Lipid nanoparticles for hepatic delivery

Abstract

Provided herein are lipid nanoparticles composition comprising a lipid component which comprises: (i) a first lipid wherein the first lipid is an ionizable cationic lipid, (ii) a second lipid wherein the second lipid is separate from the first lipid, (iii) a phospholipid, and (iv) a PEG-lipid wherein the second lipid is a lipid having a structural formula S-I'a, S-I'b, or S-I'c. Also, provided herein is a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering an effective amount of the lipid nanoparticle composition disclosed herein.

Description

LIPID NANOPARTICLES FOR HEPATIC DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 647,251 filed May 14, 2024, and U.S. Provisional Patent Application No. 63 / 737,003 filed December 20, 2024, each of which is incorporated by reference herein in its entirety..SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. The XML file, created on May 12, 2025, is named 061529-513001WO.xml and is 17,789 bytes in size.BACKGROUND

[0003] There is an unmet need for lipid nanoparticles for systematical delivery to the liver to the subject in need thereof. The present disclosure provides lipid nanoparticle compositions specifically delivered to the liver for treatment of liver disease.SUMMARY

[0004] In some aspects, disclosed herein are lipid nanoparticle compositions (LNPs) comprising a lipid component which comprises: (i) a first lipid wherein the first lipid is an ionizable cationic lipid, (ii) a second lipid wherein the second lipid is separate from the first lipid, (iii) a phospholipid, and (iv) a polyethylene glycol liquid (PEG-lipid); wherein: the second lipid is a lipid having a structural formula:wherein R1and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group, and R3and R3' are each independently C1-C6alkyl or a substituted version thereof.

[0005] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component which comprises: (i) a first lipid wherein the first lipid is an ionizable cationic lipid, (ii) a second lipid wherein the second lipid is separate from the first lipid, (iii) a phospholipid, and (iv) a PEG-lipid wherein: the second lipid is a lipid having a structural formula:wherein R1and R2are each independently C8-C24alkyl, C8- C24alkenyl, or a substituted version of either group, and R3and R3are each independently C1-C6alkyl or a substituted version thereof; and / or the phospholipid is a phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, or a derivative or analog thereof.

[0006] In some embodiments, in the compound of formula (S-I’a), R3and R3are each independently unsubstituted C1-C6alkyl. In some embodiments, in the compound of formula (S-I’a), R3and R3are each independently methyl, ethyl, or propyl. In some embodiments, in the compound of formula (S-I’a), R3 and R3' are each methyl. In some embodiments, in the compound of formula (S-I’a), R1and R2are each independently unsubstituted C8-C24alkenyl. In some embodiments, in the compound of formula (S-I’a), R1and R2are each independently unsubstituted C8-C24alkyl. In some embodiments, the second lipid is 1,2-dioleoyl-3- dimethylammonium-propane (18: 1 DODAP). In some embodiments, the second lipid is 1,2- dimyristoyl-3-dimethylammonium-propane (14:0 DAP).

[0007] In other aspects, disclosed herein are lipid nanoparticle compositions comprising a lipid component which comprises: (i) a first lipid wherein the first lipid is an ionizable cationic lipid, (ii) a second lipid wherein the second lipid is separate from the first lipid, (iii) a phospholipid, and (iv) a PEG-lipid, wherein: the second lipid is a lipid having a structural formula:wherein RT1aand RT1bare each independently C4-C18alkyl, C4-C18alkenyl, or a substituted version of either group, RC1a, RC1band, RC1care each independently C1-C6alkyl or a substituted version thereof; and xl and x2 are each independently 1, 2, 3, or 4.

[0008] In some embodiments, in the compound of formula (S-I’b) RT1aand RT1bare each independently unsubstituted C4-C18alkyl or C4-C18alkenyl. In some embodiments, in the compound of formula (S-I’b) RT1aand RT1bare each independently unsubstituted C5-C8alkyl or C6-C20alkenyl. In some embodiments, in the compound of formula (S-I’b) RC1a, RC1b, andRC1care each independently unsubstituted C1-C6alkyl. In some embodiments, in the compound of formula (S-I’b) RC1a, RC1b, and RC1care each independently methyl. In some embodiments, in the compound of formula (S-I’b) RC1a, RC1b, and RC1care each independently ethyl. In some embodiments, in the compound of formula (S-I’b) x 11and x2 are each 1. In some embodiments, in the compound of formula (S-I’b) x1and x2 are each 2. In some embodiments, the compound of formula (S-I’b) is 2A11-SC7, having the following structure:

[0009] In yet another aspect, disclosed herein are lipid nanoparticle compositions comprising a lipid component which comprises: (i) a first lipid wherein the first lipid is an ionizable cationic lipid, (ii) a second lipid wherein the second lipid is separate from the first lipid, (iii) a phospholipid, and (iv) a PEG-lipid, wherein: the second lipid is a lipid having a structural formula:wherein RT2aand RT2bare each independently C4-C18alkyl, C4-C18alkenyl, or a substituted version of either group, RC2aand, RC2bare each independently C1-C6alkyl or a substituted version thereof, and yl is 1, 2, 3, or 4.

[0010] In some embodiments, in the compound of formula (S-I’c) RT2aand RT2bare each independently unsubstituted C4-C18alkyl or C4-C18alkenyl. In some embodiments, in the compound of formula (S-I’c) RT2aand RT2bare each independently unsubstituted C5-C8alkyl or C6-C20alkenyl. In some embodiments, in the compound of formula (S-I’c) RC2aand RC2bare each independently unsubstituted C1-C6alkyl. In some embodiments, in the compound of formula (S-I’c) RC2aand RC2bare each independently methyl. In some embodiments, in the compound of formula (S-I’c) RC2aand RC2bare each independently ethyl. In some embodiments, in the compound of formula (S-I’c) y1is 1. In some embodiments, in the compound of formula (S-I’c) y1is 2. In some embodiments, in the compound of formula (S-I’c) RT2aand RT2bare each a group having the following structure: wheredenotes the point of attachment. In some embodiments, the compound of formula (S-I’c) is a 2A9-Cit, having the following structure:

[0011] In some embodiments of the lipid nanoparticle composition disclosed herein, the phospholipid is selected from 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), 1-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-sn-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 -phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2- diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl -phosphatidyl-ethanolamine(DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), 1- stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, and lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) sphingomyelin.

[0012] In some embodiments of the lipid nanoparticle composition disclosed herein the PEG-lipid is present in an amount of from about 0.5 mol % to about 5 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid is present in an amount of from about 2 mol % to about 4 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid is present in an amount of about 3 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid is present in an amount of about 4 mol % of the total lipids in the lipid component. In some embodiments, the first lipid is present in an amount of from about 5 mol % to about 40 mol % of the total lipids in the lipid component. In some embodiments, the first lipid is present in an amount of from about 5 mol % to about 30 mol % of the total lipids in the lipid component. In some embodiments, the first lipid is present in an amount of from about 10 mol % to about 20 mol % of the total lipids in the lipid component. In some embodiments, the first lipid is present in an amount of from about 15 mol % to about 20 mol % of the total lipids in the lipid component. In some embodiments, the first lipid is present in an amount of about 20 mol % of the total lipids in the lipid component. In some embodiments, the first lipid is present in an amount of about 19 mol % of the total lipids in the lipid component. In some embodiments, the phospholipid is present in an amount of from about 5 mol % to about 25 mol % of the total lipids in the lipid component. In some embodiments, the phospholipid is present in an amount of from about 10 mol % to about 20 mol % of the total lipids in the lipid component. In some embodiments, the phospholipid is present in an amount of about 20 mol % of the total lipids in the lipid component. In some embodiments, the phospholipid is present in an amount of about 19 mol % of the total lipids in the lipid component. In some embodiments, the second lipid separate from the first ionizable cationic lipid is present in an amount of from about 5 mol % to about 40 mol % of the total lipids in the lipid component. In some embodiments, the second lipid separate from the first ionizable cationic lipid is present in an amount of from about 10 mol % to about 40 mol % of the total lipids in the lipid component. In some embodiments, the second lipid separate from the first ionizable cationic lipid is present in an amount of from about 10 mol % to about 30 mol % of the total lipids in the lipid component. In some embodiments, the second lipidseparate from the first ionizable cationic lipid is present in an amount of about 20 mol % of the total lipids in the lipid component.

[0013] In some embodiments of the lipid nanoparticle composition described herein, the first lipid is present in an amount from about 10 mol % up to 20 mol % of the total lipids in the lipid component; the PEG-lipid is present in a total amount of about 4 mol % of the total lipids in the lipid component; the second lipid separate from the first ionizable cationic lipid is present in an amount of about 10 mol % to about 30 mol % of the total lipids in the lipid component; and the phospholipid is present in an amount of about 10 mol % up to 20 mol % of the total lipids in the lipid component. In some embodiments, the first lipid is present in an amount from about 10 mol % up to 20 mol % of the total lipids in the lipid component; the PEG-lipid is present in a total amount of about 3 mol % of the total lipids in the lipid component; the second lipid separate from the first ionizable cationic lipid is present in an amount of about 10 mol % or about 30 mol % of the total lipids in the lipid component; and the phospholipid is present in an amount of about 10 mol % up to 20 mol % of the total lipids in the lipid component.

[0014] In some embodiments, the lipid nanoparticle composition described herein further comprises a sterol. In some embodiments, the sterol is present in an amount of from about 20 mol % to about 50 mol % of the total lipids in the lipid component. In some embodiments, the sterol is present in an amount of from about 30 mol % to about 40 mol % of the total lipids in the lipid component. In some embodiments, the sterol is present in an amount of about 38 mol % of the total lipids in the lipid component. In some embodiments, the sterol is cholesterol.

[0015] In some embodiments of the lipid nanoparticle composition described herein, the phospholipid IS 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). In some embodiments, the phospholipid is 1,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE). In some embodiments, the phospholipid is 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC). In some embodiments, the phospholipid is dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the phospholipid is 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE). In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0016] In some embodiments of the lipid nanoparticle composition described herein, thePEG-lipid is 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG).

[0017] In some embodiments the lipid nanoparticle composition described herein, the first lipid is a compound selected from Table 4. In some embodiments, the first lipid is a compoundselected from Table 5A. In some embodiments, the first lipid is a compound selected fromTable 5B

[0018] In some embodiments, the first lipid is a compound having the following structure:wherein:RD1is a C1-C4alkyl; z1 and z2 are each independently 1, 2, or 3; and z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0019] In some embodiments, the first lipid is:

[0020] In some embodiments, the lipid nanoparticle composition described herein further comprises a payload. In some embodiments, the payload comprises a polypeptide or a protein. In some embodiments, the polypeptide or protein is selected from: alpha- 1 -antitrypsin (A1AT), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose-6-phosphatase (G6Pase), glucose-6- phosphate translocase (G6PT), lysosomal glucocerebrosidase (GB), Niemann-Pick Cl protein (NPC1), Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor IX, galactose- 1 -phosphate uridylyltransferase, galactokinase, UDP-galactose 4-epimerase, transthyretin, phenylalanine hydroxylase (PAH), homogentisate 1,2-di oxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT),argininosuccinate lyase (ASL), argininosuccinate synthetase (AS SI), P-type ATPase protein FIC-1, alpha-galactosidase A, acid ceramidase, acid α-L-fucosidase, acid β-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid α-mannosidase, β- mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid β-galactosidase, acid α-glucosidase, β-hexosaminidase B, heparan-N-sulfatase, alpha-N- acetylglucosaminidase, acetyl-CoA:α-glucosaminide N-acetyltransferase, N- acetylglucosamine-6-sulfate sulfatase, alpha-N-acetylgalactosaminidase, sialidase, β- glucuronidase, β-hexosaminidase A. In some embodiments, the payload comprises a nucleic acid. In some embodiments, the nucleic acid is selected from a small interfering ribonucleic acid (siRNA), a microRNA (miRNA), a primary microRNA (pri-miRNA), a messenger RNA (mRNA), a cluster regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer RNA (tRNA), an antisense oligonucleotide (ASO), a guide RNA, a double stranded DNA (dsDNA), a single stranded DNA (ssDNA), a single stranded RNA (ssRNA), and a double stranded RNA (dsRNA). In some embodiments, the payload comprises a small interfering RNA (siRNA). In some embodiments, the payload comprises an mRNA. In some embodiments, the mRNA encodes a gene-editing system or component thereof. In some embodiments, the gene-editing system or component thereof comprises a cluster regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), and a guide RNA. In some embodiments, the mRNA encodes a protein selected from: alpha- 1 -antitrypsin (Al AT), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose- 6-phosphatase (G6Pase), glucose-6-phosphate translocase (G6PT), lysosomal glucocerebrosidase (GB), Niemann-Pick Cl protein (NPC1), Niemann -Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor IX, galactose- 1 -phosphate uridylyltransferase, galactokinase, UDP -galactose 4-epimerase, transthyretin phenylalanine hydroxylase (PAH), homogentisate 1,2-di oxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL), argininosuccinate synthetase (AS SI), P-type ATPase protein FIC-1, alpha-galactosidase A, acid ceramidase, acidα-L-fucosidase, aacciidd β-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid α-mannosidase, β-mannosidase, arylsulfatase B, arylsulfatase A, N- acetylgalactosamine-6-sulfate sulfatase, acid β-galactosidase, acid α-glucosidase, β- hexosaminidase B, heparan-N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:α- glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alpha-N- acetylgalactosaminidase, sialidase, β-glucuronidase, β-hexosaminidase A. In some embodiments, the payload comprises a guide RNA. In some embodiments, the first lipid is present in an amount of about 19 mol % of the total lipids in the lipid component; the PEG- lipid is present in a total amount of about 4 mol % of the total lipids in the lipid component; the second lipid separate from the first ionizable cationic lipid is present in an amount of about 20 mol % of the total lipids in the lipid component; the phospholipid is present in an amount of about 19 mol % of the total lipids in the lipid component; and the cholesterol is present in an amount of about 38 mol % of the total lipids in the lipid component. In some embodiments, the first lipid is present in an amount about 19 mol % of the total lipids in the lipid component; the PEG-lipid is present in a total amount of about 3 mol % of the total lipids in the lipid component; the second lipid separate from the first ionizable cationic lipid is present in an amount of about 20 mol % of the total lipids in the lipid component; and the phospholipid is present in an amount of about 19 mol % of the total lipids in the lipid component; and the cholesterol is present in an amount of about 39 mol % of the total lipids in the lipid component.

