Ionizable lipids and compositions thereof for delivery of therapeutic agents

Abstract

The disclosure features novel lipids and compositions involving the same. Lipid nanoparticles include a novel ionizable lipid as well as additional lipids such as cationic lipids, phospholipids, structural lipids, and PEG lipids. Lipid nanoparticles 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 THERAPEUTICAGENTSCROSS-REFERENCE TO RELATED .APPLICATIONS

[0001] This application claims priority to and the benefit of U.S, Provisional Application No. 63 / 624,028, filed on January 23, 2024, the contents of which are incorporated by reference herein in their entirety for all purposes.FIELD OF DISCLOSURE

[0002] The present disclosure provides novel ionizable 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 cationic 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.SUMMARYIn an aspect, the present disclosure provides a compound of Formula (I*):or a pharmaceutically acceptable salt thereof, wherein: one is a single bond, and the otheris a double bond; n is 3, 4, 5, or 6;M is -()-('( ())-* and -C(:::O)-O~*, wherein * indicates attachment to L;L is a bond or -CH2-;R1is H, C1-12 alkyl, or C2-12 alkenyl;R2is H, C1-12 alkyl, or 62-52 alkenyl;R3is C1-12 alkyl, or C2-12 alkenyl; wherein at least one of R1, R2, and R3is a C1-12 alkyl or C2-12 alkenyl; m is 3, 4, 5, or 6;each is a single bond or a double bond, wherein no more than one is a double bond;M’ is -O~C(=;O)~* and -6(:;=O)-O-*, wherein * indicates attachment to L’;L’ is a bond or -6H2-;Rris H, C1-12 alkyl, or C2-12 alkenyl;R2is H, C1-12 alkyl, or C2-12 alkenyl;R3’ is H, C1-12 alkyl, or C2-12 alkenyl; wherein at least one of R{”, R2, and R3is a C1-12 alkyl or C2-12 alkenyl;RHis -(CH2)P-Q, -(CH2)P-NRG-T-Q, -(CI Oh-T-Q. -(CH2)p-NRG-C(-O)H or -(CH2)P-NRG-6(=O)-T-Q; p is 1, 2, 3, 4, or 5; each RGindependently is H, C 1-6 alkyl, or C2-6 alkenyl,T is a bond, C1-3 alkylene, C2-3 alkenylene, or 62-3 alkynylene;Q is -OH, -O-(C 1-6 alkyl), C 1-6 alkyl, C2-5 alkenyl, C2-6 alkynyl, C3-10 cycloalkyl, 5-10 membered heteroaryl, C6-10 and, or 3-12 membered heterocycloalkyl, wherein the -O-(C 1-6 alkyl), C 1-6 alkyl, C2-6 alkenyl, C3-10 cycloalkyl, or 3-12 membered heterocycloalkyl is optionally substituted with one or more RQ; andeach RQindependently is oxo, cyano, -OH, -O-(C 1-6 alkyl), -NH?, -M hC « alkyl), -N(C 1-6 alkyl ):-. -C(=O)-(C 1-6 alkyl), -O-C(=O)-(C 1-6 alkyl), -NR-C(=O)-(C 1-6 alkyl), C 1-6 alkyl, C2-6 alkenyl, -(C 1-6 alkyl)-OH, C2-6 alkenyl, or C3-10 cycloalkyl.In some embodiments, the compound is of Formula (I**):or a pharmaceutically acceptable salt thereof, wherein: one is a single bond, and the otheris a double bond; n is 3, 4, 5, or 6;M is -O-C(=O)-* or -C(=O)-O-*, wherein * indicates attachment to L;L is -CH2~;R1is H;R2is C3-5 alkyl;R ’ is C3..5 alkyl; m is 3, 4, 5, or 6;each is a single bond or a double bond, wherein no more than one is a double bond;M’ is -C(=O)-O-*, wherein * indicates attachment to L’;L’ is a bond;R1is C7-10 alkyl;R ' is C 5-8 alkyl;R3is H;RHis -(CH2)P-Q; p is 1, 2, 3, 4, or 5; and Q is -OH.In some embodiments, the compound is of Formula (I):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (II):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula ( HI):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (IV):or a pharmaceutically acceptable salt thereofIn some embodiments, the compound is of Formula ( V):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (VI):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (1-2):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (II-2):(II-2) or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (III-2):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (IV -2):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (V-2):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (VI-2):(VI-2) or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (VI-2):(VI-2) or a pharmaceutically acceptable salt thereofIn some embodiments, the compound is of Formula (1-3):or a pharmaceutically acceptable salt thereofIn some embodiments, the compound is of Formula (1-3):(1-3)or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (II-3):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (III-3):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (IV-3):(IV-3) or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula ( V-3):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (VI-2):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (1-4):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (11-4):(H-4)or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (III-4):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (IV-4):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (V-4):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (VI-4):(VI-4)or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (1-5):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (II-5):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (III-5):(III-5) or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (IV-5):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (V-5):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (VI-5):(VI-5) or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (1-6)or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (II-6):(II-6) or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (III-6):(III-6) or a pharmaceutically acceptable salt thereof.In some embodiments the compound is of Formula (IV-6):or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (V-6):(V-6) or a pharmaceutically acceptable salt thereof.In some embodiments, the compound is of Formula (VI-6):or a pharmaceutically acceptable salt thereof.In some embodiments, RHis -(Cltyty-Q or -(CIty)P-NRG~T-Q. IN some embodiments, p is 2 or 3. In some embodiments, RGis H. In some embodiments, T is a bond. In some embodiments, Q is OH or C3-6 cycloalkyl substituted with one or more oxo, -NH2, -NH(CI-6 alkyl), or -N(C 1-6 alkylty In some embodiments, Q is cyclobutenyl substituted with one or more oxo, -NH2, -NH(C 1-6 alkyl), or -N(C 1-6 alkylty.In some embodiments, n is 5. In some embodiments, M is -C(=O)-O-*, wherein * indicates attachment to L. In some embodiments, m is 5. In some embodiments M’ is - C(=O)-O-*, wherein * indicates attachment to L’. In some embodiments, L is -CH2-.In some embodiments, R1is H, methyl, or ethyl.In some embodiments, R2is Ck-n alkyl. In some embodiments, R2is H, propyl, butyl, or heptyl. In some embodiments, R2is propyl, butyl, or heptyl. In some embodiments, R2is pentenyl .In some embodiments, R3is methyl, butyl, or pentyl. In some embodiments, R3is hexenyl ,In some embodiments, L’ is -City-. In some embodiments, L’ is a bond.In some embodiments, M’ is -C(=O)-O-*, wherein * indicates attachment to L’, and L’ is -City-. In some embodiments, M’ is -C(=O )-O -*, wherein * indicates attachment to L’, and L’ is a bond.In some embodiments, R1is H, methyl, or ethyl.In some embodiments, R2is C2-12 alkyl. In some embodiments, R2is H, propyl, butyl, or heptyl. In some embodiments, R2” is propyl, butyl, or heptyl. In some embodiments, R2is pentenyl.In some embodiments, R3’ is H, methyl, butyl, or pentyl. In some embodiments, R3’ is hexenyl.In some aspects, the present disclosure provides a compound having the any one of the structures shown in Table 1, or a pharmaceutically acceptable salt thereof.In some aspects, the present disclosure provides a lipid nanoparticle ( LNP) comprising a lipid of any one the present disclosure, a phospholipid, a structural lipid, and a PEG lipid.In some aspects, the present disclosure provides a lipid nanoparticle (LNP) comprising a lipid of the present disclosure, a phospholipid, a structural lipid, a cationic lipid, and a PEG lipid.In some embodiments, the LNP comprises about 40 mol % to about 60 mol % said lipid, about 0 mol % to about 20 mol % phospholipid, about 30 mol % to about 50 mol % structural lipid, and about 0 mol % to about 5 mol % PEG lipid. In some embodiments, the phospholipid is selected from the group consisting of:1.2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC),1.2-dimyri stoyl-sn-glycero-phosphocholine (DMPC),1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1.2-diundecanoyl -sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1.2-di-O-octadecenyl-s«-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-5w-glycero-3-phosphocholine (OChemsPC), l-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-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1.2-diphytanoyl-sn-glycero~3-phosphoethanolamme (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),sphingomyelin, and mixtures thereof. In some embodiments, the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, and mixtures thereof. In some embodiments, 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. In some embodiments, the PEG lipid is selected from PEG-III and PEG-II:and 100, , (PL-II), wherein rFEGis an integer between 1 and 100, and mixtures thereof. In some embodiments, the PEG lipid is selected from PEGzk-DMG and PEG-1 :and mixtures thereof.In some embodiments, the LNP further comprises one or more therapeutic and / or prophylactic agents. In some embodiments, the one or more therapeutic and / or prophylactic agents is a nucleic acid. In some embodiments, the nucleic acid is an RNA, and wherein the RNA is selected from the group consisting of a short interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), an RNA interference (RNAi) molecule, a microRNA (miRNA), an antagomir, an antisense RNA, a ribozyme, a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is an MRNA.In some embodiments, the present disclosure provides a pharmaceutical composition comprising the LNP of the present disclosure and a pharmaceutically acceptable carrier.In some embodiments, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a cell within a subject, the method comprising administering to the subject the LNP of the present disclosure.In some embodiments, the present disclosure provides a method of specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, the method comprising administering to the subject a LNP of the present disclosure.In some embodiments, the present disclosure provides a method of producing a polypeptide of interest in a cell within a subject, the method comprising administering to the subject a LNP of the present disclosure.In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a LNP of the present disclosure.In some embodiments, the present disclosure provides a LNP of the present disclosure for use in delivering a therapeutic and / or prophylactic agent to a cell within a subject.In some embodiments, the present disclosure provides a LNP of the present disclosure for use in specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject.In some embodiments, the present disclosure provides a LNP of the present disclosure for use in producing a polypeptide of interest in a cell within a subject.In some embodiments, the present disclosure provides a LNP of the present disclosure for use in treating or preventing a disease or disorder in a subject in need thereof.In some embodiments, the present disclosure provides the use of the LNP of the present disclosure in the preparation of a medicament for delivering a therapeutic and / or prophylactic agent to a cell within a subject.In some embodiments, the present disclosure provides the use of the LNP of the present disclosure in the preparation of a medicament for delivering a therapeutic and / or prophylactic agent to an organ of a subject.In some embodiments, the present disclosure provides the use of the LNP of the present disclosure in the preparation of a medicament for producing a polypeptide of interest in a cell within a subject.In some embodiments, the present disclosure provides the use of a LNP of the present disclosure in the preparation of a medicament for treating or preventing a disease or disorder in a subject in need thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 provides a graph showing the expression of human erythropoietin (hEPO) in the serum of female CD-I mice effectuated by LNPs comprising hEPO mRNA and compounds of the present disclosure at 6 and 24 hours following administration.

[0006] FIG. 2 provides a graph showing the expression of human erythropoietin (hEPO) in the serum of female CD-I mice effectuated by LNPs comprising hEPO mRNA and compounds of the present disclosure at 6 and 24 hours following administration.

[0007] FIG. 3 provides a graph showing the expression of human erythropoietin (hEPO) in the serum of female CD-I mice effectuated by LNPs comprising hEPO mRNA and compounds of the present disclosure at 6 and 24 hours following administration.DET AILED DESCRIPTION

[0008] The present disclosure provides novel ionizable lipids, e.g., including a central amine moiety and at least one biodegradable group. Without wishing to be bound by theory, the ionizable lipids described herein may be advantageously used in lipid nanoparticles for the delivery of therapeutic and / or prophylactic agents to mammalian cells or organs. For example, the ionizable lipids described herein may be advantageously used in lipid nanoparticles for the delivery of therapeutic and / or prophylactic agents to specific mammalian cells or organs. In some embodiments, the ionizable lipids described herein may be advantageously used in lipid nanoparticles for the delivery of therapeutic and / or prophylactic agents to endothelial cells or the lung.Definitions

[0009] 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 “Cni4 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.

[0010] ,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, Cis alkenyl may include one or more double bonds. A C18 alkenyl 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.

[0011] 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-C6 for straight chain, C3-C6 for 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-C6 alkenylene linker” or “C2-C6 alkynylene linker” is intended to include C2, C3, C4, Cs or C6 chain (linear or branched) divalent unsaturated aliphatic hydrocarbon groups. For example, C2- C6alkenylene linker is intended to include C2, C3, C4, C5 and C6 alkenylene linker groups. The term “alkynylene” refers to a divalent alkynyl group. Unless otherwise specified, an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.

[0012] 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 “C3-6 carbocycle” 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.

[0013] As used herein, the term “cycloal kyl” 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,cyclobutenyl, cyclopentenyl, cyclohex enyl, 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.

[0014] 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 sulfur atoms. 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- 1 , 1 '-isobenzofuran]-yl, 7'H-spiro[cyclohexane- l,5‘-furo[3,4-b]pyridin]-yl, 3‘H-spiro[cyclohexane-l,r-furo[3,4-c]pyridin]-yl, 3- azabicyclop .1.0]hexanyl, 3-azabicyclo[3.1 ,0]hexan-3-yl, 1 ,4,5,6-tetrahydropyrrolo[3,4- c]pyrazolyl, 3,4,5,6,7,8-hexahydropyrido[4,3~d]pyritnidinyl, 4,5,6,7-tetrahydro-IH- 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.

[0015] As used herein, the term “hydroxy” or “hydroxyl” includes groups with an -OH or -O’

[0016] As used herein, the term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

[0017] The term “haloalkyl” or “haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or more halogen atoms, respectively.

[0018] 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, aryl carbonyl oxy, alkoxy carbonyl oxy, aryloxycarbonyloxy, carboxylate, alkyl carbonyl, 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 chloromethoxy.

[0019] 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.

[0020] As used herein, an “and 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, fund, 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.

[0021] 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, oriodide group), a carboxylic acid (e.g, -C(O)OH), an alcohol (e.g, a hydroxyl, -OH), an ester (e.g., -C(O)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 (e.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)?.OH), a thial (e.g., -C(S)H), a sulfate (e.g., S(O) r ). a sulfonyl (e.g., -S(O)2-), an amide (e.g., -C(O)NR2, or -N(R)C(O)R), an azido (e.g, -Na), 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 -Nth), 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 C1-6 alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.

[0022] 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 an LNP, “about” may mean +!- 10% of the recited value. For instance, an LNP including a lipid component having about 40% of a given compound may include 30-50% of the compound,

[0023] 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.

[0024] 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.

[0025] As used herein, the term “contacting” means establishing a physical connection between two or more entities. In some embodiments, contacting a mammalian cell with an 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 an LNP and a mammalian cell disposed within 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 an LNP.

[0026] 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.

[0027] 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 an LNP including the therapeutic and / or prophylactic agent to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of an LNP to a mammal or mammalian cell may involve contacting one or more cells with the lipid nanoparticle,

[0028] 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 / orprophylactic 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 that, the 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).

[0029] 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).

[0030] As used herein, “encapsulation efficiency” refers to the amount of a therapeutic and / or prophylactic agent that becomes part of an LNP, relative to the initial total amount of therapeutic and / or prophylactic agent used in the preparation of an LNP. In some embodiments, if 97 mg of therapeutic and / or prophylactic agent are encapsulated in an 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%.

[0031] 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.

[0032] 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.

[0033] 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).

[0034] 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).

[0035] 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.

[0036] 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 cis! trans 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.

[0037] 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.

[0038] 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 atriphosphate group or by a chain including two phosphate moieties and a boranophosphate moiety.

[0039] As used herein, “methods of administration” may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. A method of administration may be selected to target delivery' (e.g., to specifically deliver) to a specific region or system of a body.

[0040] 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.

[0041] 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 an LNP including a lipid component and an RNA.

[0042] 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, an LNP may be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0043] As used herein, “naturally occurring” means existing in nature without artificial aid.

[0044] 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.

[0045] As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.

[0046] 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.

[0047] 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 withoutexcessive toxicity, irritation, allergic response, or other problems or complication, commensurate with a reasonable benefit / risk ratio.

[0048] The phrase “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than the compounds described herein (for example, a vehicle capable of suspending, 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.

[0049] 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, digluconate, 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.

[0050] 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.

[0051] As used herein, the “polydispersity' 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 narrowparticle size distribution.

[0052] 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.

[0053] 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.

[0054] 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 poly A 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.

[0055] 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.

[0056] As used herein, a “split dose” is the division of a single unit, dose or total daily dose into two or more doses.

[0057] 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.

[0058] 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.

[0059] As used herein, “Tx” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of an 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, “T8o%” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of an LNP, LNP solution, lyophilized LNP composition, or LNP 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, “Ti / ?” refers to the amount of time lasted for thenucleic acid integrity (e.g., mRNA integrity) of an 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] As used herein, the term “zeta potential” refers to the electrokinetic potential of a lipid, e.g., in a particle composition.

[0068] As used herein, the term “polydispersity”, “polydispersity index”, or “PDF’ refers to a measurement of the distribution of molecular mass in a given sample. The polydispersity 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).

[0069] The term “spread”, as used herein, refers to the width at half height of a peak (e.g., a mobility peak).

[0070] 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)).

[0071] 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 consisti ng 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 consi sting of A, B, and / or C, i.e., one or more As, one or more Bs, one or more Cs, or any combination thereof, unless indicated otherwise.

[0072] It is understood that, when more than two of a variable (e.g., more than two variable X) are present, the phrase “tw'o 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.

[0073] 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.

[0074] 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.

[0075] 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 publi cati ons 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 written description, 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 - Ionizable Lipids

[0076] 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).

[0077] In some aspects, the present disclosure provides a compound of Formula (I*):or a pharmaceutically acceptable salt thereof, wherein: one is a single bond, and the otheris a double bond; n is 3, 4, 5, or 6;M is -O-C(=O)-* and -C(=O)-O-*, wherein * indicates attachment to L;L is a bond or -CH2-;R1is H, C1-12 alkyl, or C2-12 alkenyl;R2is H, C1-12 alkyl, or C2-12 alkenyl;R- is C1-12 alkyl, or C2-12 alkenyl; wherein at least one of R1, R2, and R3is a C1-12 alkyl or C2-12 alkenyl; m is 3, 4, 5, or 6;each is a single bond or a double bond, wherein no more than one is a double bond,M’ is -O-C(=O)-* and -C(=O)-O-*, wherein * indicates attachment to L’;L’ is a bond or -CH2-,R1” is H, C1.12 alkyl, or C2-12 alkenyl;R2is H, C1-12 alkyl, or C2-12 alkenyl;RJis H, C1-12 alkyl, or C2-12 alkenyl; wherein at least one of R:', R2, and R3is a C1-12 alkyl or C2-12 alkenyl;RHis -(CH2)P-Q, ~(CH2)P-NRG-T-Q, -( C1-12),-XRG-S( O).’-T-Q. -(CH2)p-NRG-C(:==O)H or -(CH2)P-NRG-C(=O)-T-Q; p is 1, 2, 3, 4, or 5; each RGindependently is H, Ci-e alkyl, or C2-6 alkenyl;T is a bond, C1.3 alkylene, C2-3 alkenylene, or C2-3 alkynylene;Q is -OH, -O-(C 1-6 alkyl), C1-6 alkyl, C2-6 alkenyl, €2-6 alkynyl, C3-10 cycloalkyl, 5-10 membered heteroaryl, C6-io aryl, or 3-12 membered heterocycloalkyl, wherein the -O-(C 1-6 alkyl), C 1-6 alkyl, C2-6 alkenyl, C3-10 cycloalkyl, or 3-12 membered heterocycloalkyl is optionally substituted with one or more RQ; and each RQindependently is oxo, cyano, -OH, -O-(C 1-6 alkyl), -NH2, -NH(C 1-6 alkyl), - N(C 1-6 alkyl)2, -C(=O)-(C 1-6 alkyl), -O-C(=O)-(C 1-6 alkyl), -NR-C(=O)-(C 1-6 alkyl), C 1-6 alkyl, C2-6 alkenyl, -(C 1-6 alkyl)-OH, C2-6 alkenyl, or C3-10 cycloalkyl.Variables RH, RG, R' i. p, and Q

[0078] In some embodiments, RHis -(CH2)P-Q.

