid solution is free of PEG lipid.

[0390] In some embodiments, the lipid solution further comprises a PEG lipid.

[0391] In some embodiments, the PEG lipid is PEG-DMG or PEG-1.

[0392] In some embodiments, the PEG lipid is present in the lipid solution at a concentration of about 0.25±0.2 mol%, about 0.25±0.15 mol%, about 0.25±0.1 mol%, about 0.25±0.09 mol%, about 0.25±0.08 mol%, about 0.25±0.07 mol%, about 0.25±0.06 mol%, about 0.25±0.05 mol%, about 0.25±0.04 mol%, about 0.25±0.03 mol%, about 0.25±0.02 mol%, or about 0.25±0.01 mol% (e.g., about 0.25 mol%), as compared to the total amount of the lipids present in the lipid solution.

[0393] In some embodiments, the total amount of the lipids is present in the lipid solution at a concentration of about 12.5±5 mM, about 12.5±4 mM, about 12.5±3 mM, about 12.5±2 mM, about 12.5±1 mM, about 12.5±0.5 mM, about 12.5±0.4 mM, about 12.5±0.3 mM, about 12.5±0.2 mM, or about 12.5±0.1 mM (e.g., about 12.5 mM).

[0394] In some embodiments, the lipid solution comprises alcohol.

[0395] In some embodiments, the lipid solution comprises ethanol.Aqueous Buffer Solutions

[0396] In some embodiments, the aqueous buffer solution is free of nucleic acid.

[0397] In some embodiments, the aqueous buffer solution further comprises a nucleic acid.

[0398] In some embodiments, the nucleic acid is an RNA.

[0399] In some embodiments, the nucleic acid is an mRNA.

[0400] In some embodiments, the buffering agent is citrate, acetate, phosphate, tris(hydroxymethyl)aminomethane (tris), or 2-[4-(2-Hydroxyethyl)piperazin-l-yl]ethane-l- sulfonic acid (HEPES).

[0401] In some embodiments, the buffering agent is acetate (e.g., sodium acetate).

[0402] In some embodiments, the acetate (e.g., sodium acetate) is present in the aqueous buffer solution at a concentration of about 25±10 mM, about 25±9 mM, about 25±8 mM, about 25±7 mM, about 25±6 mM, about 25±5 mM, about 25±4 mM, about 25±3 mM, about 25±2 mM, or about 25±1 mM (e.g., about 25 mM).

[0403] In some embodiments, the aqueous buffer solution has a pH value of about 5.0±2.0, about 5.0±1.5, about 5.0±1.0, about 5.0±0.9, about 5.0±0.8, about 5.0±0.7, about 5.0±0.6, about 5.0±0.5, about 5.0±0.4, about 5.0±0.3, about 5.0±0.2, or about 5.0±0.1 (e.g., about 5.0).Mixings

[0404] In some embodiments, the lipid solution and the aqueous buffer solution are mixed at a volumetric ratio of about 1 :5, about 1 :4, about 1 :3, about 1 :2, or about 1 : 1.

[0405] In some embodiments, the total amount of the lipids and the nucleic acid are present in the intermediate lipid nanoparticle solution at an N / P ratio of about 4.9±2.0, about 4.9±1.5, about4.9±1.0, about4.9±0.9, about4.9±0.8, about4.9±0.7, about4.9±0.6, about4.9±0.5, about 4.9±0.4, about 4.9±0.3, about 4.9±0.2, or about 4.9±0.1 (e.g., about 4.9).

[0406] In some embodiments, the intermediate LNP solution has a pH value of about 5.0±2.0, about 5.0±1.5, about 5.0±1.0, about 5.0±0.9, about 5.0±0.8, about 5.0±0.7, about 5.0±0.6, about 5.0±0.5, about 5.0±0.4, about 5.0±0.3, about 5.0±0.2, or about 5.0±0.1 (e.g., about 5.0).Diluting Solutions, Residence Times, and pH-Adjusting Solutions

[0407] In some embodiments, the diluting solution comprises citrate, acetate, phosphate, tris, or HEPES.

[0408] In some embodiments, the diluting solution comprises citrate.

[0409] In some embodiments, the diluting solution comprises citrate-buffered saline (CBS).

[0410] In some embodiments, the ionic strength of the diluting solution is from about 0.15 molar to about 0.2 molar.

[0411] In some embodiments, the ionic strength of the diluting solution is from about 0.15 molar to about 0.25 molar.

[0412] In some embodiments, the ionic strength of the diluting solution is about 0.15 molar.

[0413] In some embodiments, the ionic strength of the diluting solution is 0.15 molar.

[0414] In some embodiments, the ionic strength of the diluting solution is about 0.2 molar.

[0415] In some embodiments, the ionic strength of the diluting solution is 0.2 molar.

[0416] In some embodiments, the residence time is less than about 1 minute.

[0417] In some embodiments, the residence time is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 1 hour.

[0418] In some embodiments, the residence time is about 30±20 minutes, about 30±15 minutes, about 30±10 minutes, about 30±9 minutes, about 30±8 minutes, about 30±7 minutes, about 30±6 minutes, about 30±5 minutes, about 30±4 minutes, about 30±3 minutes, about 30±2 minutes, or about 30±l minutes (e.g., about 30 minutes).

[0419] In some embodiments, the pH-adjusting solution comprises citrate, acetate, phosphate, tris, or HEPES.

[0420] In some embodiments, the pH-adjusting solution comprises phosphate.

[0421] In some embodiments, the pH-adjusting solution comprises phosphate-buffered saline (PBS).

[0422] In some embodiments, the pH-adjusting solution comprises tris.

[0423] In some embodiments, the pH-adjusting solution comprises tris-buffered saline (TBS).

[0424] In some embodiments, the pH-adjusting solution comprises HEPES.PEG Lipid Solutions

[0425] In some embodiments, the PEG lipid solution comprises a PEG lipid.

[0426] In some embodiments, the PEG lipid is PEG-DMG or PEG-1.

[0427] In some embodiments, upon adding the PEG lipid solution to the LNP solution, the PEG lipid is present in the LNP solution at a concentration of about 1.5±1.0 mol%, about 1.5±0.9 mol%, about 1.5±0.8 mol%, about 1.5±0.7 mol%, about 1.5±0.8 mol%, about 1.5±0.6 mol%, about 1.5±0.5 mol%, about 1.5±0.4 mol%, about 1.5±0.3 mol%, about 1.5±0.2 mol%, or about 1.5±0.1 mol% (e.g., about 1.5 mol%), as compared to the total amount of the lipids present in the LNP solution.

[0428] In some embodiments, upon adding the PEG lipid solution to the LNP solution, the PEG lipid is present in the LNP solution at a concentration of about 3.0±2.0 mol%, 3.0±1.5 mol%, 3.0±1.0 mol%, about 3.0±0.9 mol%, about 3.0±0.8 mol%, about 3.0±0.7 mol%, about 3.0±0.8 mol%, about 3.0±0.6 mol%, about 3.0±0.5 mol%, about 3.0±0.4 mol%, about 3.0±0.3 mol%, about 3.0±0.2 mol%, or about 3.0±0.1 mol% (e.g., about 3.0 mol%), as compared to the total amount of the lipids present in the LNP solution.Processing Steps

[0429] In some embodiments, the processing further comprises: ii-b) adding a cryoprotectant the LNP solution.

[0430] In some embodiments, the processing further comprises: ii-c) filtering the LNP solution.

[0431] In some embodiments, the step of processing the empty-LNP solution or loaded-LNP solution comprises: ii-b) adding a cryoprotectant the LNP solution; and ii-c) filtering the LNP solution.

[0432] In some embodiments, the step of processing the empty-LNP solution or loaded-LNP solution comprises one or more of the following steps:ii-d) lyophilizing the LNP solution, thereby forming a lyophilized LNP composition; ii-e) storing the lyophilized LNP composition; and ii-f) adding a buffering solution to the lyophilized LNP composition, thereby forming the LNP formulation.Ionizable Lipids

[0433] In some embodiments, a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) of the disclosure further includes one or more ionizable lipids in addition to the cationic lipid of Formula (I).

[0434] In some aspects, the ionizable lipid is of compound of Formula (IL-A):(IL-A) or its N-oxide, or a salt or isomer thereof, wherein:R1is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;R2and R3are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;R4is selected from the group consisting of hydrogen, a C3-6carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R12)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted C1- 6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -OC(O)O-, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2,N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(CH2)nN(R)2and-C(R)N(R)2C(O)OR, NRAS(O)2RSX, and, wherein A is a 3-14membered heterocycle containing one or more heteroatoms selected from N, O and S; and a is1, 2, 3, or 4; wherein denotes a point of attachment;each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5;R8is selected from the group consisting of C3-6carbocycle and heterocycle;R9is selected from the group consisting of H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6alkenyl, C3-6carbocycle and heterocycle;R12is selected from the group consisting of H, OH, C1-3alkyl, and C2-3alkenyl; each R is independently selected from the group consisting of C1-6alkyl, C1-3alkyl-aryl, C2-3alkenyl, and H;RAis selected from H and C1-3alkyl;Rsxis selected from a C3-8carbocycle, a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, C1-6alkyl, C2-6alkenyl, (C1-3alkoxy)C1-3alkyl, (CH2)PIO(CH2)P2RSX1, and (CH2)PIRSX1, wherein the carbocycle and heterocycle are optionally substituted with one or more groups selected from oxo, C1-6alkyl, and (C1-3alkoxy)C1-3alkyl;RSX1is selected from C(O)NR14R14’, a C3-8carbocycle, and a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, wherein the carbocycle and heterocycle are each optionally substituted with one or more groups selected from oxo, halo, C1-3alkyl, (C1-3alkoxy)C1-3alkyl, C1-6alkylamino, di-(C1-6alkyl) amino, and NH2; each R13is selected from the group consisting of OH, oxo, halo, C1-6alkyl, C1-6alkoxy, C2-6alkenyl, C1-6alkylamino, di-(C1-6alkyl) amino, NH2, C(0)NH2, CN, and NO2;R14and R14are each independently selected from the group consisting of H and C1-6alkyl; pi is selected from 1, 2, 3, 4, and 5;P2 is selected from 1, 2, 3, 4, and 5; each R5is independently selected from the group consisting of OH, C1-3alkyl, C2-3alkenyl, and H; each R6is independently selected from the group consisting of OH, C1-3alkyl, C2-3alkenyl, and H;R7is selected from the group consisting of C1-3alkyl, C2-3alkenyl, and H;M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)-M”- C(O)O-,-C(O)N(RM)-, -N(RM)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(O RM)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, C1-13 alkyl or C2- 13 alkenyl; each RMis independently selected from the group consisting of H, C1-6alkyl and C2-6alkenyl; each R’ is independently selected from the group consisting of C1-18alkyl, C2-18alkenyl, -R*YR”, -YR”, (CH2)qOR*, and H, and each q’ is independently selected from 1, 2, and 3; each R” is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; each R* is independently selected from the group consisting of C1-12alkyl and C2-12alkenyl; each Y is independently a C3-6carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0435] In some aspects, the ionizable lipid is of compound of Formula (IL-B):r its N-oxide, or a salt or isomer thereof, wherein R’ais R’branched; whereindenotes a point of attachment; wherein Raα, Raβ, Raγand Raδare each independently selected from the group consisting of H, C2-12alkyl, and C2-12alkenyl;R2and R3are each independently selected from the group consisting of C1-14alkyl and C2-14alkenyl;R4is selected from the group consisting of -(CH2)nOH, wherein n is selected from the group consistingwhereindenotes a point of attachment; whereinR10is N(R)2; each R is independently selected from the group consisting of C1- 6 alkyl, C2-3alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R5is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl, and H; each R6is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl, and H;M and M’ are each independently selected from the group consisting of -C(O)O- and-0C(0)-;R’ is a C1-12 alkyl or C2-12alkenyl;1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0436] In some aspects, the ionizable lipid is of compound a compound of Formula (IL-C):salt or isomer thereof, wherein1 is selected from 1, 2, 3, 4, and 5;Mi is M’;R4 is -(CH2)nQ, in which Q is OH, and n is selected from 1, 2, 3, 4, or 5;M and M’ are independently selected from -C(O)O- and -OC(O)-;R2 and R3 are both C1-14 alkyl, or C2-14 alkenyl; andR’ is a C1-C12 linear alkyl.

[0437] In some aspects, the ionizable lipid is of compound a compound of Formula (IL-D):whereindenotes a point of attachment;wherein Rayis selected from the group consisting of C1-12alkyl and C2-12alkenyl;R2and R3are each independently selected from the group consisting of C1-14alkyl and C2-14 alkenyl;R4is -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5;R’ is a C1-12alkyl or C2-12alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0438] In some aspects, the ionizable lipid is a of compound of Formula (IL-I):r its N-oxide, or a salt or isomer thereof, wherein:R1is selected from the group consisting of C5-30alkyl, C5-20alkenyl, -R*YR”, -YR”, and -R”M’R’;R2and R3are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;R4is selected from the group consisting of hydrogen, a C3-6carbocycle, -(CH2)nQ, - (CH2)nCHQR, -(CH2)oC(R10)2(CH2)n-oQ, -CHQR, -CQ(R)2, and unsubstituted C1-6alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, - N(R)C(S)N(R)2, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR,-N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2,-N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R,-C(O)N(R)OR, and -C(R)N(R)2C(0)0R, each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; each R5is independently selected from the group consisting of OH, C1-3alkyl, C2-3alkenyl, and H; each R6is independently selected from the group consisting of OH, C1-3alkyl, C2-3alkenyl, and H;M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, C1-13 alkyl or C2- 13 alkenyl;R7is selected from the group consisting of C1-3alkyl, C2-3alkenyl, and H;R8is selected from the group consisting of C3-6carbocycle and heterocycle;R9is selected from the group consisting of H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6alkenyl, C3-6carbocycle and heterocycle;R10is selected from the group consisting of H, OH, C1-3alkyl, and C2-3alkenyl; each R is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl, (CH2)qOR*, and H, and each q is independently selected from 1, 2, and 3; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-15 alkyl and C3-15alkenyl; each R* is independently selected from the group consisting of C1-12alkyl and C2-12alkenyl; each Y is independently a C3-6carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0439] In some aspects, the ionizable lipid is a of compound of Formula (IL-IA):salt or isomer thereof, wherein1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9;M1is a bond or M’;R4 is unsubstituted C1-3alkyl, or -(CH2)nQ, in which Q isOH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8,-NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR,-N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, or heteroaryl, and each n is selected from 1,2, 3, 4, or 5;M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; andR2 and R3 are both C1-14 alkyl or C2-14 alkenyl;Rs is selected from the group consisting of C3-6carbocycle and heterocycle;R9 is selected from the group consisting of H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6alkenyl, C3-6carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl, and H; andR’ is a Cuis alkyl or C2-18 alkenyl.

[0440] In some aspects, the ionizable lipid is a compound of Formula (IL-IB):or a salt or isomer thereof, wherein1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9;R is selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; andR2and R3are independently selected from the group consisting of C1-14 alkyl, and C2- 14 alkenyl;M and M’ are independently selected from -C(O)O- and -OC(O)-;RNis H, or C1-3alkyl;Xaand Xbare each independently O or S;R10is selected from the group consisting of H, halo, -OH, R, -N(R)2, -CN, -N3, - C(O)OH, -C(O)OR, -OC(O)R, -OR, -SR, -S(O)R, -S(O)OR, -S(O)2OR, -NO2, -S(O)2N(R)2, - N(R)S(O)2R, -NH(CH2)t1N(R)2, -NH(CH2)p1O(CH2)q1N(R)2, -NH(CH2)s1OR, - N((CH2)SOR)2, -N(R)-carbocycle, -N(R)-heterocycle, -N(R)-aryl, -N(R)-heteroaryl, - N(R)(CH2)ti-carbocycle, -N(R)(CH2)ti-heterocycle, -N(R)(CH2)ti-aryl, -N(R)(CH2)ti- heteroaryl, a carbocycle, a heterocycle, aryl and heteroaryl; each R is independently selected from the group consisting of C1-12alkyl, C2-12alkenyl, and H; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; r is 0 or 1;t1is selected from 1, 2, 3, 4, and 5; p1is selected from 1, 2, 3, 4, and 5; q1is selected from 1, 2, 3, 4, and 5; and s1is selected from 1, 2, 3, 4.

[0441] In some aspects, the ionizable lipid is a compound of Formula (IL-IC):whereindenotes a point of attachment; wherein Ray, Ray, and Rayare each C1-12alkyl or C2-12alkenyl;Rbyis H, C1-12alkyl or C2-12alkenyl;R2and R3are each independently selected from the group consisting of C1-14alkyl and C2-14alkenyl;R4is -(CH2)nOH;denotes a point of attachment; each R’ independently is a C1-12alkyl or C2-12alkenyl;R10is N(R)2; each R is independently selected from the group consisting of C1-6alkyl, C2-3alkenyl, and H; and n and n2 are each selected from the group consisting of 1, 2, 3, 4, and 5;Yais a C3-6carbocycle;R*”ais selected from the group consisting of C1-15alkyl and C2-15alkenyl;1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; ands is 2 or 3.

[0442] In some embodiments, the ionizable lipid is a compound of Formula (IL*)or a salt thereof, wherein:R1is -OH, -NRN-C4-10cycloalkenyl optionally substituted with one or more oxo or - N(RN’RN”);RNis H or C1-6alkyl;RNis H or C1-6alkyl;RNis H or C1-6alkyl; o is 1, 2, 3, or 4; n is 4, 5, 6, 7, or 8; m is 4, 5, 6, 7, or 8;M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2;M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3;R2aR2bR2isor -(C1-6alkylene)-(C3-8cycloalkyl)-C1-6alkyl;R2ais -H or C1-io alkyl;R2bis -H or C1-io alkyl;R2Cis C I-8 alkyl or C2-8 alkenyl;R3ais H or C1-10alkyl;R3bis H or C1-8alkyl; andR3Cis C1-10alkyl or C2-8alkenyl.

[0443] In some embodiments, the ionizable lipid is of Formula (IL**-I):(IL** -I) or a salt thereof, wherein:R1is -OH; o is 2, 3, or 4; n is 4, 5, 6, 7, or 8;M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8;M’ is -C(=O)-O-*, wherein * indicates attachment to R3;R2Cis C4-8alkyl;R3ais C7-10alkyl; andR3Cis C3-5alkyl.

[0444] In some embodiments, the ionizable lipid is of Formula (IL**-III):(IL**-III) or a salt thereof, wherein:R1is NRN-C4 -10cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN”);RNis H;RNis C 1-2 alkyl;RN” is H; o is 2, 3, or 4; n is 6, 7, or 8;M is -C(=O)-O-*, wherein * indicates attachment to R2;m is 6, 7, or 8;M’ is -C(=O)-O-*, wherein * indicates attachment to R3;R2ais C7-10 alkyl;R2Cis C4-6 alkyl;R3ais C1-3alkyl; andR3Cis C4-6 alkyl.