[0021] In some embodiments, a lipid nanoparticle composition of the disclosure delivers a payload preferentially to a liver cell, or to both a liver cell and a lung cell.

[0022] In some embodiments, described herein are methods of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering an effective amount of a lipid nanoparticle composition described herein. In some embodiments, the method comprises selectively delivering the payload to a target organ. In some embodiments, the method comprises selectively delivering the payload to a target cell.

[0023] In other aspects, disclosed herein are methods of selectively delivering a payload to a target organ of a subject in need thereof, the method comprising administering to the subject an effective amount of a lipid nanoparticle composition described herein. In some embodiments, the target organ is the liver. In some embodiments, the payload is an mRNA and wherein the selectively delivering results in expression of a protein encoded by the mRNA in a cell of the target organ. In some embodiments, the payload is a polynucleotide encoding a gene product and wherein the selectively delivering results in expression of the gene product in a cell of the target organ and optionally wherein the gene product is functional in the cell ofthe target organ. In some embodiments, the payload comprises an mRNA encoding a geneediting system or component thereof and wherein the selectively delivering results in altered expression of a protein targeted by the gene-editing system in a cell of the target organ. In some embodiments, the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition is at least 200%, 300%, at least 400%, at least 500%, or at least 600% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

[0024] In some embodiments, the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition is about 200% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition. In some embodiments, the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition is about 300%, 400%, about 500%, or about 600% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

[0025] In some embodiments, the reference lipid nanoparticle composition comprises a second ionizable cationic lipid that does nnoott comprise aa 1,2-dimyristoyl-3- dimethylammonium-propane but is otherwise identical to the lipid nanoparticle composition administered to the subject.

[0026] In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is at least 500% or at least 1000% of the level of expression of the protein or gene product in a cell from another tissue of the body. In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is at least 500% or at least 1000% of the level of expression of the protein or gene product in a cell from lung tissue. In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is at least 500% or at least 1000% of the level of expression of the protein or gene product in a cell from spleen tissue.

[0027] In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein or gene product in another tissue of the body. In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 500% to about 20000% of thelevel of expression of the protein or gene product in a cell from lung tissue. In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein or gene product in a cell from spleen tissue. In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein or gene product in a cell from another tissue of the body. In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein or gene product in a cell from lung tissue. In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein or gene product in a cell from spleen tissue.

[0028] In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a lung cell that is less than 20%, less than 10%, or less than 5% of the level of expression of the protein or gene product in a cell from the target tissue.

[0029] In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a lung cell that is less than 20%, less than 10%, or less than 5% of the total level of expression of the protein or gene product in the subject. In some embodiments, the selectively delivering does not result in expression of the protein or gene product in a cell from lung tissue.

[0030] In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a spleen cell that is less than 20%, less than 10%, or less than 5% of the level of expression of the protein or gene product in a cell from the target tissue.

[0031] In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a spleen cell that is less than 20%, less than 10%, or less than 5% of the total level of expression of the protein or gene product in the subject. In some embodiments, the selectively delivering does not result in expression of the protein or gene product in a spleen tissue. In some embodiments, the level of expression is determined by in vivo biofluorescence imaging.

[0032] In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0033] In another aspect, disclosed herein are methods of delivering a payload to a target cell in a subject in need thereof, comprising administering to the subject an effective amountof the lipid nanoparticle composition described herein. In some embodiments, the target cell is a liver cell.

[0034] In another aspect, disclosed herein are pharmaceutical compositions comprising a lipid nanoparticle composition described herein, and a pharmaceutically acceptable excipient.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0036] FIG. 1 shows the Liver / Lung ratio of bioluminescence as measured by organ in vivo imaging (IVIS®) by formulation.

[0037] FIGs. 2A-2B show comparison of formulations with top ratios across organs. FIG. 2A shows top 10 median Liver / Lung ratio; 10 median Liver / Spleen ratio; and top 10 median Liver / (Lung+Spleen) ratio. FIG. 2B shows top 10 median Liver / Lung ratio; 10 median Liver / Spleen ratio; and top 10 median Liver / Other ratio.

[0038] FIG. 3 shows hierarchically clustered heatmap of top hepatic lipid nanoparticles.

[0039] FIGs. 4A-4F show rat in vivo imaging (IVIS®) for top 8 formulations for liver.FIG. 4A shows organ in vivo imaging (IVIS®) of Formulations 3E7, 3E5, and 3E4. FIG. 4B shows whole body in vivo imaging (IVIS®) of Formulations 3E7, 3E5, and 3E4. FIG. 4C shows organ in vivo imaging (IVIS®) of Formulations X, 3F1, and 2L. FIG. 4D shows whole body in vivo imaging (IVIS®) of Formulations X, 3F1, and 2L. FIG. 4E shows organ in vivo imaging (IVIS®) of Formulations 2M and B. FIG. 4F shows whole body in vivo imaging (IVIS®) of Formulations 2M and B.

[0040] FIG. 5 shows boxplot of relevant ratios across formulations, conditions, and replicates.

[0041] FIGs. 6A-6E show distribution of liver fluorescence by subtypes. FIG. 6A shows distribution of liver fluorescences by dendrimer subtype. FIG. 6B shows distribution of liver fluorescences by SORT subtype. FIG. 6C shows distribution of liver fluorescences by helper lipid subtype. FIG. 6D shows distribution of liver fluorescences by PEG subtype. FIG. 6E shows distribution of liver fluorescences by SORT -PEG subtype.

[0042] FIGs. 7A-7F show distribution of extrahepatic ratios by subtypes. FIG. 7 A shows distribution of extrahepatic ratios by dendrimer subtype. FIG. 7B shows distribution of extrahepatic ratios by SORT subtype. FIG. 7C shows distribution of extrahepatic ratios by helper lipid subtype. FIG. 7D shows distribution of extrahepatic ratios by PEG subtype. FIG.7E shows distribution of extrahepatic ratios by top-performing PEG subtype. FIG. 7F shows distribution of extrahepatic ratios by SORT -PEG subtype.

[0043] FIGs. 8A and 8B show Bonferroni-corrected statistically significant effect sizes of components on various dependent variables. FIG. 8A shows statistically significant effect sizes of components on logioLung variable, logioLiver variable, and logioSpleen variable. FIG. 8B shows statistically significant effect sizes of components on Liver / Lung ratio variable, Liver / Spleen ratio variable, Liver / (Lung+Spleen) ratio variable.

[0044] FIG. 9 shows Liver / Lung ratio of hepatic formulations vs. comparators.

[0045] FIG. 10 shows in bioluminescence imaging data (measured by vivo imaging system(IVIS®) ) data in Sprague Dawley rat subjects intravenously dosed with lipid nanoparticle (LNP) compositions described herein.

[0046] FIGs. 11A-11E shows the occurrence (pg / mL) of cytokines in the blood serum of Sprague Dawley rat subjects intravenously dosed with lipid nanoparticle (LNP) compositions described herein.

[0047] FIGs. 12A-12C shows body weight change in Sprague Dawley rats subjected to selected LNP compositions described herein.

[0048] FIGs. 13A-13B shows luciferase expression in Sprague Dawley rat subjects intravenously dosed with LNP 3E5 in different buffers. LNP 3E5 was used to deliver a Luc- HA mRNA and expression was analyzed using IVIS® organ imaging.

[0049] FIG. 14 shows DNAI1-HA expression and their biodistributions in Sprague Dawley rat subjects following intravenous administration of LNP compositions described herein.

[0050] FIGs. 15A-15C shows DNAI1-HA and their biodistributions in Sprague Dawley rat subjects following intravenous administration of LNP compositions described herein shown qualitatively using Western Blots.

[0051] FIGs. 16A-16C shows the reduction of DNAI1-HA protein expression in the lung (FIG. 16A), spleen (FIG. 16B), and liver (FIG. 16C) after 4 hours and 24 hours post-dosing of rat subjects with LNP compositions described herein.

[0052] FIG. 17 shows a hierarchically clustered heatmap of top cytokines expressed in lipid nanoparticle (LNP) compositions described herein. “Group” refers to the dosing groups identified in Table 14.

[0053] FIGs. 18A-18E show the occurrence (pg / mL) of liver enzymes, triglycerides and total complements (CH50), in the blood serum of Sprague Dawley rat subjects intravenously dosed with lipid nanoparticle (LNP) compositions described herein.

[0054] FIGs. 19A and 19B show the effect intravenously dosed lipid nanoparticle (LNP) compositions described herein on levels of platelets and neutrophils in the blood of Sprague Dawley rat subjects.DETAILED DESCRIPTION

[0055] Provided herein are lipid nanoparticle (LNP) compositions for delivering a payload, such as a therapeutic polypeptide, or a polynucleotide encoding a therapeutic polypeptide to a cell or tissue. In some embodiments, the LNP composition comprises a therapeutic polypeptide associated with a liver disease. The disclosure also provides for pharmaceutical compositions comprising the LNP compositions. Also provided herein are methods of use of LNP compositions, and pharmaceutical compositions.Definitions

[0056] Before the embodiments of the disclosure are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

[0057] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Various scientific dictionaries that include the terms included herein are well known and available to those in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice or testing of the disclosure, some preferred methods and materials are described. Accordingly, the terms defined immediately below are more fully described by reference to the specification as a whole.

[0058] The singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0059] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%,or up to 1% of a given value. In some cases, the term “about” refers to ±10% of a stated number or value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0060] The term “at least” followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example, “at least 1” means 1 or more than 1.

[0061] The term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40%” means 40% or less than 40%. When, in this specification, a range is given as “(a first number) to (a second number)” or “(a first number)- (a second number)” this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100 mm.

[0062] As used herein, the phrases “at least one,” “one or more,” and “and / or” are open- ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0063] As used herein, “or” may refer to “and,” “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B,” “A but not B,” “B but not A,” and “A and B”. In some cases, context may dictate a particular meaning.

[0064] As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. As also used herein, in any instance or embodiment described herein, “comprising” may be replaced with “consisting essentially of” and / or “consisting of” used herein, in any instance or embodiment described.

[0065] The terms “increased,” “increasing,” “increase,” “improved,” “improvement,” “improving” and the like, are used herein to generally means an increase by a statically significant amount. In some aspects, the terms “increased” or “improved” means an increase or improvement of at least 10% as compared to a reference level, for example an increase or improvement of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any improvement between 10- 100% as compared to a reference level, standard, or control. Other examples of “increase” or “improvement” includes an increase of at least 2 -fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0066] The terms “decreased,” “decreasing,” “decrease,” “reduced,” “reducing,” “reduce” and the like, are used herein generally to mean a decrease or reduction by a statistically significant amount. In some aspects, “decreased” or “reduced” means a reduction by at least 10% as compared to a reference level, for example a decrease or reduction by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease or reduction (e.g., absent level or non-detectable level as compared to a reference level), or any decrease or reduction between 10-100% as compared to a reference level.

[0067] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0068] As used herein, the term “lipid nanoparticle,” “LNP,” “lipid nanoparticle composition,” or “LNP composition” refers to a carrier or vehicle, formed by one or more lipid components, for payload (e.g., nucleic acid, protein, peptide, polypeptide, polynucleotide, or oligonucleotide) delivery in the context of pharmaceuticals. Further, as used herein the term “formulation” refers to a specific lipid nanoparticle composition of the disclosure. In otherwords, the terms “formulation,” lipid nanoparticle,” “LNP,” “lipid nanoparticle composition,” or “LNP composition” are used herein interchangeably. Lipid nanoparticles can have one or more lipids with at least one dimension on the order of nanometers (e.g., 1-1000 nm). Generally, lipid nanoparticle compositions for delivery are composed of one or more lipids, such as, but not limited to, a synthetic ionizable or cationic lipid, a phospholipid, a structural lipid (e.g., a sterol), and a polyethylene glycol (PEG) lipid. These compositions may also include other lipids. In various embodiments, the lipid nanoparticle composition comprises five components: (i) a first lipid (e.g., an ionizable cationic lipid); (ii) a phospholipid; (iii) a steroid or steroid derivative; (iv) a polymer-conjugated lipid; and (v) a second lipid separate from said first lipid (e.g., a selective organ targeting (SORT) lipid). In some embodiments, at least one therapeutic agent (e.g., mRNA) can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation of other undesirable effects induced by the biological mechanism of a target subject, tissue, and / or cell, e.g., an adverse immune response. In some embodiments, lipid nanoparticles comprise at least one therapeutic agent (e.g., mRNA) that is either organized within inverse lipid micelles and encased within a lipid monolayer envelop or intercalated between adjacent lipid bilayers. In some embodiments, the morphology of lipid nanoparticles is not like a traditional liposome, which are characterized by a lipid bilayer surrounding an aqueous core. In some embodiments, lipid nanoparticles are substantially non-toxic. In some embodiments, the therapeutic agent (e.g., mRNA) is resistant in aqueous solution to degradation by intracellular or intercellular enzymes by virtue of the lipid nanoparticle.