[0079] In some embodiments, RHis -(CH2)p-NRG-T-Q.

[0080] In some embodiments, RHis -(CH2)p-NRG-S(=O)2-T-Q.

[0081] In some embodiments, RHis -(CH2)P-NRG-C(-O)H.

[0082] In some embodiments, RHis -(CH2)P-NRG-C(=O)-T-Q.

[0083] In some embodiments, p is 1.

[0084] In some embodiments, p is 2.

[0085] In some embodiments, p is 3.

[0086] In some embodiments, p is 2 or 3.

[0087] In some embodiments, p is 2, 3, or 4.

[0088] In some embodiments, p is 4.

[0089] In some embodiments, p is 5.

[0090] In some embodiments, RGis H.

[0091] In some embodiments, R11is methyl

[0092] In some embodiments, RGis ethyl.

[0093] In some embodiments, RGis propyl.

[0094] In some embodiments, RGis butyl.

[0095] In some embodiments, RGis pentyl.

[0096] In some embodiments, IV' is hexyl.

[0097] In some embodiments, T is a bond.

[0098] In some embodiments, T is methylene.

[0099] In some embodiments, T is ethylene.

[0100] In some embodiments, T is propylene.

[0101] In some embodiments, Q is OH.

[0102] In some embodiments, Q is C3-6 cycloalkyl substituted with one or more oxo, -NH2, -NH(C 1-6 alkyl), or -N(C 1-6 alkyl)2.

[0103] In some embodiments, Q is cyclobutenyl substituted with one or more oxo, -NH2, - NH(C 1.6 alkyl), or -N(C 1-6 alkyl)2.

[0104] In some embodiments,

[0105] In some embodiments,

[0106] In some embodiments,

[0107] In some embodiments,

[0110] In some embodiments, Q is optionally substituted with one or more RS.

[0111] In some embodiments, Q is substituted with one or more RQ.

[0112] In some embodiment, Q is substituted with one RQ.

[0113] In some embodiment, Q is substituted with two RQ.

[0114] In some embodiment, Q is substituted with three RQ.

[0115] In some embodiments, RQis oxo.

[0116] In some embodiments, RQis -NH(C 1-6 alkyl).

[0117] In some embodiments, RQis -N(C 1-6 alkyl)?..Variables n, M, and L;

[0118] In some embodiments, n is 3.

[0119] In some embodiments, n is 4.

[0120] In some embodiments, n is 5.

[0121] In some embodiments, n is 6.

[0122] In some embodiments, M is -O-C(=O)-*, whereins kindicates attachment to L.

[0123] In some embodiments, M is -C(=O)-O-* wherein * indicates attachment to L.

[0124] In some embodiments, L is a bond.

[0125] In some embodiments, L is -CH2-.

[0126] In some embodiments, n is 5 and L is -CH2-.

[0127] In some embodiments, n is 5 and M is -C(:::O)-O~* wherein * indicates attachment to L.

[0128] In some embodiments, n is 3 and L is -CH?-,

[0129] In some embodiments, n is 3 and M is -C(=O)-O-* wherein * indicates attachment to L.

[0130] In some embodiments, L is -CH2- and M is -C(=O)-O-* wherein * indicates attachment to L.

[0131] In some embodiments, n is 5 and M is -O-C(=O)-* , wherein * indicates attachment to L.

[0132] In some embodiments, n is 5, L is a bond, and M is -O-C(“O)-* wherein * indicates attachment to L.

[0133] In some embodiments, n is 5, L is -CH2-, and M is -C(::::O)-O-* wherein * indicates attachment to L.

[0134] In some embodiments, n is 3, L is -CH?.-, and M is ~C(:::O)-O~* wherein * indicates attachment to L.Variables R’, R2, and R3

[0135] In some embodiments, R? is H.

[0136] In some embodiments, R1is C1-12 alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl).

[0137] In some embodiments, R1is methyl.

[0138] In some embodiments, R1is ethyl.

[0139] In some embodiments, R1is C2-12 alkenyl.

[0140] In some embodiments, R2is H.

[0141] In some embodiments, R2is C1-12 alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl).

[0142] In some embodiments, R2is C2-12 alkyl (e.g., ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl).

[0143] In some embodiments, R2is propyl.

[0144] In some embodiments, R2is butyl.

[0145] In some embodiments, R2is pentyl,

[0146] In some embodiments, R2is C2-12 alkenyl.

[0147] In some embodiments, R2is propenyl.

[0148] In some embodiments, R2is pentenyl.

[0149] In some embodiments, Rzis branched pentenyl.

[0150] In some embodiments, R3is H.

[0151] In some embodiments, R3is C1-12 alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl).

[0152] In some embodiments, R3is butyl.

[0153] In some embodiments, R3is pentyl.

[0154] In some embodiments, R3is hexyl.

[0155] In some embodiments, R3is heptyl.

[0156] In some embodiments, R3is octyl.

[0157] In some embodiments, R3is nonyl.

[0158] In some embodiments, R3is C2-12 alkenyl.

[0159] In some embodiments, R3is hexenyl.

[0160] In some embodiments, R3is branched hexenyl.

[0161] In some embodiments, R1is H, R2is C1-12 alkyl, and RJis C1-12 alkyl.

[0162] In some embodiments, R1is H, R2is C1.12 alkyl, and R3is C2-12 alkenyl.

[0163] In some embodiments, R1is H and R2is propyl .

[0164] In some embodiments, R1is H and R2is butyl.

[0165] In some embodiments, R:is H andR3is butyl.

[0166] In some embodiments, R1is H and R3is pentyl.

[0167] In some embodiments, R2is propyl and R3is butyl.

[0168] In some embodiments, R1is H, R2is propyl, and RJis butyl.

[0169] In some embodiments, R1 is methyl, R2is propyl, and R3is butyl.

[0170] In some embodiments, R1is ethyl, R2is propyl, and R3is butyl.

[0171] In some embodiments, R2is butyl and R3is pentyl.

[0172] In some embodiments, R1is H, R2is pentyl and R3is hexyl.

[0173] In some embodiments, R2is pentyl and R3is hexyl.

[0174] In some embodiments, R:is H, R2is hexyl, and R1is heptyl.

[0175] In some embodiments, R2is hexyl and R3is heptyl.

[0176] In some embodiments, R1is H, R2is heptyl, and R3is octyl.

[0177] In some embodiments, R3is heptyl and R3is octyl.

[0178] In some embodiments, R1is H, R2is propyl, and R3is butyl,

[0179] In some embodiments, R1is H, R2is butyl, and R3is pentyl.Variables R ", R2, and ii

[0180] In some embodiments, R1’ is H.

[0181] In some embodiments, R1is CM2 alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl).

[0182] In some embodiments, Rris methyl.

[0183] In some embodiments, R1’ is ethyl.

[0184] In some embodiments, R1is C2-12 alkenyl.

[0185] In some embodiments, R2is H.

[0186] In some embodiments, R2is C1-12 alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl).

[0187] In some embodiments, R2is C2-12 alkyl (e.g., ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl).

[0188] In some embodiments, R2’ is propyl.

[0189] In some embodiments, R2' is butyl.

[0190] In some embodiments, Rz’ is pentyl.

[0191] In some embodiments, R2is C2-12 alkenyl.

[0192] In some embodiments, R2’ is propenyl.

[0193] In some embodiments, R2” is pentenyl.

[0194] In some embodiments, R2’ is branched pentenyl.

[0195] In some embodiments, R3is H.

[0196] In some embodiments, Ryis C1-12 alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl).

[0197] In some embodiments, R3’ is butyl.

[0198] In some embodiments, R3’ is pentyl.

[0199] In some embodiments, R3is hexyl.

[0200] In some embodiments, R3” is heptyl.

[0201] In some embodiments, R3” is octyl.

[0202] In some embodiments, R3” is nonyl.

[0203] In some embodiments, R3” is C2-12 alkenyl.

[0204] In some embodiments, R3” is hexenyl.

[0205] In some embodiments, R3”is branched hexenyl.

[0206] In some embodiments, R1” is Cm alkyl and R2” is Cun alkyl.

[0207] In some embodiments, R1” is C1-12 alkyl and RJ” is H.

[0208] In some embodiments, R’!” is H and R2” is propyl.

[0209] In some embodiments, R:” is H and R3” is butyl.

[0210] In some embodiments, Rz” is propyl and R3” is butyl.

[0211] In some embodiments, Rx” is octyl and R2” is hexyl.

[0212] In some embodiments, R1” is H, R2” is propyl, and R3” is butyl,

[0213] In some embodiments, R1” is octyl, R2” is hexyl, and R3” is H.Variables m, L and A / '

[0214] In some embodiments, m is 3.

[0215] In some embodiments, m is 4.

[0216] In some embodiments, m is 5.

[0217] In some embodiments, m is 6.

[0218] In some embodiments, M’ is -O-C(=O)-*, wherein * indicates attachment to L’.

[0219] In some embodiments, M’ is -C(::::O)-O-*, wherein * indicates attachment to L’.

[0220] In some embodiments, L’ is a bond.

[0221] In some embodiments, L’ is -CH?-.

[0222] In some embodiments, n is 5 and M’ is -C(=O)-O-*, wherein * indicates attachment to L’.

[0223] In some embodiments, n is 5, M’ is -C(:=:O)-O-*, wherein * indicates attachment to L’, and L’ is -CH2-.

[0224] In some embodiments, n is 5, M’ is -C(=O)-O-*, wherein * indicates attachment to L’, and L’ is a bond.

[0225] In some embodiments, m is 5 and M’ is -C(:::O)-O-*, wherein * indicates attachment to L’.

[0226] In some embodiments, m is 5, M’ is -C(:::O)-O-*, wherein * indicates attachment to L’, and L’ is -CH2-.

[0227] In some embodiments, m is 5, M’ is -C(=O)-O-*, wherein * indicates attachment to L’, and L’ is a bond.Exemplary Embodiments of the Compounds

[0228] In some embodiments, the compound is of Formula (I**)or a pharmaceutically acceptable salt thereof, wherein: one is a single bond, and the otheris a double bond; n is 3, 4, 5, or 6;M is -O-C(=O)-* or -C(=O)-O-*, wherein * indicates attachment to L;L is -CH2-;R7' is C 3-5 alkyl, R3is C3-5 alkyl; m is 3, 4, 5, or 6; each is a single bond or a double bond, wherein no more than one is a double bond;M’ is -C(=O)-O-*, wherein * indicates attachment to LL’ is a bond;R1is C7-10 alkyl;R2is C5-8 alkyl;p is I, 2, 3, 4, or 5; andQ is -OH.

[0229] In some embodiments, the compound is of Formula (I) (II), (III), (IV), (V), or (VI):or a pharmaceutically acceptable salt thereof.

[0230] In some embodiments, the compound is of Formula (1-2), (II-2), (II-2), (III-2), (IV-2),(V-2), or (VI-2):(III-2)(VI-2) or a pharmaceutically acceptable salt thereof.^0231^ In some embodiments, the compound is of Formula (1-3), (II-3), (III-3), (IV-3), (V-3), or (VI-3):(V-3)or a pharmaceutically acceptable salt thereof.^0232^ In some embodiments, the compound is of Formula (1-4), (II-4), (III-4), (IV-4), (V-4), or (VI-4):(IV -4)(VI-4) or a pharmaceutically acceptable salt thereof.

[0233] In some embodiments, the compound is of Formula (1-5) or (11-5):(III-5)(VI-5) or a pharmaceutically acceptable salt thereof.

[0234] In some embodiments, the compound is of Formula (1-6) or (II-6):or a pharmaceutically acceptable salt thereof.

[0235] In some embodiments, RHis -(CH2)p-Q, or -(CH2)P-NRG-T-Q;

[0236] In some embodiments, IV’ is H.

[0237] In some embodiments, T is a bond.

[0238] In some embodiments p is 2 or 3.

[0239] In some embodiments, Q is hydroxyl or C3-10 carbocycle optionally substituted with one or more RQ.

[0240] In some embodiments, each RQindependently is oxo, cyano, -OH, -O-(C 1-6 alkyl), - NH2, -NH(C 1-6 alkyl), -N(C 1-6 alky 1 )2, -C(-O)-(C 1-6 alkyl), -O C C (})((('■.,.. alkyl), -NR-C(=O)-C 1-6 alkyl), Ci.6 alkyl, -C 1-6 alkyl)-OH, C2-6 alkenyl, or C3-10 cycloalkyl.

[0241] In some embodiments, each R is independently selected from H, C1-6 alkyl, and C2-6 alkenyl

[0242] In some embodiments, each RQindependently is oxo, -NH2, -NH(C 1-6 alkyd), or -N(C1.6 a I ky I )2.

[0243] In some embodiments, n is 3, 4, 5, or 6.

[0244] In some embodiments, m is 5.

[0245] In some embodiments, R1is Ci.6 alkyl.

[0246] In some embodiments, Rzis C1-6 alkyl or C1-6 alkenyl.

[0247] In some embodiments, R3is C1-6 alkyd or C 1-6 alkenyl.

[0248] In some embodiments, R1is H.

[0249] In some embodiments, R2” is C1-12 alkyl.

[0250] In some embodiments, R3’ is C1-12 alkyl.

[0251] In some embodiments, the compounds is selected from the compounds described in Table 1 and salts thereof.

[0252] In some embodiments, the compounds is selected from the compounds described in Table 1.Nanopartide compositions

[0253] In some aspects, the present disclosure provides a lipid nanoparticle) comprising an ionizable lipid described herein, e.g., according to Formula (I).

[0254] In some embodiments, the lipid nanoparticle (LNP) further comprises a second ionizable lipid.

[0255] In some embodiments, the lipid nanoparticle (LNP) further comprises a cationic lipid, a phospholipid, and a structural lipid.

[0256] In some embodiments, the lipid nanoparticle (LNP) further comprises a second ionizable lipid, a phospholipid, a structural lipid, and a PEG lipid.

[0257] In some embodiments, the lipid nanoparticle (LNP) further comprises a second ionizable lipid, a phospholipid, a structural lipid, a PEG lipid, and a cationic lipid.

[0258] In some embodiments, the lipid nanoparticle (LNP) further comprises a phospholipid, a structural lipid, and a PEG lipid.

[0259] In some embodiments, the nucleic acid is an RNA.

[0260] In some embodiments, the nucleic acid is an mRNA.

[0261] In some embodiments, the largest dimension of a nanoparticle composition is 1 um or shorter (e.g, 1 gm, 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, 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.

[0262] lipid nanoparticle of the present disclosure comprises at least one ionizable lipid according to Formula (I). In some embodiments, an LNP of the disclosure includes one or more of ionizable lipids of Table 1 , Nanoparticle compositions may also include a variety of other components. For example, in some embodiments, the LNP includes one or more ionizable lipids in addition to a lipid according to Formula (I).

[0263] 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 toabout 7:1. In some embodiments, a molar ratio of the cationic lipid to the therapeutic and / or prophylactic agent is about 1.5: 1 to about 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.

[0264] In some embodiments, the 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.Nanopartide physical properties

[0265] The characteristics of a lipid nanoparticle may depend on the components thereof. For example, a lipid nanoparticle including cholesterol as a structural lipid may have different characteristics than a lipid nanoparticle that includes a different structural lipid. Similarly, the characteristics of a lipid nanoparticle may depend on the absolute or relative amounts of its components. For instance, a lipid nanoparticle including a higher molar fraction of a phospholipid may have different characteristics than a lipid nanoparticle including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0266] Lipid nanoparticles 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, potentiometric titrations) 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 i s 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 LNP compositions lipid is measuredusing an instrument employing the principle of MP-PALS. Zeta potential can be measured on a Malvern Zetasizer (Nano ZS).

[0267] In some embodiments, the mean diameter of a lipid nanoparticle of the disclosure 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 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 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.

[0268] In some embodiments, the poly dispersity' index (“PDI”) of a plurality of lipid nanoparticles formulated with lipids of the disclosure is less than 0.3. In some embodiments, plurality of lipid nanoparticles 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 formulated with lipids of the disclosure has a poly dispersity index of from about 0.10 to about 0.20.

[0269] 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 thedisclosure 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.

[0270] The zeta potential of a lipid nanoparticle 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 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 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.

[0271] 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 after preparation, relative to the 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 an LNP before and after breaking up the 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 LNPs formulated with lipids of the disclosure, 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 efficiencyis at least 90%. In some embodiments, the encapsulation efficiency of the therapeutic and / or prophylactic agent is between 80% and 100%.Cationic Lipids

[0272] In some embodiments, the lipid nanoparticles disclosed herein further comprise a cationic lipid. Exemplar}' and non-limiting cationic lipids that may be used in the nanoparticles of the present disclosure are disclosed in U.S. Provisional Patent Application No. 63 / 508, 196, filed June 14, 2023, the cationic lipids of which are hereby incorporated by reference.Second Ionizable Lipid

[0273] In some embodiments, the lipid nanoparticles disclosed herein further comprise a second ionizable lipid, i.e., a second ionizable lipid in addition to the ionizable lipid of Formula (I). Exemplary' and non-limiting second ionizable lipids that may be used in the nanoparticles of the present disclosure are disclosed in International Patent Application Nos. PCT / US2016 / 052352, fded September 16, 2016; PCT / US2018 / 022717, filed March 15, 2018; PCT / US2018 / 037541, filed June 14, 2018, PCT / US2019 / 052009, filed September 19, 2019, PCT / US2020 / 051613, filed September 18, 2020; PCT7US2020 / 051609, filed September 18, 2020, PCT / US2020 / 051629, filed September 18, 2020, and PCT / US2022 / 021560, filed March 23, 2022.Polyethylene Glycol (PEG) Lipids

[0274] 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-diacyl oxy propan-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.

[0275] In some embodiments, the PEG lipid includes, but are not limited to, 1,2-dimyristoyl- sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-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).

[0276] 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 di acylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[0277] In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about CM to about C22, In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about CM to about CM. In some embodiments, a PEG moiety, for example an mPEG-NHb, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In one embodiment, the PEG lipid is PEGzk-DMG.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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 di alkylamines, 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.

[0282] In some embodiments, the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure:

[0283] 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.

[0284] In some embodiments, a PEG lipid useful in the present invention is a compound of Formula (PL- 1). Provided herein are compounds of Formula (PL-1):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 CMO alkylene, wherein at least one methylene of the optionally substituted Cnio 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, -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;each instance of L2is independently a bond or optionally substituted Ci-e alkylene, wherein one methylene unit of the optionally substituted C 1-6 alkylene is optionally replacedeach instance of R2is independently optionally substituted Cj.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), ('(() ;■(), (}( (() ;■, ()( (())(), OC’(O)N(RN), NRNC(O)O, C(O)S, SC(O). - C( \Rh 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.