[0445] In some embodiments, the ionizable lipid is of Formula (IL**-IV):(IL** -IV) or a salt thereof, wherein:R1is OH; o is 2, 3, or 4; n is 6, 7, or 8;M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8;M’ is -C(=O)-O-*, wherein * indicates attachment to R3;R2bis C3-5 alkyl;R2Cis C2-4 alkyl;R3ais C7-10 alkyl; andR3Cis C4-6 alkyl.

[0446] In some embodiments, the ionizable lipid is of Formula (IL*-I):(IL*-Ia) or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; and R3ais C1-8alkyl.

[0447] In some embodiments, ionizable lipid is of Formula (IL*-Ia):or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8alkyl.

[0448] In some embodiments, the ionizable lipid is of Formula (IL*-Ia’):or a salt thereof, wherein: o, M, M’, R2Cand R3care as defined for variable IL*; and R3ais C1-8alkyl.

[0449] In some embodiments, the ionizable lipid is of Formula (IL*-IIa):or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8alkyl.

[0450] In some embodiments, the ionizable lipid is of Formula (IL*-IF):or a salt thereof, wherein: o, M, M’, R2Cand R3care as defined for variable IL*; and R3ais C1-8alkyl.

[0451] In some embodiments, the ionizable lipid is of Formula (IL*-III):(IL* -III) or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;R2ais a C1-8alkyl; andR3ais C1-8alkyl.

[0452] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa):or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;R2bis a C1-8alkyl; andR3ais C1-8alkyl.

[0453] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa):or a salt thereof, wherein:R1, o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8alkyl; andR3ais C1-8alkyl.

[0454] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa’):(IL*-IIIa’) or a salt thereof, wherein:R1, o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8alkyl; andR3ais C1-8alkyl.

[0455] In some embodiments, the ionizable lipid is of Formula (IL*-IIIb):(IL*-IIIb) or a salt thereof, wherein:R1, o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8alkyl; andR3ais C1-8alkyl.

[0456] In some embodiments, the ionizable lipid is of Formula (IL*-IIIb’):(IL*-IIIb’) or a salt thereof, wherein:R1, o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8alkyl; andR3ais C1-8alkyl.

[0457] In some embodiments, the ionizable lipid is of Formula (IL*-IV):(IL*-IV) or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;R2bis a C1-8alkyl; andR3ais C1-8alkyl.

[0458] In some embodiments, the ionizable lipid is of Formula (IL*-IVa):or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;R2bis a C1-8alkyl; andR3ais C1-8alkyl.

[0459] In some embodiments, the ionizable lipid is of Formula (IL*-Iva’):(ILMVa) or a salt thereof, wherein: o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8alkyl; andR3ais C1-8alkyl.Variables o, R1, RN, RN, RNof Ionizable Lipid

[0460] In some embodiments of the ionizable lipid, o is 1.

[0461] In some embodiments of the ionizable lipid, o is 2.

[0462] In some embodiments of the ionizable lipid, o is 3.

[0463] In some embodiments of the ionizable lipid, o is 4.

[0464] In some embodiments of the ionizable lipid, R1is -OH.

[0465] In some embodiments of the ionizable lipid, RNis H.

[0466] In some embodiments of the ionizable lipid, RNis methyl.

[0467] In some embodiments of the ionizable lipid, RNis ethyl.

[0468] In some embodiments of the ionizable lipid, R1is -NRN-cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(RNRN”).

[0469] In some embodiments of the ionizable lipid, RNis H.

[0470] In some embodiments of the ionizable lipid, RNis methyl.

[0471] In some embodiments of the ionizable lipid, RNis ethyl.

[0472] In some embodiments of the ionizable lipid, RNis H.

[0473] In some embodiments of the ionizable lipid, RNis methyl.

[0474] In some embodiments of the ionizable lipid, RNis ethyl.

[0475] In some embodiments of the ionizable lipid, RNis H and RNis methyl.

[0476] In some embodiments of the ionizable lipid,

[0477] In some embodiments of the ionizable lipid,Variables m and n of the Ionizable Lipid

[0478] In some embodiments of the ionizable lipid, m is 4.

[0479] In some embodiments of the ionizable lipid, m is 5.

[0480] In some embodiments of the ionizable lipid, m is 6.

[0481] In some embodiments of the ionizable lipid, m is 7.

[0482] In some embodiments of the ionizable lipid, m is 8.

[0483] In some embodiments of the ionizable lipid, m is 4.

[0484] In some embodiments of the ionizable lipid, n is 5.

[0485] In some embodiments of the ionizable lipid, n is 6.

[0486] In some embodiments of the ionizable lipid, n is 7.

[0487] In some embodiments of the ionizable lipid, n is 8.

[0488] In some embodiments of the ionizable lipid, n is 5 and m is 7.

[0489] In some embodiments of the ionizable lipid, n is 7 and m is 7.

[0490] In some embodiments of the ionizable lipid, m is 6 and n is 6.Variables M and M ’

[0491] In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2.

[0492] In some embodiments of the ionizable lipid, M is -C(=O)-O-* wherein * indicates attachment to R2.

[0493] In some embodiments of the ionizable lipid, M’ is -O-C(=O)-* , wherein * indicates attachment to R3.

[0494] In some embodiments of the ionizable lipid, M’ is -C(=O)-O-* wherein * indicates attachment to R3.

[0495] In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2, and M’ is -C(=O)-O-* wherein * indicates attachment to R3.Variables R2, R2a, R2b, R2c

[0496] In some embodiments of the ionizable lipid, R2is

[0497] In some embodiments of the ionizable lipid, R2ais hydrogen.

[0498] In some embodiments of the ionizable lipid, R2ais methyl.

[0499] In some embodiments of the ionizable lipid, R2ais ethyl.

[0500] In some embodiments of the ionizable lipid, R2ais propyl.

[0501] In some embodiments of the ionizable lipid, R2ais butyl.

[0502] In some embodiments of the ionizable lipid, R2ais pentyl.

[0503] In some embodiments of the ionizable lipid, R2ais hexyl.

[0504] In some embodiments of the ionizable lipid, R2ais heptyl.

[0505] In some embodiments of the ionizable lipid, R2ais octyl.

[0506] In some embodiments of the ionizable lipid, R2bis hydrogen.

[0507] In some embodiments of the ionizable lipid, R2bis methyl.

[0508] In some embodiments of the ionizable lipid, R2bis ethyl.

[0509] In some embodiments of the ionizable lipid, R2bis propyl.

[0510] In some embodiments of the ionizable lipid, R2bis butyl.

[0511] In some embodiments of the ionizable lipid, R2bis pentyl.

[0512] In some embodiments of the ionizable lipid, R2bis hexyl.

[0513] In some embodiments of the ionizable lipid, R2bis heptyl.

[0514] In some embodiments of the ionizable lipid, R2bis octyl.

[0515] In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis hydrogen.

[0516] In some embodiments of the ionizable lipid, R2ais hexyl and R2bis hydrogen.

[0517] In some embodiments of the ionizable lipid, R2ais octyl and R2bis hydrogen.

[0518] In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis butyl.

[0519] In some embodiments of the ionizable lipid, R2cis methyl.

[0520] In some embodiments of the ionizable lipid, R2cis ethyl.

[0521] In some embodiments of the ionizable lipid, R2cis propyl.

[0522] In some embodiments of the ionizable lipid, R2cis butyl.

[0523] In some embodiments of the ionizable lipid, R2cis pentyl.

[0524] In some embodiments of the ionizable lipid, R2cis hexyl.

[0525] In some embodiments of the ionizable lipid, R2cis heptyl.

[0526] In some embodiments of the ionizable lipid, R2cis octyl.

[0527] In some embodiments of the ionizable lipid, R2is — (C1-6alkylene)-(C3-8cycloalkyl)-C1-6alkyl.

[0528] In some embodiments of the ionizable lipid, R2is — (C1-6alkylene)-(cyclohexyl)- C1-6alkyl.

[0529] In some embodiments of the ionizable lipid, R2is — (C1-6alkylene)-(cyclopentyl)- C1-6alkyl.Variables R3, R3a, R3b, andR3c

[0530] In some embodiments of the ionizable lipid, R3is

[0531] In some embodiments of the ionizable lipid, R3ais hydrogen.

[0532] In some embodiments of the ionizable lipid, R3ais methyl.

[0533] In some embodiments of the ionizable lipid, R3ais ethyl.

[0534] In some embodiments of the ionizable lipid, R3ais propyl.

[0535] In some embodiments of the ionizable lipid, R3ais butyl.

[0536] In some embodiments of the ionizable lipid, R3ais pentyl.

[0537] In some embodiments of the ionizable lipid, R3ais hexyl.

[0538] In some embodiments of the ionizable lipid, R3ais heptyl.

[0539] In some embodiments of the ionizable lipid, R3ais octyl.

[0540] In some embodiments of the ionizable lipid, R3bis hydrogen.

[0541] In some embodiments of the ionizable lipid, R3bis methyl.

[0542] In some embodiments of the ionizable lipid, R3bis ethyl.

[0543] In some embodiments of the ionizable lipid, R3bis propyl.

[0544] In some embodiments of the ionizable lipid, R3bis butyl.

[0545] In some embodiments of the ionizable lipid, R3bis pentyl.

[0546] In some embodiments of the ionizable lipid, R3bis hexyl.

[0547] In some embodiments of the ionizable lipid, R3bis heptyl.

[0548] In some embodiments of the ionizable lipid, R3bis octyl.

[0549] In some embodiments of the ionizable lipid, R3ais octyl and R3bis hydrogen.

[0550] In some embodiments of the ionizable lipid, R3ais ethyl and R3bis hydrogen.

[0551] In some embodiments of the ionizable lipid, R3ais hexyl and R3bis hydrogen.

[0552] In some embodiments of the ionizable lipid, R3cis methyl.

[0553] In some embodiments of the ionizable lipid, R3cis ethyl.

[0554] In some embodiments of the ionizable lipid, R3cis propyl.

[0555] In some embodiments of the ionizable lipid, R3cis butyl.

[0556] In some embodiments of the ionizable lipid, R3cis pentyl.

[0557] In some embodiments of the ionizable lipid, R3cis hexyl.

[0558] In some embodiments of the ionizable lipid, R3cis heptyl.

[0559] In some embodiments of the ionizable lipid, R3cis octyl.It is understood that, for an ionizable lipid, variables o, R1, RN, RN, RN, m, n, M, M’, R2, R2a, R2b, R2C, R3, R3a, R3b, and R3ccan each be, where applicable, selected from the groups described herein, and any group described herein for any of variables o,. R1, RN, RN, RN, m, n, M, M’, R2, R2a, R2b, R2C, R3, R3a, R3b, and R3ccan be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R1, RN, RN, RN, m, n, M, M’, R2, R2a, R2b, R2C, R3, R3a, R3b, and R3c.

[0560] In some embodiments, the ionizable lipid is a compound selected from Table IL-1.Table IL-1: Ionizable lipids

[0561] In some embodiments, the ionizable lipid is a compound selected from Table IL-2.Table IL-2: Ionizable lipids

[0562] In some aspects, the ionizable lipid is a compound of Formula (IL-IIA):(IL-IIA), or its N-oxide, or a salt or isomer thereof, wherein: m is selected from 5, 6, 7, 8, and 9;R2and R3are each independently selected from the group consisting of H, C1-14alkyl, and C2-14 alkenyl;R4is selected from -(CH2)nOH, wherein n is selected from 1, 2, 3, 4, and 5, and, wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R10is -N(R)2, wherein each R is independently selected from the group consisting of C1-6alkyl, C2-3alkenyl, and H;M is selected from -OC(O)O-, -C(O)O-, -O-M”-O-, and -N(RM)C(O)-, in which M” is -(CH2)ZC(O)-, wherein z is 1, 2, 3, or 4;M’ is selected from -OC(O)O-, -C(O)O-, -O-M”-O-, -N(RM)C(O)O-, and -O- N=C(RM)-, wherein:M” is -(CH2)ZC(O)-, C1-13 alkyl, -B(R**)-, or -Si(R**)2-; z is 1, 2, 3, or 4; each RMis independently selected from H and C1-6alkyl; each R** is independently selected from H and C1-12alkyl;R’ais C1-18alkyl, C2-18alkenyl, or -R*YR*”, wherein:each R*” is independently C1-15alkyl; each R* is independently C1-12alkyl; each Y is independently a C3-6carbocycle; and R” is a C3-C13alkyl, optionally substituted with OH.

[0563] In some aspects, the ionizable lipid is a compound of Formula (IL-IIAX):r its N-oxide, or a salt or isomer thereof, wherein:R1is -R”M’R’, wherein: each R’ is independently C1-18alkyl;M’ is selected from -C(O)O- and -O-N=C(RM)-, wherein each RMis independently selected from H and C1-6alkyl; each R” is independently C3-15 alkyl;R2and R3are each independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl;R4is selected from -(CH2)nOH, wherein n is selected from 1, 2, 3, 4, and 5, and, wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R10is -N(R)2, wherein each R is independently selected from the group consisting of C1-6alkyl, C2-3alkenyl, and H; each R5is H; each R6is H; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0564] In some embodiments, the ionizable lipid is a compound selected from Table IL-3.Table IL-3: Ionizable lipids

[0565] In some aspects, the ionizable lipid is a compound of Formula (IL-IIB):whereindenotes a point of attachment;Raβ, Ray, and Raδare each independently selected from the group consisting of H, C1-12alkyl, and C2-12alkenyl;Rbβ, Rbγ, and Rbδare each independently selected from the group consisting of H, C1-12alkyl, and C2-12alkenyl, wherein at least one of Rbβ, Rby, and Rbδis selected from the group consisting of C1-12alkyl and C2-12alkenyl;R2and R3are each independently selected from the group consisting of C1-14alkyl and C2-14 alkenyl;R4is selected from -(CH2)nNRTQ, -(CH2)nNRS(O)2TQ, -(CH2)nNRC(O)H and -(CH2)nNRC(O)TQ wherein n is selected from 1, 2, 3, 4, and 5;T is a bond or a C1-3alkyl linker, C2-3alkenyl linker, or C2-3alkynyl linker;Q is selected from 3-14 membered heterocycle containing 1-5 heteroatoms selected from N, O, and S, C3-10 carbocycle, C1-6alkyl, and C2-6alkenyl, wherein the alkyl, alkenyl, heterocycle, and carbocycle are each optionally substituted with one or more RQ; each RQindependently is selected from the group consisting of oxo, hydroxyl, cyano, amino, C1-6alkylamino, di-C1-6alkylamino, C1-6alkyl, C1-6alkoxy, C2-6alkenyl, C1-6alkanolyl, -C(O)C1-6alkyl, and -NRC(0)C1-6alkyl; each R is independently selected from H, C1-6alkyl, and C2-6alkenyl; each R’ is independently selected from C1-12alkyl and C2-12alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; and1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0566] In some embodiments, the ionizable lipid is a compound selected from Table IL-4.

[0567] In some embodiments, the ionizable lipid is a compound selected from Table IL-5.

[0568] In some aspects, the ionizable lipid is a compound of Formula (IL-IIC):r its N-oxide, or a salt or isomer thereof, wherein:R’branchedidenotes a point of attachment; wherein Raαand Raβare each independently selected from the group consisting of H and C1-2 alkyl, wherein at least one of Raαand Raβis a C1or C2alkyl;R’ is selected from the group consisting of C1-18alkyl and C2-18alkenyl;R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14alkenyl;R4is -(CH2)nQ, wherein n is independently selected from 1, 2, 3, 4, and 5, where Q is selected fromwherein A is a 3-14 memberedheterocycle containing one or more heteroatoms selected from N, O and S; and a is 1, 2, 3, or 4; wherein denotes a point of attachment;R is selected from H and C1-3alkyl;Rsxis selected from a C3-8carbocycle, a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, C1-6alkyl, C2-6alkenyl, (C1-3alkoxy)C1-3alkyl, (CH2)PIO(CH2)P2RSX1, and (CH2)PIRSX1, wherein the carbocycle and heterocycle are optionally substituted with one or more groups selected from oxo, C1-6alkyl, and (C1-3alkoxy)C1-3alkyl;RSX1is selected from C(O)NR14R14’, a C3-8carbocycle, and a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, wherein the carbocycle and heterocycle are each optionally substituted with one or more groups selected from oxo, halo, C1-3alkyl, (C1-3alkoxy)C1-3alkyl, C1-6alkylamino, di-(C1-6alkyl) amino, and NH2; each R13is selected from the group consisting of OH, oxo, halo, C1-6alkyl, C1-6alkoxy, C2-6alkenyl, C1-6alkylamino, di-(C1-6alkyl) amino, NH2, C(0)NH2, CN, and NO2;R14and R14are each independently selected from the group consisting of H and C1-6alkyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; pi is selected from 1, 2, 3, 4, and 5; andP2 is selected from 1, 2, 3, 4, and 5.

[0569] In some embodiments, the ionizable lipid is a compound selected from Table IL-6.

[0570] In some aspects, the ionizable lipid is a compound of Formula (IL-III):t is 1 or 2;Ai and A2 are each independently selected from CH or N;Z is CH2or absent wherein when Z is CH2, the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;Ri, R2, R3, R4, and R5 are independently selected from the group consisting of C5-20 alkyl, C5-20 alkenyl, -R”MR’, -R*YR”, -YR”, and -R*OR”;Rxi and Rx2 are each independently H or C1-3alkyl; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, - OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, - P(O)(OR’)O-, -S(O)2-, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group;M* is C1-C6alkyl,W1and W2are each independently selected from the group consisting of -O- and - N(Re)-; each R6is independently selected from the group consisting of H and C1-5 alkyl;X1, X2, and X3are independently selected from the group consisting of a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -(CH2)n-C(O)-, -C(O)-(CH2)n-, -(CH2)n- C(O)O-, -OC(O)-(CH2)n-, -(CH2)n-OC(O)-, -C(O)O-(CH2)n-, -CH(OH)-, -C(S)-, and - CH(SH)-; each Y is independently a C3-6carbocycle; each R* is independently selected from the group consisting of C1-12alkyl and C2-12alkenyl; each R is independently selected from the group consisting of C1-3alkyl and a C3-6carbocycle; each R’ is independently selected from the group consisting of C1-12alkyl, C2-12alkenyl, and H; each R” is independently selected from the group consisting of C3-12alkyl, C3-12alkenyl and -R*MR’; and n is an integer from 1-6.

[0571] In some aspects, the ionizable lipid is a compound of Formula (IL-IIIA):or a salt or isomer thereof, whereinRi, R2, R3, R4, and R5 are independently selected from the group consisting of C5-20alkyl, C5-20alkenyl, -R”MR’, -R*YR”, -YR”, and -R*OR”; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group;X1, X2, and X3are independently selected from the group consisting of a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)-CH2-, -CH2-C(O)O-, -CH2-OC(O)-, -CH(OH)-, -C(S)-, and -CH(SH)-; each Y is independently a C3-6carbocycle; each R* is independently selected from the group consisting of C1-12alkyl and C2-12alkenyl; each R is independently selected from the group consistin0 g of C1-3alkyl and a C3-6carbocycle; each R’ is independently selected from the group consisting of C1-12alkyl, C2-12alkenyl, and H; and each R” is independently selected from the group consisting of C3-12alkyl and C3-12alkenyl.