[0069] The term Selective Organ Targeting (SORT) lipid, as used herein, refers to a component of a lipid nanoparticle (LNP) composition that provides predictable cell-, tissue-, and / or organ-specific targeting of the LNP (for example as described in Cheng et al. Nat Nanotechnol 15:313-320 (2020); Wang et al. Nat Protoc 18:265-291 (2023); and U.S. Pat. Nos. 11,766,408 and 11,229,609, the entire contents of each of which is incorporated herein by reference). A selected SORT lipid provides accurate and specific delivery of the cargo from a rationally-designed LNP based, in part, on the biophysical properties of the selected SORT lipid and its prevalence in the LNP. In some cases, specificity is modulated by a LNP’s surface's acid dissociation constant (pKa), which may be affected by the proportion of charged and uncharged ionizable lipids at the LNP surface and may depend on the type of SORT used in the LNP formulation. Without wishing to be bound by theory, the SORT lipid directs tissue specificity of a rationally-design LNP by adjusting surface properties and / or physicochemicalcharacteristics of the LNP. Illustrative SORT lipids include, but are not limited to, permanently cationic lipids, anionic lipids, zwitterionic lipids, and ionizable cationic lipids. See, e.g., Table 6 and Table 7. In some embodiments, anionic SORT lipids generally favor delivery to the spleen, at least when administered intravenously; ionizable cationic SORT lipids or ionizable amino SORT lipids generally favor delivery to the liver; permanently cationic SORT lipids generally favor delivery to the lungs; and zwitterionic SORT lipids favor delivery to the spleen.

[0070] As used herein, the term “ionizable cationic lipid” refers to lipid and lipid-like molecules having at least one pKa in the range of about 4.5-8, such that, without being bound by theory, they may facilitate release of LNP payloads upon uptake into the endosomal compartment of a cell. The ionizable cationic lipid may maintain a neutral charge in pH above the pKa of the lipid; it becomes positively charged in a pH lower than its pKa which facilitates membrane fusion and subsequent cytosolic release of an LNP. Illustrative ionizable cationic lipids have one or more nitrogen atoms having pKa’s in the range of about 4.5-8, such are tertiary amine groups.

[0071] As used herein, the term “permanently cationic lipid” refers to lipid or lipid-like molecules that are positively charged in physiologically relevant solutions, regardless of a pH. Illustrative permanently cationic lipids may include a quaternary ammonium group, and lack a negatively charged phosphate group. Without being bound by theory, a permanently cationic lipid may act as a SORT lipid by raising the apparent pKa of an LNP, as described, e.g., in Billiard et al., Proc Natl Acad Sci USA 118:e2109256118 (2021).

[0072] As used herein, the term “anionic lipid” refers to a lipid that is negatively charged in physiologically relevant solutions, regardless of a pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), ethylphosphocholines, and other anionic modifying groups joined to neutral lipids.

[0073] As used herein, the term “phospholipid” refers to lipids that comprise a phosphate group. The lipid component of a lipid nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0074] As used herein, the term “sterol” refers to a subgroup of steroids with a hydroxyl group at the 3 -position of the A-ring of a gonane ringsystem. “Cholesterol” is an illustrativesterol that has a structure of four fused hydrocarbon rings (gonane ringsystem) with a polar hydroxyl group at one end and an eight-carbon branched aliphatic tail at the other end. The sterol component of an LNP, e.g., cholesterol influences the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers, for example in LNPs. For example, “sterol” can be cholesterol or sitosterol.

[0075] As used herein, the term “PEG-lipid” refers to a lipid modified with a polyethylene glycol (PEG) unit. In some embodiments, the PEG-lipid comprises dimyristoyl glycerol (DMG), and is referred to as PEG-DMG. In some embodiments, the PEG-lipid comprises 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE).

[0076] As used herein, the phrase “N / P ratio” refers to a molar ratio of nitrogen in the lipid composition to phosphate in the payload, e.g., a polynucleotide payload.

[0077] As used herein, the term “apparent pKa” refers to the overall dissociation constant of all titratable groups in the lipid nanoparticles of an LNP. Apparent pKa is an experimentally determined value of molecules or nanoparticles. Apparent pKa can be expressed as the pH at which the number of ionized (protonated) and deionized groups are equal in a system. The surface charge and ionic interaction of assembled nanomaterials in nanoparticles can be estimated according to apparent pKa. The apparent pKa of a nanoparticle can be the result of the average ratio of all the ionized to deionized groups in the nanoparticle. Thus, apparent pKa is not the intrinsic pKa value for any individual molecule. The apparent pKa of nanoparticles can be measured by various techniques. For example, acid-base titration of 2-(p-toluidino)-6- naphthalene sulfonic acid (TNS) fluorescent methods are widely used in determination of apparent pKa of blank nanoparticles.

[0078] As used herein, the phrase “lipid:RNA ratio” refers to milligram of lipid for each milligram of RNA payload. This ratio influences the encapsulation efficiency of RNA- containing lipid nanoparticles.

[0079] The term “encapsulation,” as used herein refers to the process of confining a payload within an LNP. For example, “encapsulation” refers to confining an mRNA molecule within an LNP. The term “encapsulation efficiency” refers to the fraction of a payload that is encapsulated within or otherwise coupled with a lipid nanoparticle composition when LNPs are formed. Encapsulation efficiency may be determined by comparing the amount of input payload to the amount of payload in a sample of LNPs, or by comparing the amount of payload in the LNPs to the free excess payload in the sample. For example, a fluorescence detection assay (e.g., RiboGreen™) is used to determine encapsulation efficiency by measuring the freeRNA in a sample with intact LNPs compared with the total RNA in a sample treated to disrupt the LNPs.

[0080] The term “payload” refers to a bioactive molecule or molecules, such as a small molecule, biomolecule, nucleic acid (e.g., DNA, RNA, siRNA, shRNA), protein, polypeptide, or peptide, which is associated with an LNP composition. For example, the payload can be bound covalently or non-covalently to the LNP, encapsulated in the LNP, coupled to the LNP, or complexed with the LNP within the LNP composition.

[0081] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro RNA (miRNA), transfer RNA, ribosomal RNA, a ribozyme, an antisense RNA, a guide RNA (gRNA), cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolated RNA, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences. When the polynucleotides are chemically and / or structurally modified the polynucleotides may be referred to as “modified polynucleotides.”

[0082] As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression, or chemically synthesized. Where appropriate, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, or backbone modifications. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.

[0083] As used herein, the term “shRNA” or “short hairpin RNA” refers to a short sequence of RNA, which can make a tight hairpin turn and can be used to silence gene expression.

[0084] As used herein, the term “microRNA” refers to noncoding RNA consisting of about 22 ribonucleotides which regulates gene expression in the post transcriptional stage bysilencing messenger RNA by base-pairing with a complementary sequence in its targeted mRNA.

[0085] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and optionally one or more post-translational modifications (e.g., glycosylation) and / or other modifications known in the art.

[0086] As used herein, the term “gene-editing system” refers to a DNA or RNA editing system that comprises one or more guide RNA elements and one or more RNA-guided endonuclease elements. The guide RNA element comprises a target RNA comprising a nucleotide sequence substantially complementary to a nucleotide sequence at the one or more target genomic regions or a nucleic acid comprising a nucleotide sequence(s) encoding the target RNA. The RNA-guided endonuclease element comprises an endonuclease that is guided or brought to a target genomic region(s) by a guide RNA element or a nucleic acid comprising a nucleotide sequence(s) encoding such endonuclease.

[0087] The terms “identity,” “identical,” and “sequence identity” refer to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can readily be calculated by known methods, including, but not limited to, those described in Needleman and Wunsch, J. Mol. Biol. 48(3):443 (1970), as such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. The term “percent sequence identity,” “percent identity,” or “identical to” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence. For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. The term “reference sequence” refers to a molecule to which a test sequence is compared. Methods of sequence alignment for comparison and determination of percent sequence identity are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443.

[0088] The term “isolated” when applied to a polynucleotide or polypeptide, denotes that the polynucleotide or polypeptide is essentially free of other cellular components with which it is associated in the natural state or components present during chemical synthesis. It can be,for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A polynucleotide or polypeptide that is the predominant species present in a preparation is substantially purified.

[0089] The term “variant” refers to a polypeptide or polynucleotide having one or more insertions, deletions, or amino acid substitutions relative to a reference polypeptide or polynucleotide.

[0090] The terms “subject” refers to a living organism to which any of the compositions as described herein may be administered. The subject may be suffering from or be at risk for a disease or condition that can be treated by administration of pharmaceutical composition as provided herein or by a therapeutic method disclosed herein. Non-limiting examples of subjects include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the subject is a primate, e.g., a human.

[0091] The term “therapeutically effective amount,” as used herein, refers to an amount of an LNP and / or an LNP comprising a therapeutic agent sufficient to treat a disease, a disorder, or a condition. For example, with regard to the use of LNPs with mRNA payload to treat e.g., cystic fibrosis (CF) or primary ciliary dyskinesia (PCD), a therapeutically effective amount is the dosage or concentration of the LNP and / or the LNP comprising the mRNA (e.g., CFTR or PCD mRNA) capable of eradicating, inhibiting, preventing, slowing down the progression of all or part of e.g., CF or PCD respiratory symptoms or some combination thereof. For the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100% of symptoms in a subject in need. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2 -fold, 5- fold, or more effect over a control. The “therapeutically effective amount” can vary depending, for example, but not limited to, on the compound, the disease, or the condition and / or symptoms thereof, severity of the disease or the condition and / or symptoms thereof, the age, weight, and / or health of the subject to be treated, and the judgment of the prescribing physician. An appropriate amount in any given instance can be ascertained by those skilled in the art or capable of determination by routine experimentation.

[0092] The term “administering” refers to providing a composition to a subject in a manner that permits the composition to have its intended effect. Administration may be performed byintramuscular injection, intravenous injection, intraperitoneal injection, inhalation, or any other suitable route.

[0093] “Co-administer” means that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies. The compositions provided herein can be administered alone or can be co-administered to the subject. Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination. Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce degradation of an LNP or the payload of the LNP).

[0094] As used herein, the term “delivering” means causing, through chemical or biophysical properties of a composition (e.g., an LNP composition) and / or the payload (e.g., a polynucleotide) of an LNP to pass from a site of administration to a subject to a target organ, target tissue, or target cell. In some cases, “delivering” is equivalent to “administering,” e.g., to a subject in need thereof. As used herein, the term “selectively delivering” refers to the delivery to a target organ, tissue, or cell at a greater rate or in a greater amount than delivered to a reference, non-target organ, tissue, or cell, or that a greater fraction of total the amount of LNP or payload administered to a subject is delivered to a target organ, tissue, or cell by the composition than delivered by a reference composition. For example, selective delivery may mean that at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the total amount administered is delivered to the target organ, tissue, or cell. “Selective delivery” is determined by comparing the fraction of an LNP composition or payload that is delivered to a target organ (e.g., the lung) by an LNP composition comprises a selected lipid (e.g., SORT lipid) compared to a reference LNP composition in which the selected lipid is replaced by a control lipid.

[0095] “Treating” or “treatment” as used herein (and as well-understood in the art) also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, "treatment" as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve thedisease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.

[0096] “Prevention” or “preventing” refers to inhibiting the onset of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and / or delaying the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and / or predisposed to the disease but has not yet experienced or displayed any of the pathology or symptomatology of the disease. Prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.

[0097] The term “pharmaceutically acceptable excipients” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of a herein-disclosed composition and absorption by a subject of the same. A pharmaceutically acceptable excipients do not cause a significant adverse toxicological effect on the subject. These excipients are usually approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Non-limiting examples of pharmaceutically acceptable excipients include water, a sodium chloride (NaCl) solution, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure. One of skill in the art will recognize that other pharmaceutically acceptable excipients are useful in the present disclosure.

[0098] The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, PCR, and immunohistochemistry).

[0099] Chemical moi eties referred to as univalent chemical moi eties (e.g., alkyl, aryl, etc.) also encompass structurally permissible multivalent moieties, as understood by those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g.,CH3CH2-), in appropriate circumstances an “alkyl” moiety can also refer to a divalent radical (e.g., -CH2CH2-, which is equivalent to an “alkylene” group). Similarly, under circumstances where a divalent moiety is required, those skilled in the art will understand that the term “aryl” refers to the corresponding divalent arylene group.

[0100] As used herein, “Alkyl” refers to optionally substituted, straight and branched chain aliphatic groups having from 1 to 30 carbon atoms. For example, “C1, C2, C3, C4, C5or C6alkyl,” “C1-C6alkyl,” “alkyl(C≤6),” or “alkyl(C1-C6),” is intended to include C1, C2, C3, C4, C5or C6straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5or C6branched saturated aliphatic hydrocarbon groups. Examples of alkyl include, moi eties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n- butyl, s-butyl, t-butyl, n-pentyl, i -pentyl, or n-hexyl. In some embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g., C1-C6for straight chain, C3-C6for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms. Analogously, for example “C18, C19, C20, C21, C22, C23or C24alkyl,” “C18-C24alkyl,” “alkyl(C≤24),” or “alkyl(C8-C24)” is intended to include C18, C19, C20, C21, C22, C23 or C24straight chain (linear) saturated aliphatic hydrocarbon groups and C18, C19, C20, C21, C22, C23 or C24branched saturated aliphatic hydrocarbon groups. Examples of “C18-C24alkyl” include octadecyl, nonadecyl, didecyl, henicosyl, docosyl, tricosyl, tetracosyl, 5-butylpentadecanyl, 4- methyl-5-(pentan-2-yl)hexadecanyl, 7-methylhenicosanyl, 2,15,15-trimethylhenicosanyl, 8,9- dimethyldocosanyl, 6-ethyl-8-methylnonadecanyl, and 6,7-dimethyl-8-propyltridecanyl.