[0285] In some embodiments, the compound of Formula (PL-I) is a PEG-OH lipid (i.e., RJis -OR°, and R° is hydrogen). In some embodiments, the compound of Formula (PL-I) is of Formula (PL-I -OH):or a salt thereof.

[0286] 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 C 10-40 alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted Cw-40 alkynyl; and optionally one or more methylene groups of R’ are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene,optionally substituted arylene, optionally substituted heteroaryiene, 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( \R' ). 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.

[0287] In some embodiments, the compound of Formula (PL-II) is of Formula (PL-II-OH):OHO J v (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 Cw-40 alkynyl; and optionally one or more methylene groups of R’ are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroaryiene, 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( \R'" ). 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.

[0288] In some embodiments, r is an integer between 10 to 80, between 20 to 70, between 30 to 60, or between 40 to 50.

[0289] In some embodiments, r is 45.

[0290] In some embodiments, R3is C17 alkyl.

[0291] In yet other embodiments the compound of Formula (PL-II) is:or a salt thereof.

[0292] In one embodiment, the compound of Formula (PL-II) is

[0293] In some aspects, the lipid composition of the pharmaceutical compositions described herein does not comprise a PEG lipid.

[0294] In some embodiments, the PEG lipids may be one or more of the PEG lipids described in U.S. Application No. 62 / 520,530.

[0295] 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.

[0296] In some embodiments, the PEG lipid is a compound of the following formula:or a salt or isomer thereof.Structural Lipids

[0297] As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moi eties.

[0298] 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 someembodiments, 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.

[0299] In some embodiments, the structural lipids may be one or more structural lipids described in P.C.T. Application No. PCT / US 18 / 37922.

[0300] 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.

[0301] In some embodiments, the structural lipid isor a salt thereof.

[0302] In some embodiments, the structural lipid is SL-1.

[0303] In some embodiments, the structural lipid isor a salt thereof.

[0304] 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 %.

[0305] 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 moi %, about 36.6±8 moi %, 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

[0306] 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-G5 alkyl, C2-C6 alkenyl, and (C= =NH)N(Rioi h wherein each Rioi is independently selected from the group consisting of H, Ci-C6 alkyl, and C2-C6 alkenyl;R203 is selected from the group consisting of C1-C20 alkyl and C2-C20 alkenyl;R204 is selected from the group consisting of FI, C1-C20 alkyl, C2-C20 alkenyl, C(O)(OCi- C20 alkyl), C(O)(OC2-C20 alkenyl), C(0)(NHCi-C?o alkyl), and C(O)(NHC2-C20 alkenyl); nl is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0307] In some embodiments, R?OJ and R202 are each independently selected from the group consisting of H and CH3.

[0308] In some embodiments, R201 and R202 are each independently selected from the group consisting of (C=NH)NH2 and (C=NH)N(CH3)2

[0309] In some embodiments, R203 is selected from the group consisting of C1-C20 alkyl, Cs- Cig alkyl, and C12-C16 alkyl.

[0310] In some embodiments, R204 is selected from the group consisting of H, C1-C20 alkyl, C2-C20 alkenyl, C(O)(OCi-C20 alkyl), CXOXOC2-C20 alkenyl), C(O)(NHCi-C20 alkyd), and C(0)(NHC2-C2O alkenyl), Cs-Ci8 alkyl, Cs-Cis alkenyl, C(O)(OC8-Ci8alkyl), C(O)(OC8-C58alkenyl), C(O)(NHC8-CI8alkyl), and C(O)(NHC8-CI8alkenyl), and C12-C16 alkyl, C12-C16 alkenyl, C(O)(OCi2-Ci6 alkyl), C(O)(OCi2-Ci6 alkenyl), C(O)(NHCi2-Ci6 alkyl), and C(O)(NHCj 2-C16 alkenyl);

[0311] 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.

[0312] In some embodiments, nl is 3.

[0313] In some embodiments of the present disclosure, the encapsulation agent is a compound of Formula (EA-II):or salts or isomers thereof, whereinX loi is a bond, NH, or O;R101 and R102 are each independently selected from the group consisting of H, C1-C6 alkyl, and C2-C6 alkenyl;R103 and RIG4 are each independently selected from the group consisting of C1-C20 alkyl and C2-C20 alkenyl; and nl is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0314] In some embodiments, X101 is a bond.

[0315] In some embodiments, X101 is NH.

[0316] In some embodiments, X101 is O.

[0317] In some embodiments, Run and Rw2 are each independently selected from the group consisting of H and CH?,.

[0318] In some embodiments, R103 is selected from the group consisting of C1-C20 alkyl, C8- C18 alkyl, and C12-C16 alkyl.

[0319] In some embodiments, R104 is selected from the group consisting of C1-C20 alkyl, C8-C18 alkyl, and C12-C16 alkyl.

[0320] 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.

[0321] In some embodiments, nl is 3.

[0322] Exemplar}' 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.

[0323] In certain embodiments, the encapsulation agent is ethyl lauroyl arginate,salt or isomer thereof.

[0324] 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

[0325] Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0326] 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.

[0327] 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.

[0328] 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., deliver}' of the one or more elements to a target tissue.

[0329] 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).

[0330] 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.

[0331] 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 i, 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 Cue alkylene, wherein one methylene unit of the optionally substituted Ci-e alkylene is optionally replacedeach instance of R2is independently optionally substituted Ciuo 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)-,-SC(O)-, ••(■( XR \)-. ■(•( =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)X(R' >. -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(())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 I or 2.

[0332] In some embodiments, the phospholipids is not of the formula:wherein each instance of R2is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0333] In some embodiments, the phospholipids may be one or more of the phospholipids described in U.S. Application No. 62 / 520,530.

[0334] In some embodiments, the phospholipids may be selected from the non-limiting group consisting of l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanol amine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine ( DM PC ), l,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), l,2-di-O-octadecenyl-57?-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, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3 -phosphoethanol amine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanol amine, l,2-dilinolenoyl-sn-glycero~3-phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3 -phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt(DOPG), and sphingomyelin. In some embodiments, an LNP includes DSPC. In some embodiments, an LNP includes DOPE. In some embodiments, an LNP includes both DSPC and DOPE. i) Phospholipid Head ModifK:alions

[0335] 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.

[0336] 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 moiety7. In some embodiments, the compound of Formula (PhL-I) is of Formula (PhL-I-b):or a salt thereof. ii) Phospholipid Tail Modifications

[0337] 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)-, -XRX'CO.H -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 )X(RX)-.-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(RX)-, -OS(O)2N(RN)-, or -N(RN)S( O )2()-.

[0338] 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~, -(•(O)S-. -SC(O)-, -C( =NRN)-, -( ■( =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.

[0339] 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 phosphor) / ! 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 (Ph.L-1), 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

[0340] 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

[0341] A lipid nanoparticle may include one or more components in addition to those described in the preceding sections. For example, a lipid nanoparticle may include one or more small hydrophobic molecules such as a vitamin (c.g., vitamin A or vitamin E) or a sterol.

[0342] Lipid nanoparticles 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).

[0343] 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, poly acrylates, 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 polyethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), poly siloxanes, 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), polyfethyl(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 andits copolymers, polyhydroxy alkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, polyoxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly( / V-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[0344] 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 poloxamer), mucolytic agents (e.g, acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, doniiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin [34, 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, by coating, adsorption, covalent linkage, or other process).

[0345] lipid nanoparticle 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 may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane penneation are well known in the art.

[0346] In addition to these components, lipid nanoparticles may include any substance useful in pharmaceutical compositions. For example, the lipid nanoparticle 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, disintegrates, 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.

[0347] 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, corn starch, tapioca starch, sodium starch glycolate,clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microciystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0348] 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 distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [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 [BRU® 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.

[0349] 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.

[0350] 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 hydroxy anisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenedi aminetetraacetic 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 (SEES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™, KATHON™, and / or EUXYL®.

[0351] 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., Hl .Ph'S), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. Lubricating agents may be 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.

[0352] 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, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury', sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils 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.Adjuvants

[0353] In some embodiments, an 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), polyfEC ), aluminum hydroxide, and Pam3CSK4.Therapeutic Agents

[0354] Lipid nanoparticles may include one or more therapeutic and / or prophylactics agents.The disclosure features methods of delivering a therapeutic and / or prophylactic agent to amammalian 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 including a therapeutic and / or prophylactic agent.

[0355] 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.

[0356] 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.

[0357] 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

[0358] 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.

[0359] 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.

[0360] 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 some embodiments, the DNA molecule is a recombinant molecule. Non-limiting exemplar}' DNA therapeutic agents include plasmid expression vectors and viral expression vectors.

[0361] 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 regulator}' 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.

[0362] In some embodiments, in the 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).

[0363] 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.

[0364] In some embodiments, in the 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

[0365] For example, in some embodiments, when the therapeutic and / or prophylactic agents is an 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.

[0366] 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), a small hairpin RNA (shRNA), a messenger RNA (mRNA), locked nucleic acids (LNAs) and CRISPR / Cas9 technology, and mixtures thereof.

[0367] For example, in some embodiments, when the therapeutic and / or prophylactic agents is an 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.

[0368] 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).

[0369] 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 poly A sequence, a poly adenylation signal, and / or a 5’ cap structure.

[0370] 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.

[0371] 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 anysuitable 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.

[0372] 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 poly A sequence, and / or a polyadenylation signal.

[0373] A 5' cap structure or cap species is a compound including two nucleoside moieties 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, m 27,03 'GppppG, m27,O2'GppppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27, 03 'GppppG, and m27,O2'GppppG.

[0374] 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' deoxy adenosine (cordycepin), 3’ deoxyuridine, 3' deoxy cytosine, 3’ deoxyguanosine, 3' deoxythymine, and 2', 3' dideoxynucleosides, such as 2', 3' dideoxyadenosine, 2', 3’ dideoxyuridine, 2', 3' dideoxycytosine, 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.

[0375] 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 apoly 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 .

[0376] 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 deoxy thy mi dine, 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 incorporated herein by reference. A polyA sequence may be a tail located adjacent to a 3' untranslated region of an mRNA. In some embodiments, a polyA sequence may affect the nuclear export, translation, and / or stability of an mRNA.

[0377] ,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 of mRNAs, ie., 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 incorporated herein by reference.

[0378] 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 2 A peptide. IRES sequences and 2 A 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.

[0379] 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 efficiencyof protein production, intracellular retention of nucleic acids, as well as possess reduced immunogenicity.

[0380] 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.

[0381] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (v), 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 -carboxymethyl - pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl~2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl -uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio- uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl -pseudouridine, 5-taurinomethyl-uridine (rm5U), 1 -taurinomethyl-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 (ml q / ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl- 4-thio-pseudouridine (m 1 s4w), 4-thio- 1 -methyl -pseudouridine, 3 -methyl -pseudouridine (m.3q / ), 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-thio- 1 -methyl- 1 - deaza-pseudouridine, di hydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5- methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- meth oxy -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 (acp35-(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 (cmnm5Um), 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.

[0382] 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- 1 -methyl- 1 -deaza- pseudoisocytidine, I -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- 1 -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-di methylcytidine (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.

[0383] 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- di aminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyl -adenosine (mlA), 2-methyl- adenine (m2A), N6-m ethyl -adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl -adenosine (i6A), 2-methylthio-N6-isopentenyl -adenosine (nis2i6A), 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-hydroxynorvalyl carbamoyladenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6- acetyl -adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-m ethoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), l,2'~O-dimethyl-adenosine (mlAm), 2'-0- 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- pentaoxanonadecy l)-aden osi ne .

[0384] 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), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQO), 7-aminomethyl-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- m ethoxy -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, 1 -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-m ethyl -guanosine (m22Gm), 1 -methyl-2’-O-methyl-guanosine (ml Gm), N2,7-dimethyl- 2'-O-methyl-guanosine (m2,7Gm), 2 '-O-m ethyl -inosine (Im), l,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.

[0385] 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.)

[0386] In some embodiments, the modified nucleobase is pseudouridine (iji), NI- methylpseudouridine (mh[ / ), 2-thiouridine, 4 ’-thiouridine, 5-methylcytosine, 2 -thio- 1 -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- 1 -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 theaforementioned modified nucleobases.) In some embodiments, the modified nucl eobase i s N1 - methylpseudouridineand the mRNA of the disclosure is fully modified with Nl- methylpseudouridine (mlrg). In some embodiments, N1 -methylpseudouridine (mlw) represents from 75-100% of the uracils in the mRNA. In some embodiments, Nl- methylpseudouridine (m lw) represents 100% of the uracils in the mRN A.

[0387] 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 the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0388] In some embodiments, the modified nucleobase is a modified adenine. Exemplary' nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl- adenosine (mlA), 2-methyl -adenine (m2A), 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.)

[0389] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m i l), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza- guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQi), 7-methyl-guanosine (m7G), I -methyl -guanosine (mIG), 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.)

[0390] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (rn by). 5- methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ig), 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.)

[0391] In some embodiments, the mRNA comprises pseudouridine (y). In some embodiments, the mRNA comprises pseudouridine (t| / ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (mlw). In some embodiments, the mRNAcomprises I -methyl-pseudouridine 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 N6- methyl-adenosine (m6A). In some embodiments, the mRNA comprises N6-m ethyl -adenosine (m6A) and 5-methyl-cytidine (m5C).103921 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 (mh|i) or 5- methyl-cytidine (m5C), meaning that all uridines or all cytosine nucleosides in the mRNA sequence are replaced with Nl-methylpseudouridine (mlw) 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.

[0393] 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.

[0394] 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.

[0395] 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 isused 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.

[0396] 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 or shuffle 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.

[0397] 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.

[0398] 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.

[0399] Non-natural modified nucleobases may be introduced into polynucleotides, e.g., mRNA, during synthesis or post-synthesis. In certain embodiments, modifications may be on internucleoside 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.

[0400] Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or their regions with different functional moi eties, such as targeting or deliveryagents, fluorescent labels, liquids, nanoparticles, etc. Therapeutic Agents for Reducing Protein Expression

[0401] 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.Peptide / Polypeptide Therapeutic Agents

[0402] In some embodiments, the therapeutic agent is a peptide therapeutic agent. In some embodiments the therapeutic agent is a polypeptide therapeutic agent.

[0403] 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).

[0404] In some embodiments, in the LNP of the disclosure, the one or more therapeutic and / or prophylactic agents is a polynucleotide or a polypeptide.Genome Editing Techniques

[0405] In some embodiments, the nucleic acid is suitable for a genome editing technique.

[0406] In some embodiments, the genome editing technique is clustered regularly interspaced short palindromic repeats (CRISPR) or transcription activator-like effector nuclease (TALEN).

[0407] 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.V accines

[0408] 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.

[0409] 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 sy stem in a more native fashion.

[0410] 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,

[0411] In some embodiments the vaccine is a personalized vaccine and wherein the cancer antigen is a subject specific antigen.

[0412] 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,

[0413] In some embodiments, the vaccines further comprise an adjuvant.

[0414] 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.

[0415] 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- thiopseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l- methylpseudouridine, 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 vaccin e formul ati on .

[0416] In some embodiments, a lipid nanoparticle comprises a cationic lipid, an ionizable lipid, a PEG-modified lipid, a sterol and a phospholipid lipid.

[0417] In some embodiments the lipid nanoparticle formulation includes an immune potentiator (e.g., TLR agonist) to enhance immunogenicity of the vaccine (formulation).

[0418] 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.

[0419] 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 CUT A, 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.

[0420] 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.

[0421] 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.

[0422] 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.

[0423] 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] 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.

[0428] In some embodiments the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to the RNA vaccine.

[0429] In other embodiments the immune response is assessed by determining antibody titer in the subject.

[0430] 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 prophy lactical I y effective dose of a traditional vaccine against the cancer antigen. In some embodiments the immune response in the subject is inducedin a subject vaccinated with a prophyiacticaliy effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to the RNA vaccine.

[0431] 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 prophyiacticaliy effective dose of a traditional vaccine.

[0432] 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 prophyiacticaliy 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 prophyiacticaliy effective dose of a traditional vaccine.

[0433] 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 prophyiacticaliy effective dose of a traditional vaccine.

[0434] 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 prophyiacticaliy effective dose of a traditional vaccine.

[0435] 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 prophyiacticaliy effective dose of a traditional vaccine.

[0436] 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 prophyiacticaliy effective dose of a traditional vaccine.

[0437] In some embodiments the nucleic acid vaccines described herein are chemically modified. In other embodiments the nucleic acid vaccines are unmodified.

[0438] 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.

[0439] 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, 10-15ug, 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.

[0440] 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.

[0441] 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.

[0442] 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 exemplar}' 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 titer is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)

[0443] 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 exemplar} / 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 exemplar}' 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, hi some embodiments, the adjuvant is selected from a cationic peptide and an immunostimulator}' nucleic acid. In some embodiments, the cationic peptide is protamine.

[0444] .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.

[0445] 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.

[0446] 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.

[0447] 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 .

[0448] .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.

[0449] 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.

[0450] 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

[0451] concatemeric polypeptide, wherein the vaccine has at least 10 fold less RNA polynucleotide than is required for an unmodified mRNA vaccine not formulated in an LNP to produce an equivalent antibody titer. In some embodiments, the RN A polynucleotide is present in a dosage of 25-100 micrograms.

[0452] 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.

[0453] 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.

[0454] 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.

[0455] 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 someembodiments, 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 RNA polynucleotide 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.

[0456] 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.

[0457] 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 niRN A 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.LNP Solutions, LNP Compositions, and LNP Formulations

[0458] In some embodiments, the present disclosure provides a lipid nanoparticle solution (LNP solution) comprising an ionizable lipid described herein, e.g., according to Formula (I).

[0459] In some embodiments, the present disclosure provides a lipid nanoparticle composition (LNP composition) comprising an ionizable lipid described herein, e.g., according to Formula (I).

[0460] In some embodiments, the present disclosure provides a lipid nanoparticle formulation (LNP formulation) comprising an ionizable lipid described herein, e.g., according to Formula (I).

[0461] In some embodiments, the LNP Solution, LNP composition, or LNP formulation further comprises a cationic lipid.

[0462] In some embodiments, the LNP Solution, LNP composition, or LNP formulation further comprises a second ionizable lipid.

[0463] In some embodiments, the LNP Solution, LNP composition, or LNP formulation further comprises a cationic lipid, a phospholipid, and a structural lipid.

[0464] In some embodiments, the LNP Solution, LNP composition, or LNP formulation further comprises a cationic lipid, a second ionizable lipid, a phospholipid, and a structural lipid.

[0465] In some embodiments, the LNP Solution, LNP composition, or LNP formulation further comprises a cationic lipid, a phospholipid, a structural lipid, and a PEG lipid.

[0466] In some embodiments, the LNP Solution, LNP composition, or LNP formulation further comprises a cationic lipid, a second ionizable lipid, a phospholipid, a structural lipid, and a PEG lipid.

[0467] In some embodiments, the LNP Solution, LNP composition, or LNP formulation further comprises a phospholipid, and a structural lipid.

[0468] In some embodiments, the LNP Solution, LNP composition, or LNP formulation further comprises a phospholipid, a structural lipid, and a PEG lipid.