[0572] In some embodiments, the ionizable lipid is a compound selected from Table IL-7.Table IL-7: Ionizable lipids

[0573] In some embodiments, the ionizable lipid is a compound selected from:

[0574] In some embodiments, the ionizable lipid is a lipid disclosed in Published International Patent Application Nos. WO / 2017 / 049245, WO / 2017 / 112865, WO / 2018 / 170306, WO / 2018 / 232120, WO / 2021 / 055835, WO / 2021 / 055833, and WO / 2021 / 055849, each of which is incorporated by reference herein in its entirety.Polyethylene Glycol (PEG) Lipids

[0575] As used herein, the term “PEG lipid” refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG- modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0576] In some embodiments, the PEG lipid includes, but are not limited to, 1,2-dimyristoyl- sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-di stearoyl -sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEGDAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2- dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[0577] In one embodiment, the PEG lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[0578] In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about C14to about C22, In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about C14to about C16. In some embodiments, a PEG moiety, for example an mPEG-NEE, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In one embodiment, the PEG lipid is PEG2k-DMG.

[0579] In one embodiment, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG- DSG and PEG-DSPE.

[0580] PEG lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015 / 130584 A2, which are incorporated herein by reference in their entirety.

[0581] In general, some of the other lipid components (e.g., PEG lipids) of various formulae, described herein may be synthesized as described International Patent Application No. PCT / US2016 / 000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety.

[0582] The lipid component of a lipid nanoparticle or lipid nanoparticle formulation may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG- modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG- modified dialkylglycerols, and mixtures thereof. In some embodiments, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0583] In some embodiments, the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure:

[0584] In one embodiment, PEG lipids useful in the present invention can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In some embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy -PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In some embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In some embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0585] In some embodiments, a PEG lipid useful in the present invention is a compound of Formula (PL-I). Provided herein are compounds of Formula (PL-I):or salts thereof, wherein:R3is -OR°;R° is hydrogen, optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive;L1is optionally substituted C1-10 alkylene, wherein at least one methylene of the optionally substituted C1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, - OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN);D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;A is of the formula:each instance of of L2is independently a bond or optionally substituted C1-6alkylene, wherein one methylene unit of the optionally substituted C1-6alkylene is optionally replaced with O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), - NRNC(O)O, or NRNC(O)N(RN); each instance of R2is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), - NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), - C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O) , OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), - S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O; each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2.

[0586] In some embodiments, the compound of Formula (PL-I) is a PEG-OH lipid (i.e., R3is -OR°, and R° is hydrogen). In some embodiments, the compound of Formula (PL-I) is of Formula (PL-I-OH):or a salt thereof.

[0587] In some embodiments, a PEG lipid useful in the present invention is a PEGylated fatty acid. In some embodiments, a PEG lipid useful in the present invention is a compound of Formula (PL-II). Provided herein are compounds of Formula (PL-II):or a salt thereof, wherein:R3is-OR°;R° is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between 1 and 100, inclusive;R5is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; and optionally one or more methylene groups of R5are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), - C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), - C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, - OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, - N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O; and each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[0588] In some embodiments, the compound of Formula (PL-II) is of Formula (PL-II-OH):or a salt thereof, wherein: r is an integer between 1 and 100;R5is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; and optionally one or more methylene groups of R5arereplaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), - C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), - C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, - OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, - N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O; and each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[0589] In some embodiments, r is an integer between 10 to 80, between 20 to 70, between 30 to 60, or between 40 to 50.

[0590] In some embodiments, r is 45.

[0591] In some embodiments, R5is C17 alkyl.

[0592] In yet other embodiments the compound of Formula (PL-II) is:or a salt thereof.

[0593] In one embodiment, the compound of Formula (PL-II) is

[0594] In some aspects, the lipid composition of the pharmaceutical compositions described herein does not comprise a PEG lipid.

[0595] In some embodiments, the PEG lipids may be one or more of the PEG lipids described in U.S. Application No. 62 / 520,530.

[0596] In some embodiments, the PEG lipid is a compound of Formula (PL-III):or a salt or isomer thereof, wherein s is an integer between 1 and 100.

[0597] In some embodiments, the PEG lipid is a compound of the following formula:or a salt or isomer thereof.Structural Lipids

[0598] As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties.

[0599] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a mixture of two or more components each independently selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alphatocopherol, hopanoids, phytosterols, and steroids. In some embodiments, the structural lipid is a sterol. In some embodiments, the structural lipid is a mixture of two or more sterols. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is alpha-tocopherol.

[0600] In some embodiments, the structural lipids may be one or more structural lipids described in P.C.T. Application No. PCT / US 18 / 37922.

[0601] As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is alpha-tocopherol.

[0602] In some embodiments, the structural lipid isI l lor a salt thereof.

[0603] In some embodiments, the structural lipid is SL-1.

[0604] In some embodiments, the structural lipid is(SL-2) or a salt thereof.

[0605] In some embodiments, the structural lipid (e.g., SL-2) is present at a concentration ranging from about 15 mol% to about 70 mol %, from about 20 mol% to about 60 mol %, from about 25 mol% to about 50 mol %, from about 30 mol% to about 45 mol %, from about 35 mol% to about 40 mol %, or from about 36 mol% to about 38 mol %.

[0606] In some embodiments, the structural lipid (e.g., SL-2) is present at a concentration of about 36.6±25 mol %, about 36.6±20 mol %, about 36.6±15 mol %, about 36.6±10 mol %, about 36.6±9 mol %, about 36.6±8 mol %, about 36.6±7 mol %, about 36.6±6 mol %, about 36.6±5 mol %, about 36.6±4 mol %, about 36.6±3 mol %, about 36.6±2 mol %, about 36.6±1 mol %, about 36.6±0.8 mol %, about 36.6±0.6 mol %, about 36.6±0.5 mol %, about 36.6±0.4 mol %, about 36.6±0.3 mol %, about 36.6±.2 mol %, or about 36.6±0.1 mol % (e.g., about 36.6 mol %).Encapsulation Agent

[0607] In some embodiments of the present disclosure, the encapsulation agent is a compound of Formula (EA-I):or salts or isomers thereof, whereinR201 and R202 are each independently selected from the group consisting of H, C1-C6alkyl, C2-C6alkenyl, and (C=NH)N(RIOI)2 wherein each R101 is independently selected from the group consisting of H, C1-C6alkyl, and C2-C6alkenyl;R203 is selected from the group consisting of C1-C20alkyl and C2-C20alkenyl;R204 is selected from the group consisting of H, C1-C20alkyl, C2-C20alkenyl, C(O)(OC1-C20alkyl), C(0)(OC2-C20alkenyl), C(0)(NHC1-C20alkyl), and C(0)(NHC2-C20alkenyl); nl is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0608] In some embodiments, R201 and R202 are each independently selected from the group consisting of H and CH3.

[0609] In some embodiments, R201 and R202 are each independently selected from the group consisting of (C=NH)NH2and (C=NH)N(CH3)2.

[0610] In some embodiments, R203 is selected from the group consisting of C1-C20alkyl, C8- C18alkyl, and C12-C16alkyl.

[0611] In some embodiments, R204 is selected from the group consisting of H, C1-C20alkyl, C2-C20alkenyl, C(0)(OC1-C20alkyl), C(O)(OC2-C20alkenyl), C(0)(NHC1-C20alkyl), and C(0)(NHC2-C2O alkenyl); C8-C18alkyl, C8-C18alkenyl, C(O)(OC8-C18alkyl), C(O)(OC8-C18alkenyl), C(O)(NHC8-C18alkyl), and C(O)(NHC8-C18alkenyl); and C12-C16alkyl, C12-C16alkenyl, C(O)(OC12-C16alkyl), C(O)(OC12-C16alkenyl), C(O)(NHC12-C16alkyl), and C(O)(NHC12-C16alkenyl).

[0612] In some embodiments, nl is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; nl is selected from 1, 2, 3, 4, 5, and 6; nl is selected from 2, 3, and 4.

[0613] In some embodiments, nl is 3.

[0614] In some embodiments of the present disclosure, the encapsulation agent is a compound of Formula (EA-II):or salts or isomers thereof, whereinX101 is a bond, NH, or O;Rioi and R102 are each independently selected from the group consisting of H, C1-C6alkyl, and C2-C6alkenyl;R103 and R104 are each independently selected from the group consisting of C1-C20alkyl and C2-C20alkenyl; and nl is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0615] In some embodiments, X101 is a bond.

[0616] In some embodiments, X101 is NH.

[0617] In some embodiments, X101 is O.

[0618] In some embodiments, R101 and R102 are each independently selected from the group consisting of H and CH3.

[0619] In some embodiments, R103 is selected from the group consisting of C1-C20alkyl, C8- C18alkyl, and C12-C16alkyl.

[0620] In some embodiments, R104 is selected from the group consisting of C1-C20alkyl, C8- C18alkyl, and C12-C16alkyl.

[0621] In some embodiments, n1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; nl is selected from 1, 2, 3, 4, 5, and 6; nl is selected from 2, 3, and 4.

[0622] In some embodiments, n1 is 3.

[0623] Exemplary encapsulation agents include, but are not limited to, ethyl lauroyl arginate, ethyl myristoyl arginate, ethyl palmitoyl arginate, ethyl cholesterol-arginate, ethyl oleic arginate, ethyl capric arginate, and ethyl carprylic arginate.

[0624] In certain embodiments, the encapsulation agent is ethyl lauroyl arginate,salt or isomer thereof.

[0625] In certain embodiments, the encapsulation agent is at least one compound selected from the group consisting of:or salts and isomers thereof, such as, for example free bases, TFA salts, and / or HC1 salts.Phospholipids

[0626] Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0627] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[0628] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0629] Particular phospholipids can facilitate fusion to a membrane. In some embodiments, a cationic phospholipid can interact with one or more negatively charged phospholipids of amembrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

[0630] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. In some embodiments, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0631] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[0632] In some embodiments, a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC. In some embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (PhL-I):or a salt thereof, wherein: each R1is independently optionally substituted alkyl; or optionally two R1are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R1are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;A is of the formula:each instance of L2is independently a bond or optionally substituted C1-6alkylene, wherein one methylene unit of the optionally substituted C1-6alkylene is optionally replaced with -O-, -N(RN)-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, or -NRNC(O)N(RN)-; each instance of R2is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(RN)-, -N(RN)S(O)O-, -S(O)2-, -N(RN)S(O)2-, -S(O)2N(RN)-, -N(RN)S(O)2N(RN)-, -OS(O)2N(RN)-, or -N(RN)S(O)2O-; each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2.

[0633] In some embodiments, the phospholipids is not of the formula:wherein each instance of R2is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0634] In some embodiments, the phospholipids may be one or more of the phospholipids described in U.S. Application No. 62 / 520,530.

[0635] In some embodiments, the phospholipids may be selected from the non-limiting group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3 -phosphocholine(DOPC), 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn- glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di-O-octadecenyl-sw-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sw-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn- glycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt(DOPG), and sphingomyelin. In some embodiments, a LNP includes DSPC. In some embodiments, a LNP includes DOPE. In some embodiments, a LNP includes both DSPC and DOPE. i) Phospholipid Head Modifications

[0636] In some embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified phospholipid head (e.g., a modified choline group). In some embodiments, a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine. In some embodiments, in embodiments of Formula (PhL-I), at least one of R1is not methyl. In some embodiments, at least one of R1is not hydrogen or methyl. In some embodiments, the compound of Formula (PhL-I) is one of the following formulae:or a salt thereof, wherein: each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each v is independently 1, 2, or 3.In some embodiments, a compound of Formula (PhL-I) is of Formula (PhL-I-a):or a salt thereof.

[0637] In some embodiments, a phospholipid useful or potentially useful in the present invention comprises a cyclic moiety in place of the glyceride moiety. In some embodiments, a phospholipid useful in the present invention is DSPC, or analog thereof, with a cyclic moiety in place of the glyceride moiety. In some embodiments, the compound of Formula (PhL-I) is of Formula (PhL-I-b):or a salt thereof. ii) Phospholipid Tail Modifications

[0638] In some embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified tail. In some embodiments, a phospholipid useful or potentially useful in the present invention is DSPC, or analog thereof, with a modified tail. As described herein, a “modified tail” may be a tail with shorter or longer aliphatic chains, aliphatic chains with branching introduced, aliphatic chains with substituents introduced, aliphatic chains wherein one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof. In some embodiments, In some embodiments, the compound of (PhL-I) is of Formula (PhL-I-a), or a salt thereof, wherein at least one instance of R2is each instance of R2is optionally substituted C1-30 alkyl, wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-,-NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(RN)-, -N(RN)S(O)O-, -S(O)2-, -N(RN)S(O)2-, -S(O)2N(RN)-, -N(RN)S(O)2N(RN)-, -OS(O)2N(RN)-, or -N(RN)S(O)2O-.

[0639] In some embodiments, the compound of Formula (PhL-I) is of Formula (PhL-I-c):or a salt thereof, wherein: each x is independently an integer between 0-30, inclusive; and each instance is G is independently selected from the group consisting of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(RN)-, -N(RN)S(O)O-, -S(O)2-, -N(RN)S(O)2-, -S(O)2N(RN)-, -N(RN)S(O)2N(RN)-, -OS(O)2N(RN)-, or -N(RN)S(O)2O-. Each possibility represents a separate embodiment of the present invention.

[0640] In some embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in some embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (PhL-I), wherein n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a compound of Formula (PhL-I) is of one of the following formulae:or a salt thereof.Alternative lipids

[0641] In some embodiments, an alternative lipid is used in place of a phospholipid of the present disclosure. Non-limiting examples of such alternative lipids include the following:Other Components

[0642] A lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may include one or more components in addition to those described in the preceding sections. For example, a lipidnanoparticle (e.g., an empty LNP or a loaded LNP) may include one or more small hydrophobic molecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol.

[0643] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may also include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents, or other components. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

[0644] A polymer may be included in and / or used to encapsulate or partially encapsulate a nanoparticle composition. A polymer may be biodegradable and / or biocompatible. A polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, a polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co- glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co- caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co- PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, polyoxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), trimethylene carbonate, poly(7V-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[0645] Surface altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and pol oxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, domase alfa, neltenexine, and erdosteine), and DNases (e.g., rhDNase). A surface altering agent may be disposed within a nanoparticle and / or on the surface of a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) (e.g, by coating, adsorption, covalent linkage, or other process).

[0646] A lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may also comprise one or more functionalized lipids. For example, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction. In particular, a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition, or imaging. The surface of a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0647] In addition to these components, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may include any substance useful in pharmaceutical compositions. For example, the lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included.

[0648] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and / or combinations thereof. Granulating and dispersing agents may be selected from the nonlimiting list consisting of potato starch, com starch, tapioca starch, sodium starch glycolate,clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0649] Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol di stearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0650] A binding agent may be starch (e.g cornstarch and starch paste); gelatin; sugars (e.g sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof, or any other suitable binding agent.

[0651] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMAB EN®II, NEOLONE™, KATHON™, and / or EUXYL®.

[0652] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate,d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. Lubricating agents may selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0653] Examples of oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils as well as butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.Nanoparticle compositions

[0654] In some aspects, the present disclosure provides a lipid nanoparticle (an LNP, e.g., an empty LNP or a loaded LNP) comprising a cationic lipid described herein, e.g., according to Formula (I).

[0655] In some embodiments, the lipid nanoparticle (LNP) further comprises an ionizable lipid.

[0656] In some embodiments, the lipid nanoparticle (LNP) further comprises an ionizable lipid, a phospholipid, and a structural lipid.

[0657] In some embodiments, the lipid nanoparticle (LNP) further comprises an ionizable lipid, a phospholipid, a structural lipid, and a PEG lipid.

[0658] In some embodiments, the lipid nanoparticle (LNP) is an empty LNP. In some embodiments, the lipid nanoparticle (LNP) is free of therapeutic agent, e.g., free of nucleic acid.

[0659] In some embodiments, the lipid nanoparticle (LNP) is a loaded LNP. In some embodiments, the lipid nanoparticle (LNP) further comprises a therapeutic agent, e.g., a nucleic acid.

[0660] In some embodiments, the nucleic acid is an RNA.

[0661] In some embodiments, the nucleic acid is an mRNA.

[0662] In some embodiments, the largest dimension of a nanoparticle composition is 1 pm or shorter (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter), e.g., when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. Nanoparticle compositions include, for example, lipid nanoparticles (LNPs; e.g., empty LNPs or loaded LNPs), liposomes, lipid vesicles, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and / or cross-linked to one another. Lipid bilayers may include one or more ligands, proteins, or channels.

[0663] A lipid nanoparticle (e.g., an empty LNP or a loaded LNP) of the present disclosure comprises at least one cationic lipid according to Formula (I). In some embodiments, an empty LNP or a loaded LNP of the disclosure includes one or more of cationic lipids of Table 1. Nanoparticle compositions may also include a variety of other components. For example, in some embodiments, the empty LNP or a loaded LNP includes one or more ionizable lipids in addition to a lipid according to Formula (I).

[0664] In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 0.1 : 1 to about 20: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 1.5: 1 to about 10: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 1.5: 1 to about 9: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 1.5: 1 to about 8: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 1.5: 1 to about 7: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / orprophylactic agent is about 1.5: 1 to about 6: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 1.5: 1 to about 5: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 1.5: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 2: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 3: 1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 4: 1. In some embodiments, a molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is about 5: 1.

[0665] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) has a zeta potential of about 5 mV to about 20 mV. In some embodiments, the nanoparticle has a zeta potential of about 5 mV to about 15 mV. In some embodiments, the nanoparticle has a zeta potential of about 5 mV to about 12 mV. In some embodiments, the nanoparticle has a zeta potential of about 5 mV to about 10 mV.

[0666] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises 3.5% of PEG lipid PL-II. In some embodiments, the LNP comprises 3.25% PL-II. In some embodiments, the LNP comprises 3.0% PL-II. In some embodiments, the LNP comprises 2.75% PL-II. In some embodiments, the LNP comprises 2.5% PL-II. In some embodiments, the LNP comprises 2.25% PL-II. In some embodiments, the LNP comprises 2.0% PL-II. In some embodiments, the LNP comprises 1.75% PL-II. In some embodiments, the LNP comprises 1.5% PL-II.

[0667] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises 3.5% of PEG lipid PEG-1. In some embodiments, the LNP comprises 3.25% PEG-1. In some embodiments, the LNP comprises 3.0% PEG-1. In some embodiments, the LNP comprises 2.75% PEG-1. In some embodiments, the LNP comprises 2.5% PEG-1. In some embodiments, the LNP comprises 2.25% PEG-1. In some embodiments, the LNP comprises 2.0% PEG-1. In some embodiments, the LNP comprises 1.75% PEG-1. In some embodiments, the LNP comprises 1.5% PL-II.