[0101] “Alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term “alkenyl” includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenyl groups. The term “C2, C3, C4, C5or C6alkenyl,” “C2-C6alkenyl,” “alkenyl(C≤6),” or “alkenyl(C2-C6)” includes alkenyl groups containing two to six carbon atoms. The term “C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23or C24alkenyl,” “C8-C24alkenyl,” or “alkenyl(C8-C24)” includes alkenyl groups containing eight to twenty-four carbon atoms. Examples of “C8-C24alkenyl” include 2,6-dimethylhept-2-enyl, 2,6-dimethylhept-2-enyl, 2,8- dimethylnon-2-enyl, 2,7-dimethyldec-2-ene, 3-ethyl-8-methylundec-3-ene, and 2,9,9- trimethyltridec-2-ene.

[0102] The term “optionally substituted alkyl” or “optionally substituted alkenyl” refers to an alkyl or alkenyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example,alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0103] Any composition or method disclosed herein is applicable to any herein-disclosed composition or method. In other words, any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.Lipid nanoparticle compositionIonizable cationic lipids

[0104] . In some embodiments, the ionizable cationic lipids of the lipid component of the lipid nanoparticles of the disclosure contain one or more groups which is protonated at physiological pH but may deprotonate and has no charge at a pH above the pKa of the lipid. The ionizable group may contain one or more protonatable amines which are able to form a cationic group at physiological pH. The ionizable cationic lipid compound may also further comprise one or more lipid components such as two or more fatty acids with C6-C24alkyl or alkenyl carbon groups. These lipid groups may be attached through an ester linkage or may be further added through a Michael addition to a sulfur atom. In some embodiments, these compounds may be a dendrimer, a dendron, a polymer, or a combination thereof.

[0105] The lipid component may include one or more ionizable (e.g., ionizable amino) lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH). Ionizable cationic lipids may be selected from the non-limiting group consisting of 3- (didodecylamino)-N 1 ,N1 ,4-tridodecy 1-1 -piperazineethanamine (KL 10), N1-[2-(didodecylamino)ethyl]N1,N4,N4-tridodecyl-l,4-piperazinedi ethanamine (KL22), 14,25- ditridecy 1-15,18,21 ,24-tetraaza-octatriacontane (KL25), 1 ,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2, 2-dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), heptatriaconta-6,9,28,3 l-tetraen-19-yl-4-(dimethylamino)butanoate (DLin- MC3-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 1 ,2-dioleyloxy-N,Ndimethylaminopropane (DODMA), 2-({ 8 [(3 (3)-cholest-5 -en-3 -yloxy]octylIoxy)N,Ndimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), (2R)-2-({8-[(3(3)-cholest-5-en-3-yloxy]octylIoxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine(Octyl-CLinDMA (2R)), and (2S) 2- ({8-[(3(3)-chol e st-5-en-3 -yloxy] octyl } oxy)-N,N-dimethyl-3 -[(9Z,12Z)-octadeca-9,12-di en-1 -yloxy]propan-l-amine (Octyl-CLinDMA (2S)), 4-hydroxybutyl ) azanediyl)bis (hexane- 6,l-diyl)bis(2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-((2 -hydroxy ethyl) (6-oxo-6- (undecyloxy) hexyl) amino) ooccttaannooaattee (SM-102), (9Z,12Z)-3-((4,4-Bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate (LP01) . In addition to these, an ionizable cationic lipid may also be a lipid including a cyclic amine group.

[0106] Ionizable cationic lipids can also be the compounds disclosed in International Publication No. WO2017075531, hereby incorporated by reference in its entirety. Ionizable cationic lipids can also be the compounds disclosed in International Publication No. WO201 5199952, hereby incorporated by reference in its entirety. In one embodiment, the ionizable cationic lipid may be selected from, but not limited to, an ionizable cationic lipid described i inn International Publication Nos. W02012040184, WO2011153120,WO201 1149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373 and WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent Publication No. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and S20130225836; the contents of each of which are herein incorporated by reference in their entirety.

[0107] In some embodiments, an ionizable cationic lipid comprises between 2 and 6 hydrophobic chains, often alkyl or alkenyl such as C6-C24alkyl or alkenyl groups, but may have at least 1 or more that 6 tails.Dendrimers

[0108] In some embodiments, the first lipid (e.g., the ionizable cationic lipid) is a dendrimer. A dendrimer is a polymer exhibiting regular dendritic branching, formed by the sequential or generational addition of branched layers to or from a core and are characterized by a core, at least one interior branched layer, and a surface branched layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August 19941) In other embodiments, the term “dendrimer” as used herein is intended to include, but is not limited to, a molecular architecture with an interior core, interior layers (or “generations”) of repeatingunits regularly attached to this initiator core, and an exterior surface of terminal groups attached to the outermost generation. A “dendron” is a species of dendrimer having branches emanating from a focal point which is or can be joined to a core, either directly or through a linking moiety to form a larger dendrimer. In some embodiments, the dendrimer structures have radiating repeating groups from a central core which doubles with each repeating unit for each branch. In some embodiments, the dendrimers described herein may be described as a small molecule, medium-sized molecules, lipids, or lipid-like material. These terms may be used to describe compounds described herein which have a dendron like appearance (e.g., molecules which radiate from a single focal point).

[0109] While dendrimers are polymers, dendrimers may be preferable to traditional polymers because they have a controllable structure, a single molecular weight, numerous and controllable surface functionalities, and traditionally adopt a globular conformation after reaching a specific generation. Dendrimers can be prepared by sequentially reactions of each repeating unit to produce monodisperse, tree-like and / or generational structure polymeric structures. Individual dendrimers consist of a central core molecule, with a dendritic wedge attached to one or more functional sites on that central core. The dendrimeric surface layer can have a variety of functional groups disposed thereon including anionic, cationic, hydrophilic, or lipophilic groups, according to the assembly monomers used during the preparation.

[0110] Modifying the functional groups and / or the chemical properties of the core, repeating units, and the surface or terminating groups, their physical properties can be modulated. Some properties which can be varied include, but are not limited to, solubility, toxicity, immunogenicity and bioattachment capability. Dendrimers are often described by their generation or number of repeating units in the branches. A dendrimer consisting of only the core molecule is referred to as Generation 0, while each consecutive repeating unit along all branches is Generation 1, Generation 2, and so on until the terminating or surface group. In some embodiments, half generations are possible resulting from only the first condensation reaction with the amine and not the second condensation reaction with the thiol.

[0111] Preparation of dendrimers requires a level of synthetic control achieved through series of stepwise reactions comprising building the dendrimer by each consecutive group. Dendrimer synthesis can be of the convergent or divergent type. During divergent dendrimer synthesis, the molecule is assembled from the core to the periphery in a stepwise process involving attaching one generation to the previous and then changing functional groups for the next stage of reaction. Functional group transformation is necessary to prevent uncontrolled polymerization. Such polymerization would lead to a highly branched molecule that is notmonodisperse and is otherwise known as a hyperbranched polymer. Due to steric effects, continuing to react dendrimer repeat units leads to a sphere shaped or globular molecule, until steric overcrowding prevents complete reaction at a specific generation and destroys the molecule's monodispersity. Thus, in some embodiments, the dendrimers of G1-G10 generation are specifically contemplated. In some embodiments, the dendrimers comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein. In some embodiments, the dendrimers used herein are G0, G1, G2, or G3. However, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) may be increased by reducing the spacing units in the branching polymer.

[0112] Additionally, dendrimers have two major chemical environments: the environment created by the specific surface groups on the termination generation and the interior of the dendritic structure which due to the higher order structure can be shielded from the bulk media and the surface groups. Because of these different chemical environments, dendrimers have found numerous different potential uses including in therapeutic applications.

[0113] In some embodiments of the lipid composition of the present application, the dendrimers are assembled using the differential reactivity of the acrylate and methacrylate groups with amines and thiols. The dendrimers may include secondary or tertiary amines and thioethers formed by the reaction of an acrylate group with a primary or secondary amine and a methacrylate with a mercapto group. Additionally, the repeating units of the dendrimers may contain groups which are degradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diethers, esters, amides, or disulfides groups. In some embodiments, the core molecule is a monoamine which allows dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine with multiple different dendritic branches which each may comprise one or more repeating units. The dendrimer may be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on a heteroatom such as a nitrogen atom. In some embodiments, the terminating group is a lipophilic group such as a long chain alkyl or alkenyl group. In other embodiments, the terminating group is a long chain haloalkyl or haloalkenyl group. In other embodiments, the terminating group is an aliphatic or aromatic group containing an ionizable group such as an amine (-NH2) or a carboxylic acid (-CO2H). In still other embodiments, the terminating group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.

[0114] The ionizable cationic lipids of the present application may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically activeor racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Ionizable cationic lipids may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the ionizable cationic lipids of the present application can have the S or the R configuration. Furthermore, it is contemplated that one or more of the ionizable cationic lipids may be present as constitutional isomers. In some embodiments, the compounds have the same formula but different connectivity to the nitrogen atoms of the core. Without wishing to be bound by any theory, it is believed that such ionizable cationic lipids exist because the starting monomers react first with the primary amines and then statistically with any secondary amines present. Thus, the constitutional isomers may present the fully reacted primary amines and then a mixture of reacted secondary amines.

[0115] Chemical formulas used to represent ionizable cationic lipids of the present application will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given formula, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.

[0116] The ionizable cationic lipids of the present disclosure may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than, and / or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.

[0117] In addition, atoms making up the ionizable cationic lipids of the present application are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include13C and14C.

[0118] It should be recognized that the particular anion or cation forming a part of any salt form of an ionizable cationic lipids provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptablesalts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0119] In some embodiments of the lipid composition of the present application, the ionizable lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises an ammonium group which is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH from about 6 to about 8. In some embodiments, the ionizable cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises at least two C6-C24alkyl or alkenyl groups.Dendrimers of Formula (I)

[0120] In some embodiments of the lipid composition, the first lipid (e.g., the ionizable cationic lipid) comprises at least two C8-C24alkyl groups. In some embodiments, the first lipid (e.g., the ionizable cationic lipid) is a dendrimer further defined by the formula:Core-(Repeating Unit)n-Terminating Group (D-I) wherein one or more hydrogen atoms of the core are replaced with a repeating unit and wherein: the core has the formula:wherein:X1is amino or C1-C12alkylamino, C1-C12dialkylamino, C3-C12heterocycloalkyl, C8-C12heteroaryl, or a substituted version thereof;R1is amino, hydroxy, mercapto, C1-C12alkylamino, or C1-C12dialkylamino, or a substituted version of either of these groups; and a is 1, 2, 3, 4, 5, or 6; or the core has the formula:wherein:X2is N(R5)y;R5is hydrogen, C1-C18alkyl, or substituted C1-C18alkyl; and y is 0, 1, or 2, provided that the sum of y and z is 3;R2is amino, hydroxy, mercapto, C1-C12alkylamino, or C1-C12dialkylamino, or a substituted version of either of these groups;b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, or 3; provided that the sum of z and y is 3; or the core has the formula:wherein:X3is -NR6- , wherein R6is hydrogen, C1-C8alkyl, or C1-C8substituted alkyl, -O-, or C1-C8alkylaminodiyl, C1-C8alkoxy diyl, C6-C8arenediyl, C5-C8heteroarenediyl, C3-C8heterocycloalkanediyl, or a substituted version of any of these groups;R3and R4are each independently amino, hydroxy, mercapto, C1-C12alkylamino, or C1-C12dialkylamino, or a substituted version of either of these groups; or a group of the formula: -N(Rf)f(CH2CH2N(Rc))eRd,wherein: e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3;Rc, Rd, and Rfare each independently hydrogen, C1-C6alkyl, or substituted C1-C6alkyl; c and d are each independently 1, 2, 3, 4, 5, or 6; or the core is C1-C18alkylamine, C1-C36dialkylamine, C3-C12heterocycloalkane, or a substituted version of any of these groups; wherein the repeating unit comprises a degradable diacyl or a degradable diacyl and a linker; the degradable diacyl group has the formula:wherein:A1and A2are each independently -O-, -S-, or -NRa- , wherein: Rais hydrogen, C1-C6alkyl, or substituted C1-C6alkyl;Y3is C1-C12alkanediyl, C1-C12alkenediyl, C6-C12arenediyl, or a substituted version of any of these groups; or a group of the formula:wherein:X3and X4are C1-C12alkanediyl, C2-C12alkenediyl, C6-C12arenediyl, or a substituted version of any of these groups;Y5is a covalent bond, C1-C12alkanediyl, C1-C12alkenediyl, C6- C12arenediyl, or a substituted version of any of these groups; andR9is C1-C8alkyl or substituted C1-C8alkyl; the linker group has the formula:wherein:Y1is C1-C12alkanediyl, C1-C12alkenediyl, C6-C12arenediyl, or a substituted version of any of these groups; and wherein eachindependently denotes a point of attachment to another repeating unit or a terminating group; and the terminating group has the formula:wherein:Y4is alkanediyl or an C1-C18alkanediyl wherein one or more of the hydrogen atoms on the C1-C18alkanediyl has been replaced with -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3;R10is hydrogen, carboxy, hydroxy, C6-C12aryl, C1-C12alkylamino, C1-C12dialkylamino, C3-C12N- heterocycloalkyl, -C(O)N(R11)- C1-C6alkanediyl- C3-C12heterocycloalkyl, -C(O)- C1-C12alkylamino, -C(O)- C1-C12dialkylamino, or -C(O)- C3-C12N-heterocycloalkyl, wherein: R11is hydrogen, C1-C6alkyl, or substituted C1-C6alkyl; wherein the final degradable diacyl in the chain is attached to a terminating group; n is 0, 1, 2, 3, 4, 5, or 6; or a pharmaceutically acceptable salt thereof.

[0121] In some embodiments, the terminating group is further defined by the formula:wherein:Y4is C1-C18alkanediyl; andR10is hydrogen. In some embodiments, A1and A2are each independently -O- or-NRa-.