[0469] In some embodiments, the LNP Solution, LNP composition, or LNP formulation is free of therapeutic agent, e.g., free of nucleic acid.

[0470] In some embodiments, the LNP Solution, LNP composition, or LNP formulation further comprises a therapeutic agent, e.g., a nucleic acid.

[0471] In some embodiments, the nucleic acid is an RN A.

[0472] In some embodiments, the nucleic acid is an mRNA.Pharmaceutical compositions

[0473] Formulations comprising lipid nanoparticles may be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may include one or more lipid nanoparticles. In some embodiments, a pharmaceutical composition may include one or more lipid nanoparticles including one or more different therapeutics and / or prophylactics. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington’s The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and accessory' ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory / ingredient may be incompatible with one or more components of an LNP in the formulation of the disclosure. An excipient or accessory ingredient may be incompatible with a component of an LNP of the formulation if its combination with the component or LNP may result in any undesirable biological effect or otherwise deleterious effect.

[0474] In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including an LNP, In some embodiments, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary' use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0475] Relative amounts of the one or more lipid nanoparticles, the one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary', depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition comprises between 0.1% and 100% (wt / wt) of one or more lipid nanoparticles. As anotherexample, a pharmaceutical composition comprises between 0.1% and 15% (wt / vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% w / v).

[0476] In some embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and / or shipment (e.g., being stored at a temperature of 4 °C or lower, such as a temperature between about -150 °C and about 0 °C or between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C). For example, the pharmaceutical composition comprising one or more lipid nanoparticles is a solution or solid (e.g., via lyophilization) that is refrigerated for storage and / or shipment at, for example, about -20 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, or -80 °C. In certain embodiments, the disclosure also relates to a method of increasing stability of the lipid nanoparticles and by storing the lipid nanoparticles and / or pharmaceutical compositions thereof at a temperature of 4 °C or lower, such as a temperature between about -150 °C and about 0 °C or between about -80 °C and about -20 °C, e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C).

[0477] Lipid nanoparticles and / or pharmaceutical compositions including one or more lipid nanoparticles may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery of a therapeutic and / or prophylactic agent to one or more particular cells, tissues, organs, or systems or groups thereof, such as the renal system. Although the descriptions provided herein of lipid nanoparticles and pharmaceutical compositions including lipid nanoparticles are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other mammal. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary', if any, experimentation. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and / or rats.

[0478] A pharmaceutical composition including one or more lipid nanoparticles may be prepared by any method known or hereafter developed in the art. of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with anexcipient and / or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0479] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g, lipid nanoparticle). The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0480] Pharmaceutical compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration. In some embodiments, pharmaceutical compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0481] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include additional therapeutics and / or prophylactics, additional agents such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0482] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluentsand / or solvents, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0483] Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.(G4841 In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactidepolyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of daig release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[0485] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

[0486] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. In such solid dosage forms, an active ingredient is mixed with at least, one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or fillers or extenders (e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia), humectants (e.g, glycerol), disintegrating agents (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retardingagents (e.g, paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite clay, silicates), and lubricants (e.g:, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

[0487] Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only. In some embodiments, the solid compositions may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0488] Dosage forms for topical and / or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and / or any needed preservatives and / or buffers as may be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and / or dispensing the compound in the proper medium. Alternatively or additionally, rate may be controlled by either providing a rate controlling membrane and / or by dispersing the compound in a polymer matrix and / or gel.

[0489] Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short, needle devices such as those described in U.S. Patents 4,886,499; 5,190,521 ; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99 / 34850 and functional equivalents thereof. Jet injection devices which deliver liquid compositions to the dermis via a liquid jet injector and / or via a needle w'hich pierces the stratum corneum andproduces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Patents 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569, 189; 5,704,911; 5,383,851; 5,893,397; 5,466,220, 5,339,163; 5,312,335; 5,503,627; 5,064,413, 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97 / 37705 and WO 97 / 13537. Ballistic powder / particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes may be used in the classical mantoux method of intradermal administration.

[0490] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi liquid preparations such as liniments, lotions, oil in water and / or water in oil emulsions such as creams, ointments and / or pastes, and / or solutions and / or suspensions. Topical ly-administrable formulations may, for example, comprise from about 1% to about 10% (wt / wt) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

[0491] A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and / or using a self- propelling solvent / powder dispensing container such as a device comprising the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0492] Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50% to 99.9% (wt / wt) of the composition, and active ingredient may constitute 0.1% to 20% (wt / wt) of the composition. A propellant may further comprise additional ingredients such as a liquid nonionic and / or solid anionic surfactant and / or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

[0493] Pharmaceutical compositions formulated for pulmonary' delivery' may provide an active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions, optionally sterile, comprising active ingredient, and may conveniently beadministered using any nebulization and / or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and / or a preservative such as methylhydroxybenzoate. Droplets provided by this route of administration may have an average diameter in the range from about 1 nm to about 200 nm. 104941 Formulations described herein as being useful for pulmonary deliver}' are useful for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

[0495] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (wr-'wi) and as much as 100% (wt / wt) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges made using conventional methods, and may, for example, 0.1% to 20% (vvi / vvi ) active ingredient, the balance comprising an orally dissolvable and / or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.

[0496] A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1 / 1.0% (wt / wt) solution and / or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and / or one or more other of any additional ingredients described herein. Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or eye drops are contemplated as being within the scope of this present disclosure.Methods of Use

[0497] In some aspects, the disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a cell (e.g, a mammalian cell). This method includes the step of contacting the cell with an LNP or a pharmaceutical composition of the disclosure, whereby the therapeutic and / or prophylactic agent is delivered to the cell. In some embodiments, the cell is in a subject and the contacting comprises administering the cell to the subject. In some embodiments, the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I) a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, whereby the therapeutic and / or prophylactic agent is delivered to the cell.

[0498] In some embodiments, the disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a cell within a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEG?k-DMG, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an Rfs'A).

[0499] In some embodiments, the disclosure provides a method of delivering a therapeutic and / or prophylactic agent to a cell within a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA).

[0500] In some aspects, the disclosure provides a method of delivering (e.g, specifically delivering) a therapeutic and / or prophylactic agent to a mammalian organ or tissue (e.g.. a liver, kidney, spleen, or lung). This method includes the step of contacting the cell with an LNP or a pharmaceutical composition of the disclosure, whereby the therapeutic and / or prophylactic agent is delivered to the target organ or tissue. In some embodiments, the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, whereby the therapeutic and / or prophylactic agent is delivered to the target organ or tissue. In some embodiments, the target organ is the lung or the target tissue is tissue is the pulmonary endothelium.

[0501] In some embodiments, the disclosure provides a method of specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEGsk-DMG, and one or more therapeutic and / orprophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA).

[0502] In some embodiments, the disclosure provides a method of specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA).

[0503] In some aspects, the disclosure features a method for the enhanced delivery' of a therapeutic and / or prophylactic agent (e.g., an mRNA) to a target tissue (e.g., a liver, spleen, or lung). This method includes the step of contacting the cell with an LNP or a pharmaceutical composition of the disclosure, whereby the therapeutic and / or prophylactic agent is delivered to the target tissue (e.g. , a liver, kidney, spleen, or lung). In some embodiments, the target tissue is the pulmonary' endothelium. In some embodiments, the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, whereby the therapeutic and / or prophylactic agent is delivered to the target tissue (e.g, a liver, kidney, spleen, or lung). In some embodiments, the target tissue is the pulmonary endothelium.

[0504] In some embodiments, the disclosure provides a method for the enhanced delivery of a therapeutic and / or prophylactic agent to a target tissue, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEGik-DMG, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA).

[0505] In some embodiments, the disclosure provides a method for the enhanced delivery of a therapeutic and / or prophylactic agent to a target tissue, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEG- 1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA). In some embodiments, the target tissue is the pulmonary' endothelium.

[0506] In some aspects, the disclosure provides a method of producing a polypeptide of interest in a cell (e.g, a mammalian cell). This method includes the step of contacting the cell with an LNP or a pharmaceutical composition of the disclosure, wherein the LNP or pharmaceutical composition comprises an mRNA, whereby the mRNA is capable of being translated in the cell to produce the polypeptide. In some embodiments, the cell is in a subject and thecontacting comprises administering the ceil to the subject. In some embodiments, the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), a phospholipid, a structural lipid, a PEG lipid, and an mRNA, whereby the mRNA is capable of being translated in the cell to produce the polypeptide.

[0507] In some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), cholesterol, and PEGzk-DMG, and an mRNA. For example, in some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Table 1, DSPC, cholesterol, and PEG2k-DMG, and an mRNA.

[0508] In some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), cholesterol, and PEG-1, and an mRNA. For example, in some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Table 1, DSPC, cholesterol, and PEG-1, and an mRNA.

[0509] In some aspects, the disclosure provides a method of treating a disease or disorder in a mammal (e.g., a human) in need thereof. The method includes the step of administering to the mammal a therapeutically effective amount of LNP or a pharmaceutical composition of the disclosure. In some embodiments, the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents, whereby the therapeutic and / or prophylactic agent is delivered to the cell . In some embodiments, the disease or disorder is characterized by dysfunctional or aberrant protein or polypeptide activity. For example, the disease or disorder is selected from the group consisting of rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases.

[0510] In some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEG2k- DMG, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, apolypeptide, and a nucleic acid (e.g., an RNA). For example, in some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Table 1, DSPC, cholesterol, and PEG2k-DMG, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA).

[0511] In some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEG-I, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA). For example, in some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Table 1, DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA).

[0512] In yet another aspect, the disclosure features a method of lowering immunogenicity comprising introducing LNP or a pharmaceutical composition of the disclosure into cells, wherein the LNP or a pharmaceutical composition reduces the induction of the cellular immune response of the cells to the LNP or a pharmaceutical composition, as compared to the induction of the cellular immune response in cells induced by a reference composition. In some embodiments, the cell is in a subject and the contacting comprises administering the cell to the subject. In some embodiments, the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), a phospholipid, a structural lipid, a PEG lipid, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA), wherein the lipid nanoparticle comprising an ionizable lipid of Formula (I), reduces the induction of the cellular immune response of the cells to the lipid nanoparticle comprising an ionizable lipid of Formula (I), as compared to the induction of the cellular immune response in cells induced by a reference composition. For example, the cellular immune response is an innate immune response, an adaptive immune response, or both.

[0513] In some embodiments, the disclosure provides a method of lowering immunogenicity in a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEG2k- DMG, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, apolypeptide, and a nucleic acid (e.g., an RNA). For example, in some embodiments, the disclosure provides a method of lowering immunogenicity in a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Table 1, DSPC, cholesterol, and PEG2k-DMG, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA).

[0514] In some embodiments, the disclosure provides a method of lowering immunogenicity in a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Formula (I), DSPC, cholesterol, and PEG-I, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA). For example, in some embodiments, the disclosure provides a method of lowering immunogenicity in a subject, wherein the method comprises the step of administering to the subject a lipid nanoparticle comprising an ionizable lipid of Table 1, a DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., an RNA).

[0515] The disclosure also includes methods of synthesizing an ionizable lipid of Formula (I), and methods of making a lipid nanoparticle including a lipid component comprising the cationic lipid of Formula (I).Methods of producing polypeptides in cells

[0516] The present disclosure provides methods of producing a polypeptide of interest in a mammalian cell. Methods of producing polypeptides involve contacting a cell with a formulation of the disclosure comprising an LNP including an mRNA encoding the polypeptide of interest. Upon contacting the cell with the lipid nanoparticle, the mRNA may be taken up and translated in the cell to produce the polypeptide of interest.

[0517] In general, the step of contacting a mammalian cell with an LNP including an mRNA encoding a polypeptide of interest may be performed / >? vivo, ex vivo, in culture, or in vitro. The amount of lipid nanoparticle contacted with a cell, and / or the amount of mRNA therein, may depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics of the lipid nanoparticle and the mRNA (e.g., size, charge, and chemical composition) therein, and other factors. In general, an effective amount of the lipid nanoparticle will allow7for efficient polypeptide production in the cell. Metrics for efficiency may include polypeptide translation (indicated by polypeptide expression), level of mRNA degradation, and immune response indicators.

[0518] The step of contacting an LNP including an mRNA with a cell may involve or cause transfection. A phospholipid including in the lipid component of an LNP may facilitate transfection and / or increase transfection efficiency, for example, by interacting and / or fusing with a cellular or intracellular membrane. Transfection may allow for the translation of the mRNA within the cell,

[0519] In some embodiments, the lipid nanoparticles described herein may be used therapeutically. For example, an mRNA included in an LNP may encode a therapeutic polypeptide (e.g., in a translatable region) and produce the therapeutic polypeptide upon contacting and / or entry (e.g., transfection) into a cell. In other embodiments, an mRNA included in an LNP may encode a polypeptide that may improve or increase the immunity' of a subject. In some embodiments, an mRNA may encode a granulocyte-colony stimulating factor or trastuzumab.

[0520] In some embodiments, an mRNA included in an LNP may encode a recombinant polypeptide that may replace one or more polypeptides that may be substantially absent in a cell contacted with the lipid nanoparticle. The one or more substantially absent polypeptides may be lacking due to a genetic mutation of the encoding gene or a regulatory pathway thereof. Alternatively, a recombinant polypeptide produced by translation of the mRNA may antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell. An antagonistic recombinant polypeptide may be desirable to combat deleterious effects caused by activities of the endogenous protein, such as altered activities or localization caused by mutation. In another alternative, a recombinant polypeptide produced by translation of the mRNA may indirectly or directly antagonize the activity of a biological moiety present in, on the surface of, or secreted from the cell. Antagonized biological moi eties may include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins. Recombinant polypeptides produced by translation of the mRNA may be engineered for localization within the cell, such as within a specific compartment such as the nucleus, or may be engineered for secretion from the cell or for translocation to the plasma membrane of the cell.

[0521] In some embodiments, contacting a cell with an LNP including an mRNA may reduce the innate immune response of a cell to an exogenous nucleic acid. A cell may be contacted with a first lipid nanoparticle including a first amount of a first exogenous mRNA including a translatable region and the level of the innate immune response of the cell to the first exogenous mRNA may be determined. Subsequently, the cell may be contacted with a second composition including a second amount of the first exogenous mRNA, the second amountbeing a lesser amount of the first exogenous mRNA compared to the first amount. Alternatively, the second composition may include a first amount of a second exogenous mRNA that is different from the first exogenous mRNA. The steps of contacting the cell with the first and second compositions may be repeated one or more times. Additionally, efficiency of polypeptide production (e.g., translation) in the cell may be optionally determined, and the cell may be re-contacted with the first and / or second composition repeatedly until a target protein production efficiency is achieved.Methods of delivering therapeutic agents to ceils and organs(G522J The present disclosure provides methods of delivering a therapeutic and / or prophylactic agent, such as a nucleic acid, to a mammalian cell or organ. Delivery of a therapeutic and / or prophylactic agent to a cell involves administering a formulation of the disclosure that comprises an LNP including the therapeutic and / or prophylactic agent, such as a nucleic acid, to a subject, where administration of the composition involves contacting the cell with the composition. In some embodiments, a protein, cytotoxic agent, radioactive ion, chemotherapeutic agent, or nucleic acid (such as an RNA, e.g., mRNA) may be delivered to a cell or organ. In the instance that a therapeutic and / or prophylactic agent is an mRNA. upon contacting a cell with the lipid nanoparticle, a translatable mRNA may be translated in the cell to produce a polypeptide of interest. However, mRNAs that are substantially not translatable may also be delivered to cells. Substantially non-translatable mRNAs may be useful as vaccines and / or may sequester translational components of a cell to reduce expression of other species in the cell.

[0523] In some embodiments, an LNP may target a particular type or class of cells (e.g, cells of a particular organ or system thereof). In some embodiments, an LNP including a therapeutic and / or prophylactic agent of interest may be specifically delivered to a mammalian liver, kidney, spleen, femur, or lung. Specific delivery to a particular class of cells, an organ, or a system or group thereof implies that a higher proportion of lipid nanoparticles including a therapeutic and / or prophylactic agent are delivered to the destination (e.g., tissue) of interest relative to other destinations, e.g., upon administration of an LNP to a mammal. In some embodiments, specific delivery may result in a greater than 2 fold, 5 fold, 10 fold, 15 fold, or 20 fold increase in the amount of therapeutic and / or prophylactic agent per 1 g of tissue of the targeted destination (e.g., tissue of interest, such as a liver) as compared to another destination (e.g., the spleen). In some embodiments, the tissue of interest is selected from the groupconsisting of a liver, kidney, a lung, a spleen, a femur, vascular endothelium in vessels (e.g, intra-coronary or intra-femoral) or kidney, and tumor tissue (e.g., via intratumoral injection).

[0524] As another example of targeted or specific delivery', an mRNA that encodes a proteinbinding partner (e.g., an antibody or functional fragment thereof, a scaffold protein, or a peptide) or a receptor on a cell surface may be included in an LXP An mRNA may additionally or instead be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties. Alternatively, other therapeutics and / or prophylactics or elements (e.g., lipids or ligands) of an LNP may be selected based on their affinity for particular receptors (e.g, low density lipoprotein receptors) such that, an LNP may more readily interact with a target cell population including the receptors. In some embodiments, ligands may include, but are not limited to, members of a specific binding pair, antibodies, monoclonal antibodies, Fv fragments, single chain Fv (scFv) fragments. Fab’ fragments, F(ab’)2 fragments, single domain antibodies, camelized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof, multivalent binding reagents including mono- or bispecific antibodies such as disulfide stabilized Fv fragments, scFv tandems, diabodies, tribodies, or tetrabodies, and aptamers, receptors, and fusion proteins.

[0525] In some embodiments, a ligand may be a surface-bound antibody, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site. In one embodiment, multiple antibodies are expressed on the surface of a cell, and each antibody can have a different specificity for a desired target. Such approaches can increase the avidity and specificity of targeting interactions.

[0526] ,A ligand can be selected, e.g., by a person skilled in the biological arts, based on the desired localization or function of the cell.

[0527] In some embodiments, an LNP may target hepatocytes. Apolipoproteins such as apolipoprotein E (apoE) have been shown to associate with neutral or near neutral lipid- containing lipid nanoparticles in the body, and are known to associate with receptors such as low-density lipoprotein receptors (LDLRs) found on the surface of hepatocytes. Thus, an LNP including a lipid component with a neutral or near neutral charge that is administered to a subject may acquire apoE in a subject’s body and may subsequently deliver a therapeutic and / or prophylactic agent (e.g., an RNA) to hepatocytes including LDLRs in a targeted manner.Methods of treating diseases and disorders

[0528] In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof an LNP described herein.

[0529] In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof an LNP solution described herein.

[0530] In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof an LNP formulation described herein.

[0531] In some embodiments, the administering is performed parenterally.

[0532] In some embodiments, the administering is performed intramuscularly, intradermally, subcutaneously, and / or intravenously.

[0533] In some aspects, the present disclosure provides an LNP disclosed herein for use in treating or preventing a disease or disorder in a subject.

[0534] In some aspects, the present disclosure provides an LNP solution disclosed herein for use in treating or preventing a disease or disorder in a subject.

[0535] In some aspects, the present disclosure provides an LNP formulation disclosed herein for use in treating or preventing a disease or disorder in a subject.