[0668] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises 0.5% of PEG-DSG. In some embodiments, the LNP comprises 0.25% PEG-DSG. In some embodiments, the LNP comprises 0.5% PEG-DSG. In some embodiments, the LNP comprises 0.75% PEG-DSG. In some embodiments, the LNP comprises 1.0% PEG-DSG. In some embodiments, the LNP comprises 1.25% PEG-DSG. In some embodiments, the LNP comprises 1.5% PEG-DSG. In some embodiments, the LNP comprises 1.75% PEG-DSG. In some embodiments, the LNP comprises 2.0% PEG-DSG.

[0669] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises 0% of PEG-DSG and 3% PEG-II. In some embodiments, the LNP comprises 0.25% PEG-DSG and 2.75% PEG-II. IN some embodiments, the LNP comprises 0.5% PEG-DSG and 2.5% PEG-II. In some embodiments, the LNP comprises 0.75% PEG-SG and 2.25% PEG-II. In some embodiments, the LNP comprises 1.0% PEG-DSG and 2.0% PEG-II. In some embodiments, the LNP comprises 1.25% PEG-DSG and 1.75% PEG-II. In some embodiments, the LNP comprises 1.5% PEG-DSG and 1.5% PEG-II.

[0670] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises 0% of PEG-DSG and 3% PEG-1. In some embodiments, the LNP comprises 0.25% PEG-DSG and 2.75% PEG-1. IN some embodiments, the LNP comprises 0.5% PEG-DSG and 2.5% PEG-1. In some embodiments, the LNP comprises 0.75% PEG-SG and 2.25% PEG-1. In some embodiments, the LNP comprises 1.0% PEG-DSG and 2.0% PEG-1. In some embodiments, the LNP comprises 1.25% PEG-DSG and 1.75% PEG-1. In some embodiments, the LNP comprises 1.5% PEG-DSG and 1.5% PEG-1.

[0671] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises about 3.5% of PEG lipid PL-II. In some embodiments, the LNP comprises about 3.25% PL-II. In some embodiments, the LNP comprises about 3.0% PL-II. In some embodiments, the LNP comprises about 2.75% PL-II. In some embodiments, the LNP comprises about 2.5% PL-II. In some embodiments, the LNP comprises about 2.25% PL-II. In some embodiments, the LNP comprises about 2.0% PL-II. In some embodiments, the LNP comprises about 1.75% PL-II. In some embodiments, the LNP comprises about 1.5% PL-II.

[0672] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises about 3.5% of PEG lipid PEG-1. In some embodiments, the LNP comprises about 3.25% PEG-1. In some embodiments, the LNP comprises about 3.0% PEG-1. In some embodiments, the LNP comprises about 2.75% PEG-1. In some embodiments, the LNP comprises about 2.5% PEG- 1. In some embodiments, the LNP comprises about 2.25% PEG-1. In some embodiments, the LNP comprises about 2.0% PEG-1. In some embodiments, the LNP comprises about 1.75% PEG-1. In some embodiments, the LNP comprises about 1.5% PL-II.

[0673] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises about 0.5% of PEG-DSG. In some embodiments, the LNP comprises about 0.25% PEG-DSG. In some embodiments, the LNP comprises about 0.5% PEG-DSG. In some embodiments, the LNP comprises about 0.75% PEG-DSG. In some embodiments, the LNP comprises about 1.0% PEG-DSG. In some embodiments, the LNP comprises about 1.25% PEG-DSG. In some embodiments, the LNP comprises about 1.5% PEG-DSG. In some embodiments, the LNPcomprises about 1.75% PEG-DSG. In some embodiments, the LNP comprises about 2.0% PEG-DSG.

[0674] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises about0% of PEG-DSG and about 3% PEG-II. In some embodiments, the LNP comprises about0.25% PEG-DSG and about 2.75% PEG-II. IN some embodiments, the LNP comprises about0.5% PEG-DSG and about 2.5% PEG-II. In some embodiments, the LNP comprises about0.75% PEG-SG and about 2.25% PEG-II. In some embodiments, the LNP comprises about1.0% PEG-DSG and about 2.0% PEG-II. In some embodiments, the LNP comprises about1.25% PEG-DSG and about 1.75% PEG-II. In some embodiments, the LNP comprises about1.5% PEG-DSG and about 1.5% PEG-II.

[0675] In some embodiments, the LNP (e.g., an empty LNP or loaded LNP) comprises about0% of PEG-DSG and about 3% PEG-1. In some embodiments, the LNP comprises about0.25% PEG-DSG and about 2.75% PEG-1. IN some embodiments, the LNP comprises about0.5% PEG-DSG and about 2.5% PEG-1. In some embodiments, the LNP comprises about0.75% PEG-SG and about 2.25% PEG-1 In some embodiments, the LNP comprises about1.0% PEG-DSG and about 2.0% PEG-1. In some embodiments, the LNP comprises about1.25% PEG-DSG and about 1.75% PEG-1. In some embodiments, the LNP comprises about1.5% PEG-DSG and about 1.5% PEG-1.Physical properties

[0676] The characteristics of a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may depend on the components thereof. For example, a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) including cholesterol as a structural lipid may have different characteristics than a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) that includes a different structural lipid. Similarly, the characteristics of a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may depend on the absolute or relative amounts of its components. For instance, a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) including a higher molar fraction of a phospholipid may have different characteristics than a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0677] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometrictitrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Zeta potential can be measured on a Wyatt Technologies Mobius Zeta Potential instrument. This instrument characterizes the mobility and zeta potential by the principle of “Massively Parallel Phase Analysis Light Scattering” or MP- PALS. Without wishing to be bound by theory, this measurement is more sensitive and less stress inducing than ISO Method 13099-1 :2012 which only uses one angle of detection and required higher voltage for operation. In some embodiments, the zeta potential of the herein described empty LNP compositions lipid is measured using an instrument employing the principle of MP -PALS. Zeta potential can be measured on a Malvern Zetasizer (Nano ZS).

[0678] In some embodiments, the mean diameter of a lipid nanoparticle of the disclosure (e.g., an empty LNP or a loaded LNP) is between 10s of nm and 100s of nm as measured by dynamic light scattering (DLS). For example, in some embodiments, the mean diameter of a lipid nanoparticle of the disclosure is from about 40 nm to about 150 nm. In some embodiments, the mean diameter of a lipid nanoparticle of the disclosure is about 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, or 150 nm. In some embodiments, the mean diameter of a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) is from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 150 nm, from about 70 nm to about 130 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 150 nm, from about 80 nm to about 130 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, from about 90 nm to about 150 nm, from about 90 nm to about 130 nm, or from about 90 nm to about 100 nm. In certain embodiments, the mean diameter of a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) of the disclosure is from about 70 nm to about 130 nm or from about 70 nm to about 100 nm. In some embodiments, the mean diameter of a nanoparticle of the disclosure is about 80 nm. In some embodiments, the mean diameter of a nanoparticle of the disclosure is about 100 nm. In some embodiments, the mean diameter of a nanoparticle of the disclosure is about 110 nm. In some embodiments, the mean diameter of a nanoparticle of the disclosure is about 120 nm.

[0679] In some embodiments, the poly dispersity index (“PDI”) of a plurality of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) formulated with lipids of the disclosure is less than 0.3. In some embodiments, plurality of lipid nanoparticles (e.g., empty LNPs orloaded LNPs) formulated with lipids of the disclosure has a poly dispersity index of from about 0 to about 0.25. In some embodiments, plurality of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) formulated with lipids of the disclosure has a poly dispersity index of from about 0.10 to about 0.20.

[0680] Surface hydrophobicity of nanoparticles of the disclosure can be measured by Generalized Polarization by Laurdan (GPL). In this method, Laurdan, a fluorescent aminonaphthalene ketone lipid, is post-inserted into the nanoparticle surface and the fluorescence spectrum of Laurdan is collected to determine the normalized Generalized Polarization (N-GP). In some embodiments, nanoparticles formulated with lipids of the disclosure have a surface hydrophobicity expressed as N-GP of between about 0.5 and about 1.5. For example, in some embodiments, nanoparticles formulated with lipids of the disclosure have a surface hydrophobicity expressed as N-GP of about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, or about 1.5. In some embodiments, nanoparticles formulated with lipids of the disclosure have a surface hydrophobicity expressed as N-GP of about 1.0 or about 1.1.

[0681] The zeta potential of a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of colloidal dispersions, e.g., a nanoparticle composition. Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles in the dispersion. In some embodiments, the zeta potential of a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0682] The efficiency of encapsulation of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent that is encapsulated or otherwise associated with a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) after preparation, relative tothe initial amount provided. The encapsulation efficiency is desired to be high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing a loaded LNP before and after breaking up the loaded LNP with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in a solution. For the loaded LNPs formulated with lipids of the di closure, the encapsulation efficiency of a therapeutic and / or prophylactic agent is at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency is at least 80%. In some embodiments, the encapsulation efficiency is at least 90%. In some embodiments, the encapsulation efficiency of the therapeutic and / or prophylactic agent is between 80% and 100%.Adjuvants

[0683] In some embodiments, a LNP that includes one or more lipids described herein may further include one or more adjuvants, e.g., Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.Therapeutic Agents

[0684] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may include one or more therapeutic and / or prophylactics agents. The disclosure features methods of delivering a therapeutic and / or prophylactic agent to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof comprising administering to a mammal and / or contacting a mammalian cell with a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) including a therapeutic and / or prophylactic agent.

[0685] Therapeutic and / or prophylactic agents include biologically active substances and are alternately referred to as “active agents.” A therapeutic and / or prophylactic agent may be a substance that, once delivered to a cell or organ, brings about a desirable change in the cell, organ, or other bodily tissue or system. Such species may be useful in the treatment of one or more diseases, disorders, or conditions. In some embodiments, a therapeutic and / or prophylactic agent is a small molecule drug useful in the treatment of a particular disease, disorder, or condition.

[0686] In some embodiments, a therapeutic and / or prophylactic agent is a vaccine, a compound (e.g., a polynucleotide or nucleic acid molecule that encodes a protein or polypeptide or peptide or a protein or polypeptide or protein) that elicits an immune response, and / or another therapeutic and / or prophylactic agent. Vaccines include compounds and preparations that are capable of providing immunity against one or more conditions related to infectious diseases and can include mRNAs encoding infectious disease derived antigens and / or epitopes. Vaccines also include compounds and preparations that direct an immune response against cancer cells and can include mRNAs encoding tumor cell derived antigens, epitopes, and / or neoepitopes. In some embodiments, a vaccine and / or a compound capable of eliciting an immune response is administered intramuscularly via a composition of the disclosure.

[0687] In other embodiments, a therapeutic and / or prophylactic agent is a protein, for example a protein needed to augment or replace a naturally-occurring protein of interest. Such proteins or polypeptides may be naturally occurring, or may be modified using methods known in the art, e.g., to increase half life. Exemplary proteins are intracellular, transmembrane, or secreted.Polynucleotides and Nucleic Acids

[0688] In some embodiments, the therapeutic agent is an agent that enhances (i.e., increases, stimulates, upregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used for enhancing protein expression include RNAs, mRNAs, dsRNAs, CRISPR / Cas9 technology, ssDNAs and DNAs (e.g., expression vectors). The agent that upregulates protein expression may upregulate expression of a naturally occurring or non- naturally occurring protein (e.g., a chimeric protein that has been modified to improve half life, or one that comprises desirable amino acid changes). Exemplary proteins include intracellular, transmembrane, or secreted proteins, peptides, or polypeptides.

[0689] In some embodiments, the therapeutic agent is a DNA therapeutic agent. The DNA molecule can be a double-stranded DNA, a single-stranded DNA (ssDNA), or a molecule that is a partially double-stranded DNA, i.e., has a portion that is double-stranded and a portion that is single-stranded. In some cases the DNA molecule is triple-stranded or is partially triplestranded, i.e., has a portion that is triple stranded and a portion that is double stranded. The DNA molecule can be a circular DNA molecule or a linear DNA molecule.

[0690] A DNA therapeutic agent can be a DNA molecule that is capable of transferring a gene into a cell, e.g., that encodes and can express a transcript. In other embodiments, the DNA molecule is a synthetic molecule, e.g., a synthetic DNA molecule produced in vitro. In someembodiments, the DNA molecule is a recombinant molecule. Non-limiting exemplary DNA therapeutic agents include plasmid expression vectors and viral expression vectors.

[0691] The DNA therapeutic agents described herein, e.g., DNA vectors, can include a variety of different features. The DNA therapeutic agents described herein, e.g., DNA vectors, can include a non-coding DNA sequence. For example, a DNA sequence can include at least one regulatory element for a gene, e.g., a promoter, enhancer, termination element, polyadenylation signal element, splicing signal element, and the like. In some embodiments, the non-coding DNA sequence is an intron. In some embodiments, the non-coding DNA sequence is a transposon. In some embodiments, a DNA sequence described herein can have a non-coding DNA sequence that is operatively linked to a gene that is transcriptionally active. In other embodiments, a DNA sequence described herein can have a non-coding DNA sequence that is not linked to a gene, i.e., the non-coding DNA does not regulate a gene on the DNA sequence.

[0692] In some embodiments, in the loaded LNP of the disclosure, the one or more therapeutic and / or prophylactic agents is a nucleic acid. In some embodiments, the one or more therapeutic and / or prophylactic agents is selected from the group consisting of a ribonucleic acid (RNA) and a deoxyribonucleic acid (DNA).

[0693] For example, in some embodiments, when the therapeutic and / or prophylactic agents is a DNA, the DNA is selected from the group consisting of a double-stranded DNA, a singlestranded DNA (ssDNA), a partially double-stranded DNA, a triple stranded DNA, and a partially triple-stranded DNA. In some embodiments, the DNA is selected from the group consisting of a circular DNA, a linear DNA, and mixtures thereof.

[0694] In some embodiments, in the loaded LNP of the disclosure, the one or more therapeutic and / or prophylactic agents is selected from the group consisting of a plasmid expression vector, a viral expression vector, and mixtures thereof.

[0695] For example, in some embodiments, when the therapeutic and / or prophylactic agents is a RNA, the RNA is selected from the group consisting of a single-stranded RNA, a doublestranded RNA (dsRNA), a partially double-stranded RNA, and mixtures thereof. In some embodiments, the RNA is selected from the group consisting of a circular RNA, a linear RNA, and mixtures thereof.

[0696] For example, in some embodiments, when the therapeutic and / or prophylactic agents is a RNA, the RNA is selected from the group consisting of a short interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a RNA interference (RNAi) molecule, a microRNA (miRNA), an antagomir, an antisense RNA, a ribozyme, a Dicer-substrate RNA (dsRNA), asmall hairpin RNA (shRNA), a messenger RNA (mRNA), locked nucleic acids (LNAs) and CRISPR / Cas9 technology, and mixtures thereof.

[0697] For example, in some embodiments, when the therapeutic and / or prophylactic agents is a RNA, the RNA is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof.

[0698] In some embodiments, the one or more therapeutic and / or prophylactic agents is an mRNA. In some embodiments, the one or more therapeutic and / or prophylactic agents is a modified mRNA (mmRNA).

[0699] In some embodiments, the one or more therapeutic and / or prophylactic agents is an mRNA that incorporates a micro-RNA binding site (miR binding site). Further, in some embodiments, an mRNA includes one or more of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and / or a 5’ cap structure.

[0700] An mRNA may be a naturally or non-naturally occurring mRNA. An mRNA may include one or more modified nucleobases, nucleosides, or nucleotides, as described below, in which case it may be referred to as a “modified mRNA” or “mmRNA.” As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group.

[0701] An mRNA may include a 5' untranslated region (5'-UTR), a 3' untranslated region (3'- UTR), and / or a coding region (e.g., an open reading frame). An mRNA may include any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900) or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) of base pairs. Any number (e.g., all, some, or none) of nucleobases, nucleosides, or nucleotides may be an analog of a canonical species, substituted, modified, or otherwise non-naturally occurring. In certain embodiments, all of a particular nucleobase type may be modified. In some embodiments, all uracils or uridines are modified. When all nucleobases, nucleosides, or nucleotides are modified, e.g., all uracils or uridines, the mRNA can be referred to as “fully modified”, e.g., for uracil or uridine.

[0702] In some embodiments, an mRNA as described herein may include a 5' cap structure, a chain terminating nucleotide, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem loop, a polyA sequence, and / or a polyadenylation signal.

[0703] A 5' cap structure or cap species is a compound including two nucleoside moi eties joined by a linker and may be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog (ARCA). A cap species may include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap may include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position joined by a triphosphate linkage at their 5' positions, e.g., m7G(5')ppp(5')G, commonly written as m7GpppG. A cap species may also be an anti-reverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, m27,O2'GppppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, and m27,O2'GppppG.

[0704] An mRNA may instead or additionally include a chain terminating nucleoside. For example, a chain terminating nucleoside may include those nucleosides deoxygenated at the 2’ and / or 3' positions of their sugar group. Such species may include 3' deoxyadenosine (cordycepin), 3' deoxyuridine, 3' deoxy cytosine, 3' deoxyguanosine, 3' deoxythymine, and 2', 3' dideoxynucleosides, such as 2', 3' dideoxyadenosine, 2', 3' dideoxyuridine, 2', 3' dideoxy cytosine, 2', 3' dideoxyguanosine, and 2', 3' dideoxythymine. In some embodiments, incorporation of a chain terminating nucleotide into an mRNA, for example at the 3 '-terminus, may result in stabilization of the mRNA.

[0705] An mRNA may instead or additionally include a stem loop, such as a histone stem loop. A stem loop may include 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, a stem loop may include 4, 5, 6, 7, or 8 nucleotide base pairs. A stem loop may be located in any region of an mRNA. For example, a stem loop may be located in, before, or after an untranslated region (a 5' untranslated region or a 3' untranslated region), a coding region, or a poly A sequence or tail. In some embodiments, a stem loop may affect one or more function(s) of an mRNA, such as initiation of translation, translation efficiency, and / or transcriptional termination.

[0706] An mRNA may instead or additionally include a polyA sequence and / or polyadenylation signal. A polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof. A poly A sequence may also comprise stabilizing nucleotides or analogs. For example, a poly A sequence can include deoxythymidine, e.g., inverted (or reverse linkage) deoxythymidine (dT), as a stabilizing nucleotide or analog. Details on using inverted dT and other stabilizing poly A sequence modifications can be found, for example, in WO2017 / 049275 A2, the content of which is incoported herein by reference. A polyA sequence may be a tail located adjacent to a 3' untranslated region of an mRNA. Insome embodiments, a polyA sequence may affect the nuclear export, translation, and / or stability of an mRNA.