[0122] In some embodiments of the dendrimer of formula (D-I), the terminating group is a structure selected from the structures in Table 3.

[0123] In some embodiments of the dendrimer of formula (D-I), the core is further defined by the formula:wherein:X2is N(R5)y;R5is hydrogen or C1-C8alkyl, or substituted C1-C18alkyl; and y is 0, 1, or 2, provided that the sum of y and z is 3; R2is amino, hydroxy, or mercapto, or C1-C12alkylamino, C1-C12dialkylamino, or a substituted version of either of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, 3; provided that the sum of z and y is 3.

[0124] In some embodiments of the dendrimer of formula (D-I), the core is further defined by the formula:wherein:X3is -NR6-, wherein R6is hydrogen, C1-C8alkyl, or substituted C1-C8alkyl, -O-, or C1-C8alkylaminodiyl, C1-C8alkoxydiyl, C1-C8arenediyl, C1-C8heteroarenediyl, C1-C8heterocycloalkanediyl, or a substituted version of any of these groups;R3and R4are each independently amino, hydroxy, or mercapto, or C1-C12alkylamino, dialkylamino, or a substituted version of either of these groups; or a group of the formula: N(Rf)f(CH2CH2N(Rc))eRd,wherein: e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3;Rc, Rd, and Rfare each independently hydrogen, C1-C6alkyl, or substituted C1-C6alkyl; c and d are each independently 1, 2, 3, 4, 5, or 6.

[0125] In some embodiments of the dendrimer of formula (I), the terminating group is represented by the formula:wherein:Y4is alkanediyl(C≤18); andR10is hydrogen.

[0126] In some embodiments of the dendrimer of formula (D-I), a core of the structure of formula (D-IV) is:or a pharmaceutically acceptable salt thereof.

[0127] In some embodiments of the dendrimer of formula (D-I), the core comprises a structural formula set forth in Table 2 and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a repeating unit (i.e., where a hydrogen of the core is replaced with a repeating unit).

[0128] In some embodiments of the dendrimer of formula (D-I), the degradable diacyl is further defined as:

[0129] In some embodiments of the dendrimer of formula (D-I), the linker is further defined aswherein Y1is C1-C8alkanediyl or substituted C1-C12alkanediyl.

[0130] In some embodiments, in the core of formula (D-IV), R6is H. In some embodiments, in the core of formula (D-IV), R6is C1-C8alkyl. In some embodiments, in the core of formula (D-IV), R6is substituted alkyl (e.g., alkyl substituted with -NH2, alkyl substituted with -NHCH3, or alkyl substituted with -NHCH2CH3).

[0131] In some embodiments one or two hydrogen atoms of the core are replaced with a repeating unit. In some embodiments three or four hydrogen atoms of the core is replaced with a repeating unit. In some embodiments five hydrogen atoms of the core is replaced with a repeating unit. In some embodiments six hydrogen atoms of the core is replaced with a repeating unit.

[0132] In some embodiments of the dendrimer of formula (D-I), the dendrimer is selected from the group consisting of:and pharmaceutically acceptable salts thereof.

[0133] In some embodiments an ionizable lipid of the disclosure (e.g., a dendrimer of formula (D-I)) is a compound of formula (D-A):wherein:RD1is a C1-C4alkyl; z1 and z2 are each independently 1, 2, or 3; and z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.Dendrimers of Formula (X)

[0134] In some embodiments of the lipid composition, the ionizable cationic lipid is a dendrimer of the formula. In some embodiments, the ionizable cationic lipid is a dendrimer of the formula:

[0135] In some embodiments of the lipid composition, the ionizable cationic lipid is a dendrimer of a generation (g) having a structural formula:or a pharmaceutically acceptable salt thereof, wherein:(a)the core comprises a structural formula (XCore):wherein:Q is independently at each occurrence a covalent bond, -O-, -S-, -NR2-, or - CR3aR3b-;R2is independently at each occurrence R1gor -L2-NR1eR1f;R3aand R3bare each independently at each occurrence hydrogen or an optionally substituted (e.g., C1-C6, such as C1-C3) alkyl; R1a, R1b, R1c, R1d, R1e, R1f, and R1g(if present) are each independently at each occurrence a point of connection to a branch, hydrogen, or an optionally substituted (e.g., C1-C12) alkyl; L0, L1, and L2are each independently at each occurrence selected from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or, alternatively, part of L1form a (e.g., C4-C6) heterocycloalkyl (e.g., containing one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur) with one of R1cand R1d; and x1is 0, 1, 2, 3, 4, 5, or 6; and(b)each branch of the plurality (N) of branches independently comprises a structural formula (XBranch):wherein:* indicates a point of attachment of the branch to the core; g is 1, 2, 3, or 4;Z = 2(g-1);G=0, when g=1; or(c) each diacyl group independently comprises a structural formulawherein:* indicates a point of attachment of the diacyl group at the proximal end thereof;** indicates a point of attachment of the diacyl group at the distal end thereof;Y3is independently at each occurrence an optionally substituted (e.g., C1-C12); alkylene, an optionally substituted (e.g., C1-C12) alkenylene, or an optionally substituted (e.g., C1-C12) arenylene;A1and A2are each independently at each occurrence -O-, -S-, or - NR4-, wherein:R4is hydrogen or optionally substituted (e.g., C1-C6) alkyl; m1and m2are each independently at each occurrence 1, 2, or 3; and R3c, R3d, R3eand R3fare each independently at each occurrence hydrogen or an optionally substituted (e.g., C1-C8) alkyl; and(d) each linker group independently comprises a structural formulawherein:** indicates a point of attachment of the linker to a proximal di acyl group;*** indicates a point of attachment of the linker to a distal di acyl group; andY1is independently at each occurrence an optionally substituted (e.g., C1-C12) alkylene, an optionally substituted (e.g, C1-C12) alkenylene, or an optionally substituted (e.g, C1-C12) arenylene; and(e) each terminating group is independently selected from optionally substituted (e.g., C1-C18, such as C4-C18) alkylthiol, and optionally substituted (e.g., C1- C18, such as C4-C18) alkenylthiol.

[0136] In some embodiments of XCore, Q is independently at each occurrence a covalent bond, -O-, -S-, -NR2-, or -CR3aR3b. In some embodiments of XCore, Q is independently at each occurrence a covalent bond. In some embodiments of XCore, Q is independently at each occurrence an -O-. In some embodiments of XCore, Q is independently at each occurrence a -S- . In some embodiments of XCore, Q is independently at each occurrence a -NR2and R2is independently at each occurrence R1gor -L2-NR1eR1f. In some embodiments of XCore, Q is independently at each occurrence a -CR3aR3bR3a, and R3aand R3bare each independently at each occurrence hydrogen or an optionally substituted alkyl (e.g., C1-C6, such as C1-C3).

[0137] In some embodiments of XCore, R1a, R1b, R1c, R1d, R1e, R1f, and R1g(if present) are each independently at each occurrence a point of connection to a branch, hydrogen, or an optionally substituted alkyl. In some embodiments of XCore, R1a, R1b, R1c, R1d, R1e, R1f, and R1g(if present) are each independently at each occurrence a point of connection to a branch, hydrogen. In some embodiments of XCore, R1a, R1b, R1c, R1d, R1e, R1f, and R1g(if present) are each independently at each occurrence a point of connection to a branch an optionally substituted alkyl (e.g., C1-C12).

[0138] In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence selected from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or, alternatively, part of L1form a heterocycloalkyl (e.g., C4-C6and containing one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur) with one of R1cand R1d. In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence can be a covalent bond. In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence can be a hydrogen. In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence can be an alkylene (e.g., C1-C12, such as C1- C6or C1-C3). In some embodiments of XCore, L0, L1, and L2are each independently at eachoccurrence can be a heteroalkylene (e.g., C1-C12, such as C1-C8or C1-C6). In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence can be a heteroalkylene (e.g., C2-C8alkyleneoxide, such as oligo(ethyleneoxide)). In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence can be a [alkylene]-[heterocycloalkyl]-[alkylene] [(e.g., C1-C6) alkylene]-[(e.g., C4-C6) heterocycloalkyl]-[(e.g., C1-C6) alkylene]. In some embodiments of Xc ore, L0, L1, and L2are each independently at each occurrence can be a [alkylene]-(arylene)-[alkylene] [(e.g., C1-C6) alkylene]-(arylene)-[(e.g., C1-C6) alkylene]. In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence can be a [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C1- C6) alkylene]-phenylene-[(e.g., C1-C6) alkylene]). In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence can be a heterocycloalkyl (e.g., C4-C6heterocycloalkyl). In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence can be an arylene (e.g., phenylene). In some embodiments of XCore, part of L1form a heterocycloalkyl with one of R1cand R1d. In some embodiments of XCore, part of L1form a heterocycloalkyl (e.g., C4-C6heterocycloalkyl) with one of R1cand R1dand the heterocycloalkyl can contain one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur.

[0139] In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence selected from a covalent bond, C1-C6alkylene (e.g., C1-C3alkylene), C2-C12(e.g., C2-C8) alkyleneoxide (e.g., oligo(ethyleneoxide), such as -(CH2CH2O)I-4-(CH2CH2)-), [(C1- C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g.,and[(C1-C4) alkylene]-phenylene-[(C1-C4) alkylene] (e.g.,In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence selected from C1-C6alkylene (e.g., C1-C3alkylene), -(C1-C3alkylene-O)1-4-(C1-C3alkylene), -(C1-C3alkylene)-phenylene-(C1-C3alkylene)-, and -(C1-C3alkylene)-piperazinyl-(C1-C3alkylene)-. In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence C1- C6alkylene (e.g., C1-C3alkylene). In some embodiments, L0, L1, andL2are each independently at each occurrence C2-C12(e.g., C2-C8) alkyleneoxide (e.g., -(C1-C3alkylene-O)1-4-(C1-C3alkylene)). In some embodiments of XCore, L0, L1, and L2are each independently at each occurrence selected from [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene](e.g., -(C1-C3alkylene)-phenylene-(C1-C3alkylene)-) and [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3alkylene)-piperazinyl-(C1-C3alkylene)-).

[0140] In some embodiments of XCore,, x1is 0, 1, 2, 3, 4, 5, or 6. In some embodiments of XCore, x1is 0. In some embodiments of XCore, x1is 1. In some embodiments of XCore, x1is 2. In some embodiments of XCore, x1is 0, 3. In some embodiments of XCore, x1is 4. In some embodiments of XCore, x1is 5. In some embodiments of XCore, x1is 6.

[0141] In some embodiments of XCore, the core comprises a structural formula:). In some embodiments of XCore, the core comprises a structural formula:. In some embodiments of XCore, the core comprises a structural formula:In some embodiments of XCore, the core comprises a structural formula:In some embodiments of XCore, the core comprises a structural formula:In some embodiments of XCore, the core comprises a structural formula:In some embodiments of XCore,the core comprises a structural formula:, such asIn some embodiments of XCore, the core comprises a structural formula:wherein Q’ is -NR2- or -CR3aR3b-; q1and q2are each independently 1 or 2. In some embodiments of XCore, the core comprises a structural formula:In some embodiments of Xc.the core comprises a structural formulawherein ring A is an optionally substituted aryl or an optionally substituted (e.g., C3-C12, such as C3-C5) heteroaryl. In some embodiments of XCore, the core comprises has a structural formula

[0142] In some embodiments of XCore, the core comprises a structural formula set forth in Table 7 and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a branch of the plurality of branches.

[0143] In some embodiments, the plurality (N) of branches comprises at least 3 branches, at least 4 branches, at least 5 branches. In some embodiments, the plurality (N) of branches comprises at least 3 branches. In some embodiments, the plurality (N) of branches comprises at least 4 branches. In some embodiments, the plurality (N) of branches comprises at least 5 branches.

[0144] In some embodiments of XBranch, g is 1, 2, 3, or 4. In some embodiments of XBranch, g is 1. In some embodiments of XBranch, g is 2. In some embodiments of XBranch, g is 3. In some embodiments of XBranch, g is 4.

[0145] In some embodiments of XBranch, Z = 2(g-1)and when g=1, G=0. In some embodiments of XBranch, Z 2(g-1)and

[0146] In some embodiments of XBranch, g=1, G=0, Z=1, and each branch of the plurality of branches comprises a structural formula each branch of the plurality of branches comprises a structural formula

[0147] In some embodiments of XBranch, g=2, G=1, Z=2, and each branch of the plurality of branches comprises a structural formula

[0148] In some embodiments of XBranch, g=3, G=3, Z=4, and each branch of the plurality of branches comprises a structural formula

[0149] In some embodiments of XBranch, g=4, G=7, Z=8, and each branch of the plurality of branches comprises a structural formula

[0150] In some embodiments, the dendrimers described herein with a generation (g) = 1 has the structure:

[0151] In some embodiments, the dendrimers described herein with a generation (g) 1 has the structure:

[0152] An example formulation of the dendrimers described herein for generations 1-4 is shown in Table 1. The number of diacyl groups, linker groups, and terminating groups can be calculated based on g.Table 1. Formulation of Dendrimer Groups Based on Generation (g)

[0153] In some embodiments, the diacyl group independently comprises a structural formula* indicates a point of attachment of the diacyl group at the proximal end thereof, and ** indicates a point of attachment of the diacyl group at the distal end thereof.

[0154] In some embodiments of the diacyl group of XBranch, Y3is independently at each occurrence an optionally substituted; alkylene, an optionally substituted alkenylene, or an optionally substituted arenylene. In some embodiments of the diacyl group of XBranch, Y3is independently at each occurrence an optionally substituted alkylene (e.g., C1-C12). In some embodiments of the diacyl group of XBranch, Y3is independently at each occurrence an optionally substituted alkenylene (e.g., C1-C12). In some embodiments of the diacyl group of XBranch, Y3is independently at each occurrence an optionally substituted arenylene (e.g., C1- C12).