[0536] In some aspects, the present disclosure provides a use of an LNP disclosed herein in the manufacture of a medicament for treating or preventing a disease or disorder.

[0537] In some aspects, the present disclosure provides a use of an LNP solution disclosed herein in the manufacture of a medicament for treating or preventing a disease or disorder.

[0538] In some aspects, the present disclosure provides a method of administering an LNP disclosed herein to a subject.

[0539] In some aspects, the present disclosure provides a method of administering an LNP solution disclosed herein to a subject.

[0540] In some aspects, the present disclosure provides a method of administering an LNP formulation disclosed herein to a subject.

[0541] Lipid nanoparticles may be useful for treating a disease, disorder, or condition. In particular, such compositions may be useful in treating a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity. In some embodiments, a formulation of the disclosure that comprises an LNP including an mRNA encoding a missing or aberrant polypeptide may be administered or delivered to a cell. Subsequent translation of the mRNA may produce the polypeptide, thereby reducing or eliminating an issue caused bythe absence of or aberrant activity caused by the polypeptide. Because translation may occur rapidly, the methods and compositions may be useful in the treatment of acute diseases, disorders, or conditions such as sepsis, stroke, and myocardial infarction. A therapeutic and / or prophylactic agent included in an LNP may also be capable of altering the rate of transcription of a given species, thereby affecting gene expression.|0542| The disclosure provides methods involving administering lipid nanoparticles including one or more therapeutic and / or prophylactic agents, such as a nucleic acid, and pharmaceutical compositions including the same. The terms therapeutic and prophylactic can be used interchangeably herein with respect to features and embodiments of the present disclosure. Therapeutic compositions, or imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any reasonable amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and / or condition and / or any other purpose. The specific amount administered to a given subject may vary depending on the species, age, and general condition of the subject; the purpose of the administration; the particular composition; the mode of administration; and the like. Compositions in accordance with the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of a composition of the present, disclosure will be decided by an attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or otherwise appropriate dose level (e.g., for imaging) for any particular patient will depend upon a variety of factors including the severity and identify of a disorder being treated, if any; the one or more therapeutics and / or prophylactics employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific pharmaceutical composition employed, the duration of the treatment, drugs used in combination or coincidental with the specific pharmaceutical composition employed; and like factors well known in the medical arts.|0543| An LNP including one or more therapeutics and / or prophylactics, such as a nucleic acid, may be administered by any route. In some embodiments, compositions, including prophylactic, diagnostic, or imaging compositions including one or more lipid nanoparticles described herein, are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, trans- or intra-dermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powders, ointments, creams, gels, lotions, and / or drops), mucosal, nasal, buccal,enteral, intravitreal, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray and / or powder, nasal spray, and / or aerosol, and / or through a portal vein catheter. In some embodiments, a composition may be administered intravenously, intramuscularly, intradermally, intra-arterially, intratumorally, subcutaneously, or by inhalation. However, the present disclosure encompasses the delivery' or administration of compositions described herein by any appropriate route taking into consideration likely advances in the sciences of drug delivery. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the lipid nanoparticle including one or more therapeutics and / or prophylactics (e.g, its stability in various bodily environments such as the bloodstream and gastrointestinal tract), the condition of the patient ty.g., whether the patient is able to tolerate particular routes of administration), etc.

[0544] In certain embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about. 5 mg / kg, from about 0,05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 2.5 mg / kg, from about 0.001 mg / kg to about 2.5 mg / kg, from about 0.005 mg / kg to about 2.5 mg / kg, from about 0.01 mg / kg to about 2.5 mg / kg, from about 0.05 mg / kg to about 2.5 mg / kg, from about 0.1 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about. 2,5 mg / kg, from about 2 mg / kg to about 2.5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg. from about 0.001 mg / kg to about 1 mg / kg. from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about. 1 mg / kg, from about 0.05 mg / kg to about 1 mg / kg, from about 0. 1 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.25 mg / kg, from about 0.001 mg / kg to about 0.25 mg / kg, from about 0,005 mg / kg to about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg, or from about 0.1 mg / kg to about 0.25 mg / kg of a therapeutic and / or prophylactic agent (e.g, an mRNA) in a given dose, where a dose of 1 mg / kg (mpk) provides 1 mg of a therapeutic and / or prophylactic agent per 1 kg of subject body weight. In some embodiments, a dose of about 0.001 mg / kg to about 10 mg / kg of a therapeutic and / or prophylactic agent (e.g, mRNA)of an LNP may be administered, hi other embodiments, a dose of about 0.005 mg / kg to about 2.5 mg / kg of a therapeutic and / or prophylactic agent may be administered. In certain embodiments, a dose of about 0.1 mg / kg to about 1 mg / kg may be administered. In other embodiments, a dose of about 0.05 mg / kg to about 0.25 mg / kg may be administered. A dose may be administered one or more times per day, in the same or a different amount to obtain a desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered, for example, three times a day, two times a day, once a day, every other day, every' third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In some embodiments, a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute disease, disorder, or condition.

[0545] Lipid nanoparticles including one or more therapeutics and / or prophylactics, such as a nucleic acid, may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. In some embodiments, one or more lipid nanoparticles including one or more different therapeutics and / or prophylactics may be administered in combination. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and / or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery' of compositions, or imaging, diagnostic, or prophylactic compositions thereof in combination with agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution within the body.

[0546] It will further be appreciated that therapeutically, prophylactically, diagnostically, or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination may be lower than those utilized individually.

[0547] The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and / orprocedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, a composition useful for treating cancer may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g. , control of any adverse effects, such as infusion related reactions).

[0548] An LNP may be used in combination with an agent to increase the effectiveness and / or therapeutic window of the composition. Such an agent may be, for example, an antiinflammatory compound, a steroid (e.g., a corticosteroid), a statin, an estradiol, a BTK inhibitor, an SIP 1 agonist, a glucocorticoid receptor modulator (GRM), or an anti -histamine. In some embodiments, an LNP may be used in combination with dexamethasone, methotrexate, acetaminophen, an Hl receptor blocker, or an H2 receptor blocker. In some embodiments, a method of treating a subject in need thereof or of delivering a therapeutic and / or prophylactic agent to a subject (e.g, a mammal) may involve pre-treating the subject with one or more agents prior to administering an LNP. In some embodiments, a subject may be pre-treated with a useful amount (e.g, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 nig, 90 mg, 100 mg, or any other useful amount) of dexamethasone, methotrexate, acetaminophen, an Hl receptor blocker, or an H2 receptor blocker. Pre- treatment may occur 24 or fewer hours (e.g, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes) before administration of the lipid nanoparticle and may occur one, two, or more times in, for example, increasing dosage amounts.

[0549] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be iimited to the above Description, but rather is as set forth in the appended claims.

[0550] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary' or otherwise evident from the context. Claims or descriptions that, include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary' or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all, of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0551] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the terms “consisting essentially of” and “consisting of’ are thus also encompassed and disclosed. Throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0552] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0553] In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art. may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein.

[0554] All cited sources, for example, references, publications, patent applications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

[0555] The disclosure having been described, the following examples are offered by way of illustration and not limitation.EXEMPLARY EMBODIMENTS

[0556] Exemplary Embodiment No. 1. A compound of Formula (I*):(F}or a pharmaceutically acceptable salt thereof, wherein: one is a single bond, and the otheris a double bond; n is 3, 4, 5, or 6;M is -O-C(=O)-* and -C(=O)-O-*, wherein * indicates attachment to L;L is a bond or -CH2-;R1is H, C1-12 alkyl, or C2-12 alkenyl;R2is H, C1-12 alkyl, or C2-12 alkenyl,R ’ is C1-12 alkyl or C2-12 alkenyl; wherein at least one of R!, R2, and R3is a C1-12 alkyl or C2-12 alkenyl; m is 3, 4, 5, or 6;each is a single bond or a double bond, wherein no more than one is a double bond;M’ is -O-C(==O)-* and -C(=O)-O-*, wherein * indicates attachment to L’;L’ is a bond or -CH2-;R1’ is H, C1-12 alkyl, or C2-12 alkenyl,R2is H, C1-12 alkyl, or C2-12 alkenyl;R3’ is H, C1-12 alkyl, or C2-12 alkenyl; wherein at least one of R1, R2”, and R3is a C1..12 alkyl or C2-12 alkenyl;p is 1, 2, 3, 4, or 5; each R&independently is H, C1-6 alkyl, or C2-6 alkenyl;T is a bond, C1.3 alkylene, C2-3 alkenylene, or C2-3 alkynylene;Q is -OH, -0-(C 1-6 alkyl), C 1-6 alkyl, C2-6 alkenyl, C2-s alkynyl, C3-10 cycloalkyl, 5-10 membered heteroaryl, C6-io aryl, or 3- 12 membered heterocycloalkyl, wherein the -O-(C 1-6 alkyl), C 1-6 alkyl, C2-5 alkenyl, C3-10 cycloalkyl, or 3-12 membered heterocycloalkyl is optionally substituted with one or more RQ; and each RQindependently is oxo, cyano, -OH, -O-(C 1-6 alkyl), -NHz, -NH(C 1-6 alkyl), - N(C 1-6 alkyl ):. -C(=O)-(C 1-6 alkyl), -O-C(=O)-(C 1-6 alkyl), -NR-C(=O)-(C 1-6 alkyl), Ci- 6 alkyl, C2-6 alkenyl, -(C1-6 alkyl)-OH, C2-6 alkenyl, or C3-10 cycloalkyl.

[0557] Exemplary Embodiment No. 2. The compound of embodiment I, wherein the compound is of Formula (I**):or a pharmaceutically acceptable salt thereof, wherein: one is a single bond, and the otheris a double bond; n is 3, 4, 5, or 6;M is -O-C(=O)-* or -C(=O)-O-*, wherein * indicates attachment to L;L is -Cl b:R1is H;R2is C3-5 alkyl;R3is C3-5 alkyl; m is 3, 4, 5, or 6;each is a single bond or a double bond, wherein no more than one is a double bond;M’ is -C(::::O)-O-*, wherein * indicates attachment to L’;If is a bond;R1is G7-10 alkyl;R2is Cs-8 alkyl;R3” is H;RHis -(CH2)P-Q; p is 1, 2, 3, 4, or 5; andQ is -OH.

[0558] Exemplary Embodiment No. 3. The compound of any one of the previous embodiments, wherein the compound is of Formula ( I):or a pharmaceutically acceptable salt thereof.

[0559] Exemplary Embodiment No. 4. The compound of any one of the previous embodiments, wherein the compound is of Formula (II):or a pharmaceutically acceptable salt thereof.

[0560] Exemplary Embodiment No. 5. The compound of any one of the previous embodiments, wherein the compound is of Formula (III):or a pharmaceutically acceptable salt thereof.

[0561] Exemplary Embodiment. No. 6. The compound of any one of the previous embodiments, wherein the compound is of Formula (IV):or a pharmaceutically acceptable salt thereof.

[0562] Exemplary Embodiment No. 7. The compound of any one of the previous embodiments, wherein the compound is of Formula (V):or a pharmaceutically acceptable salt thereof.

[0563] Exemplary Embodiment No. 8. The compound of any one of the previous embodiments, wherein the compound is of Formula (VI):or a pharmaceutically acceptable salt thereof.

[0564] Exemplary Embodiment No. 9. The compound of any one of the previous embodiments, wherein the compound is of Formula (1-2):or a pharmaceutically acceptable salt thereof.

[0565] Exemplary Embodiment No. 10. The compound of any one of the previous embodiments, wherein the compound is of Formula (11-2):(11-2) or a pharmaceutically acceptable salt thereof.

[0566] Exemplary Embodiment No. 11. The compound of any one of the previous embodiments, wherein the compound is of Formula (III-2):(III-2) or a pharmaceutically acceptable salt thereof.

[0567] Exemplary Embodiment No. 12. The compound of any one of the previous embodiments, wherein the compound is of Formula (TV -2):or a pharmaceutically acceptable salt thereof.

[0568] Exemplary Embodiment No. 13. The compound of any one of the previous embodiments, wherein the compound is of Formula (V-2):(V-2) or a pharmaceutically acceptable salt thereof.

[0569] Exemplary Embodiment No. 14. The compound of any one of the previous embodiments, wherein the compound is of Formula (VI- 2):(VI-2) or a pharmaceutically acceptable salt thereof.

[0570] Exemplary Embodiment No. 15. The compound of any one of the previous embodiments, wherein the compound is of Formula (1-3):or a pharmaceutically acceptable salt thereof.

[0571] Exemplary Embodiment No. 16. The compound of any one of the previous embodiments, wherein the compound is of Formula (11-3):or a pharmaceutically acceptable salt thereof.

[0572] Exemplary Embodiment No. 17. The compound of any one of the previous embodiments, wherein the compound is of Formula (III-3):(III-3) or a pharmaceutically acceptable salt thereof

[0573] Exemplary Embodiment No. 18. The compound of any one of the previous embodiments, wherein the compound is of Formula (IV-3):(IV-3) or a pharmaceutically acceptable salt thereof.

[0574] Exemplary Embodiment No. 19. The compound of any one of the previous embodiments, wherein the compound is of Formula (V-3):or a pharmaceutically acceptable salt thereof.

[0575] Exemplary Embodiment No. 20. The compound of any one of the previous embodiments, wherein the compound is of Formula (VI-2):or a pharmaceutically acceptable salt thereof.

[0576] Exemplary Embodiment No. 21. The compound of any one of the previous embodiments, wherein the compound is of Formula (1-4):(1-4) or a pharmaceutically acceptable salt thereof.

[0577] Exemplary Embodiment No. 22. The compound of any one of the previous embodiments, wherein the compound is of Formula (11-4):(11-4) or a pharmaceutically acceptable salt thereof

[0578] Exemplary Embodiment No. 23. The compound of any one of the previous embodiments, wherein the compound is of Formula (III-4):(III-4) or a pharmaceutically acceptable salt thereof.

[0579] Exemplary Embodiment No. 24. The compound of any one of the previous embodiments, wherein the compound is of Formula (lV-4):(IV-4) or a pharmaceutically acceptable salt thereof.

[0580] Exemplary Embodiment No. 25. The compoundany one of the previous embodiments, wherein the compound is of Formula (V-4):(V-4) or a pharmaceutically acceptable salt thereof.

[0581] Exemplary Embodiment No. 26. The compound of anv one of the previous embodiments, wherein the compound is of Formula (VI-4):or a pharmaceutically acceptable salt thereof.

[0582] Exemplary Embodiment No. 27. The compound of any one of the previous embodiments, wherein the compound is of Formula (1-5):or a pharmaceutically acceptable salt thereof.

[0583] Exemplary Embodiment No. 28. The compound of anv one of the previous embodiments, wherein the compound is of Formula (II-5):or a pharmaceutically acceptable salt thereof.

[0584] Exemplary Embodiment No. 29. The compound of any one of the previous embodiments, wherein the compound is of Formula (111-5):(III-5) or a pharmaceutically acceptable salt thereof

[0585] Exemplary Embodiment No. 30. The compound of any one of the previous embodiments, wherein the compound is of Formula (IV-5):or a pharmaceutically acceptable salt thereof.

[0586] Exemplary Embodiment No. 31. The compound of any one of the previous embodiments, wherein the compound is of Formula (V-5):(V-5) or a pharmaceutically acceptable salt thereof.

[0587] Exemplary Embodiment No. 32. The compound of any one of the previous embodiments, wherein the compound is of Formula (VI-5):or a pharmaceutically acceptable salt thereof.

[0588] Exemplary Embodiment No. 33. The compound of anv one of the previous embodiments, wherein the compound is of Formula (1-6)(1-6) or a pharmaceutically acceptable salt thereof.

[0589] Exemplary Embodiment No. 34. The compound of any one of the previous embodiments, wherein the compound is of Formula (11-6):(II-6) or a pharmaceutically acceptable salt thereof.

[0590] Exemplary Embodiment No. 35. The compound of any one of the previous embodiments, wherein the compound is of Formula (III-6):or a pharmaceutically acceptable salt thereof.

[0591] Exemplary Embodiment No. 36. The compound of any one of the previous embodiments, wherein the compound is of Formula (IV-6):(IV-6) or a pharmaceutically acceptable salt thereof.

[0592] Exemplary Embodiment No. 37. The compound of any one of the previous embodiments, wherein the compound is of Formula (V-6):or a pharmaceutically acceptable salt thereof.

[0593] Exemplary Embodiment No. 38. The compound of any one of the previous embodiments, wherein the compound is of Formula (VI-6):or a pharmaceutically acceptable salt thereof.

[0594] Exemplary Embodiment No. 39. The compound of any one of the previous embodiments, wherein RHis -(CHsjp-Q or “(CH2.)P-NRG-T-Q.

[0595] Exemplary Embodiment No. 40. The compound of any one of the previous embodiments, wherein p is 2 or 3.

[0596] Exemplary Embodiment No. 41. The compound of any one of the previous embodiments, wherein RGis H.

[0597] Exemplary Embodiment No. 42. The compound of any one of the previous embodiments, wherein T is a bond.

[0598] Exemplary Embodiment No. 43. The compound of any one of the previous embodiments, wherein Q is OH or C3-6 cycloalkyl substituted with one or more oxo, -NH?, -NH(C 1-6 alkyl), or -N(C 1-6 alkyl)?.

[0599] Exemplary Embodiment No. 44. The compound of any one of the previous embodiments, wherein Q is cyclobutenyl substituted with one or more oxo, -NH?, -NH(CI-6 alkyl), or -N(C 1-6 alkyl)?.

[0600] Exemplary Embodiment No. 45. The compound of any one of the previous embodiments, wherein n is 5.

[0601] Exemplary Embodiment No. 46. The compound of any one of the previous embodiments, wherein M is -C(=O)-O-*, wherein * indicates attachment to L.

[0602] Exemplary Embodiment No. 47. The compound of any one of the previous embodiments, wherein m is 5,

[0603] Exemplary Embodiment No. 48. The compound of any one of the previous embodiments, wherein M’ is -C(::::O)-O-*, wherein * indicates attachment to L’.

[0604] Exemplary Embodiment No. 49. The compound of any one of the previous embodiments, wherein L is -CH2-.

[0605] Exemplary Embodiment No. 50. The compound of any one of the previous embodiments, wherein R1is H, methyl, or ethyl .

[0606] Exemplary Embodiment No. 51. The compound of any one of the previous embodiments, wherein R2is C2-12 alkyl.

[0607] Exemplary Embodiment No. 52. The compound of any one of the previous embodiments, wherein R2is H, propyl, butyl, or heptyl.

[0608] Exemplary Embodiment No. 53. The compound of any one of the previous embodiments, wherein R2is propyl, butyl, or heptyl.

[0609] Exemplary Embodiment No. 54. The compound of any one of the previous embodiments, wherein R2is pentenyl.

[0610] Exemplary Embodiment No. 55. The compound of any one of the previous embodiments, wherein R3is methyl, butyl, or pentyl.

[0611] Exemplary Embodiment No. 56. The compound of any one of the previous embodiments, wherein R3is hexenyl.

[0612] Exemplary Embodiment No. 57. The compound of anv one of the previous embodiments, wherein L’ is -CH2-.

[0613] Exemplary Embodiment No. 58. The compound of any one of the previous embodiments, wherein L’ is a bond.

[0614] Exemplary Embodiment No. 59. The compound of any one of the previous embodiments, wherein M’ is -C(::::O)~O-*, wherein * indicates attachment to L’, and L’ is -CH2~.