[0707] An mRNA may instead or additionally include a microRNA binding site. MicroRNA binding sites (or miR binding sites) can be used to regulate mRNA expression in various tissues or cell types. In exemplary embodiments, miR binding sites are engineered into 3’ UTR sequences of an mRNA to regulate, e.g., enhance degradation of mRNA in cells or tissues expressing the cognate miR. Such regulation is useful to regulate or control “off-target” expression ir mRNAs, i.e., expression in undesired cells or tissues in vivo. Details on using mir binding sites can be found, for example, in WO 2017 / 062513 A2, the content of which is incoported herein by reference.

[0708] In some embodiments, an mRNA is a bicistronic mRNA comprising a first coding region and a second coding region with an intervening sequence comprising an internal ribosome entry site (IRES) sequence that allows for internal translation initiation between the first and second coding regions, or with an intervening sequence encoding a self-cleaving peptide, such as a 2A peptide. IRES sequences and 2A peptides are typically used to enhance expression of multiple proteins from the same vector. A variety of IRES sequences are known and available in the art and may be used, including, e.g., the encephalomyocarditis virus IRES.

[0709] In some embodiments, an mRNA of the disclosure comprises one or more modified nucleobases, nucleosides, or nucleotides (termed “modified mRNAs” or “mmRNAs”). In some embodiments, modified mRNAs may have useful properties, including enhanced stability, intracellular retention, enhanced translation, and / or the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced, as compared to a reference unmodified mRNA. Therefore, use of modified mRNAs may enhance the efficiency of protein production, intracellular retention of nucleic acids, as well as possess reduced immunogenicity.

[0710] In some embodiments, an mRNA includes one or more (e.g., 1, 2, 3 or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, an mRNA includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified mRNA may have reduced degradation in a cell into which the mRNA is introduced, relative to a corresponding unmodified mRNA.

[0711] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (Ψ), pyridin-4- one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5 -bromo-uridine), 3 -methyl -uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5 -aminomethyl -2 -thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl -uridine (cmnm5U), 5-carboxymethylaminomethyl -2 -thiouridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5 -taurinom ethyl -uridine (rm5U), 1 -taurinom ethyl -pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1- taurinomethyl-4-thio-pseudouridine, 5 -methyl -uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1Ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl- 4-thio-pseudouridine (mls4y), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3y), 2-thio- 1-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-thio-l-methyl-l- deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5- methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, N1 -methyl -pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1- methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 y), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2 -thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O- methyl-pseudouridine (ym), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl- 2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2'-O-methyl -uridine (cmnnriUm), 3, 2'-O-dimethyl -uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2’-F-ara-uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)]uridine.

[0712] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5 -aza-cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl- cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5- iodo-cytidine), 5 -hydroxymethyl -cytidine (hm5C), 1 -methyl -pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-l -methyl -pseudoisocytidine, 4-thio-l-methyl-l-deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l -methyl - pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O- dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl- cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1 -thio-cytidine, 2’-F-ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

[0713] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include a-thio-adenosine, 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo- purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyl -adenosine (mlA), 2-methyl- adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis- hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl -adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl- adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl- adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6- acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a- thio-adenosine, 2 '-O-m ethyl -adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (mlAm), 2'-O- ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido- adenosine, 2’-F-ara-adenosine, 2’-F-adenosine, 2’-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

[0714] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include a-thio-guanosine, inosine (I), 1 -methyl -inosine (mil), wyosine (imG), methyl wyosine (mimG), 4-dem ethyl -wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxy wybutosine (o2yW), hydroxy wybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQO), 7-aminom ethyl -7-deaza-guanosine (preQi), archaeosine (G+), 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl -guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl -inosine, 6- methoxy-guanosine, 1 -methyl -guanosine (mlG), N2-methyl-guanosine (m2G), N2,N2- dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl- guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl- 2'-O-methyl-guanosine (m22Gm), l-methyl-2'-O-methyl-guanosine (ml Gm), N2,7-dimethyl- 2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (mllm), 2'-O-ribosylguanosine (phosphate) (Gr(p)) , 1 -thio-guanosine, O6-methyl-guanosine, 2’-F-ara-guanosine, and 2’-F-guanosine.

[0715] In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0716] In some embodiments, the modified nucleobase is pseudouridine (Ψ), Nl- methylpseudouridine (m1Ψ), 2-thiouridine, 4’ -thiouridine, 5-methylcytosine, 2-thio-l-methyl- 1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine , 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2’-O-methyl uridine. In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.) In some embodiments, the modified nucleobase isN1- methylpseudouridine (m1Ψ) and the mRNA of the disclosure is fully modified with Nl- methylpseudouridine (m1Ψ). In some embodiments, N1 -methylpseudouridine (m1Ψ) represents from 75-100% of the uracils in the mRNA. In some embodiments, Nl- methylpseudouridine (m1Ψ) represents 100% of the uracils in the mRNA.

[0717] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5- methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine. In some embodiments, an mRNA of the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0718] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1 -methyladenosine (ml A), 2-methyl-adenine (m2 A), N6-methyl-adenosine (m6A). In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0719] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methyl wyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza- guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQi), 7-methyl-guanosine (m7G), 1 -methyl -guanosine (mlG), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0720] In some embodiments, the modified nucleobase is 1 -methyl -pseudouridine (m1Ψ), 5- methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (Ψ), a-thio-guanosine, or a-thio-adenosine. In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0721] In some embodiments, the mRNA comprises pseudouridine (Ψ). In some embodiments, the mRNA comprises pseudouridine (Ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1 -methyl -pseudouridine ( (m1Ψ). In some embodiments, the mRNA comprises 1 -methyl -pseudouridine (m1Ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5- methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2’-O-methyl uridine. In some embodiments, the mRNA comprises 2’-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises comprises N6-methyl-adenosine (m6A). In some embodiments, the mRNA comprises N6-methyl- adenosine (m6A) and 5-methyl-cytidine (m5C).

[0722] In certain embodiments, an mRNA of the disclosure is uniformly modified (i.e., fully modified, modified through-out the entire sequence) for a particular modification. For example, an mRNA can be uniformly modified with N1 -methylpseudouridine (m1Ψ) or 5- methyl-cytidine (m5C), meaning that all uridines or all cytosine nucleosides in the mRNA sequence are replaced with N1 -methylpseudouridine (m1Ψ) or 5-methyl-cytidine (m5C). Similarly, mRNAs of the disclosure can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0723] In some embodiments, an mRNA of the disclosure may be modified in a coding region (e.g., an open reading frame encoding a polypeptide). In other embodiments, an mRNA may be modified in regions besides a coding region. For example, in some embodiments, a 5'-UTR and / or a 3'-UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region.

[0724] The mmRNAs of the disclosure can include a combination of modifications to the sugar, the nucleobase, and / or the internucleoside linkage. These combinations can include any one or more modifications described herein.

[0725] Where a single modification is listed, the listed nucleoside or nucleotide represents 100 percent of that A, U, G or C nucleotide or nucleoside having been modified. Where percentages are listed, these represent the percentage of that particular A, U, G or C nucleobase triphosphate of the total amount of A, U, G, or C triphosphate present. For example, the combination: 25 % 5-Aminoallyl-CTP + 75 % CTP / 25 % 5-Methoxy-UTP + 75 % UTP refers to a polynucleotide where 25% of the cytosine triphosphates are 5-Aminoallyl-CTP while 75% of the cytosines are CTP; whereas 25% of the uracils are 5-methoxy UTP while 75% of the uracils are UTP. Where no modified UTP is listed then the naturally occurring ATP, UTP, GTP and / or CTP is used at 100% of the sites of those nucleotides found in the polynucleotide. In this example all of the GTP and ATP nucleotides are left unmodified.

[0726] The mRNAs of the present disclosure, or regions thereof, may be codon optimized. Codon optimization methods are known in the art and may be useful for a variety of purposes: matching codon frequencies in host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove proteins trafficking sequences, remove / add post translation modification sites in encoded proteins (e.g., glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, adjust translation rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art; non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA) and / or proprietary methods. In some embodiments, the mRNA sequence is optimized using optimization algorithms, e.g., to optimize expression in mammalian cells or enhance mRNA stability.

[0727] In certain embodiments, the present disclosure includes polynucleotides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the polynucleotide sequences described herein.

[0728] mRNAs of the present disclosure may be produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods may be utilized. In some embodiments, mRNAs are made using IVT enzymatic synthesis methods. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors that may be used to in vitro transcribe an mRNA described herein.

[0729] Non-natural modified nucleobases may be introduced into polynucleotides, e.g., mRNA, during synthesis or post-synthesis. In certain embodiments, modifications may be on intemucleoside linkages, purine or pyrimidine bases, or sugar. In particular embodiments, the modification may be introduced at the terminal of a polynucleotide chain or anywhere else in the polynucleotide chain; with chemical synthesis or with a polymerase enzyme.

[0730] Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or their regions with different functional moi eties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc.

[0731] In some embodiments, the therapeutic agent is a therapeutic agent that reduces (i.e., decreases, inhibits, downregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used for reducing protein expression include mRNAs that incorporate a micro-RNA binding site(s) (miR binding site), microRNAs (miRNAs), antagomirs, small (short) interfering RNAs (siRNAs) (including shortmers and dicer-substrate RNAs), RNA interference (RNAi) molecules, antisense RNAs, ribozymes, small hairpin RNAs (shRNAs), locked nucleic acids (LNAs) and CRISPR / Cas9 technology.Sensor Sequences and MicroRNA (miRNA) Binding Sites

[0732] Sensor sequences include, for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and / or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof. Non-limiting examples of sensor sequences are described in U.S. Publication 2014 / 0200261, the contents of which are incorporated herein by reference in their entirety.

[0733] In some embodiments, a polyribonucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the disclosure comprising an open reading frame (ORF) encoding a polypeptide further comprises a sensor sequence. In some embodiments, the sensor sequence is a miRNA binding site.

[0734] A miRNA is a 19-25 nucleotide long noncoding RNA that binds to a polyribonucleotide and down-regulates gene expression either by reducing stability or by inhibiting translation of the polyribonucleotide. A miRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature miRNA. A miRNA seed can comprise positions 2-8 or 2-7 of the mature miRNA. In some embodiments, a miRNA seed can comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA binding site is flanked by an adenosine (A) opposed to miRNA position 1. In some embodiments, a miRNA seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA binding site is flanked by an adenosine (A) opposed to miRNA position 1. See, for example, Grimson A, Farh KK, Johnston WK, Garrett-Engel e P, Lim LP, Bartel DP; Mol C6ll. 2007 Jul 6;27(1 ): 91 - 105. miRNA profiling of the target cells or tissues can be conducted to determine the presence or absence of miRNA in the cells or tissues. In some embodiments, a polyribonucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the disclosure comprises one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences can correspond to any known microRNA such as those taught in US Publication US2005 / 0261218 and US Publication US2005 / 0059005, the contents of each of which are incorporated herein by reference in their entirety.

[0735] As used herein, the term “microRNA (miRNA or miR) binding site” refers to a sequence within a polyribonucleotide, e.g., within a DNA or within an RNA transcript, including in the 5'UTR and / or 3'UTR, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA. In some embodiments, a polyribonucleotide of the disclosure comprising an ORF encoding a polypeptide furthercomprises a miRNA binding site. In exemplary embodiments, a 5'UTR and / or 3'UTR of the polyribonucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) comprises a miRNA binding site.

[0736] A miRNA binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polyribonucleotide, e.g., miRNA-mediated translational repression or degradation of the polyribonucleotide. In exemplary aspects of the disclosure, a miRNA binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the polyribonucleotide, e.g., miRNA-guided RNA- induced silencing complex (RlSC)-mediated cleavage of mRNA. The miRNA binding site can have complementarity to, for example, a 19-25 nucleotide miRNA sequence, to a 19-23 nucleotide miRNA sequence, or to a 22 nucleotide miRNA sequence. A miRNA binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally-occurring miRNA sequence. In some embodiments, the desired regulation is mRNA degradation. In some embodiments, the miRNA binding site has full or complete complementarity (e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally-occurring miRNA). In some embodiments, the mRNA degradation has full or complete complementarity.

[0737] In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with an miRNA seed sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with an miRNA sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA sequence. In some embodiments, a miRNA binding site has complete complementarity with a miRNA sequence but for 1, 2, or 3 nucleotide substitutions, terminal additions, and / or truncations.

[0738] In some embodiments, the miRNA binding site is the same length as the corresponding miRNA. In some embodiments, the miRNA binding site is one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5' terminus, the 3' terminus, or both. In still other embodiments, the microRNA binding site is two nucleotides shorter than the corresponding microRNA at the 5' terminus, the 3' terminus, or both. The miRNA binding sites that are shorter than the corresponding miRNAsare still capable of degrading the mRNA incorporating one or more of the miRNA binding sites or preventing the mRNA from translation.

[0739] In some embodiments, the miRNA binding site binds to the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA binding site or prevents the mRNA from being translated. In some embodiments, the miRNA binding site has sufficient complementarity to miRNA so that a RISC complex comprising the miRNA cleaves the polyribonucleotide comprising the miRNA binding site. In some embodiments, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA induces instability in the polyribonucleotide comprising the miRNA binding site. In another embodiment, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA represses transcription of the polyribonucleotide comprising the miRNA binding site.

[0740] In some embodiments, the miRNA binding site has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.

[0741] In some embodiments, the miRNA binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one, respectively, contiguous nucleotides of the corresponding miRNA.

[0742] By engineering one or more miRNA binding sites into a polyribonucleotide of the disclosure, the polyribonucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the polyribonucleotide. In some embodiments, if a polyribonucleotide of the disclosure is not intended to be delivered to a tissue or cell but ends up there, then a miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5'UTR and / or 3'UTR of the polyribonucleotide.

[0743] Conversely, miRNA binding sites can be removed from polyribonucleotide sequences in which they naturally occur in order to increase protein expression in specific tissues. In some embodiments, a binding site for a specific miRNA can be removed from a polyribonucleotide to improve protein expression in tissues or cells containing the miRNA.

[0744] In one embodiment, a polyribonucleotide of the disclosure can include at least one miRNA-binding site in the 5'UTR and / or 3'UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and / or cancerous cells. In another embodiment, a polyribonucleotide of the disclosure can include two, three, four, five, six, seven, eight, nine, ten, or more miRNA-binding sites in the 5'-UTR and / or 3 '-UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and / or cancerous cells.

[0745] Regulation of expression in multiple tissues can be accomplished through introduction or removal of one or more miRNA binding sites. The decision whether to remove or insert a miRNA binding site can be made based on miRNA expression patterns and / or their profilings in diseases. Identification of miRNAs, miRNA binding sites, and their expression patterns and role in biology have been reported (e.g., Bonauer et al., Curr Drug Targets 2010 11 :943-949; Anand and Cheresh Curr Opin Hematol 2011 18: 171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20. doi: 10.1038 / leu.2011.356); Bartel C6ll 2009 136:215-233; Landgraf et al, C6ll, 2007 129: 1401-1414; Gentner andNaldini, Tissue Antigens. 2012 80:393- 403 and all references therein; each of which is incorporated herein by reference in its entirety).

[0746] miRNAs and miRNA binding sites can correspond to any known sequence, including non-limiting examples described in U.S. Publication Nos. 2014 / 0200261, 2005 / 0261218, and 2005 / 0059005, each of which are incorporated herein by reference in their entirety.

[0747] Examples of tissues where miRNA are known to regulate mRNA, and thereby protein expression, include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR- 208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR- 16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-ld, miR- 149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR- 126).

[0748] Specifically, miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen presenting cells (APC8) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc. Immune cell specific miRNAs are involved in immunogenicity, autoimmunity, the immune-response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue / organ transplantation. Immune cells specific miRNAs also regulate many aspects of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells). In some embodiments, miR-142 and miR-146 are exclusively expressed in immune cells, particularly abundant in myeloid dendritic cells. It has been demonstratedthat the immune response to a polyribonucleotide can be shut-off by adding miR-142 binding sites to the 3'-UTR of the polyribonucleotide, enabling more stable gene transfer in tissues and cells. miR-142 efficiently degrades exogenous polyribonucleotides in antigen presenting cells and suppresses cytotoxic elimination of transduced cells (e.g., Annoni A et al., blood, 2009, 114, 5152-5161; Brown BD, et al., Nat med. 2006, 12(5), 585-591; Brown BD, et al., blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by reference in its entirety).

[0749] An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen presenting cells and displayed on the surface of the antigen presenting cells. T cells can recognize the presented antigen and induce a cytotoxic elimination of cells that express the antigen.

[0750] Introducing a miR-142 binding site into the 5'UTR and / or 3'UTR of a polyribonucleotide of the disclosure can selectively repress gene expression in antigen presenting cells through miR-142 mediated degradation, limiting antigen presentation in antigen presenting cells (e.g., dendritic cells) and thereby preventing antigen-mediated immune response after the delivery of the polyribonucleotide. The polyribonucleotide is then stably expressed in target tissues or cells without triggering cytotoxic elimination.

[0751] In one embodiment, binding sites for miRNAs that are known to be expressed in immune cells, in particular, antigen presenting cells, can be engineered into a polyribonucleotide of the disclosure to suppress the expression of the polyribonucleotide in antigen presenting cells through miRNA mediated RNA degradation, subduing the antigen- mediated immune response. Expression of the polyribonucleotide is maintained in non- immune cells where the immune cell specific miRNAs are not expressed. In some embodiments, in some embodiments, to prevent an immunogenic reaction against a liver specific protein, any miR-122 binding site can be removed and a miR-142 (and / or mirR-146) binding site can be engineered into the 5'UTR and / or 3'UTR of a polyribonucleotide of the disclosure.

[0752] To further drive the selective degradation and suppression in APC8and macrophage, a polyribonucleotide of the disclosure can include a further negative regulatory element in the 5'UTR and / or 3'UTR, either alone or in combination with miR-142 and / or miR-146 binding sites. As a non-limiting example, the further negative regulatory element is a Constitutive Decay Element (CDE).

[0753] Immune cell specific miRNAs include, but are not limited to, hsa-let-7a-2-3p, hsa-let- 7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa- let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-l--3p, hsa-let-7f-2--5p, hsa-let-7f-5p, miR-125b-l-3p, miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-143-3p, miR-143-5p, miR-146a-3p, miR-146a-5p, miR-146b-3p, miR-146b-5p, miR-147a, miR-147b, miR-148a- 5p, miR-148a-3p, miR-150-3p, miR-150-5p, miR-151b, miR-155-3p, miR-155-5p, miR-15a- 3p, miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-l-3p, miR-16-2-3p, miR-16-5p, miR-17- 5p, miR-181a-3p, miR-181a-5p, miR-18 la-2-3 p, miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p, miR-21-5p, miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p, miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-l-5p,miR-24-2-5p, miR-24-3p, miR-26a-l-3p, miR-26a-2-3p, miR-26a-5p, miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a- 5p, miR-27b-3p,miR-27b-5p, miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-l-5p, miR-29b-2-5p, miR-29c-3p, miR-29c-5p„ miR-30e-3p, miR-30e-5p, miR-331- 5p, miR-339-3p, miR-339-5p, miR-345-3p, miR-345-5p, miR-346, miR-34a-3p, miR-34a-5p, , miR-363-3p, miR-363-5p, miR-372, miR-377-3p, miR-377-5p, miR-493-3p, miR-493-5p, miR-542, miR-548b-5p, miR548c-5p, miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935, miR-99a-3p, miR-99a-5p, miR-99b-3p, and miR-99b-5p. Furthermore, novel miRNAs can be identified in immune cell through micro-array hybridization and microtome analysis (e.g., Jima DD et al, Blood, 2010, 116:el l8-el27; Vaz C et al., BMC Genomics, 2010, 11,288, the content of each of which is incorporated herein by reference in its entirety.)