[0155] In some embodiments of the diacyl group of XBranch, A1and A2are each independently at each occurrence -O-, -S-, or -NR4-. In some embodiments of the diacyl group of XBranch, A1and A2are each independently at each occurrence -O-. In some embodiments of the diacyl group of XBranch, A1and A2are each independently at each occurrence -S-. In someembodiments of the diacyl group of XBranch, A1and A2are each independently at each occurrence -NR4- and R4is hydrogen or optionally substituted alkyl (e.g, C1-C6). In some embodiments of the diacyl group of XBranch, m1and m2are each independently at each occurrence 1, 2, or 3. In some embodiments of the diacyl group of XBranch, m1and m2are each independently at each occurrence 1. In some embodiments of the diacyl group of XBranch, m1and m2are each independently at each occurrence 2. In some embodiments of the diacyl group of XBranch, m1and m2are each independently at each occurrence 3. In some embodiments of the diacyl group of XBranch, R3c, R3d, R3e, and R3fare each independently at each occurrence hydrogen or an optionally substituted alkyl. In some embodiments of the diacyl group of XBranch, R3c, R3d, R3e, and R3fare each independently at each occurrence hydrogen. In some embodiments of the diacyl group of XBranch, R3c, R3d, R3e, and R3fare each independently at each occurrence an optionally substituted (e.g., C1-C8) alkyl.

[0156] In some embodiments of the diacyl group, A1is -O- or -NH-. In some embodiments of the diacyl group, A1is -O-. In some embodiments of the diacyl group, A2is -O- or -NH-. In some embodiments of the diacyl group, A2is -O-. In some embodiments of the diacyl group, Y3is C1-C12(e.g., C1-C6, such as C1-C3) alkylene.

[0157] In some embodiments of the diacyl group, the diacyl group independently at each occurrence comprises a structural formula(e.g., such as and optionally R3c, R3d,R3e, and R3fare each independently at each occurrence hydrogen or C1-C3alkyl.

[0158] In some embodiments, linker group independently comprises a structural formula** indicates a point of attachment of the linker to a proximal diacyl group,and *** indicates a point of attachment of the linker to a distal diacyl group.

[0159] In some embodiments of the linker group of XBranch, if present, Y1is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted arenylene. In some embodiments of the linker group of XBranch, if present, Y1is independently at each occurrence an optionally substituted alkylene (e.g, C1- C12). In some embodiments of the linker group of XBranch, if present, Y1is independently ateach occurrence an optionally substituted alkenylene (e.g., C1-C12). In some embodiments of the linker group of XBranch, if present, Y1is independently at each occurrence an optionally substituted arenylene (e.g., C1-C12).

[0160] In some embodiments of the terminating group of XBranch, each terminating group is independently selected from optionally substituted alkylthiol and optionally substituted alkenylthiol. In some embodiments of the terminating group of XBranch, each terminating group is an optionally substituted alkylthiol (e.g., C1-C18, such as C4-C18). In some embodiments of the terminating group of XBranch, each terminating group is optionally substituted alkenylthiol (e.g., C1-C18, such as C4-C18).

[0161] In some embodiments of the terminating group of XBranch, each terminating group is independently C1-C18alkenylthiol or C1-C18alkylthiol, and the alkyl or alkenyl moiety is optionally substituted with one or more substituents each independently selected from halogen, C6-C12aryl, C1-C12alkylamino, C4-C6N-heterocycloalkyl , -OH, -C(O)OH, -C(O)N(C1-C3alkyl)-(C1-C6alkylene)-(C1-C12alkylamino), -C(O)N(C1-C3alkyl)-(C1-C6alkylene)-(C4-C6N-heterocycloalkyl), -C(O)-(C1-C12alkylamino), and -C(O)-(C4-C6N-heterocycloalkyl), and the C4-C6N-heterocycloalkyl moiety of any of the preceding substituents is optionally substituted with C1-C3alkyl or C1-C3hydroxy alkyl.In some embodiments of the terminating group of XBranch, each terminating group is independently C1-C18(e.g., C4-C18) alkenylthiol or C1-C18(e.g., C4-C18) alkylthiol, wherein the alkyl or alkenyl moiety is optionally substituted with one or more substituents each independently selected from halogen, C6-C12aryl (e.g., phenyl), C1-C12(e.g., C1-C8) alkylamino (e.g., C1-C6mono-alkylamino (such as -NHCH2CH2CH2CH3) or C1-C8di- alkylamino (such as C4-C6N-heterocycloalkyl (e.g.,N-pyrrolidinylN-piperidinyl N-azepanyl -OH, -C(O)OH,-C(O)N(C1-C3alkyl)-(C1-C6alkylene)-(C1-C12alkylamino (e.g., mono- or di-alkylamino)) (e.g.,C(O)N(C1-C3alkyl)-(C1-C6alkylene)-(C4-C6N- heterocycloalkyl) (e.g.,-C(O)-(C1-C12alkylamino (e.g., mono- or dialkylamino)), and -C(O)-(C4-C6N-heterocycloalkyl) (e.g., wherein theC4-C6N-heterocycloalkyl moiety of any of the preceding substituents is optionally substituted with C1-C3alkyl or C1-C3hydroxyalkyl. In some embodiments of the terminating group of XBranch, each terminating group is independently C1-C18(e.g., C4-C18) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent -OH. In some embodiments of the terminating group of XBranch, each terminating group is independently C1-C18(e.g., C4-C18) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent selected from C1-C12(e.g., C1-C8) alkylamino (e.g., C1-C6mono-alkyl amino (such as -NHCH2CH2CH2CH3) or C1-C8di-alkylamino (such asand C4-C6N- heterocycloalkyl (e.g., N-pyrrolidinyl N-piperidinyl N-azepanyl)). In some embodiments of the terminating group of XBranch, each terminating group is independently C1-C18(e.g., C4-C18) alkenylthiol or C1-C18(e.g., C4-C18) alkylthiol. In some embodiments of the terminating group of XBranch, each terminating group is independently C1-C18(e.g., C4-C18) alkylthiol.Table 2. Example core structuresID# Structure

[0162] In some embodiments of XCore, the core comprises a structural formula selected from the group consisting of:, and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a branch of the plurality of branches.

[0163] In some embodiments of the terminating group of XBranch, each terminating group is independently a structure selected from the structure in Table 3. In some embodiments, the dendrimers described herein can comprise a terminating group or pharmaceutically acceptable salt, or thereof selected in Table 3.Table 3. Example terminating group / peripheries structures

[0164] In some embodiments, in the dendrimer of Formula (D-I) or (X) , the core of Formula D-II, D-III, D-IV or XCorehas a structure of Table 2 and the terminating group inXBranchor of formula D-VII has a structure of Table 3. In each of the structures of Table 2 andTable 3,denotes a point of attachment to the following structure:wherein the core of Table 2 and the terminating group of Table 3 are attached at opposite ends of the structure.

[0165] In some embodiments, the dendrimer of Formula (D-I) or (X) is selected from those set forth in Table 4 and pharmaceutically acceptable salts thereof.Table 4. Example ionizable cationic lipids (dendrimers)Other Ionizable cationic lipids

[0166] In some embodiments of the lipid composition, the ionizable cationic lipid comprises a structural formula (D-I'):wherein: a is 1 and b is 2, 3, or 4; or, alternatively, b is 1 and a is 2, 3, or 4; m is 1 and n is 1; or, alternatively, m is 2 and n is 0; or, alternatively, m is 2 and n is1; andR1, R2, R3, R4, R5, and R6are each independently selected from the group consisting of H, -CH2CH(OH)R7, -CH(R7)CH2OH, -CH2CH2C(=O)OR7, -CH2CH2C(=O)NHR7, and - CH2R7, wherein R7is independently selected from C3-C18alkyl, C3-C18alkenyl having one C=C double bond, a protecting group for an amino group, -C(=NH)NH2, a poly(ethylene glycol) chain, and a receptor ligand; provided that at least two moieties among R1to R6are independently selected from - CH2CH(OH)R7, -CH(R7)CH2OH, -CH2CH2C(=O)OR7, -CH2CH2C(=O)NHR7, or -CH2R7, wherein R7is independently selected from C3-C18alkyl or C3-C18alkenyl having one C=C double bond; and wherein one or more of the nitrogen atoms indicated in formula (D-I') may be protonated to provide an ionizable cationic lipid.

[0167] In some embodiments of the ionizable cationic lipid of formula (D-I'), a is 1. In some embodiments of the ionizable cationic lipid of formula (D-I'), b is 2. In some embodiments of the ionizable cationic lipid of formula (D-I'), m is 1. In some embodiments of the ionizable cationic lipid of formula (D-I'), n is 1. In some embodiments of the ionizable cationic lipid of formula (D-I'), R1, R2, R3, R4, R5, and R6are each independently H or - CH2CH(OH)R7. In some embodiments of the ionizable cationic lipid of formula (D-I'), R1, R2,R3, R4, R5, and R6are each independently H orIn some embodiments of the ionizable cationic lipid of formula (D-I'), R1, R2, R3, R4, R5, and R6are each independently H orIn some embodiments of the ionizable cationic lipid of formula (D-I'), R7is C3- C18alkyl (e.g., C6-C12alkyl).

[0168] In some embodiments, the ionizable cationic lipid of formula (D-I') is 13,16,20- tris(2 -hydroxy dodecyl)- 13,16,20,23 -tetraazap entatricontane- 11 ,25-diol :

[0169] In some embodiments, the ionizable cationic lipid of formula (D-I') is (11R,25R)- 13,16, 20-tris((R)-2-hydroxy dodecyl)- 13,16,20,23 -tetraazapentatricontane- 11 ,25-diol :

[0170] Additional ionizable cationic lipids that can be used in the compositions and methods of the present application include those ionizable cationic lipids as described inInternational Patent Publication W02010144740, WO2013149140, WO2016118725,WO2016118724, WO2013063468, WO2016205691, WO2015184256, WO2016004202,WO2015199952, W02017004143, WO2017075531, WO2017117528, WO2017049245,WO2017173054 and W02015095340, which are incorporated herein by reference for allpurposes. Examples of those ionizable cationic lipids include but are not limited to those as shown in Table 5 A and Table 5B.Table 5A. Example Ionizable cationic lipidsTable 5B. Selected Example Ionizable Cationic Lipids

[0171] In some embodiments, the ionizable cationic lipid is a lipid selected from Table 5A or Table 5B. In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present in the composition at a molar percentage from about 10% to about 25%.

[0172] In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present in the composition at a molar percentage about 5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.

[0173] In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present in the composition at a molar percentage from about 5% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 20% to about 60%, from about 20% toabout 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 10% to about 25%.

[0174] In some embodiments of the lipid composition of the present application, the ionizable lipid is present at a molar percentage of at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, or at least (about) 30%. In some embodiments of the lipid composition of the present application, the ionizable lipid is present at a molar percentage of at most (about) 5%, at most (about) 10%, at most (about) 15%, at most (about) 20%, at most (about) 25%, or at most (about) 30%.

[0175] In some embodiments, the lipid nanoparticle composition comprises a first ionizable cationic lipid. In some embodiments, the lipid nanoparticle composition comprises a first ionizable cationic lipid, wherein the first ionizable cationic lipid is a lipid selected from Table 4. In some embodiments, the lipid nanoparticle composition comprises a first ionizable cationic lipid, wherein the first ionizable cationic lipid is a lipid selected from Table 5A or Table 5B. In some embodiments the lipid component comprises the first ionizable cationic lipid in an amount of from about 10 mol % to about 55 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the first ionizable cationic lipid in an amount of from about 10 mol % to about 30 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the first ionizable cationic lipid in an amount of from about 10 mol % to about 20 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the first ionizable cationic lipid in an amount of from about 12 mol % to about 20 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the first ionizable cationic lipid in an amount of from about 14 mol % to about 17 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the first ionizable cationic lipid in an amount of from about 14 mol % to about 16 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the first ionizable cationic lipid in an amount of about 16 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the first ionizable cationic lipid in an amount of about 14 mol % of the total lipids in the lipid component.

[0176] In some embodiments, the first ionizable cationic lipid comprises:

[0177] In some embodiments, the first ionizable cationic lipid isSelective organ targeting (SORT) lipids

[0178] The lipid composition may further include a second lipid separate from the first lipid, wherein the second lipid is an anionic lipid, an ionizable cationic lipid, or permanently cationic lipid. In some embodiments of the lipid composition of the present application, the lipid (e.g., nanoparticle) composition is preferentially delivered to a target organ. The second lipid separate from the first lipid is also referred to herein as a “SORT lipid.”

[0179] In some embodiments of the lipid composition disclosed herein, the lipid composition further includes a lipid comprising a permanently positively charged moiety (i.e., is a permanently cationic lipid). The permanently positively charged moiety may be positively charged at a physiological pH such that the lipid comprises a positive charge upon delivery of a polynucleotide to a cell. In some embodiments the positively charged moiety is a quaternary amine or quaternary ammonium ion. In some embodiments, the lipid composition further comprises a lipid that comprises, or is otherwise complexed to or interacting with, a counterion.

[0180] In some embodiments of the lipid composition disclosed herein, the lipid composition further comprises a permanently cationic lipid (i.e., a lipid comprising one or more hydrophobic components and a permanently cationic group). The permanently cationic lipid may contain a group which has a positive charge regardless of the pH. One permanently cationic group that may be used in the permanently cationic lipid is a quaternary ammoniumgroup. The permanently cationic lipid may comprise a structural formula:wherein:Y1, Y2, or Y3are each independently X1C(O)R1or X2N+R3R4R5; provided at least one of Y1, Y2, and Y3is X2N+R3R4R5;R1is C1-C24alkyl, C1-C24substituted alkyl, C1-C24alkenyl, C1-C24substituted alkenyl;X1is O or NRa, wherein Rais hydrogen, C1-C4alkyl, or C1-C4substituted alkyl;X2is C1-C6alkanediyl or C1-C6substituted alkanediyl;R3, R4, and R5are each independently C1-C24alkyl, C1-C24substituted alkyl, C1-C24alkenyl, C1-C24substituted alkenyl; andA1is an anion with a charge equal to the number of X2N+R3R4R5groups in the compound.