[0615] Exemplary Embodiment No. 60. The compound of any one of the previous embodiments, wherein M’ is -C(=O)-O-*, wherein * indicates attachment to L’, and L’ is a bond.

[0616] Exemplary Embodiment No. 61. The compound of any one of the previous embodiments, wherein Rl’ is H, methyl, or ethyl.

[0617] Exemplary Embodiment No. 62. The compound of any one of the previous embodiments, wherein R2is C2-12 alkyl.

[0618] Exemplary Embodiment No. 63. The compound of any one of the previous embodiments, wherein R2is H, propyl, butyl, or heptyl.

[0619] Exemplary Embodiment No. 64. The compound of any one of the previous embodiments, wherein R2is propyl, butyl, or heptyl.

[0620] Exemplary Embodiment No. 65. The compound of any one of the previous embodiments, wherein R2” is pentenyl.

[0621] Exemplary Embodiment No. 66. The compound of any one of the previous embodiments, wherein R3is H, methyl, butyl, or pentyl.

[0622] Exemplary Embodiment No. 67. The compound of any one of the previous embodiments, wherein R3’ is hexenyl.

[0623] Exemplary Embodiment No. 68. A compound having the any one of the structures shown in Table 1, or a pharmaceutically acceptable salt thereof.

[0624] Exemplary Embodiment No. 69. A lipid nanoparticle (LNP) comprising the lipid of any one of the preceding embodiments, a phospholipid, a structural lipid, and a PEG lipid.

[0625] Exemplary Embodiment No. 70. An LNP comprising the lipid of any one of the preceding embodiments, a phospholipid, a structural lipid, a cationic lipid, and a PEG lipid.

[0626] Exemplary Embodiment No. 71. The LNP of any one of the preceding embodiments, comprising about 40 mol % to about 60 mol % said lipid, about 0 mol % to about 20 mol % phospholipid, about 30 mol % to about 50 mol % structural lipid, and about 0 mol % to about 5 mol % PEG lipid.

[0627] Exemplary Embodiment No. 72. The LNP of any one of the preceding embodiments, wherein the phospholipid is selected from the group consisting of: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-distearoyl-sn-glycero-3-phosphocholine (DSPC),1.2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1.2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-57?-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (C l 6 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-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1.2-diphytanoyl-sn-glycero-3-phosphoethanol amine (ME 16.0 PE),1.2-distearoyl-sn-glycero-3-phosphoethanolamine,1.2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine,1.2-dilinolenoyl-sn-glycero-3-phosphoethanol amine,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), sphingomyelin, and mixtures thereof.

[0628] Exemplary Embodiment No. 73. The LNP of any one of the preceding embodiments, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigniasterol, brassicasterol, and mixtures thereof.

[0629] Exemplary Embodiment No. 74. The LNP of any one of the preceding embodiments, wherein the PEG lipid is selected from the group consi sting 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.

[0630] Exemplary Embodiment No. 75. The LNP of any one of the preceding embodiments, wherein the PEG lipid is selected from PEG-HI and PEG-II:and 100 , (PL-II), wherein rPE(jis an integer between 1 and 100, and mixtures thereof.

[0631] Exemplary Embodiment No. 76. The LNP of any one of the preceding embodiments, wherein the PEG lipid is selected from PEG2k-DMG and PEG-1 :and mixtures thereof.

[0632] Exemplary Embodiment No. 77. The LNP of any one of the preceding embodiments, wherein the LNP further comprises one or more therapeutic and / or prophylactic agents.

[0633] Exemplary Embodiment No. 78. The LNP of any one of the preceding embodiments, wherein the one or more therapeutic and / or prophylactic agents is a nucleic acid.

[0634] Exemplary Embodiment No. 79. The LNP of any one of the preceding embodiments, wherein the nucleic acid is an RNA, and wherein the RNA is selected from the group consisting of a short interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), an RNA interference (RNAi) molecule, a microRNA (miRNA), an antagomir, an antisense RNA, a ribozyme, a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof.

[0635] Exemplary Embodiment No. 80. The LNP of any one of the preceding embodiments, wherein the RNA is an mRNA.

[0636] Exemplary Embodiment No. 81. A pharmaceutical composition comprising the LNP of any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0637] Exemplary Embodiment No. 82. A method of delivering a therapeutic and / or prophylactic agent to a cell within a subject, the method comprising administering to the subject the LNP of any one of the preceding embodiments.

[0638] Exemplary Embodiment No. 83. A method of specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, the method comprising administering to the subject the LNP of any one of the preceding embodiments.

[0639] Exemplary Embodiment No. 84. A method of producing a polypeptide of interest in a cell within a subject, the method comprising administering to the subject the LNP of anyone of the preceding embodiments.

[0640] Exemplary Embodiment No. 85. A method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject the LNP of any one of the preceding embodiments.

[0641] Exemplary Embodiment No. 86. The LNP of any one of the previous embodiments for use in delivering a therapeutic and / or prophylactic agent to a cell within a subject.

[0642] Exemplary Embodiment No. 87. The LNP of any one of the previous embodiments for use in specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject.

[0643] Exemplary Embodiment No. 88. The LNP of any one of the previous embodiments for use in producing a polypeptide of interest in a cell within a subject.

[0644] Exemplary Embodiment No. 89. The LNP of any one of the previous embodiments for use in treating or preventing a disease or disorder in a subject in need thereof.

[0645] Exemplary Embodiment No. 90. Use of the LNP of any one of the preceding embodiments in the preparation of a medicament for delivering a therapeutic and / or prophylactic agent to a cell within a subject.

[0646] Exemplary Embodiment No. 91. Use of the LNP of any one of the preceding embodiments in the preparation of a medicament for delivering a therapeutic and / or prophylactic agent to an organ of a subject.

[0647] Exemplary Embodiment No. 92. Use of the LNP of any one of the preceding embodiments in the preparation of a medicament for producing a polypeptide of interest in a cell within a subject.

[0648] Exemplary Embodiment No. 93. Use of the LNP of any one of the preceding embodiments in the preparation of a medicament for treating or preventing a disease or disorder in a subject in need thereof.EXAMPLES

[0649] It is understood that, unless specified otherwise, values presented in the examples are approximate values, and are subject to instrumental and experimental variations.Example 1. Synthesis of Exemplary Compounds

[0650] Heptadecan-9-yl 8-bromooctanoateChemical Formula: C25H49BrO2Molecular Weight: 461.57

[0651] To a solution of 8-bromooctanoic acid (1.04 g, 4.6 mmol) and heptadecan-9-ol (1.5 g, 5.8 mmol) in dichloromethane (20 mL) were added N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC.HCl, 1.1 g, 5.8 mmol), N,N-diisopropylethylamine (DIPEA, 3.3 mL, 18.7 mmol) and DMAP (0.114 g, 0.9 mmol). The reaction was allowed to stir at room temperature for 18 hours. The reaction was diluted with di chloromethane and extracted with saturated NaHCCL (aq.). The organic layer was separated and washed with brine, then dried over MgSO4, filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% EtOAc in Hex to obtain heptadecan-9-yl 8- bromooctanoate (875 mg, 41% yield).1H NMR (300 MHz, CDC13) δ ppm 4.89 (m, 1H), 3.42 (m, 2H), 2.31 (m, 2H), 1.89 (m, 2H), 1.73-1.18 (br. m, 36H), 0.88 (t, 6H).

[0652] Heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoateMolecular Weight: 441 .74

[0653] A solution of heptadecan-9-yl 8-bromooctanoate (3.8 g, 8.2 mmol) and 2- aminoethan-1-ol (15 mL, 248 mmol) in ethanol (3 mL) was allowed to stir at 62 °C for 18 hours. The reaction mixture was concentrated under vacuum and the residue was extracted with EtOAc and water. The organic layer was separated and washed with water, brine and dried over NasSCri, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in di chloromethane) to obtain heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate (3.1 g, 85% yield).!H Will (300 MHz, CDC13) δ ppm 4.89 (quin, 1H), 3.67 (t, 2H), 2.81 (t, 2H), 2.65 (t, 2H), 2.30 (t, 2H), 2.05 (br.m, 2H), 1.72-1.41 (br. m, 8H), 1.40-1.20 (br. m, 30H), 0.88 (t, 6H).

[0654] Ethyl 3-hexylnon-2~enoate

[0655] A suspension ofNaH (3.22 g, 80.67 mmol), in THF (202 mL) was chilled to 0 °C and added ethyl 2-(diethoxyphosphoryl)acetate (18.08 g, 80.67 mmol) slowly. The reaction was allowed to warm to 20 °C. The reaction was stirred at rt for 20 min and added tridecan-7- one (8.000 g, 40.33 mmol). The reaction was stirred at 75 °C for 6 hours. The reaction was cooled to rt and quenched with Sat. sodium carbonate. The reaction was evaporated under vac and the residue was dissolved in diethyl ether and extracted with water. The organic layer was separated and dried with MgSO< filtered, and evaporated under vac. Purified by silica gel chromatography with 0-20% EtOAc in Hex to obtain the product in (8.1 g, 74.8% yield).!H NMR (300 MHz, CDCh) δ ppm 5.63 (s, 1H); 4.16 (q, 2H); 2.60 (m, 2H); 2.15 (rn, 2H); 1.57- 1.23 (m, 19H); 0.91 (m, 6H).

[0656] 3-Hexylnon-2-en-l-ol

[0657] In a round-bottom flask equipped with stir bar and back-filled with nitrogen, a solution of ethyl 3-hexylnon-2-enoate (4.000 g, 14,90 mmol) in DCM (37 mL) was set stirringin a dry-ice acetone bath. Once the solution was brought to -78°C, DIBAL-H (IM in DCM) (44.70 mL, 44.70 mmol) was added dropwise to the cold mixture. The reaction was stirred at - 78°C for 2.5h. The reaction was quenched with Sat. Roselle salt. The reaction was warm to rt and diluted with DCM. The mixture was diluted with DCM and the organic layer was separated. The aqueous layer was extracted with DCM, and all organic layers were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated to a colorless oil. The oil was taken up in minimal Hex and purified by flash chromatography with (0-20%) EtOAc in Hex to obtain the product (3.37g, 70% yield).]H NMR (300 MHz, CDCh) δ ppm 5.41 (t, 1 H), 4.17 (d, 2H), 2,05 (m, 4H); 1.49-1.11 (m, 17H); 0.91 (m, 6H).

[0658] 3-Butylhept-2-enoic acid

[0659] To a suspension of lithium hydroxide (0.45 g, 18,8 mmol) in water (7.5 mL) stirring at room temperature was added a solution of ethyl 3-butylhept-2-enoate (1.0 g, 4.71 mmol) in ethanol (1.0 mL). The mixture was heated to 70 °C and allowed to stir overnight. The reaction mixture was cooled to room temperature and acidified to pH 4 with slow addition of IN HC1. The aqueous phase was extracted with EtOAc, and all organic phases were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated to an oil, confirmed as clean without further purification (0.80 g, 92% yield). UPLC / ELSD: RT = 1.01 min. MS (ES): m / z (MIL) 185.2 for Ci]H2o02. ’H NMR (301 MHZ, CDC13) 3 ppm 11.59 (br. s, 1 H), 5.67 (s, 1H), 2.69 - 2.58 (m, 2H), 2.20 (t, ,7= 7.5 Hz, 2H), 1.42 (m, 9H), 0.94 (t, J= 7.2 Hz, 6H).

[0660] 8-Bromooctyl 3-butylhept-2-enoate

[0661] To a solution of 8-bromooctan-l-ol (0.91 g, 4.34 mmol) and 3-butylhept-2-enoic acid (0.80 g, 4.34 mmol) in dry' DCM (21.7 mL) stirring under nitrogen was added 4- dimethylaminopyridine (0.53 g, 4.34 mmol) and 1 -ethyl -3 -(3- dimethylaminopropyl)carbodiimide hydrochloride (1.00 g, 5.21 mmol). The reaction was allowed to proceed at room temperature for 3 h and was then diluted with 10 mL DCM and quenched with 10 mL water. The organic layer was separated, washed with 5% aqueous HC1, saturated aqueous sodium bicarbonate, and brine, dried over sodium sulfate, filtered, andconcentrated to an oil. The oil was taken up in DCM and purified on silica in Hex with a 0- 30% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (0.75 g, 46% yield). UPLC / ELSD: RT == 2.58 min. MS (ES): m / z (MH;) 375.9 for Ci9H35BrO2.;H NMR (301 MHz, CDC13) δ ppm 5.64 (s, 1H), 4.09 (t, J= 6.7 Hz, 2H), 3.43 (t, .. / 6.8 Hz, 2H), 2.61 (t, J - 7.7 Hz, 2H), 2.16 (t, 7 8.2 Hz, 2H), 1.88 (m, 5H), 1.69 (m, 2H), 1.37 (m, 16H), 0.94 (t, J= 73 Hz, 6H).

[0662] Ethyl 3-pentyloct-2-enoateChemical Formula: C15H28O2Molecular Weight: 240.39

[0663] To a suspension of sodium hydride (4.70 g, 60% w / w, 117.44 mmol) in dry THF (58.7 niL) set stirring under nitrogen at 0°C was added ethyl 2-(diethoxyphosphoryi)acetate (23.3 mL, 1 17.44 mmol) dropwise over 20 minutes, eliciting gas evolution. The mixture stirred at room temperature until gas evolution ceased. The mixture was then cooled back to 0 °C and undecan-6-one (10.00 g, 58.72 mmol) was added. The mixture was allowed to gradually warm to room temperature and then refluxed at 80 °C overnight. The mixture was then cooled to room temperature and quenched slowly with 30 mL saturated aqueous ammonium chloride. The solution was diluted with 50 mL diethyl ether and the aqueous layer was separated and extracted with ether. The organic layers were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated to a light yellow oil. The oil was taken up in minimal Hex and purified on silica in Hex with a 0-10% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (7.13 g, 51% yield). UPLC / ELSD: RT = 2.01 min. MS (ES): m / z (MH4) 241.4 for C15H28O2. ‘HNMR (301 MHz, CDCh) δ ppm 5.63 (s, 1H), 4.16 (q, 7 7.2 Hz, 2H), 2.66 - 2.55 (m, 2H), 2.20 - 2.09 (m, 2H), 1.47 (m, 4H), 1.35 - 1.20 (m, UH), 1.00 - 0.84 (m, 6H).

[0664] 3 -Pentyl oct-2-en- 1 -olChemical Formula: C13H26O Molecular Weight: 198.35

[0665] To a solution of ethyl 3 -pentyl oct-2-enoate (7.12 g, 29.62 mmol) in dry DCM (74. 1 mL) stirring at -78 °C under nitrogen was added diisobutyl aluminum hydride (IM in DCM, 88.86 ml..., 88.86 mmol ) dropwise. The mixture stirred for 3 hours at -78 °C and then reactioncompletion was confirmed by TLC (9: 1 Hex / EtOAc, KMnCfi stain). The mixture was quenched with 50 niL saturated aqueous Rochelle's salt, and allowed to gradually warm to room temperature overnight. The aqueous layer was then separated and extracted with DCM, and all organic layers were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated to a colorless oil. The oil was taken up in minimal hexanes and purified on silica in Hex with a 0-20% EtOAc gradient. The product was concentrated to a colorless oil (5.25 g, 89% yield). UPLC / ELSD: RT === 1.42 min. MS (ES): m / z (MH ) 198.1 for Cfo h-O. ’H NMR (301 MHz, CDCI3) δ ppm 5.41 (t, J= 7.0 Hz, 1H), 4.17 (d, J= 7.0 Hz, 2H), 2.04 (dt, . / = 11.9, 7.5 Hz, 4H), 1 .55 - 1 . 19 (m, 1 TH) 1.16 (s, 1 H), 0.91 (t, . / - 6.9 Hz, 6H).[0666| 3-Pentyloct-2-en-l-yl 6-bromohexanoateChemical Formula: G19H35BrO2Molecular Weight: 375.39

[0667] To a solution of 6-bromohexanoic acid (0.98 g, 5.04 mmol) and 3-pentyloct-2-en~ l-ol (1.00 g, 5.04 mmol) in dry DCM (25.21 mL) stirring under nitrogen was added 4- dimethylaminopyridine (0.62 g, 5.04 mmol) and 1 -ethyl- 3 -(3- dimethylanunopropyllcarbodiirnide hydrochloride (1.16 g, 6,05 mmol). The reaction was allowed to proceed at room temperature for 3 hours and was diluted with DCM and quenched with 20 mL water. The organic layer was separated, washed with 5% aqueous HC1, saturated aqueous sodium bicarbonate, and brine, dried over sodium sulfate, filtered, and concentrated to an oil. The oil was taken up in Hex and purified on silica in hexanes with a 0-20% EtOAc gradient. The product was concentrated to a colorless oil (1.08 g, 57% yield). UPLC / ELSD: RT = 2.40 min. MS (ES): m / z (MH j 375.1 for Ci9H35BrO2.;H NMR (301 MHz, CDCI3) δ ppm 5.24 (t, .7 - 7.2 Hz, 1H), 4.52 (d, J== 7.2 Hz, 2H), 3.33 (t, J-= 6.8 Hz, 2H), 2.25 (t, J== 7.4 Hz, 2H), 2.05 - 1.89 (m, 4H), 1.88 - 1.73 (m, 2H), 1.59 (dt, J= 14.4, 7.1 Hz, 2H), 1.49 - 1.10 (m, 14H), 0.82 (t, J ------ 6.9 Hz, 6H ).

[0668] 3-Pentyloct-2-en-l-yl 8-bromooctanoateChemical Formula: C21H39BrO2Molecular Weight: 403.44

[0669] To a solution of 8-bromooctanoic acid (1.13 g, 5.04 mmol) and 3 -pentyl oct-2-en-l- ol (1.00 g, 5.04 mmol) in dry' DCM (25.21 mL) stirring under nitrogen was added 4-dimethylaminopyridine (0.62 g, 5.04 mmol) and 1 -ethyl-3-(3~ dimethylaminopropyljcarbodiimide hydrochloride (1.16 g, 6.05 mmol). The reaction was allowed to proceed at room temperature for 3 h and was diluted with DCM and quenched with water. The organic layer was separated, washed with 5% aqueous HC1, saturated aqueous sodium bicarbonate, and brine, dried over sodium sulfate, filtered, and concentrated to an oil. The oil was taken up in hexanes and purified on silica in hexanes with a 0-20% EtOAc gradient. The product was concentrated to a colorless oil (1.68 g, 83% yield). UPLC / ELSD: RT = 2.61 min. MS (ES): m / z (MH+) 404.1 for C21H39B1-O2.1H NMR (301 MHz, CDCI3) δ ppm 5.24 (t, J--- 7.1 Hz, H l). 4.52 (d, J ------ 7.2 Hz, 2H), 3.33 (t, J -- 6.8 Hz, 2H), 2.23 (t, . / - 7.5 Hz, 2H), 2.05 -- 1.89 (m, 4H), 1.78 (p, , / = 6.8 Hz, 2H), 1.56 (m, 2H), 1.37 - 1.20 (m, 18H), 0.82 (t, J = 6.9 Hz, 6H).