[0754] miRNAs that are known to be expressed in the liver include, but are not limited to, miR- 107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR- 1303, miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR- 199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p. miRNA binding sites from any liver specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the liver. Liver specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polyribonucleotide of the disclosure.

[0755] miRNAs that are known to be expressed in the lung include, but are not limited to, let- 7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a- 3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-l-5p, miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, and miR-381-5p. MiRNA binding sites from any lung specific miRNA can be introduced to or removed from apolyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the lung. Lung specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polyribonucleotide of the disclosure.

[0756] miRNAs that are known to be expressed in the heart include, but are not limited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a- 5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p. MiRNA binding sites from any heart specific microRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the heart. Heart specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polyribonucleotide of the disclosure.

[0757] miRNAs that are known to be expressed in the nervous system include, but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-l-3p, miR-125b-2-3p, miR- 125b-5p,miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p, miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR- 212-3p, miR-212-5p, miR-219-l-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p,miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-l-3p, miR-30c-2-3p, miR-30c-5p, miR-30d-3p, miR-30d- 5p, miR-329, miR-342-3p, miR-3665, miR-3666, miR-380-3p, miR-380-5p, miR-383, miR- 410, miR-425-3p, miR-425-5p, miR-454-3p, miR-454-5p, miR-483, miR-510, miR-516a-3p, miR-548b-5p, miR-548c-5p, miR-571, miR-7-l-3p, miR-7-2-3p, miR-7-5p, miR-802, miR- 922, miR-9-3p, and miR-9-5p. MiRNAs enriched in the nervous system further include those specifically expressed in neurons, including, but not limited to, miR-132-3p, miR-132-3p, miR- 148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, miR-922 and those specifically expressed in glial cells, including, but not limited to, miR-1250, miR- 219- 1 -3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657. MiRNA binding sites from any CNS specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the nervous system. Nervous system specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polyribonucleotide of the disclosure.

[0758] miRNAs that are known to be expressed in the pancreas include, but are not limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p,miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a- 5p, miR-375, miR-7-l-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944. MiRNA binding sites from any pancreas specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the pancreas. Pancreas specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polyribonucleotide of the disclosure.

[0759] miRNAs that are known to be expressed in the kidney include, but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-l-3p, miR-30c-2- 3p, miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR- 562. MiRNA binding sites from any kidney specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the kidney. Kidney specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polyribonucleotide of the disclosure.

[0760] miRNAs that are known to be expressed in the muscle include, but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR- 143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p. MiRNA binding sites from any muscle specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the muscle. Muscle specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polyribonucleotide of the disclosure.

[0761] miRNAs are also differentially expressed in different types of cells, such as, but not limited to, endothelial cells, epithelial cells, and adipocytes.

[0762] miRNAs that are known to be expressed in endothelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17- 3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-l-5p, miR-19b-2-5p, miR- 19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p, miR-296-5p, miR-361-3p, miR-361-5p, miR-421, miR-424-3p, miR-424-5p, miR-513a-5p, miR-92a-l-5p, miR-92a-2-5p, miR-92a-3p, miR-92b-3p, and miR-92b-5p. Many novel miRNAs are discovered in endothelial cells from deep-sequencing analysis (e.g., Voellenkle C et al., RNA, 2012, 18, 472- 484, herein incorporated by reference in its entirety). MiRNA binding sites from any endothelial cell specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the endothelial cells.

[0763] miRNAs that are known to be expressed in epithelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific in respiratory ciliated epithelial cells, let-7 family, miR-133a, miR-133b, miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5p specific in renal epithelial cells, and miR-762 specific in corneal epithelial cells. MiRNA binding sites from any epithelial cell specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the epithelial cells.

[0764] In addition, a large group of miRNAs are enriched in embryonic stem cells, controlling stem cell self-renewal as well as the development and / or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (e.g., Kuppusamy KT et al., Curr. Mol Med, 2013, 13(5), 757-764; Vidigal JA and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436; Goff LA et al., PLoS One, 2009, 4:e7192; Morin RD et al., Genome Res,2008,18, 610-621; Yoo JK et al., Stem C6lls Dev. 2012, 21(11), 2049- 2057, each of which is herein incorporated by reference in its entirety). MiRNAs abundant in embryonic stem cells include, but are not limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p, miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246, miR-1275, miR-138-l-3p, miR-138-2-3p, miR-138-5p, miR-154-3p, miR-154-5p, miR-200c-3p, miR- 200c-5p, miR-290, miR-301a-3p, miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367- 3p, miR-367-5p, miR-369-3p, miR-369-5p, miR-370, miR-371, miR-373, miR-380-5p, miR- 423-3p, miR-423-5p, miR-486-5p, miR-520c-3p, miR-548e, miR-548f, miR-548g-3p, miR- 548g-5p, miR-548i, miR-548k, miR-5481, miR-548m, miR-548n, miR-548o-3p, miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p, miR-664b-3p, miR-664b-5p, miR-766-3p, miR-766- 5p, miR-885-3p, miR-885-5p,miR-93-3p, miR-93-5p, miR-941,miR-96-3p, miR-96-5p, miR- 99b-3p and miR-99b-5p. Many predicted novel miRNAs are discovered by deep sequencing in human embryonic stem cells (e.g., Morin RD et al., Genome Res, 2008, 18, 610-621; GoffLA et al., PLoS One, 2009, 4:e7192; Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each of which is incorporated herein by reference in its entirety).

[0765] In one embodiment, the binding sites of embryonic stem cell specific miRNAs can be included in or removed from the 3'UTR of a polyribonucleotide of the disclosure to modulate the development and / or differentiation of embryonic stem cells, to inhibit the senescence of stem cells in a degenerative condition (e.g., degenerative diseases), or to stimulate the senescence and apoptosis of stem cells in a disease condition (e.g., cancer stem cells).

[0766] Many miRNA expression studies are conducted to profile the differential expression of miRNAs in various cancer cells / tissues and other diseases. Some miRNAs are abnormally over-expressed in certain cancer cells and others are under-expressed. In some embodiments, miRNAs are differentially expressed in cancer cells (W02008 / 154098, US2013 / 0059015, US2013 / 0042333, WO2011 / 157294); cancer stem cells (US2012 / 0053224); pancreatic cancers and diseases (US2009 / 0131348, US2011 / 0171646, US2010 / 0286232, US8389210); asthma and inflammation (US8415096); prostate cancer (US2013 / 0053264); hepatocellular carcinoma (WO2012 / 151212, US2012 / 0329672, W02008 / 054828, US8252538); lung cancer cells (WO2011 / 076143, W02013 / 033640, W02009 / 070653, US2010 / 0323357); cutaneous T cell lymphoma (W02013 / 011378); colorectal cancer cells (WO2011 / 0281756, WO201 1 / 076142); cancer positive lymph nodes (W02009 / 100430, US2009 / 0263803); nasopharyngeal carcinoma (EP2112235); chronic obstructive pulmonary disease (US2012 / 0264626, US2013 / 0053263); thyroid cancer (WO2013 / 066678); ovarian cancer cells ( US2012 / 0309645, WO2011 / 095623); breast cancer cells (W02008 / 154098,W02007 / 081740, US2012 / 0214699), leukemia and lymphoma (W02008 / 073915, US2009 / 0092974, US2012 / 0316081, US2012 / 0283310, W02010 / 018563, the content of each of which is incorporated herein by reference in its entirety.)

[0767] As a non-limiting example, miRNA binding sites for miRNAs that are over-expressed in certain cancer and / or tumor cells can be removed from the 3'UTR of a polyribonucleotide of the disclosure, restoring the expression suppressed by the over-expressed miRNAs in cancer cells, thus ameliorating the corresponsive biological function, for instance, transcription stimulation and / or repression, cell cycle arrest, apoptosis and cell death. Normal cells and tissues, wherein miRNAs expression is not up-regulated, will remain unaffected.

[0768] MiRNA can also regulate complex biological processes such as angiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18: 171-176). In the polyribonucleotides of the disclosure, miRNA binding sites that are involved in such processes can be removed or introduced, in order to tailor the expression of the polyribonucleotides tobiologically relevant cell types or relevant biological processes. In this context, the polyribonucleotides of the disclosure are defined as auxotrophic polyribonucleotides.Peptide / Polypeptide Therapeutic Agents

[0769] In some embodiments, the therapeutic agent is a peptide therapeutic agent. In some embodiments the therapeutic agent is a polypeptide therapeutic agent.

[0770] In some embodiments, the peptide or polypeptide is naturally-derived, e.g., isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, e.g., a synthetic peptide or polypeptide produced in vitro. In some embodiments, the peptide or polypeptide is a recombinant molecule. In some embodiments, the peptide or polypeptide is a chimeric molecule. In some embodiments, the peptide or polypeptide is a fusion molecule. In some embodiments, the peptide or polypeptide therapeutic agent of the composition is a naturally occurring peptide or polypeptide. In some embodiments, the peptide or polypeptide therapeutic agent of the composition is a modified version of a naturally occurring peptide or polypeptide (e.g., contains less than 3, less than 5, less than 10, less than 15, less than 20, or less than 25 amino substitutions, deletions, or additions compared to its wild type, naturally occurring peptide or polypeptide counterpart).

[0771] In some embodiments, in the loaded LNP of the disclosure, the one or more therapeutic and / or prophylactic agents is a polynucleotide or a polypeptide.Genome Editing Techniques

[0772] In some embodiments, the nucleic acid is suitable for a genome editing technique.

[0773] In some embodiments, the genome editing technique is clustered regularly interspaced short palindromic repeats (CRISPR) or transcription activator-like effector nuclease (TALEN).

[0774] In some embodiments, the nucleic acid is at least one nucleic acid suitable for a genome editing technique selected from the group consisting of a CRISPR RNA (crRNA), a transactivating crRNA (tracrRNA), a single guide RNA (sgRNA), and a DNA repair template.Vaccines

[0775] In some embodiments, the therapeutic and / or prophylactic agent is a ribonucleic acid (RNA) vaccine of an RNA (e.g., messenger RNA (mRNA)) that can safely direct the body' s cellular machinery to produce nearly any protein or fragment thereof of interest. In some embodiments, the RNA is a modified RNA.

[0776] While not wishing to be bound by theory, it is believed that the RNA vaccines, as mRNA polynucleotides, are better designed to produce the appropriate protein conformation upon translation as the RNA vaccines co-opt natural cellular machinery. Unlike traditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the RNA vaccines are presented to the cellular system in a more native fashion.

[0777] Some embodiments of the present disclosure provide cancer vaccines that include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof {e.g., an immunogenic fragment capable of inducing an immune response to cancer). Other embodiments include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding two or more antigens or epitopes capable of inducing an immune response.

[0778] In some embodiments the vaccine is a personalized vaccine and wherein the cancer antigen is a subject specific antigen.

[0779] In some embodiments a single mRNA encodes the antigens. In other embodiments a plurality of mRNA encode the antigens. Each antigen is 10-50 amino acids in length in some embodiments. In other embodiments each antigen is 15- 20 amino acids in length. In other embodiments the antigen is 20-50, 25-100, 100-200, 200-300, 300-400, 400-500, 500-1,000, or 1,000-10,000 amino acids in length.

[0780] In some embodiments, the vaccines further comprise an adjuvant.

[0781] Some embodiments of the present disclosure provide a cancer vaccine that includes at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer polypeptide, at least one 5' terminal cap and at least one chemical modification, formulated within a lipid nanoparticle. In some embodiments, a 5' terminal cap is 7mG(5')ppp(5')NlmpNp.

[0782] In some embodiments, at least one chemical modification is selected from pseudouridine, Nl-methylpseudouridine, Nl-ethylpseudouridine, 2-thiouridine, 4'- thiouridine, 5-methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio-l-methyl- pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l- methyl-pseudouridine, 4-thio-pseudouridine, 5 -aza-uridine, dihydropseudouridine, 5- methyluridine, 5- methoxyuridine and 2' -O-methyl uridine. In some embodiments the extent of incorporation of chemically modified nucleotides has been optimized for improved immune responses to the vaccine formulation.

[0783] In some embodiments, a lipid nanoparticle (e.g., an empty LNP or a loaded LNP of the disclosure) comprises a cationic lipid, an ioniazlbe lipid, a PEG-modified lipid, a sterol and a phospholipid lipid.

[0784] In some embodiments the lipid nanoparticle formulation includes an immune potentiator (e.g., TLR agonist) to enhance immunogenicity of the vaccine (formulation).

[0785] In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a Nl-methyl pseudouridine.

[0786] In other embodiments a mRNA encoding an APC reprograming molecule is included in the vaccine or coadministered with the vaccine. The APC reprograming molecule may be a CIITA, a chaperone protein such as CLIP, HLA-DO, HLA-DM, a costimulatory molecule such as CD40, CD80, CD86, a CIITA fragment such as amino acids 26-137 of CIITA or a protein having 80% sequence identity to CIITA.

[0787] A method of eliciting an immune response in a subject against an antigen is provided in other aspects of the invention. The method involves administering to the subject a RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to antigenic polypeptide or an immunogenic fragment thereof, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the cancer antigen at 2 times to 100 times the dosage level relative to the RNA vaccine.

[0788] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to the RNA vaccine.

[0789] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to the RNA vaccine.

[0790] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times the dosage level relative to the RNA vaccine.

[0791] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 5 times the dosage level relative to the RNA vaccine. In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at 10 times the dosage level relative to the RNA vaccine.

[0792] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 50 times the dosage level relative to the RNA vaccine.

[0793] In some embodiments the immune response in the subject is equivalent to an immune response in a subj ect vaccinated with a traditional vaccine at 100 times the dosage level relative to the RNA vaccine.

[0794] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to the RNA vaccine.

[0795] In some embodiments the immune response in the subject is equivalent to an immune response in a subj ect vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to the RNA vaccine.

[0796] In other embodiments the immune response is assessed by determining antibody titer in the subject.

[0797] In other aspects the invention comprises a method of eliciting an immune response in a subject against a by administering to the subject a RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the antigenic polypeptide or an immunogenic fragment thereof, wherein the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the cancer antigen. In some embodiments the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to the RNA vaccine.

[0798] In some embodiments the immune response in the subject is induced 2 days earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0799] In some embodiments the immune response in the subject is induced 3 days earlier relative to an immune response induced in a subject vaccinated a prophylactically effective dose of a traditional vaccine. In some embodiments the immune response in the subject is induced 1 week earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0800] In some embodiments the immune response in the subject is induced 2 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0801] In some embodiments the immune response in the subject is induced 3 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0802] In some embodiments the immune response in the subject is induced 5 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0803] In some embodiments the immune response in the subject is induced 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0804] In some embodiments the nucleic acid vaccines described herein are chemically modified. In other embodiments the nucleic acid vaccines are unmodified.

[0805] Yet other aspects provide compositions for and methods of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and wherein an adjuvant is not coformulated or co-administered with the vaccine.

[0806] In other aspects the invention is a composition for or method of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide wherein a dosage of between 10 ug / kg and 400 ug / kg of the nucleic acid vaccine is administered to the subject. In some embodiments the dosage of the RNA polynucleotide is 1-5 ug, 5-10 ug, 10-15 ug, 15-20 ug, 10-25 ug, 20-25 ug, 20-50 ug, 30-50 ug, 40-50 ug, 40-60 ug, 60-80 ug, 60-100 ug, 50-100 ug, 80-120 ug, 40-120 ug, 40-150 ug, 50-150 ug, 50-200 ug, 80-200 ug, 100-200 ug, 120-250 ug, 150-250 ug, 180-280 ug, 200-300 ug, 50-300 ug, 80-300 ug, 100- 300 ug, 40- 300 ug, 50-350 ug, 100-350 ug, 200-350 ug, 300-350 ug, 320-400 ug, 40-380 ug, 40-100 ug, 100-400 ug, 200-400 ug, or 300-400 ug per dose. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day twenty one.

[0807] In some embodiments, a dosage of 25 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 50 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 75 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 150 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 200 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node. In other embodiments the nucleic acid vaccine is chemically modified and in other embodiments the nucleic acid vaccine is not chemically modified.

[0808] In certain aspects, vaccines of the invention (e.g., LNP-encapsulated mRNA vaccines) produce prophylactically- and / or therapeutically- efficacious levels, concentrations and / or titers of antigen- specific antibodies in the blood or serum of a vaccinated subject. As defined herein, the term antibody titer refers to the amount of antigen-specific antibody produces in s subject, e.g., a human subject. In exemplary embodiments, antibody titer is expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result. In exemplary embodiments, antibody titer is determined or measured by enzyme- linked immunosorbent assay (ELISA). In exemplary embodiments, antibody titer is determined or measured by neutralization assay, e.g., by microneutralization assay. In certain aspects, antibody titer measurement is expressed as a ratio, such as 1 :40, 1 : 100, etc.

[0809] In exemplary embodiments of the invention, an efficacious vaccine produces an antibody titer of greater than 1 :40, greater that 1 : 100, greater than 1 :400, greater than 1 : 1000, greater than 1 :2000, greater than 1 :3000, greater than 1 :4000, greater than 1 :500, greater than 1 :6000, greater than 1 :7500, greater than 1 : 10000. In exemplary embodiments, the antibody titer is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In exemplary embodiments, the titer is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the titeris produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)

[0810] In exemplary aspects of the invention, antigen- specific antibodies are measured in units of pg / ml or are measured in units of IU / L (International Units per liter) or mlU / ml (milli International Units per ml). In exemplary embodiments of the invention, an efficacious vaccine produces >0.5 pg / ml, >0.1 pg / ml, >0.2 pg / ml, >0.35 pg / ml, >0.5 pg / ml, >1 pg / ml, >2 pg / ml, >5 pg / ml or >10 pg / ml. In exemplary embodiments of the invention, an efficacious vaccine produces >10 mlU / ml, >20 mlU / ml, >50 mlU / ml, >100 mlU / ml, >200 mlU / ml, >500 mlU / ml or > 1000 mlU / ml. In exemplary embodiments, the antibody level or concentration is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In exemplary embodiments, the level or concentration is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the level or concentration is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.) In exemplary embodiments, antibody level or concentration is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody level or concentration is determined or measured by neutralization assay, e.g., by microneutralization assay. Also provided are nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, wherein the RNA polynucleotide is present in a formulation for in vivo administration to a host for eliciting a longer lasting high antibody titer than an antibody titer elicited by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide. In some embodiments, the RNA polynucleotide is formulated to produce a neutralizing antibodies within one week of a single administration. In some embodiments, the adjuvant is selected from a cationic peptide and an immunostimulatory nucleic acid. In some embodiments, the cationic peptide is protamine.