[0181] In some embodiments, the permanently cationic lipid has a structural formula:wherein: R6-R9are each independently C1-C24alkyl, C1-C24substituted alkyl, C1-C24alkenyl, C1-C24substituted alkenyl; provided at least one of R6-R9is a group of C8-C24; and A2is a monovalent anion.

[0182] In some embodiments, the permanently cationic lipid is 1,2-dilauroyl-sn-glycero-3- ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycero-3 -ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycero-3 -ethylphosphocholine (16:0 EPC), 1,2-distearoyl-sn- glycero-3 -ethylphosphocholine (18:0 EPC), 1,2-dioleoyl-sn-glycero-3 -ethylphosphocholine (18:1 EPC), 1 -palmitoyl -2-oleoyl-sn-glycero-3 -ethylphosphocholine (16:0-18:0 EPC), 1,2- dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14: 1 EPC),Dimethyldioctadecylammonium (18:0 DDAB), 1,2-dimyristoyl-3-trimethylammonium- propane(14:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), 1,2-stearoyl- 3 -trimethylammonium -propane (18:0 TAP), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 TAP, DOTAP), or 1,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA).

[0183] In some embodiments of the lipid composition disclosed herein, the second lipid is an ionizable cationic lipid (e.g., comprising one or more hydrophobic components and an ionizable group, e.g., a tertiary amino group). The ionizable positively charged moiety may be positively charged at a physiological pH. One ionizable group that may be used in the ionizablecationic lipid is a tertiary ammine group. In some embodiments of the lipid compositions disclosed herein, the second lipid has a structural formula:wherein:R1and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group; andR3and R3' are each independently C1-C6alkyl or substituted C1-C6alkyl.

[0184] In some embodiments of formula (S-I’a) R1and R2are each independently C8-C24alkenyl (e.g., hexadecane, heptadecene, or octadecene). In some embodiments of formula (S- I’a), R3and R3' are each independently C1-C6alkyl (e.g, methyl or ethyl). In some embodiments of formula (S-I’a) R1and R2are each independently C8-C24alkenyl, (e.g, hexadecane, heptadecene, or octadecene) and R3and R3' are each independently C1-C6alkyl (e.g., methyl or ethyl).

[0185] In some embodiments, the ionizable cationic lipid is 1,2-distearoyl-3- dimethylammonium-propane (18:0 DAP), 1,2-dipalmitoyl-3 -dimethylammonium -propane (16:0 DAP), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), 1,2-dioleoyl-3- dimethylammonium-propane (18: 1 DAP, DODAP), or 1,2-dioleyloxy-3- dimethylaminopropane (DODMA).

[0186] In some embodiments of the lipid composition disclosed herein, the ionizable cationic lipid or permanently cationic lipid comprises a head group of a particular structure. In some embodiments, the permanently cationic lipid comprises a headgroup having a structural formula: wherein L is a linker; Z+is positively charged moiety and X- is acounterion. In some embodiment, the linker is a biodegradable linker. The biodegradable linker may be degradable under physiological pH and temperature. The biodegradable linker may be degraded by proteins or enzymes from a subject. In some embodiments, the positively charged moiety is a quaternary ammonium ion or quaternary amine.

[0187] In some embodiments of the lipid composition disclosed herein, the permanently cationic lipid has a structural formula: wherein R1and R2are eachindependently an optionally substituted C6-C24alkyl, or an optionally substituted C6-C24alkenyl.

[0188] In some embodiments of the lipid compositions, the permanently cationic lipid has a structural formula:

[0189] In some embodiments of the lipid composition disclosed herein, the second lipid comprises a Linker (L). In some embodiments, L is wherein:p and q are each independently 1, 2, or 3; andR4is an optionally substituted C1-C6alkyl

[0190] In some embodiments of the lipid composition disclosed herein, the permanently cationic lipid has a structural formula:wherein:R1and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group;R3, R3', and R3" are each independently C1-C6alkyl or substituted C1-C6alkyl; R4is C1-C6alkyl or substituted C1-C6alkyl; andX- is a monovalent anion.

[0191] In some embodiments of the lipid composition disclosed herein, the permanently cationic lipid is a phosphatidylcholine (e.g., 14:0 EPC). In some embodiments, the phosphatidylcholine compound is further defined as:wherein:R1and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group;R3, R3', and R3" are each independently C1-C6alkyl or substituted C1-C6alkyl; andX is a monovalent anion.

[0192] In some embodiments of the lipid composition disclosed herein, the lipid composition further comprises a phosphocholine lipid. In some embodiments, the lipid composition further comprises an ethylphosphocholine. The ethylphosphocholine may be, by way of example, without being limited to, 1,2-dimyristoleoyl-sn-glycero-3- ethylphosphocholine (14: 1 EPC), 1,2-dioleoyl-sn-glycero-3 -ethylphosphocholine (18: 1 EPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), 1,2-dipalmitoyl-sn-glycero-3- ethylphosphocholine (16:0 EPC), 1,2-dimyristoyl-sn-glycero-3 -ethylphosphocholine (14:0 EPC), 1,2-dilauroyl-sn-glycero-3 -ethylphosphocholine (12:0 EPC), l-palmitoyl-2-oleoyl-sn- glycero-3 -ethylphosphocholine (16:0-18:0 EPC).

[0193] In some embodiments of the lipid compositions, the lipid composition further comprises a lipid having a structural formula wherein:R1and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group;R3, R3', and R3" are each independently C1-C6alkyl or substituted C1-Ce alkyl;X- is a monovalent anion.

[0194] By way of example, a lipid (e.g., SORT lipid)) of the structural formula of the immediately preceding paragraph is 1,2-dioleoyl-3-trimethylammonium-propane (18: 1 DOTAP) (e.g., chloride salt).

[0195] In some embodiments of the lipid compositions, the permanently cationic lipid has a structural formula:wherein: R4and R4' are each independently alkyl(C6-C24), alkenyl(C6-C24), or a substituted version of either group; R4" is alkyl(C≤24), alkenyl(C≤24), or a substituted version of either group;R4”' is alkyl(C1-C8), alkenyl(C2-C8), or a substituted version of either group; andX2is a monovalent anion.

[0196] By way of example, and without being limited thereto, a lipid (e.g., SORT lipid) of the structural formula of the immediately preceding paragraph is dimethyldioctadecylammonium (DDAB).

[0197] In some embodiments of the lipid compositions, lipid composition further comprises:1,2-dioleoyl-sn-glycero-3 -phosphate (18: 1PA).Table 6. Example second lipid (e.g., SORT lipids)Table 7. Example second lipid (e.g., SORT lipids)

[0198] In some embodiments of the lipid composition of the present application, the second (e.g., SORT) lipid is present in the composition at a molar percentage from about 5% to about 50%.

[0199] In some embodiments of the lipid composition of the present application, the second (e.g., SORT) lipid is present in the composition at a molar percentage about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.

[0200] In some embodiments of the lipid composition of the present application, the second (e.g., SORT) lipid is present in the composition at a molar percentage from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 5% to about 20%, from about 5% to about 10%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 20% to about 25%.

[0201] In some embodiments of the lipid composition of the present application, the second (e.g., SORT) lipid is present at a molar percentage of at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, or at least (about) 55%. In some embodiments of the lipid composition of the present application, the ionizable lipid is present at a molar percentage of at most (about) 60%, at most (about) 55%, at most (about) 50%, at most (about) 45%, at most (about) 40%, at most (about) 35%, at most (about) 30%, or at most (about) 25%.Helper lipids

[0202] In some embodiments, lipid compositions described herein comprise a helper lipid. In some embodiments, the helper lipid is a phospholipid. Phospholipids, as defined herein, are any lipid that comprise a phosphate group. The lipid component of a lipid nanoparticle composition may include one or more phospholipids, such as one or more (poly) unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moi eties. A phospholipid moietymay be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2- lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety may be selected 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.

[0203] Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may 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 may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may 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).

[0204] Phospholipids useful or potentially useful in the compositions and methods described herein may comprise a: phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, or a derivative or analog thereof.

[0205] Phospholipids useful or potentially useful in the compositions and methods described herein may be selected from: 1,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 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), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn- glycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3 -phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 - phosphocholine, 1,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), 1,2- diphytanoyl-sn-glycero-3 -phosphocholine (4ME 16:0 PC), 1,2-diphytanoyl-sn-glycero-3- phospho-(l'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS), 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, and 1,2-dioleoyl-sn-glycero-3- phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin.

[0206] As described herein, phosphatidylcholine and phosphocholine may be used interchangeably. In some embodiments, the phosphatidylcholine is selected from: 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1 ,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 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-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn- glycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3 -phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 - phosphocholine, and 1,2-diphytanoyl-sn-glycero-3 -phosphocholine (4ME 16:0 PC).

[0207] In some embodiments, the lipid composition comprises a phospholipid selected from the group consisting of: 1,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 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-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn- glycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3 -phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 - phosphocholine, 1,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), 1,2- diphytanoyl-sn-glycero-3 -phosphocholine (4ME 16:0 PC), 1,2-diphytanoyl-sn-glycero-3- phospho-(l'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-diphytanoyl-sn-glycero-3- phospho-L-serine (sodium salt) (4ME 16:0 PS), 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, and 1,2-dioleoyl-sn-glycero-3- phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin.

[0208] In some embodiments, the lipid composition comprises a phospholipid selected from the group consisting of: 1,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1 ,2-diundecanoyl-sn-glycero- phosphocholine (DUPC).

[0209] In some embodiments, the lipid composition comprises a phospholipid selected from the group consisting of: 1,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE).

[0210] In some embodiments, the lipid composition comprises 1,2-distearoyl-sn-glycero- 3 -phosphocholine (DSPC). In some embodiments, the lipid composition comprises 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the lipid composition comprises 1,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC). In some embodiments, the lipid composition comprises 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC). In some embodiments, the lipid composition comprises 1,2-Dimyristoyl-sn-glycero- 3 -phosphoethanolamine (DMPE). In some embodiments, the lipid composition comprises 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE).

[0211] In some embodiments, the lipid composition comprises a phospholipid selected from the group consisting of: 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1,2-distearoyl-sn- glycero-3-phosphorylethanolamine (DSPE).

[0212] In some embodiments, the lipid composition comprises a phospholipid selected from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) 1,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE).

[0213] In some embodiments, the phospholipid may contain one or two long chain (e.g., C6-C24) alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionally, a small organic molecule. The small organic molecule may be an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipid defines lipid and lipid-like molecules with both a positive charge and a negative charge.

[0214] In some embodiments of the lipid composition of the present application, the helper lipid is present in the composition at a molar percentage from about 7.5% to about 30%.

[0215] In some embodiments of the lipid composition of the present application, the helper lipid is present in the composition at a molar percentage about 5%, about 7.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.

[0216] In some embodiments of the lipid composition of the present application, the helper lipid is present in the composition at a molar percentage from about 5% to about 25%, from about 5% to about 50%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 12% to about 30%, from about 12% to about 25%, from about 12% to about 20%, from about 14% to about 30%, from about 14% to about 25%, from about 14% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 16% to about 30%, from about 16% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 10% to about 25%.

[0217] In some embodiments of the lipid composition of the present application, the helper lipid is present at a molar percentage of at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, or at least (about) 30%. In some embodiments of the lipid composition of the present application, the ionizable lipid is present at a molar percentage of at most (about) 5%, at most (about) 10%, at most (about) 15%, at most (about) 20%, at most (about) 25%, or at most (about) 30%.

[0218] In some embodiments, the helper lipid is present in an amount of about 10 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 11 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 12 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 13 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 14 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 15 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 16 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 17 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 18 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 19 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 20 mol % of the total lipids in the lipid component.Structural lipids

[0219] The lipid nanoparticle may include one or more structural lipids. Structural lipids can be steroids or steroid derivatives. In some embodiments of the lipid composition of the present application, the lipid composition further comprises a steroid or steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms. In one aspect, the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as shown in the formula:In some embodiments, a steroid derivative comprises the ring structure above with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol wherein the formula is further defined as:In someembodiments of the present application, the steroid or steroid derivative is a cholestane or cholestane derivative. In a cholestane, the ring structure is further defined by the formula:As described above, a cholestane derivative comprises one or more non-alkyl substitution of the above ring system. In some embodiments, the cholestane or cholestane derivative is a cholestene or cholestene derivative or a sterol or a sterol derivative.In other embodiments, the cholestane or cholestane derivative is both a cholesterol and a sterol or a derivative thereof.

[0220] Sterol useful or potentially useful in the compositions and methods may be selected from: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.

[0221] In some embodiments of the lipid composition of the present application, the sterol is present in the composition at a molar percentage from about 20% to about 50%.

[0222] In some embodiments of the lipid composition of the present application, the sterol is present in the composition at a molar percentage about 10%, about 15%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 50%, about 55%, or about 60%.

[0223] In some embodiments of the lipid composition of the present application, the sterol is present in the composition at a molar percentage from about 10% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 20% to about 25%, from about 25% to about 50%, from about 25% to about 40%, from about 25% to about 30%, from about 30% to about 50%, from about 30% to about 40%, from about 30% to about 35%, from about 35% to about 50%, from about 35% to about 45%, from about 35% to about 40%, from about 40% to about 50%, from about 40% to about 45%, or from about 45% to about 50%.