[0670] Heptadecan-9-yl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-((3-butylhept-2- enoyl)oxy)octyl)amino)octanoateMolecular Weight: 849.38

[0671] To a solution of heptadecan-9-yl 8-((3-((tert-butoxycarbonyl)- amino)propyl)amino)octanoate (0.25 g, 0.45 mmol) and 8-bromooctyl 3-butylhept-2-enoate (0.19 g, 0.52 mmol) in dry propionitrile (3.00 mL) stirring under nitrogen was added potassium carbonate (0.09 g, 0.68 mmol) and potassium iodide (0.01 g, 0.07 mmol). The reaction was heated to 80 °C and allowed to proceed overnight. The reaction mixture was then cooled to room temperature, diluted with 10 mL heptane and filtered through celite, washing the celite pad with additional heptane. The filtrate was concentrated to an oil. The oil was taken up in DCM and purified on silica in Hex with a 0-40% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (0.23 g, 60% yield). UPLC / ELSD: RT = 2.72 min. MS (ES): m / z (MH ) 850.1 for1H NMR (301 MHz, CDC13) δ ppm 5.70(br. s, 1H), 5.64 (s, 1H), 4.89 (m, 1H), 4.13 (m, 2H), 3.21 (m, 2H), 2.61 (t, J = 1.1 Hz, 2H), 2.52 - 2.36 (m, 2H), 2.31 (dd, J 14.0, 6.6 Hz, 6H), 2.16 (t, J ------ 7.2 Hz, 211). 1.69 - 1.61 (m, 6H), 1.46 (m, 64H), 0.99 - 0.85 (m, 12H).

[0672] Heptadecan-9-yl 8 -( ( 3 -aminopropy l)(8-((3 -butylhept-2-enoyl)oxy)octyl)ami no)- octanoate

[0673] To a solution of heptadecan-9-yl 8-((3-((tert-butoxycarbonyl)amino)propyl)(8-((3- butylhept-2-enoyl)oxy)octyl)amino)octanoate (0.23 g, 0.27 mmol) in DCM (1.37 mL) stirring under nitrogen at room temperature was added trifluoroacetic acid (0.25 mL, 3.28 mmol) was added dropwise and the mixture stirred at room temperature for 4 h. Then, the reaction was evaporated in vacuo and the residue was taken up in 9: 1 2-methyl-tetrahydrofuran / heptane (30 mL) and washed with saturated aqueous sodium bicarbonate and brine. All aqueous layers were combined and extracted with 9: 1 2-methyl-tetrahydrofuran / heptane. All organic layers were combined, dried over sodium sulfate, filtered, and concentrated to a crude colorless oil. Following, the material was taken up in DCM and purified on silica in DCM with a 0-40% (80: 19: 1 DCM / MeOH / NH4OH) gradient. Product-containing fractions were pooled and concentrated to a colorless oil (0. 17 g, 82% yield). UPLC / ELSD: RT = 2.34 min. MS (ES): m / z (Mi l ) 750.1 for C47H92N2O4.lH NMR (301 MHz, CDC13) δ ppm 5.64 (s, 1H), 4.89 (m, 1H), 4.09 (I. . / 6.8 Hz, 6H), 2.75 (t, . / 6.8 Hz, 6H), 2.61 (t, 2H). 2.47 (t, 211), 2.39 (t, 411), 2.29 (t, 2H), 2.21 (t, 3H), 1.71 - 1.19 (m, 60H), 0.99 - 0.85 (m, 12H).

[0674] Compound 1 : Heptadecan-9-yl 8-((8-((3-butylhept-2-enoyl)oxy)octyl)(3-((2- (methylamino)-3 ,4-di oxocy clobut- 1 -en- 1 -yl)amino)propyl)amino)octanoate

[0675] To a solution of heptadecan-9-yl 8-((3 -aminopropyl )(8-((3-butylhept-2- enoyl)oxy)octyl)amino)octanoate (0.17 g, 0,22 mmol) and 3-methoxy-4- (methylamino)cyclobut-3-ene- 1,2-dione (0.04 g, 0.29 mmol) in 2-methyltetrahydrofuran (1.11 mL) stirring under nitrogen at room temperature was added 10% aqueous potassium carbonate (1.1 1 mL). The mixture was heated at 45 °C for 2.5 h. Additional 3-methoxy-4-(methylamino)cyclobut-3-ene-1 ,2-dione (0.04 g, 0.29 mmol) was added in addition to 1 mL of 2-methyltetrahydofuran and 1 mL of 10% aqueous potassium carbonate. The mixture was stirred at 45 °C for additional I h. The mixture was cooled to room temperature, and diluted with heptane and water. The aqueous layer was separated and extracted with heptane. The organic layers were all combined, washed with 1 : 1 ACN / water, dried over sodium sulfate, filtered, and concentrated to an oil. The residue was azeotroped with 1 : 1 DCM / MeOH. The resulting white residue was taken up in minimal DCM and purified on silica in DCM with a 0- 50% (80: 19: 1 DCM / MeOH / NH4OH) gradient. Product-containing fractions were pooled and concentrated to an off-white waxy solid (0. 15 g, 72% yield). UPLC / ELSD: RT ~ 2,61 min. MS (E S): m / z (MH ) 859.2 for C52H95N3O6.VH NMR (301 MHz, CDC13) δ ppm 5.64 (s, 1H), 4.90 (m, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.66 (br. s, 2H), 3.29 (d, ,7 = 5.0 Hz, 3H), 2.60 (m, 4H), 2.43 (m, 4H), 2.31 (t, . / 7.4 Hz, 2H), 2.16 (t, , / 7.0 Hz, 2H), 1.77 (m, 7H), 1.28 (m, 53H), 0.92 (dt, ,7 = 11.5, 7.2 Hz, 12H).

[0676] 3-Butylhept-2-en-l-yl 8-bromooctanoateChemical Formula: C1sH35BrO2Molecular Weight: 375.39

[0677] 8-bromooctanoic acid (4.52 g, 20.26 mmol) and 3-butylhept-2-en-l-ol (3.80 g, 22.28 mmol) were dissolved into 67 mL of DCM. l-ethyl-3 -carbodiimide hydrochloride (4.66 g, 24.31 mmol) and 4-Dimethylaminopyridine (495 mg, 4.05 mmol) were added to start the reaction. The reaction was stirred at 25 °C for 11 h and washed with aqueous NaHCCL solution. The organic layer was dried over NaSCL and purified by silica, gel chromatography (0-15% EtOAc in Hex) to yield 3-butylhept-2-en-l-yl 8-bromooctanoate (5.84 g, 78% yield) in a clear oil. ’H NMR (301 MHz, CDCI3) δ ppm 5.31 {J, / 7. 1 Hz, 1H), 4.59 (d, J == 7.1 Hz, 2H), 3.40 (t, 7 = 6.9 Hz, 2H), 2.30 (t, 7 = 7.5 Hz, 2H), 2.05 (dt, J= 13.9, 7.5 Hz, 4H), 1.85 (p, J = 7.0 Hz, 2H), 1 .62 (tt, .J 9.3, 4.8 Hz, 3H), 1.54 - 1.17 (m, I3H), 0.90 (t, 7 7.0 Hz, 6H).

[0678] Compound 2: 3-Butylhept-2-en-l -yl 8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2- hydroxyethyl)amino)octanoateMolecular Weight: 736.22[06791 To a solution of heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate (864.40 mg, 1.63 mmol) in MeCN (6.92 mL) and CPME (6.92 mL) at 25 °C was added K2CO3 (898.61 mg, 6.50 mmol) and KI (350.79 mg, 2.11 mmol). To the emulsion, 3-butylhept-2-en-l-yl 8- bromooctanoate (622.4 mg, 1.658 mmol) was added and the reaction was stirred at 80 °C for 48 h. The reaction mixture was cooled down to room temperature and filtered through celite using heptane, which then was washed with MeCN. Heptane layer was separated and purified by silica gel chromatography (0-100% (mixture of 1 % NH4OH, 20% MeOH in DCM) in DCM) to give 3-butylhept-2-en-l-yl 8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2- hydroxyethyl)amino)octanoate (523,6 mg, 40% yield) as a pale yellow oil. UPLC / ELSD: RT = 2.58 min. MS (CI): m / z (Mi l ) 736.96 for CM LA'CL.1H NMR (301 MHz, CDC13) δ ppm 5.31 (t, J ------ 7.1 Hz, 1H), 4.86 (p, .. / 6.3 Hz, 1H), 4.58 (d, J--- 7.1 Hz, 21 1). 3.54 (t, . / - 5.3 Hz, 2H), 2.60 (t, J = 5.3 Hz, 2H), 2.47 (t, .7 = 7.5 Hz, 4H), 2.28 (td, .7 = 7.5, 5.0 Hz, 4H), 2.11 - 1.95 (m, 5H), 1.61 (t, J ------ 7.2 Hz, 5H), 1.54 I I I (m, 51 H), 0.89 (q, J ------ 7. 1 Hz, 121 i).

[0680] Ethyl (E)-3-butylhept-3-enoateChemical Formula: C13H24O2Molecular Weight: 212.33

[0681] Tri ethyl phosphonoacetate (9.15 mL, 45.70 mmol) was added dropwise over 20 min to a suspension of sodium hydride (1.83 g, 60% Wt, 45,70 mmol) in THF (13,52 mL) and the reaction mixture stirred at room temperature until gas evolution ceased. The reaction mixture was chilled to 0 °C and the nonan-5-one (5.00 g, 6. 1 mL, 35.15 mmol) was added. The reaction was gradually warmed to room temperature and was allowed to stir at reflux for 60 h. The reaction was quenched with saturated aqueous sodium bicarbonate. The aqueous phase was extracted with diethyl ether, and the organic extracts were washed with brine, dried (MgSO4), and concentrated. The crude material was purified by silica gel chromatography (0-20%EtOAC:Hex) to afford the product (1.16 g, 16% yield) in a clear oil. NMR (300 MHz, CDCh) δ ppm 5.27 (t, J = 9.0 Hz, 1H), 4.13 (q, ,7 = 9.0 Hz, 2H), 2.97 (s, 2H), 2.09 (t, J = 6.0 Hz, 211), 2.01 (q, J 6.0 Hz, 2H), 1.44-1.24 (m, 6H), 1.25 (t, . / 6.0 Hz, 3H), 0.90 (t, .7 6.0 Hz, 6H).

[0682] (E)-3-Butylhept-3-en-l-olMolecular Weight: 170.30

[0683] To a mixture of lithium aluminum hydride (249.4 mg, 6.57 mmol) in dry ether (7 mL) under ice bath and N2 (g) was added dropwise ethyl (E)-3-butylhept-3-enoate (1.16 g, 1 Eq, 5.48 mmol ) in dry ether (4 mL). The mixture was stirred at room temperature for 2.5 h then re-cooled to 0 °C. Water (1 mL per g of LAH) was added, followed by 15% sodium hydroxide (1 mL per g of LAH) and additional water (3 mL per g of LAH). The solution was stirred for a few7minutes at room temperature, filtered through a celite pad, washed with diethyl ether, dried, and concentrated. Silica gel chromatography (0-40% EtOAc.Hex) was used for purification to obtain the product (702 mg, 75% yield) in a clear oil formation.]H NMR (300 MHz, CDCh) δ ppm 5.23 (t, J 7.5 Hz, 1H), 3.64 (t, J ------ 6.0 Hz, 2H), 2.25 (dt, J ------ 6.0, 3.0 Hz, 2H), 2.08-1.94 (m, 4H), 1.44-1.21 (m, 7H), 0.90 (dt, J= 9.0, 3.0 Hz, 6H).

[0684] (2))-3-Butylhept-3-en-1 -yl 8-bromooctanoateChemical Formula: C19H35BrO?Molecular Weight: 375.39

[0685] 8-bromooctanoic acid (1.10 g, 4.95 mmol) and (7yi-3-butylhept-3-en-l -ol (702.0 mg, 4.12 mmol) were dissolved in DCM (8.24 mL). To the solution EDC (1.03 g, 5.36 mmol) and DMAP (100.7 mg, 824.4 pmol) were added to the flask sequentially. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water and was extracted with DCM. The combined organic layer was dried over MgSCh, filtered, and concentrated. The crude residue was purified using silica gel chromatography (0-20% EtOAc.Hex) to yield the product (1.24 g, 80% yiel d) in a clear oil . (300 MH1Hz, N CMDRCh) 5 ppm 5.17 (t, 7.5 Hz, 1 H), 4.12 (t, . / 6.0 Hz, 211), 3.40 (t, .7 == 6.0 Hz, 2H), 2.28 (dt, J 9.0, 3.0 Hz, 4H), 2.07-1.92 (m, 4H), 1.91-1.79 (m, 2H), 1.68-1.55 (m, 2H), 1.49-1.22 (m, 12H), 0.95-0.84 (m, 6H).

[0686] Compound 3: (E)-3-Butylhept-3-en-l-yl 8-((8-(heptadecan-9-yloxy)-8- oxooctyl)(2-hydroxyethyl)amino)octanoateChemical Formula: C46H89NO5Molecular Weight: 736.22

[0687] To a solution of heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate (600.0 mg, 1.36 mmol) and (£)-3-butylhept~3-en-l-yl 8-bromooctanoate (535.4 mg, 1.43 mmol) in MeCN (6.04 ml) and CPME (6.04 ml) was added K2CO3 (1.13 g, 8.15 mmol) and KI (248.0 mg, 1.50 mmol). The reaction was allowed to stir at 80 °C for 16 h. The volatiles were evaporated under vacuum. The residue was diluted with DCM and extracted with water. The organic layer was separated, washed with brine, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (0-20% MeOH:DCM:l% NH4OH in DCM). The product was obtained in a clear oil (585 mg, 56% yield). UPLC / ELSD: RT = 2.55 min. MS (CI): m / z iMH j 736.71 for CM bAO-, ’H NMR (300 MHz, CDC13) δ ppm 5.17 (t, J - 7.5 Hz, IH), 4.86 (pentet, J= 6.0 Hz, 1H), 4.12 (t, J= 7.5 Hz, 2H), 3.56 (br t, J= 6.0 Hz, 2H), 2.62 (br s, 2H), 2.49 (br s, 4H), 2.33-2.22 (m, 6H), 2.06-1.92 (m, 4H), 1.68-1.55 (br m, 411 ), 1.55-1.42 (br m, 8H), 1 .39-1.17 (m, 42H), 0.95-0.82 (m, 1211 ).

[0688] Ethyl 2-methyl-3-oxoheptanoateChemical Formula: C10H18O3Molecular Weight: 186.25

[0689] To a suspension of sodium hydride (2.51 g, 60% w / w, 62.71 mmol) in THF (104.5 mL) set stirring under nitrogen at room temperature was added ethyl 3-oxoheptanoate (9.30 mL, 62.71 mL) dropwise over 30 min, eliciting gas evolution. The mixture turned from a gray slurry to a clear yellow solution over 30 min of stirring. Then, iodomethane (3.90 mL, 62.71 mmol) was added and the mixture refluxed at 68 °C overnight. Then, the mixture was cooled to room temperature and quenched with slow addition of saturated aqueous ammonium chloride (30 mL). The white cloudy aqueous layer was separated from the yellow organic layer and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to a yellow oil. Theoil was taken up in minimal Hex and purified on silica in Hex with a 0-20% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (6.15 g, 57% yield). UPLC / ELSD: RT === 0.59 min. MS (ES): m / z (MIL) 187.1 for CioHjsOa. ' l l NMR (301 MHz, CDCh) δ ppm 4.20 (q, J = 7.1 Hz, 2H), 3.52 (q, J = 7.2 Hz, 1H), 2.68 - 2.40 (m, 2H), 1,64-1,54 (m, 2H), 1.41 - 1.21 (m, 8H), 0,91 (t, J ------ 7.3 Hz, 3H).

[0690] Ethyl fZ)-2-methyl-3-(((trifluoromethyl)sulfonyl)oxy)hept-2-enoateChemical Formula: C11H17F3O5S Molecular Weight: 318.31

[0691] To a suspension of sodium hydride (2.30 g, 60% w / w, 57.48 mmol) in dry toluene (33.0 niL) set stirring under nitrogen at room temperature was added a solution of ethyl 2- methyl-3-oxoheptanoate (5.95 g, 31.94 mmol) in 33.0 mL dry toluene dropwise over 30 minutes, eliciting gas evolution. The mixture was then stirred at 85 °C for 1.5 h, turning from a gray slurry to a clear yellow solution. The mixture was then cooled to 0 °C and trifluoromethanesulfonic anhydride (8.06 mL, 47.90 mmol) was added dropwise. The mixture was stirred at 0 °C for 2 h, and then quenched with 25 mL cold water while still at 0 °C. The aqueous layer was separated from the yellow organic layer and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with water and brine, dried over sodium sulfate, fdtered, and concentrated to a yellow oil. The oil was taken up in minimal Hex and purified on silica in Hex with a 0-10% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (8.30 g, 82% yield). UPLC / ELSD: RT = 1.32 min. MS (ES): m / z (MH i 318.7 for C11H17F3O5S. H NMR (301 MHz, CDCh) δ ppm 4.29 (q, J 7.2 Hz, 21 1), 2.44 (t, . / 7.6 Hz, 2H), 2.00 (s, 311 ), 1.67 - 1.51 (m, 21 1), 1.48 - 1.27 (m, 511), 0.96 (t, J = 7.3 Hz, 3H).

[0692] Ethyl 3-butyl-2-methylhept-2-enoateCMolecular Weight: 226.36

[0693] A solution of copper cyanide (8.39 g, 93,64 mmol) in diethyl ether (125,0 mL) was cooled to -78 °C. A solution of butylmagnesium chloride (2.0M in THF, 30.0 mL, 60.0 mmol) was added dropwise and after addition the mixture was stirred for 5 min at -78 °C and then foran hour at 0°C. Then, a solution of ethyl (Z>-2-methyl-3-(((trifluoromethyl)sulfonyl)oxy)hept- 2 -enoate (5.96 g, 18.73 mmol) in 10 mL diethyl ether was added slowly at 0 °C and the mixture was allowed to stir at 0°C for 2 h. The reaction was quenched with slow addition of 30 mL saturated aqueous ammonium chloride at 0 °C. The mixture was filtered through celite, washing the celite pad with additional diethyl ether. The organic layer was separated and the aqueous layer was extracted with diethyl ether. All organic layers were combined, washed with water and brine, dried over sodium sulfate, and concentrated to a colorless oil. The oil was taken up in minimal Hex and purified on silica in Hex with a 0-15% EtOAc gradient. Productcontaining fractions were pooled and concentrated to a colorless oil (3.01 g, 71% yield). UPLC / ELSD: RT = 1.77 min. MS (ES): m / z (MH+) 227.3 for C14H26O2.SH NMR (301 MHz, CDCh) δ ppm 4.11 (q, J = 7.1 Hz, 2H), 2.22 (t, J = 7.7 Hz, 2H), 2.03 (t, J= 7.8 Hz, 2H), 1.78 (s, 311), 1.42 - 1.16 (m, 1 1 H), 0.84 (q, J 7.1 Hz, 6H).