[0811] Aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally no nucleotide modification, the open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, wherein the RNA polynucleotide is present in the formulation for in vivo administration to a host such that the level of antigen expression in the host significantly exceeds a level of antigen expression produced by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide.

[0812] Other aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally no nucleotide modification, the open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, wherein the vaccine has at least 10 fold less RNA polynucleotide than is required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dosage of 25- 100 micrograms.

[0813] Aspects of the invention also provide a unit of use vaccine, comprising between 10 ug and 400 ug of one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally no nucleotide modification, the open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, and a pharmaceutically acceptable carrier or excipient, formulated for delivery to a human subject. In some embodiments, the vaccine further comprises a cationic lipid nanoparticle.

[0814] Aspects of the invention provide methods of creating, maintaining or restoring antigenic memory to a tumor in an individual or population of individuals comprising administering to said individual or population an antigenic memory booster nucleic acid vaccine comprising (a) at least one RNA polynucleotide, said polynucleotide comprising at least one chemical modification or optionally no nucleotide modification and two or more codon-optimized open reading frames, said open reading frames encoding a set of reference antigenic polypeptides, and (b) optionally a pharmaceutically acceptable carrier or excipient. In some embodiments, the vaccine is administered to the individual via a route selected from the group consisting of intramuscular administration, intradermal administration and subcutaneous administration. In some embodiments, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition. In some embodiments, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition in combination with electroporation.

[0815] Aspects of the invention provide methods of vaccinating a subject comprising administering to the subject a single dosage of between 25 ug / kg and 400 ug / kg of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide in an effective amount to vaccinate the subject.

[0816] Other aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification,the open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, wherein the vaccine has at least 10 fold less RNA polynucleotide than is required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dosage of 25-100 micrograms.

[0817] Other aspects provide nucleic acid vaccines comprising an LNP formulated RNA polynucleotide having an open reading frame comprising no nucleotide modifications (unmodified), the open reading frame encoding a first antigenic polypeptide or a

[0818] concatemeric polypeptide, wherein the vaccine has at least 10 fold less RNA polynucleotide than is required for an unmodified mRNA vaccine not formulated in a LNP to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dosage of 25-100 micrograms.

[0819] In other aspects the invention encompasses a method of treating an elderly subject age 60 years or older comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide or a concatemeric polypeptide in an effective amount to vaccinate the subject.

[0820] In other aspects the invention encompasses a method of treating a young subject age 17 years or younger comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide or a concatemeric polypeptide in an effective amount to vaccinate the subject.

[0821] In other aspects the invention encompasses a method of treating an adult subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide or a concatemeric polypeptide in an effective amount to vaccinate the subject.

[0822] In some aspects the invention comprises a method of vaccinating a subject with a combination vaccine including at least two nucleic acid sequences encoding antigens wherein the dosage for the vaccine is a combined therapeutic dosage wherein the dosage of each individual nucleic acid encoding an antigen is a sub therapeutic dosage. In some embodiments, the combined dosage is 25 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 100 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments the combined dosage is 50 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 75 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 150 micrograms of the RNApolynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 400 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the sub therapeutic dosage of each individual nucleic acid encoding an antigen is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 micrograms. In other embodiments the nucleic acid vaccine is chemically modified and in other embodiments the nucleic acid vaccine is not chemically modified.

[0823] Aspects of the invention provide a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or a concatemeric polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and a pharmaceutically acceptable carrier or excipient, wherein an adjuvant is not included in the vaccine. In some embodiments, the stabilization element is a histone stem- loop. In some embodiments, the stabilization element is a nucleic acid sequence having increased GC content relative to wild type sequence.

[0824] Aspects of the invention provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is present in the formulation for in vivo administration to a host, which confers an antibody titer superior to the criterion for seroprotection for the first antigen for an acceptable percentage of human subjects. In some embodiments, the antibody titer produced by the mRNA vaccines of the invention is a neutralizing antibody titer. In some embodiments the neutralizing antibody titer is greater than a protein vaccine. In other embodiments the neutralizing antibody titer produced by the mRNA vaccines of the invention is greater than an adjuvanted protein vaccine. In yet other embodiments the neutralizing antibody titer produced by the mRNA vaccines of the invention is 1,000- 10,000, 1,200- 10,000, 1,400- 10,000, 1,500- 10,000, 1,000- 5,000, 1,000- 4,000, 1,800- 10,000, 2000-10,000, 2,000- 5,000, 2,000- 3,000, 2,000- 4,000, 3,000- 5,000, 3,000- 4,000, or 2,000- 2,500. A neutralization titer is typically expressed as the highest serum dilution required to achieve a 50% reduction in the number of plaques.Cancer Vaccines

[0825] In some embodiments, the therapeutic and / or prophylactic agent is a ribonucleic acid (RNA) cancer vaccine of an RNA (e.g., messenger RNA (mRNA)) that can safely direct the body' s cellular machinery to produce nearly any cancer protein or fragment thereof of interest. In some embodiments, the RNA is a modified RNA. The RNA vaccines of the present disclosure may be used to induce a balanced immune response against cancers, comprisingboth cellular and humoral immunity, without risking the possibility of insertional mutagenesis, for example.

[0826] The RNA vaccines may be utilized in various settings depending on the prevalence of the cancer or the degree or level of unmet medical need. The RNA vaccines may be utilized to treat and / or prevent a cancer of various stages or degrees of metastasis. The RNA vaccines have superior properties in that they produce much larger antibody titers and produce responses earlier than alternative anti -cancer therapies including cancer vaccines.

[0827] While not wishing to be bound by theory, it is believed that the RNA vaccines, as mRNA polynucleotides, are better designed to produce the appropriate protein conformation upon translation as the RNA vaccines co-opt natural cellular machinery. Unlike traditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the RNA vaccines are presented to the cellular system in a more native fashion.

[0828] Some embodiments of the present disclosure provide cancer vaccines that include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer antigenic polypeptide or an immunogenic fragment thereof {e.g., an immunogenic fragment capable of inducing an immune response to cancer). Other embodiments include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding two or more antigens or epitopes capable of inducing an immune response to cancer.

[0829] The invention in some aspects is a vaccine of a mRNA having an open reading frame encoding a cancer antigen and a mRNA having an open reading frame encoding an immune checkpoint modulator. In some embodiments the immune checkpoint modulator is an inhibitory checkpoint polypeptide. In some embodiments, the inhibitory checkpoint polypeptide is an antibody or fragment thereof that specifically binds to a molecule selected from the group consisting of PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. The inhibitory checkpoint polypeptide is an anti-CTLA4 or anti-PDl antibody in some embodiments. Optionally the vaccine includes a lipid nanoparticle. In some embodiments a vaccine of a mRNA having an open reading frame encoding a cancer antigen is administered to a subject. In other embodiments a checkpoint inhibitor 3-10 weeks later. In some embodiments the checkpoint inhibitor is administered 4 weeks later.

[0830] In other aspects the invention is a personalized cancer vaccine of a mRNA having an open reading frame encoding at least 2 cancer antigens, wherein the at least 2 cancer antigens are patient specific cancer antigens, and a lipid nanoparticle carrier. In some embodiments the lipid nanoparticle has a mean diameter of 50-200 nm.

[0831] In yet other aspects, the invention is a personalized cancer vaccine of a mRNA having an open reading frame encoding at least 2 cancer antigens wherein the at least 2 cancer antigens are representative of antigens of a patient. In some embodiments, the antigens of a patient are exosome identified antigens of the patient. In some embodiments a single mRNA encodes the cancer antigens. In other embodiments a plurality of mRNA encode the cancer antigens.

[0832] Each mRNA may encode 5-10 cancer antigens or a single cancer antigen in other embodiments. In some embodiments the mRNA encodes 2-100 cancer antigens. In other embodiments mRNA encodes 10-100, 20-100, 50-100, 100-200, 300-400, 500-600, 600-700, 700-800, 900-1,000, or 1,000-10,000 cancer antigens.

[0833] In some embodiments, a) the mRNA encoding each cancer antigen is interspersed by cleavage sensitive sites; b) the mRNA encoding each cancer antigen is linked directly to one another without a linker ; c) the mRNA encoding each cancer antigen is linked to one another with a single nucleotide linker; d) each cancer antigen comprises a 25-35 amino acids and includes a centrally located SNP mutation; e) at least 30% of the cancer antigens have a highest affinity for class I MHC molecules from the subject; f) at least 30% of the cancer antigens have a highest affinity for class II MHC molecules from the subject; g) at least 50% of the cancer antigens have a predicted binding affinity of IC >500nM for HL A- A, HLA-B and / or DRB 1; h) the mRNA encodes 20 cancer antigens; i) 50% of the cancer antigens have a binding affinity for class I MHC and 50% of the cancer antigens have a binding affinity for class II MHC; and / or j) the mRNA encoding the cancer antigens is arranged such that the cancer antigens are ordered to minimize pseudo-epitopes.

[0834] In some embodiments, each cancer antigen comprises 31 amino acids and includes a centrally located SNP mutation with 15 flanking amino acids on each side of the SNP mutation.

[0835] In some embodiments the vaccine is a personalized cancer vaccine and wherein the cancer antigen is a subject specific cancer antigen. In some embodiments, the subject specific cancer antigen may be representative of an exome of a tumor sample of the subject, or of atranscriptome of a tumor sample of the subject. In some embodiments, the subject specific cancer antigen may be representative of an exosome of the subject.

[0836] In some embodiments, the open reading frame further encodes one or more traditional cancer antigens. In some embodiments, the traditional cancer antigen is a non-mutated antigen. In some embodiments, the traditional cancer antigen is a mutated antigen.

[0837] In some embodiments, the mRNA vaccine further comprises an mRNA having an open reading frame encoding one or more traditional cancer antigens.

[0838] In some embodiments a single mRNA encodes the cancer antigens. In other embodiments a plurality of mRNA encode the cancer antigens. Each cancer antigen is 10-50 amino acids in length in some embodiments. In other embodiments each cancer antigen is 15- 20 amino acids in length. In other embodiments the cancer antigen is 20-50, 25-100, 100-200, 200-300, 300-400, 400-500, 500-1,000, or 1,000-10,000 amino acids in length.

[0839] In other aspects a method of eliciting an immune response in a subject by identifying at least 2 cancer antigens from a sample of a subject, wherein the at least 2 cancer antigens include mutations selected from the group consisting of frame-shift mutations and recombinations, and administering a mRNA vaccine having an open reading frame encoding the at least 2 cancer antigens to the subject is provided.

[0840] In some embodiments, the cancer antigens are identified from an exosome of the subject. In some embodiments 2-100 antigens are identified from the exosome. In other embodiments the mRNA vaccine has an open reading frame encoding the 2-100 antigens. A single mRNA or a plurality of mRNA may encode the antigens.

[0841] In some embodiments the antigens are cancer antigens. The cancer antigens may have mutations selected from point mutations, frame-shift mutations and recombinations. The method may further involve confirming that the cancer antigens are subject specific by exome analysis.

[0842] In some embodiments the method may further involve confirming that the cancer antigens are subject specific by transcriptome analysis.

[0843] In some embodiments the method also involves at least one month after the administration of the mRNA vaccine, identifying at least 2 cancer antigens from a sample of the subj ect to produce a second set of cancer antigens, and administering to the subj ect a mRNA vaccine having an open reading frame encoding the second set of cancer antigens to the subject.

[0844] In other embodiments the sample of the subject is a tumor sample.

[0845] In other aspects the invention comprises a method of eliciting an immune response in a subject by identifying at least 2 cancer antigens from a sample of a subject to produce a firstset of cancer antigens, administering to the subject a mRNA vaccine having an open reading frame encoding the first set of cancer antigens to the subject, at least one month after the administration of the mRNA vaccine, identifying at least 2 cancer antigens from a sample of a subject to produce a second set of cancer antigens, and administering to the subject a mRNA vaccine having an open reading frame encoding the second set of cancer antigens to the subject.

[0846] The mRNA vaccine having an open reading frame encoding second set of antigens, in some embodiments, is administered to the subject 6 months to 1 year after the mRNA vaccine having an open reading frame encoding first set of cancer antigens. In other embodiments the mRNA vaccine having an open reading frame encoding second set of antigens is administered to the subject 1-2 years after the mRNA vaccine having an open reading frame encoding first set of cancer antigens.

[0847] In some embodiments a single mRNA has an open reading frame encoding the cancer antigens. In other embodiments a plurality of mRNA encode the antigens. In some embodiments the second set of cancer antigens includes 2-100 antigens. In other embodiments the cancer antigens have mutations selected from point mutations, frame-shift mutations and recombinations.

[0848] In other aspects the invention comprises a method of eliciting an immune response in a subject, by identifying at least 2 cancer antigens from a sample of a subject, administering a mRNA having an open reading frame encoding the at least 2 cancer antigens to the subject, and administering a cancer therapeutic agent to the subject. In some embodiments the cancer therapeutic agent is a targeted therapy. The targeted therapy may be a BRAF inhibitor such as vemurafenib (PLX4032) or dabrafenib.

[0849] In other embodiments the cancer therapeutic agent is a T-cell therapeutic agent. The T- cell therapeutic agent may be a checkpoint inhibitor such as an anti-PD- 1 antibody or an anti- CTLA-4 antibody. In some embodiments the anti-PD- 1 antibody is BMS-936558 (nivolumab). In other embodiments the anti-CTLA-4 antibody is ipilimumab. The T-cell therapeutic agent in other embodiments is OX40L. In yet other embodiments the cancer therapeutic agent is a vaccine comprising a population based tumor specific antigen.

[0850] In other embodiments the cancer therapeutic agent is a vaccine comprising an mRNA having an open reading frame encoding one or more traditional cancer antigens.

[0851] In some embodiments, the mRNA having an open reading frame encoding the at least 2 cancer antigens is administered to the subject simultaneously with the cancer therapeutic agent. In some embodiments, the mRNA having an open reading frame encoding the at least 2 cancer antigens is administered to the subject before administration of the cancer therapeuticagent. In some embodiments, the mRNA having an open reading frame encoding the at least 2 cancer antigens is administered to the subject after administration of the cancer therapeutic agent.

[0852] A method comprising mixing a mRNA having an open reading frame encoding a cancer antigen with a lipid nanoparticle formulation to produce a mRNA cancer vaccine, and administering the mRNA cancer vaccine to a subject within 24 hours of mixing is provided in other aspects of the invention. In some embodiments the mRNA cancer vaccine is administered to the subject within 12 hours of mixing. In other embodiments the mRNA cancer vaccine is administered to the subject within 1 hour of mixing. The mRNA cancer vaccine encodes 2-100 cancer antigens or 10-100 cancer antigens in some embodiments.

[0853] In some embodiments the vaccine is a personalized cancer vaccine and wherein the cancer antigen is a subject specific cancer antigen.

[0854] In some embodiments a single mRNA encodes the cancer antigens. In other embodiments a plurality of mRNA encode the cancer antigens. Each mRNA encodes 5-10 cancer antigens or a single cancer antigen in other embodiments. In yet other embodiments each cancer antigen is 10-50 amino acids in length or 15-20 amino acids in length.

[0855] Further provided herein are uses of cancer vaccines in the manufacture of a medicament for use in a method of inducing an antigen specific immune response in a subject, the method comprising administering the cancer vaccine to the subject in an amount effective to produce an antigen specific immune response.

[0856] A method of treating cancer in a subject in need thereof by identifying at least 2 cancer antigens from an exosome isolated from the subject; producing, based on the identified antigens, a mRNA vaccine having an open reading frame encoding the antigens; and administering the mRNA vaccine to the subject, wherein the mRNA vaccine induces a tumorspecific immune response in the subject, thereby treating cancer in the subject is provided in other aspects. The invention in other aspects is a RNA vaccine preparable according to a method involving identifying at least 2 cancer antigens from an exosome isolated from a subject; producing, based on the identified antigens, a mRNA vaccine having an open reading frame encoding the antigens.

[0857] A method of eliciting an immune response in a subject against a cancer antigen is provided in aspects of the invention. The method involves administering to the subject a RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the antigenic polypeptide or an immunogenic fragmentthereof, wherein the anti-antigenic polypeptide antibody titer in the subject is increased following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the cancer. An "anti-antigenic polypeptide antibody" is a serum antibody the binds specifically to the antigenic polypeptide.

[0858] A prophylactically effective dose is a therapeutically effective dose that prevents advancement of cancer at a clinically acceptable level. In some embodiments the therapeutically effective dose is a dose listed in a package insert for the vaccine. A traditional vaccine, as used herein, refers to a vaccine other than the mRNA vaccines of the invention. For instance, a traditional vaccine includes but is not limited to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, etc. In exemplary embodiments, a traditional vaccine is a vaccine that has achieved regulatory approval and / or is registered by a national drug regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA.)

[0859] In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the cancer.

[0860] In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 1 log following vaccination relative to anti -antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the cancer.

[0861] In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 2 log following vaccination relative to anti -antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the cancer.

[0862] In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 3 log following vaccination relative to anti -antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the cancer.

[0863] In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 5 log following vaccination relative to anti -antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the or cancer.

[0864] In some embodiments the anti-antigenic polypeptide antibody titer in the subject is increased 10 log following vaccination relative to anti -antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the or cancer.

[0865] A method of eliciting an immune response in a subject against a cancer antigen is provided in other aspects of the invention. The method involves administering to the subject a RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to antigenic polypeptide or an immunogenic fragment thereof, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the cancer antigen at 2 times to 100 times the dosage level relative to the RNA vaccine.

[0866] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to the RNA vaccine.

[0867] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to the RNA vaccine.

[0868] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times the dosage level relative to the RNA vaccine.

[0869] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 5 times the dosage level relative to the RNA vaccine. In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times the dosage level relative to the RNA vaccine.

[0870] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 50 times the dosage level relative to the RNA vaccine.

[0871] In some embodiments the immune response in the subject is equivalent to an immune response in a subj ect vaccinated with a traditional vaccine at 100 times the dosage level relative to the RNA vaccine.

[0872] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to the RNA vaccine.

[0873] In some embodiments the immune response in the subject is equivalent to an immune response in a subj ect vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to the RNA vaccine.

[0874] In other embodiments the immune response is assessed by determining antibody titer in the subject.

[0875] In other aspects the invention comprises a method of eliciting an immune response in a subject against a by administering to the subject a RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one cancer antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the antigenic polypeptide or an immunogenic fragment thereof, wherein the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the cancer antigen. In some embodiments the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to the RNA vaccine.

[0876] In some embodiments the immune response in the subject is induced 2 days earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0877] In some embodiments the immune response in the subject is induced 3 days earlier relative to an immune response induced in a subject vaccinated a prophylactically effective dose of a traditional vaccine. In some embodiments the immune response in the subject is induced 1 week earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0878] In some embodiments the immune response in the subject is induced 2 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0879] In some embodiments the immune response in the subject is induced 3 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0880] In some embodiments the immune response in the subject is induced 5 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0881] In some embodiments the immune response in the subject is induced 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0882] A method of eliciting an immune response in a subject against a cancer by administering to the subject a cancer RNA vaccine having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and wherein an adjuvant is not coformulated or co-administered with the vaccine.

[0883] In yet other aspects the invention comprises a method of producing an mRNA encoding a concatemeric cancer antigen comprising between 1000 and 3000 nucleotides, the method by(a) binding a first polynucleotide comprising an open reading frame encoding the concatemeric cancer antigen and a second polynucleotide comprising a 5'-UTR to a polynucleotide conjugated to a solid support;(b) ligating the 3 '-terminus of the second polynucleotide to the 5 '-terminus of the first polynucleotide under suitable conditions, wherein the suitable conditions comprise a DNA Ligase, thereby producing a first ligation product;(c) ligating the 5' terminus of a third polynucleotide comprising a 3'-UTR to the 3'- terminus of the first ligation product under suitable conditions, wherein the suitable conditions comprise an RNA Ligase, thereby producing a second ligation product; and(d) releasing the second ligation product from the solid support, thereby producing an mRNA encoding the concatemeric cancer antigen comprising between 1000 and 3000 nucleotides. In some embodiments of any one of the provided compositions or methods, the mRNA encodes one or more recurrent polymorphisms. In some embodiments, the one or more recurrent polymorphisms comprises a recurrent somatic cancer mutation in p53. In some such embodiments, the one or more recurrent somatic cancer mutation in p53 are selected from the group consisting of:(1) mutations at the canonical 5' splice site neighboring codon p.T125;(2) mutations at the canonical 5' splice site neighboring codon p.331 ;(3) mutations at the canonical 3' splice site neighboring codon p.126;(4) mutations at the canonical 5' splice site neighboring codon p.224, inducing a cryptic alternative intronic 5' splice site.

[0884] In some embodiments, the invention provides a cancer therapeutic vaccine comprising mRNA encoding an open reading frame (ORF) coding for one or more of neoantigen peptides (1) through (4). In one embodiment, the invention provides the selective administration of a vaccine containing or coding for one or more of peptides (l)-(4), based on the patient's tumor containing any of the above mutations. In one embodiment, the invention provides the selective administration of the vaccine based on the dual criteria of the subject's tumor containing any of the above mutations and the subject's normal HLA type containing the corresponding HLA allele predicted to bind to the resulting neoantigen.

[0885] A method for treating a subject with a personalized mRNA cancer vaccine, by isolating a sample from a subject, identifying a set of neoepitopes by analyzing a patient transcriptome and / or a patient exome from the sample to produce a patient specific mutanome, selecting a set of neoepitopes for the vaccine from the mutanome based on MHC binding strength, MHC binding diversity, predicted degree of immunogenicity, low self reactivity, and / or T cell reactivity, preparing the mRNA vaccine to encode the set of neoepitopes and administering the mRNA vaccine to the subject within two months of isolating the sample from the subject is provided in other aspects of the invention. In some embodiments the mRNA vaccine is administered to the subject within one month of isolating the sample from the subject.

[0886] In other aspects the invention comprises a method of identifying a set of neoepitopes for use in a personalized mRNA cancer vaccine having one or more polynucleotides that encode the set of neoepitopes by a. identifying a patient specific mutanome by analyzing a patient transcriptome and a patient exome, b. selecting a subset of 15-500 neoepitopes from the mutanome using a weighted value for the neoepitopes based on at least three of: an assessment of gene or transcript-level expression in patient RNA-seq; variant call confidence score; RNA- seq allele- specific expression; conservative vs. non-conservative amino acid substitution; position of point mutation (C6ntering Score for increased TCR engagement); position of point mutation (Anchoring Score for differential HLA binding); Selfness: <100% core epitope homology with patient WES data; HLA-A and -B IC50 for 8mers-l Imers; HLA-DRB 1 IC50 for 15mers-20mers; promiscuity Score (i.e. number of patient HLAs predicted to bind); HLA- C IC50 for 8mers-l lmers;HLA-DRB3-5 IC50 for 15mers-20mers; HLA-DQB 1 / A1 IC50 for 15mers-20mers; HLA-DPB 1 / A1 IC50 for 15mers-20mers; Class I vs Class II proportion; Diversity of patient HLA-A, -B and DRB 1 allotypes covered; proportion of point mutation vs complex epitopes (e.g. frameshifts); and / or pseudo-epitope HLA binding scores, and c. selecting the set of neoepitopes for use in a personalized mRNA cancer vaccine from the subsetbased on the highest weighted value, wherein the set of neoepitopes comprise 15-40 neoepitopes.Other Components

[0887] A LNP (e.g., an empty LNP or a loaded LNP of the disclosure) may include one or more components in addition to those described in the preceding sections. In some embodiments, a LNP (e.g., an empty LNP or a loaded LNP of the disclosure)may include one or more small hydrophobic molecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol.

[0888] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs of the disclosure) may also include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents, or other components. A permeability enhancer molecule may be a molecule described by U.S. patent application publication No. 2005 / 0222064, for example. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

[0889] A polymer may be included in and / or used to encapsulate or partially encapsulate a LNP. A polymer may be biodegradable and / or biocompatible. A polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. In some embodiments, a polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co- caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co- PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyan...

Claims

CLAIMS1. A compound of Formula (I):or an ionic form or a salt thereof, wherein: q is 1, 2, 3, 4, 5, or 6;Q is -ORqor -NHRq;Rqis H, or C3-6cycloalkyl substituted one or more oxo, -NH2, -NH(C1-6alkyl), or -N(C1-6alkyl)2;T1is (i) C1-24 alkyl optionally substituted with one or more -OH, -C(=O)OH or 3- to 10- membered heterocyclyl, or (ii) -M1-L1-R1;M1is C 1-12 alkylene optionally substituted with one or more Rm1; each Rm1independently is oxo, halogen, -OH, -O(C1-6alkyl), -NH2, - NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl; or two Rm1, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6-membered heterocycloalkyl;cycloalkylene)-**, wherein * denotes attachment to T1and ** denotes attachment to R1;R1is W1or -UkykW1;U1is C1-6alkylene optionally substituted with one or more oxo, halogen, -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl;V1is *-C(=O)-O-**, *-O-C(=O)-**, *-S-S-**, *-(C6-10arylene)-**, *- 0-(C6-10arylene)-**, *-(C6-10arylene)-O-**, *-NH-(C6-10arylene)-**, *-O-**, or *-(C6-10arylene)-NH-**, wherein * denotes attachment to U1and ** denotes attachment to W1;W1isC5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl, wherein the C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl is optionally substituted with one or more Rwl;each Rwlindependently is oxo, halogen, -OH, -O(C1-6alkyl), - NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1-6alkyl, C2-6alkenyl, or C2-6alkynyl; or two Rwl, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6-membered heterocycloalkyl; b is 1, 2, 3, 4, 5, or 6;L2is -O-C(=O)-** or -C(=O)-O-** , wherein ** denotes attachment to R2;R21is H, C 1-12 alkyl, C2-12alkenyl, or C2-12alkynyl;R2bis C5 -12alkyl, C5 -12alkenyl, or C 5- 12 alkynyl; c is 1, 2, 3, 4, 5, or 6;L3is -O-C(=O)-** or -C(=O)-O-** , wherein ** denotes attachment to R3;r3 is 0, 1, 2, 3, 4, 5, or 6;R3ais H, V3a, or -U3a-V3a;U3ais -O-C(=O)-** or -C(=O)-O-**, wherein ** denotes attachment toV3a;V3ais C1-12 alkyl, C2-12alkenyl, or C2-12alkynyl;R3bis V3b, or -U3b-V3b;U3bis -O-C(=O)-** or -C(=O)-O-** , wherein ** denotes attachment toV3b; andV3bis C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl.

2. The compound of claim 1, wherein T1is -M1-L1-R1, wherein:(i) M1is C1-12 alkylene substituted with one or more Rm1;(ii) R1is -U1-V1-W1;; or(iii) R1is W1, wherein W1is C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl, wherein the C1- 12 alkyl, C2-12alkenyl, or C2-12alkynyl is substituted with one or more Rwl, wherein each Rwlindependently is oxo, halogen, -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), -N(C1-6alkyl)2, C1- 3 alkyl, C5-6 alkyl, C2-6alkenyl, or C2-6alkynyl.The compound of any one of the preceding claims, wherein q is 2 or 3.

4. The compound of any one of the preceding claims, wherein Q is -OH.

5. The compound of any one of the preceding claims, whereinThe compound of any one of the preceding claims, wherein T1is C1-24alkyl optionally substituted with one or more -C(=O)OH.

7. The compound of any one of the preceding claims, wherein T1is -M1-L1-R1.

8. The compound of any one of the preceding claims, wherein M1is C3-8alkylene (e.g., propylene, butylene, pentylene, hexylene, heptylene, or octylene) optionally substituted with one or more Rm1.

9. The compound of any one of the preceding claims, wherein at least one (e.g., each) Rm1is -OH, -O(C1-6alkyl), -NH2, -NH(C1-6alkyl), or -N(C1-6alkyl)2.

10. The compound of any one of the preceding claims, wherein at least two Rm1, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6- membered heterocycloalkyl.

11. The compound of any one of the preceding claims, wherein L1is *-O-C(=O)-** or *- C(=O)-O-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

12. The compound of any one of the preceding claims, wherein L1is *-C(=O)-NH-** or *- NH-C(=O)-**, wherein * denotes attachment to T1and ** denotes attachment to R1.

13. The compound of any one of the preceding claims, wherein L1is *-(C3-6cycloalkylene)- **, wherein * denotes attachment to T1and ** denotes attachment to R1.

14. The compound of any one of the preceding claims, wherein R1is W1.

15. The compound of any one of the preceding claims, wherein R1is -L1-V1-W1.

16. The compound of any one of the preceding claims, wherein U1is C1-6alkylene.

17. The compound of any one of the preceding claims, wherein V1is *-C(=O)-O-**, *-O-C(=O)-**, wherein * denotes attachment to U1and ** denotes attachment to W.

18. The compound of any one of the preceding claims, wherein V1is *-O-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

19. The compound of any one of the preceding claims, wherein V1is *-S-S-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

20. The compound of any one of the preceding claims, wherein V1is *-(C6-10arylene)-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

21. The compound of any one of the preceding claims, wherein V1is *-0-(C6-10arylene)- ** or *-(C6-10arylene)-O-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

22. The compound of any one of the preceding claims, wherein V1is *-NH-(C6-10arylene)-** or *-(C6-10arylene)-NH-**, wherein * denotes attachment to U1and ** denotes attachment to W1.

23. The compound of any one of the preceding claims, wherein W1is hydrogen.

24. The compound of any one of the preceding claims, wherein W1is C5 -12alkyl optionally substituted with one or more Rwl.

25. The compound of any one of the preceding claims, wherein at least one (e.g., each) RW1is -OH or -O(C1-6alkyl).

26. The compound of any one of the preceding claims, wherein at least two Rwl, together with the one or more intervening atoms they are attached to, form C3-6cycloalkyl or 3- to 6- membered heterocycloalkyl.

27. The compound of any one of the preceding claims, wherein b is 3, 4, or 5.

28. The compound of any one of the preceding claims, wherein L2is -O-C(=O)-**, wherein ** denotes attachment to R2.

29. The compound of any one of the preceding claims, wherein L2is -C(=O)-O-** , wherein ** denotes attachment to R2.

30. The compound of any one of the preceding claims, wherein31. The compound of any one of the preceding claims, wherein32. The compound of any one of the preceding claims, wherein R2ais H.

33. The compound of any one of the preceding claims, wherein R2ais C1-12alkyl, C2-12alkenyl, or C2-12alkynyl.

34. The compound of any one of the preceding claims, wherein c is 3, 4, or 5.

35. The compound of any one of the preceding claims, wherein L3is -O-C(=O)-**, wherein ** denotes attachment to R3.

36. The compound of any one of the preceding claims, wherein L3is -C(=O)-O-** , wherein ** denotes attachment to R3.

37. The compound of any one of the preceding claims, wherein R3is38. The compound of any one of the preceding claims, wherein R3is39. The compound of any one of the preceding claims, wherein R3isR3b40. The compound of any one of the preceding claims, wherein R3is R3b41. The compound of any one of the preceding claims, wherein R3ais H.

42. The compound of any one of the preceding claims, wherein R3ais V3a.

43. The compound of any one of the preceding claims, wherein R3ais -U3a-V3a.

44. The compound of any one of the preceding claims, wherein U3ais -O-C(=O)-** , wherein ** denotes attachment to V3a.

45. The compound of any one of the preceding claims, wherein U3ais -C(=O)-O-** , wherein ** denotes attachment to V3a.

46. The compound of any one of the preceding claims, wherein V3ais C1-12alkyl, C2-12alkenyl, or C2-12alkynyl.

47. The compound of any one of the preceding claims, wherein R3bis V3b.

48. The compound of any one of the preceding claims, wherein R3bis -U3b-V3b.

49. The compound of any one of the preceding claims, wherein U3bis -O-C(=O)-** , wherein ** denotes attachment to V3b.

50. The compound of any one of the preceding claims, wherein U3bis -C(=O)-O-**, wherein ** denotes attachment to V3b.

51. The compound of any one of the preceding claims, wherein V3bis C5 -12alkyl, C5 -12alkenyl, or C5 -12alkynyl.

52. The compound of any one of the preceding claims, wherein the compound is of Formula(la):or a salt thereof.

53. The compound of any one of the preceding claims, wherein the compound is of Formula(lb):or a salt thereof.

54. The compound of any one of the preceding claims, wherein the compound is of Formula(Ic):or a salt thereof.

55. The compound of any one of the preceding claims, wherein the compound is of Formula(Id):or a salt thereof.

56. The compound of any one of the preceding claims, wherein the compound is of Formula(le):or a salt thereof.

57. The compound of any one of the preceding claims, wherein the compound is of Formula(If):or a salt thereof.

58. The compound of any one of the preceding claims, wherein the compound is of Formula(Ig):or a salt thereof.

59. The compound of any one of the preceding claims, wherein the compounds is selected from the compounds described in Table 1 and salts thereof.

60. A method of preparing a lipid nanoparticle formulation (LNP formulation), comprising: i) a nanoprecipitation step, comprising: i-a) mixing a lipid solution comprising a cationic lipid, an ionizable lipid, a structural lipid, and a phospholipid, with an aqueous buffer solution comprising a buffering agent, thereby forming an intermediate lipid nanoparticle solution (intermediate LNP solution); i-b) adding a diluting solution to the intermediate LNP solution; i-c) holding the intermediate LNP solution for a residence time; i-d) adding a pH-adjusting solution to the intermediate LNP solution, thereby forming a lipid nanoparticle solution (LNP solution); and ii) processing the LNP solution, thereby forming an LNP formulation, wherein the processing comprises: ii-a) adding a PEG lipid solution to the LNP solution.

61. The method of claim 60, wherein the cationic lipid is a compound of any one of the previous claims.

62. The method of any one of the preceding claims, wherein the lipid solution is free of PEG lipid.

63. The method of any one of the preceding claims, wherein the lipid solution further comprises a PEG lipid.

64. The method of any one of the preceding claims, wherein the aqueous buffer solution is free of nucleic acid.

65. The method of any one of the preceding claims, wherein the aqueous buffer solution further comprises a nucleic acid.

66. The method of any one of the preceding claims, wherein the nucleic acid is an RNA.

67. The method of any one of the preceding claims, wherein the nucleic acid is an mRNA.

68. The method of any one of the preceding claims, wherein the diluting solution comprises citrate or citrate buffered saline.

69. The method of any one of the preceding claims, further comprising the step of: iii) storing the LNP formulation in a buffered storage solution.

70. The method of any one of the preceding claims, wherein the buffered storage solution comprises a mixture of tris, acetate, citrate, and / or sodium chloride.

71. The method of any one of the preceding claims, wherein the ionic strength of the buffered storage solution is about 0.15 molar (M) to about 0.2 M.

72. A lipid nanoparticle (LNP) made by the method of any one of the preceding claims.

73. A lipid nanoparticle (LNP) comprising a cationic lipid.

74. The LNP of claim 73, wherein the cationic lipid is the compound of any one of the preceding claims.

75. The LNP of any one of the preceding claims, further comprises an ionizable lipid.

76. The LNP of any one of the preceding claims, further comprises an ionizable lipid, a phospholipid, and a structural lipid.

77. The LNP of any one of the preceding claims, further comprises an ionizable lipid, a phospholipid, a structural lipid, and a PEG lipid.

78. The LNP of any one of the preceding claims, wherein the LNP comprises about 1% PEG-DSG and about 2% PEG-1.

79. A pharmaceutical composition comprising the LNP of any one of the preceding claims.

80. A method of delivering a therapeutic and / or prophylactic agent to a cell, comprising administering to the cell the LNP of any one of the preceding claims.

81. The LNP of any one of the preceding claims for use in delivering a therapeutic and / or prophylactic agent to a cell.

82. Use of the LNP of any one of the preceding claims in the manufacture of a medicament for delivering a therapeutic and / or prophylactic agent to a cell.

83. A method of delivering a therapeutic and / or prophylactic agent to a cell, comprising administering to the cell a LNP comprising the compound of any one of the preceding claims.

84. A LNP comprising the compound of any one of the preceding claims for use in delivering a therapeutic and / or prophylactic agent to a cell.

85. Use of the compound of any one of the preceding claims in the manufacture of a medicament for delivering a therapeutic and / or prophylactic agent to a cell.

86. A method of delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject, comprising administering to the subject the LNP of any one of the preceding claims.

87. The LNP of any one of the preceding claims for use in delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject.

88. Use of the LNP of any one of the preceding claims in the manufacture of a medicament for delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject.

89. A method of delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject, comprising administering to the subject a LNP comprising the compound of any one of the preceding claims.

90. A LNP comprising the compound of any one of the preceding claims for use in delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject.

91. Use of the compound of any one of the preceding claims in the manufacture of a medicament for delivering a therapeutic and / or prophylactic agent to an organ or a tissue in a subject.

92. A method of treating or preventing a disease or disorder in a subject, comprising administering to the LNP of any one of the preceding claims.

93. The LNP of any one of the preceding claims for use in treating or preventing a disease or disorder in a subject.

94. Use of the LNP of any one of the preceding claims in the manufacture of a medicament for treating or preventing a disease or disorder in a subject.

95. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a LNP comprising the compound of any one of the preceding claims.

96. A LNP comprising the compound of any one of the preceding claims for use in treating or preventing a disease or disorder in a subject.

97. Use of the compound of any one of the preceding claims in the manufacture of a medicament for treating or preventing a disease or disorder in a subject.

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