[0694] 3 -Butyl -2-methylhept-2-en- 1 -olChemical Formula:Molecular Weight: 184.32

[0695] To a solution of ethyl 3-butyl-2-methylhept-2-enoate (3.01 g, 13.30 mmol) in dry' DCM (33.2 mL) stirring at -78°C under nitrogen was added diisobutyl aluminum hydride (IM in DCM, 39.89 mL, 39.89 mmol) dropwise. The mixture stirred for 3 hours at -78 °C. The mixture was quenched with 25 mL saturated aqueous Rochelle's salt and allowed to gradually warm to room temperature. The aqueous layer was separated and extracted with DCM and all organic layers were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated to a colorless oi 1. The oil was stored over night at -20 °C prior to purifi cation. The oil was taken up in minimal Hex and purified on silica in Hex with a 0-15% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (2.06 g, 83% yield). UPLC / ELSD: RT = 1.19 min. MS (ES): m / z (MH+) 167.1 for C12H24O. H W1R (301 MHz, CDCI3) δ ppm 4.13 (s, 2H), 2.06 (m, 4H), 1.77 (s, 3H), 1.32 (m, 8H), 1.10 (hr. s, 1H), 0.92 (m, 6H).

[0696] 3-Butyl-2-methylhept-2-en-1 -yl 8-bromooctanoateChemical Formula: C2QH37BrO2Molecular Weight: 389.42

[0697] To a solution of 8-bromooctanoic acid (2.49 g, 11.18 mmol) and 3-butyl-2- methylhept-2-en-l-ol (2.06 g, 11.18 mmol) in dry DCM (74.51 mL) stirring under nitrogen was added 4-dimethyl aminopyridine (1.37 g, 11.18 mmol) and EDC.HC1 (2.57 g, 13.41 mmol). The reaction was allowed to proceed at room temperature for 2.5 h and was diluted with DCM and quenched with 25 mL water. The organic layer was separated, washed with 5% aqueous HC1, saturated aqueous sodium bicarbonate, and brine, dried over sodium sulfate, filtered, and concentrated to an oil. The oil was then taken up in Hex and purified on silica in Hex with a 0- 10% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (3.73 g, 84% yield). UPLC / ELSD: RT = 2.48 min. MS (ES): w / z (MH ) 388.1 for (%H vBrO.x ' H NMR (301 MHz, CDCH) 6 ppm 4.48 (s, 2H), 3.33 (t, . / 6.8 Hz, 2H), 2.24 (t, J 7.4 Hz, 2H), 2.05 - 1.91 (m, 4H), 1.78 (m, 2H), 1.31 (m, 5H), 1.30 (m, 14H), 0.84 (m, 6H).

[0698] Compound 4: 3-Butyl-2-methylhept-2-en-l-yl 8-((8-(heptadecan-9-yloxy)-8- oxooctyl)(2 -hydroxy ethyl)amino)octanoateMolecular Weight: 750.25

[0699] To a solution of 8-(heptadecan-9-y loxy)-N-(2 -hydroxy ethyl)-8-oxooctan-l - aminium carboxyformate (0.50 g, 0.94 mmol) and 3-butyl-2-methylhept-2-en-l-yl 8- bromooctanoate (0,40 g, 1 .03 mmol) in dry? propionitrile (4.70 mL) stirring under nitrogen was added potassium carbonate (0.78 g, 5.64 mmol) and potassium iodide (0.20 g, 1.22 mmol). The reaction was heated to 85 °C and allowed to proceed overnight. The reaction mixture was then cooled to room temperature, diluted with 10 mL heptane and filtered through celite, washing the celite pad with additional heptane. The filtrate was concentrated to an oil. The oil was taken up in minimal Hex and purified on silica in Hex with a 0-40% EtOAc gradient. Productcontaining fractions were pooled and concentrated to a colorless oil (0.35 g, 47% yield).UPLC / ELSD: RT == 2.60 min. MS ( ES): m / z (MH+) 750.8 for C ri b; NO5.]H NMR (301 MHz, CDCh) δ ppm 4.88 (p, J = 6.3 Hz, IH), 4.59 (s, 2H), 3.55 (br. s, 2H), 2.60 (m, 2H), 2.46 (m, 4H), 2.38 - 2.24 (m, 4H), 2.14 - 2.00 (m, 4H), 1.70 (s, 311), 1.64 (m, 4H), 1.52 (m, 8H), 1.32 (m, 44H), 0.93 (m, 12H).

[0700] Ethyl 2-ethyl-3-oxoheptanoateChemical Formula:Molecular Weight: 200.28

[0701] To a suspension of sodium hydride (1.39 g, 60% w / w, 34.84 mmol) in THF (69.7 mL) set stirring under nitrogen at room temperature was added ethyl 3-oxoheptanoate (6.00 g,34.84 mL) dropwise over 30 minutes, eliciting gas evolution. The mixture turned from a gray slurry to a clear yellow7solution over 30 minutes of stirring. Then, bromoethane (2.58 mL,34.84 mmol) was added and the mixture refluxed at 68 °C overnight. Then, the mixture was cooled to room temperature and quenched with slow7addition of saturated aqueous ammonium chloride (30 mL). The white cloudy aqueous layer was separated from the yellow organic layer and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to a yellow oil. The oil was taken up in minimal Hex and purified on silica in Hex with a 0-20% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (3.26 g, 47% yield).{H NMR (301 MHz, CDCh) δ ppm 4.20 (q, J ----- 7.2 Hz, IH), 3.37 (t, J 7.4 Hz, IH), 2,66 - 2.34 (m, 2H), 2.00 - 1.79 (m, 2H), 1.67 - 1.49 (m, 2H), 1.42 -- 1.22 (m, 5H), 1.04 - 0.85 (m, 6H).

[0702] Ethyl fZ)-2-ethyl-3-(((trifluoromethyl)sulfonyl)oxy)hept-2-enoateChemical Formula: C12H1sF3O5S Molecular Weight: 332.33

[0703] To a suspension of sodium hydride (1.17 g, 60% w / w, 29.31 mmol) in dry7toluene (16.0 mL) set stirring under nitrogen at room temperature was added a solution of ethyl 2-ethyl- 3-oxoheptanoate (3.26 g, 16.28 mmol) in 16.0 mL dry / toluene dropwise over 30 min, eliciting gas evolution. The mixture was then stirred at 85 °C for 1.5 h, turning from a gray slurry to a clear yellow7solution. The mixture w7as then cooled to 0 °C and trifluoromethanesulfonic anhydride (4. 11 mL, 24.42 mmol) was added dropwise. The mixture w7as stirred at 0 °C for 2h, and then quenched with 25 mL cold water while still at 0 °C. The aqueous layer was separated from the yellow organic layer and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with water and brine, dried over sodium sulfate, filtered, and concentrated to a yellow oil. The oil was taken up in minimal Hex and purified on silica in Hex with a 0-20% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (4.60 g, 85% yield). UPLC / ELSD and MS data was not obtained for this compound. H NMR (301 MHz, CDCI3) δ 4.36 - 4.23 (m, 2H), 2.41 (dt., . / 18.5, 7.4 Hz, 4H), 1.67 - 1.51 (m, 2H), 1.49 -- 1.23 (m, 6H), 1.11 (t, J= 7.5 Hz, 3H), 0.96 (t, ,7 = 7.3 Hz, 3H).

[0704] Ethyl 3-butyl-2-ethylhept-2-enoateChemical Formula: C^^gCL Molecular Weight: 240.39

[0705] A solution of copper cyanide (6.20 g, 69.21 mmol) in diethyl ether (100.0 mL) was cooled to -78 °C. A solution of butylmagnesium chloride (2.0M in THF, 22.15 mL, 44.29 mmol) was added dropwise and after addition the mixture was stirred for 5 min at -78 °C and then for an hour at 0°C. Then, a solution of ethyl (zp-2~ethyl-3- (((trifluoromethyl)sulfonyl)oxy)hept-2-enoate (4.60 g, 13.84 mmol) in 10 mL diethyl ether was added slowly at 0 °C and the mixture was allowed to stir at 0 °C for 2 h. The reaction was quenched with slow addition of 25 mL saturated aqueous ammonium chloride at 0CC. The mixture was filtered through celite, washing the celite pad with additional diethyl ether. The organic layer was separated and the aqueous layer was extracted with diethyl ether. All organic layers were combined, washed with water and brine, dried over sodium sulfate, and concentrated to a colorless oil. The oil was taken up in minimal Hex and purified on silica in Hex with a 0-10% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (2.12 g, 64% yield). UPLCZELSD; RT - 1.91 min. MS (ES): m / z (MH+) 241.2 for C15H28O2. ’HNMR (301 MHz, CDCI3) δ ppm 4.12 (q, ,7 = 7.1 Hz, 2H), 2.22 (q, J= 1A Hz, 2H), 2.13 (q, J 8.5 Hz, 2H), 2.01 (t, . / 7.8 Hz, 2H), 1.29 (m, 5H), 1.23 (t, . / 14.2 Hz, 6H), 0.91 (t, J= 7.5 Hz, 3H), 0.84 (q, J= 7.1 Hz, 6H).

[0706] 3-Butyl-2-ethylhept-2-en-l-olChemical Formula: C13H26O Molecular Weight: 198.35

[0707] To a solution of ethyl 3-butyl-2-ethylhept-2-enoate (2.12 g, 8.82 mmol) in dry DCM (22. 1 mL) stirring at -78°C under nitrogen was added diisobutyl aluminum hydride (IM in DCM, 26,46 mL, 26.46 mmol) dropwise. The mixture stirred for 3 hours at -78 °C. The mixture was quenched with 25 mL saturated aqueous Rochelle's salt, and allowed to gradually warm to room temperature. The aqueous layer was separated and extracted with DCM, and all organic layers were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated to a colorless oil. The oil was taken up in minimal Hex and purified on silica in Hex with a 0-20% EtOAc gradient to obtain the product in a colorless oil (1.23 g, 69% yield). UPLC. / ELSD: RT = 1.41 min. MS (ES): m / z (MH ) 181.1 for (' i.d h-O. H \MR (301 MHz, CDCI3) 5 ppm 4.14 (s, 2H), 2.17 (q, . / 7.5 Hz, 2H), 2.12 - 1.95 (m, 4H), 1.36 (m, 8H), 1.02 (t, J = 7.5 Hz, 4H), 0.94 (m, 6H).

[0708] 3-Butyl-2-ethylhept-2-en-1 ~yl 8-bromooctanoateChemical Formula: C21H3SBrO2Molecular Weight: 403.44

[0709] To a solution of 8-bromooctanoic acid (1.39 g, 6.23 mmol) and 3-butyl-2~ethylhept- 2-en-l-ol (1.24 g, 6.23 mmol) in dry DCM (41.51 mL) stirring under nitrogen \vas added 4- dimethylaminopyridine (0.76 g, 6.23 mmol) and 1 -ethyl-3-(3~ dimethylaminopropylicarbodiimide hydrochloride ( 1.43 g, 7.47 mmol). The reaction was allowed to proceed at room temperature for 2.5 h and was diluted with DCM and quenched with 25 mL water. The organic layer was separated, washed with 5% aqueous HC1, saturated aqueous sodium bicarbonate, and brine, dried over sodium sulfate, filtered, and concentrated to an oil. The oil was then taken up in Hex and purified on silica in Hex with a 0-10% EtOAc gradient. Product-containing fractions were pooled and concentrated to a colorless oil (2.09 g, 84% yield). UPLC / ELSD: RT === 2.58 min. MS (ES); m / z (MIL) 404.1 for C.fo foBrO?!H NMR (301 MHz, CDCI3) δ ppm 4.53 (s, 2H), 3.33 (t, J= 6.8 Hz, 2H), 2.23 (t, J= 7.4 Hz, 2H),2.09 - 1.91 (m, 611 ), 1.78 (p, . / 6.8 Hz, 2H), 1.55 (m, 2H), 1.31 (d, . / 30.5 Hz, 2H), 1.26 (m, 12H), 0.90 (t, J= 7.5 Hz, 3H), 0.84 (t, J= 7.0 Hz, 6H).

[0710] Compound 5: 3-Butyl-2-ethylhept-2-en-1-yl 8-((8-(heptadecan-9-yloxy)-8- oxooctyl)“(2-hydroxyethyl)amino)octanoate

[0711] To a solution of 8-(heptadecan-9-yloxy)-N-(2-hydroxyethyl)-8-oxooctan-l- aminium carboxyformate (0.50 g, 0.94 mmol) and 3-butyl-2-ethylhept-2-en-l”yl 8- bromooctanoate (0.42 g, 1 .03 mmol) in dry propionitrile (4.70 mL) stirring under nitrogen was added potassium carbonate (0.78 g, 5.64 mmol) and potassium iodide (0.20 g, 1.22 mmol). The reaction was heated to 85 °C and allowed to proceed overnight. The reaction mixture was then cooled to room temperature, diluted with 10 mL heptane and filtered through celite, washing the celite pad with additional heptane. The filtrate was concentrated to an oil. The oil was taken up in minimal DCM and purified on silica with a 0-30% (80: 19: 1 DCM / MeOH / NH.-iOH) gradient. Product-containing fractions were pooled and concentrated to a colorless oil which was taken up in Hex and purified on silica in Hex with a 0-35% EtOAc gradient. Productcontaining fractions were pooled and concentrated to a colorless oil (0.37 g, 51% yield). UPLC / ELSD: RT - 2.65 min. MS (ES): m / z (MH+) 764.8 for CM foNOs!H NMR (301 MHz, CDCI3) δ ppm 4.88 (p, J = 6.2 Hz, 1H), 4.61 (s, 2H), 3.55 (br. s, 2H), 2.60 (m, 2H), 2.46 (m, 411). 2.31 (q, J IA Hz, 4H), 2.08 (m, 6H), 1.64...

Claims

What is claimed is:

1. A compound of Formula (I*):or a pharmaceutically acceptable salt thereof, wherein: one is a single bond, and the otheris a double bond; n is 3, 4, 5, or 6;M is -<)-('( O)-::and -C( O)-O-,::, wherein * indicates attachment to L;L is a bond or -CH?-;R1is H, C1-12 alkyl, or C2-12 alkenyl;R2is H, C1-12 alkyl, or C2-12 alkenyl;RJis C1-12 alkyl or C2-1? alkenyl; wherein at least one of R\ R2, and R3is a C1-12 alkyl or C2-12 alkenyl; m is 3, 4, 5, or 6;each is a single bond or a double bond, wherein no more than one is a double bond,M’ is -O-C(=O)-* and -C(=O)-O-*, wherein * indicates attachment to L’;L’ is a bond or -CH2-,R1is H, Cm2 alkyl, or C2-12 alkenyl,R2is H, C1-12 alkyl, or C2-12 alkenyl;R’’ is H, C1-12 alkyl, or C2-J? alkenyl; wherein at least one of R:, R2, and R3is a C1-12 alkyl or C2-1? alkenyl;RHis -(CH2)P-Q, -(CH2)P-NRG-T-Q, -(CH -),-NRG-S( O)?-T-Q, -(CH2)P-NRG-C(= O)H or -(CH2)P-NRG-C(=O)-T-Q; p is 1, 2, 3, 4, or 5; each RGindependently is H, C1.6 alkyl, or C2-6 alkenyl;T is a bond, C1-3 alkylene, C2-3 alkenylene, or C2-3 alkynylene;Q is -OH, -0-(Cn6 alkyl), C 1-6 alkyl, C2-5 alkenyl, C2-6 alkynyl, C3-10 cycloalkyl, 5-10 membered heteroaryl, C6-io and, or 3-12 membered heterocycloalkyl, wherein the -O-(C 1-6 alkyl), C 1-6 alkyl, C2-5 alkenyl, C3-10 cycloalkyl, or 3-12 membered heterocycloalkyl is optionally substituted with one or more RQ; and each RQindependently is oxo, cyano, -OH, -O-(C 1-6 alkyl), -NH2, -NH(C 1-6 alkyl), - N(C 1-6 alkyl)2, -C(=O)-(Ci~6 alkyl), -O-C(=O)-(C 1-6 alkyl), -NR-C(=O)-(C 1-6 alkyl), C1-6 alkyl, C2-6 alkenyl, -(C 1-6 alkyl)-OH, C2-6 alkenyl, or C3-10 cycloalkyl.

2. The compound of claim 1, wherein the compound is of Formula (I**):or a pharmaceutically acceptable salt thereof, wherein: one is a single bond, and the otheris a double bond; n is 3, 4, 5, or 6;M is -<)-('( O)-::or -C(:::O)-O-*, wherein * indicates attachment to L;L is -CH2-;R1is H;R2is C3..5 alkyl;R3is C3.5 alkyl; m is 3, 4, 5, or 6;each is a single bond or a double bond, wherein no more than one is a double bond;M’ is -C(:::O)-O-*, wherein * indicates attachment to L’;L’ is a bond;R1is C-7-io alkyl,R2is C5-8 alkyl;R3is I I.RHis -(CH2)P-Q; p is 1, 2, 3, 4, or 5; and Q is -OH.3, The compound of any one of the previous claims, wherein the compound is ofFormula (I):or a pharmaceutically acceptable salt thereof.

4. The compound of any one of the previous claims, wherein the compound is ofFormula (II):or a pharmaceutically acceptable salt thereof.

5. The compound of any one of the previous claims, wherein the compound is of Formula (III):or a pharmaceutically acceptable salt thereof.

6. The compound of any one of the previous ciaims, wherein the compound is of Formula (IV):or a pharmaceutically acceptable salt thereof.

7. The compound of any one of the previous claims, wherein the compound is of Formula (V):or a pharmaceutically acceptable salt thereof.

8. The compound of any one of the previous claims, wherein the compound is ofFormula (VI):or a pharmaceutically acceptable salt thereof.

9. The compound of any one of the previous claims, wherein RHis -(CH2)P-Q or - (CH2)p-NRG-T-Q.

10. The compound of any one of the previous claims, wherein p is 2 or 3.

11. The compound of any one of the previous claims, wherein RGis H.

12. The compound of any one of the previous claims, wherein T is a bond.

13. The compound of any one of the previous claims, wherein Q is OH or C3-6 cycloalkyl substituted with one or more oxo, -NH2, -NH(C 1-6 alkyl), or -N(Cu-6 alkyl)2.

14. The compound of any one of the previous claims, wherein n is 5.

15. The compound of any one of the previous claims, wherein m is 5.

16. The compound of any one of the previous claims, wherein R1is H, methyl, or ethyl.

17. The compound of any one of the previous claims, wherein R2is C2-12 alkyl.

18. The compound of any one of the previous claims, wherein R3is methyl, butyl, or pentyl.

19. The compound of any one of the previous claims, wherein R3is hexenyl.

20. The compound of any one of the previous claims, wherein R1is H, methyl, or ethyl.

21. The compound of any one of the previous claims, wherein R2is C2-12 alkyl.

22. The compound of any one of the previous claims, wherein R3is H, methyl, butyl, or pentyl.

23. The compound of any one of the previous claims, wherein R3’ is hexenyl.

24. A compound having the any one of the structures shown in Table I, or a pharmaceutically acceptable salt thereof.

25. A lipid nanoparticle (LNP) comprising the lipid of any one of the preceding claims, a phospholipid, a structural lipid, and a PEG lipid.

26. The LNP of any one of the preceding claims, comprising about 40 mol % to about 60 mol % said lipid, about 0 mol % to about 20 mol % phospholipid, about 30 mol % to about 50 mol % structural lipid, and about 0 mol % to about 5 mol % PEG lipid.

27. A method of delivering a therapeutic and / or prophylactic agent to a cell within a subject, the method comprising administering to the subject the LNP of any one of the preceding claims.

28. A method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject the LNP of any one of the preceding claims.

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