[0224] In some embodiments of the lipid composition of the present application, the sterol is present at a molar percentage of at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, or at least (about) 50%. In some embodiments of the lipid composition of the present application, the ionizable lipid is present at a molarpercentage of at most (about) 60%, at most (about) 15%, at most (about) 45%, at most (about) 40%, at most (about) 35%, at most (about) 30%, at most (about) 25%, or at most (about) 20%.Polyethylene glycol lipid (PEG-lipid)

[0225] The lipid compositions of the disclosure may include lipids conjugated to polymers, such as lipids conjugated to polyethylene glycol (“PEG-lipid”). Illustrative methods for making and using PEG-lipids are described for example in Int'l Pat. Pub. No. WO2012099755 and U.S. Pat. Pub No. 2014 / 0200257.

[0226] A PEG-lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG-lipid may be PEG-c-DOMG, PEG-DMG, PEG- DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0227] In one embodiment, PEG-lipids useful in the present invention can be PEG-lipids described in Int’l Pat. Pub. No. WO 2012 / 099755, 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 certain embodiments, the PEG- lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” is a PEG-lipid having one or more hydroxyl ( — OH) groups on the lipid. In certain embodiments, the PEG-OH lipid comprises one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEG-lipid comprises an — OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0228] In some embodiments of the lipid composition of the present application, the lipid composition further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid is a PEG-lipid. In some embodiments, the PEG-lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group. In other embodiments, the PEG- lipid is a compound which contains one or more C6-C24long chain alkyl or alkenyl group or a C6-C24fatty acid group attached to a linker group with a PEG chain. Some non-limiting examples of a PEG-lipid comprises a PEG-modified phosphatidylethanolamine and phosphatidic acid, a PEG-Ceramide conjugated, PEG-modified dialkylamines and PEG- modified 1,2-diacyloxypropan-3 -amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified diastearoylphosphatidylethanolamine or PEG-modified dimyri stoy 1 -sn-glycerol .

[0229] In some embodiments, the PEG modification is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, up to about 25,000. Some non-limiting examples of lipids that may be used in the present application are taught by U.S. Patent 5,820,873, WO 2010 / 141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.

[0230] In some embodiments, the PEG-lipid is present in an amount of from about 0.5 mol % to about 5 mol % of the total lipids in the lipid component. In some embodiments, the PEG- lipid is present in an amount of from about 1.0 mol % to about 2 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid is present in an amount of from about 2 mol % to about 3 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid is present in an amount of from about 2 mol % to about 4 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid is present in an amount of from about 3 mol % to about 4 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid is present in an amount of from about 4 mol % to about 5 mol % of the total lipids in the lipid component.

[0231] In some embodiments, the PEG-lipid comprises a phosphoglyceride PEG-lipid. In some embodiments, the PEG-lipid comprises a diglyceride PEG-lipid. In some embodiments, the PEG-lipid comprises a PEG-Ceramide.

[0232] In some embodiments, the PEG-lipid is a PEG-Ceramide. In some embodiments the PEG-Ceramide is a C8 PEG-Ceramide. In some embodiments the PEG-Ceramide is a C 12PEG-Ceramide. In some embodiments the PEG-Ceramide is a C14 PEG-Ceramide. In some embodiments the PEG-Ceramide is a C16 PEG-Ceramide. In some embodiments the PEG-Ceramide is a C18 PEG-Ceramide.

[0233] In some embodiments, the PEG-Ceramide comprises N-octanoyl-sphingosine-1- { succinyl [methoxy (poly ethylene glycol)5000]} (C8 PEG5000-Ceramide), N-octanoyl- sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]}(C8 PEG2000-Ceramide), N- octanoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)750]} (C8 PEG750-Ceramide), N-palmitoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)5000]} (C16 PEG5000-Ceramide), N-palmitoyl-sphingosine-l-{succinyl[methoxy(poly ethyleneglycol)2000]} (C16 PEG200-Ceramide), oorr N-palmitoyl-sphingosine-1- { succinyl [methoxy (poly ethylene glycol)750]} (C16 PEG750-Ceramide),

[0234] In some embodiments, the PEG-Ceramide is selected from the group consisting of N-octanoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)5000]} (C8 PEG5000- Ceramide), N-octanoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]}(C8 PEG2000-Ceramide), N-octanoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)750]} (C8 PEG750-Ceramide), N-palmitoyl-sphingosine-1-{ succinyl [methoxy (poly ethylene glycol)5000]} (C16 PEG5000-Ceramide), N-palmitoyl- sphingosine-l-[succinyl[methoxy(polyethylene glycol)2000]} (C16 PEG200-Ceramide), or N-palmitoyl-sphingosine-l-[succinyl[methoxy(polyethylene glycol)750]} (C16 PEG750- Ceramide).

[0235] In some embodiments, the PEG-Ceramide is C8 PEG750-Ceramide. In some embodiments, the PEG-Ceramide is C8 PEG2000-Ceramide.

[0236] In some embodiments, the lipid composition comprises more than one PEG-lipid. In some embodiments, the lipid composition comprises a first PEG-lipid and a second PEG- lipid. In some embodiments, the PEG-lipid comprises a first PEG-Ceramide and a second PEG- Ceramide. In some embodiments, the PEG-lipid comprises a first PEG-Ceramide and a second PEG ceramide, wherein the first PEG-Ceramide and the second PEG-Ceramide are not the same. In some embodiments, the first PEG-Ceramide and the second PEG-Ceramide are each independently selected from: a PEG750-Ceramide, a PEG2000-Ceramide, and a PEG5000- Ceramide. In some embodiments, the first PEG-Ceramide and the second PEG-Ceramide are each independently a C8 PEG-Ceramide (i.e., the lipid composition comprises a dual C8- Ceramide). In some embodiments, the first PEG-lipid and the second PEG-lipid are in a ratio of about: 1 : 1, 1 :2, 1 :3, 2: 1, 2:3, 1 :4, 1 :5, 2:5, 3: 1, 3:2, 3:4, 3:5, 4: 1, or 5: 1.

[0237] In some embodiments, the first PEG-lipid or the second PEG-lipid is a DMG PEG- lipid. In some embodiments, the first PEG-lipid or the second PEG-lipid is a PEG-Ceramide lipid. In some embodiments, the first PEG-lipid or the second PEG-lipid is a diglyceride PEG- lipid. In some embodiments, the first PEG-lipid or the second PEG-lipid is a polyglyceride PEG-lipid.

[0238] In some embodiments the first PEG-lipid and the second PEG-lipid are both DMG PEG-lipids. In some embodiments the first PEG-lipid and the second PEG-lipid are both PEG- Ceramide lipids. In some embodiments the first PEG-lipid and the second PEG-lipid are both diglyceride PEG-lipids. In some embodiments the first PEG-lipid and the second PEG-lipid are both polyglyceride PEG-lipids.

[0239] In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide and C8 PEG750-Ceramide (i.e., a dual C8-Ceramide). In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide and C8 PEG5000-Ceramide. In some embodiments, the PEG-lipid comprises C8 PEG750-Ceramide and C8 PEG5000-Ceramide. In some embodiments, the C8 PEG750-Ceramide is present in an amount from about 0.1 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG750 -Ceramide is present in an amount from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG750-Ceramide is present in an amount from about 0.5 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG750 -Ceramide is present in an amount from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG750-Ceramide is present in an amount from about 1.0 mol % to about 2.0 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG750 -Ceramide is present in an amount from about 1.5 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG750-Ceramide is present in an amount of about 0.25 mol %, about 0.5 mol %, about 0.75 %, about 1.0 mol %, about 1.25 mol %, about 1.5 mol %, about 1.75 mol %, about 2.0 mol %, about 2.25 mol %, or about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG750-Ceramide is present in an amount of about 0.5 mol %, about 0.75 mol %, about 1.0 mol %, about 1.5 mol % or about 2.25 mol % of the total lipids in the lipid component.

[0240] In some embodiments, the C8 PEG2000-Ceramide is present in an amount from about 0.1 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG2000-Ceramide is present in an amount from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG2000-Ceramide is present in an amount from about 0.5 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG2000-Ceramide is present in an amount from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG2000-Ceramide is present in an amount from about 1.0 mol % to about 2.0 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG2000-Ceramide is present in an amount from about 1.5 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG2000-Ceramide is present in an amount of about 0.25 mol %, about 0.5 mol %, about 0.75 %, about 1.0 mol %, about 1.25 mol %, about 1.5 mol %, about 1.75 mol %, about 2.0 mol %, about 2.25 mol %, or about 2.5 mol % of the total lipids in the lipid component. In someembodiments, the C8 PEG2000-Ceramide is present in an amount of about 0.5 mol %, about 0.75 mol %, about 1.0 mol %, about 1.5 mol % or about 2.25 mol % of the total lipids in the lipid component.

[0241] In some embodiments, the C8 PEG5000-Ceramide is present in an amount from about 0.1 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG5000-Ceramide is present in an amount from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG5000-Ceramide is present in an amount from about 0.5 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG5000-Ceramide is present in an amount from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG5000-Ceramide is present in an amount from about 1.0 mol % to about 2.0 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG5000-Ceramide is present in an amount from about 1.5 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG5000-Ceramide is present in an amount of about 0.25 mol %, about 0.5 mol %, about 0.75 %, about 1.0 mol %, about 1.25 mol %, about 1.5 mol %, about 1.75 mol %, about 2.0 mol %, about 2.25 mol %, or about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG5000-Ceramide is present in an amount of about 0.5 mol %, about 0.75 mol %, about 1.0 mol %, about 1.5 mol % or about 2.25 mol % of the total lipids in the lipid component.

[0242] In some embodiments, the C8 PEG2000-Ceramide and the C8 PEG750-Ceramide are present in total combined amount of about 2 mol % to about 3 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG2000-Ceramide and the C8 PEG5000- Ceramide are present in total combined amount of about 2 mol % to about 3 mol % of the total lipids in the lipid component. In some embodiments, the C8 PEG750-Ceramide and the C8 PEG5000-Ceramide are present in total combined amount of about 2 mol % to about 3 mol % of the total lipids in the lipid component.

[0243] In some embodiments, the C8 PEG2000-Ceramide is present in an amount of from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component, the C8 PEG750- Ceramide is present in an amount of from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component, and the C8 PEG2000-Ceramide and the C8 PEG750-Ceramide are present in total combined amount of from about 2 mol % to about 3 mol % of the total lipids in the lipid component.

[0244] In some embodiments, the C8 PEG2000-Ceramide is present in an amount of from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component, the C8 PEG5000-Ceramide is present in an amount of from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component, and the C8 PEG2000-Ceramide and the C8 PEG5000- Ceramide are present in total combined amount of from about 2 mol % to about 3 mol % of the total lipids in the lipid component.

[0245] In some embodiments, the C8 PEG5000-Ceramide is present in an amount of from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component, the C8 PEG750- Ceramide is present in an amount of from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component, and the C8 PEG5000-Ceramide and the C8 PEG750-Ceramide are present in total combined amount of from about 2 mol % to about 3 mol % of the total lipids in the lipid component.

[0246] In some embodiments of the lipid composition of the present application, the PEG- lipid has a structural formula: wherein: R12and R13are eachindependently alkyl(C≤24), alkenyl(C≤24), or a substituted version of either of these groups; Reis hydrogen, alkyl(C≤8), or substituted alkyl(C≤8); and x is 1-250. In some embodiments, Reis alkyl(C≤8)such as methyl. R12and R13are each independently alkyl(C≤4-20). In some embodiments, x is 5-250. In one embodiment, x is 5-125 orx is 100-250. In some embodiments, the PEG-lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0247] In some embodiments of the lipid composition of the present application, the PEG- lipid has a structural formula:wherein: n1is an integer between 1 and 100 and n2and n3are each independently selected from an integer between 1 and 29. In some embodiments, n1is 5, 10, 15, 20, 25, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, or 100, or any range derivable therein. In some embodiments, n1is from about 30 to about50. In some embodiments, n2is from 5 to 23. In some embodiments, n2is 11 to about 17. In some embodiments, n3is from 5 to 23. In some embodiments, n3is 11 to about 17.

[0248] In some embodiments of the lipid composition of the present application, the PEG- lipid is selected from: 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG) (e.g., 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG)), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[0249] In some embodiments of the lipid composition of the present application, the PEG- lipid is present in the composition at a molar percentage from about 0.5% to about 10%.

[0250] In some embodiments of the lipid composition of the present application, the PEG- lipid is present in the composition at a molar percentage about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0251] In some embodiments of the lipid composition of the present application, the PEG- lipid is present in the composition at a molar percentage from about 0.5% to about 10%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, from about 0.5% to about 1%, from about 1% to about 5%, from about 1% to about 4.5%, from about 1% to about 4%, from about 1% to about 3.5%, from about 1% to about 3%, from about 1% to about 2%, from about 2% to about 5%, from about 2% to about 4.5%, from about 2% to about 4%, from about 2% to about 3.5%, from about 2% to about 3%, from about 3% to about 5%, from about 3% to about 4.5%, from about 3% to about 4%, from about 3% to about 3.5%, from about 4% to about 5%, or from about 4% to about 4.5%.

[0252] In some embodiments of the lipid composition of the present application, the PEG- lipid is present at a molar percentage of at least (about) 0.5%, at least (about) 1%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, or at least (about) 3.5%. In some embodiments of the lipid composition of the present application, the ionizable lipid is present at a molar percentage of at most (about) 10%, at most (about) 9%, at most (about) 8%, at most (about) 7%, at most (about) 6%, or at most (about) 5%.Multi-component Lipid Nanoparticle Compositions

[0253] In one aspect, the disclosure provides a lipid nanoparticle composition for delivering a payload to a cell in the liver of a subject, including a payload, a helper lipid (e.g.,a phospholipid), a sterol, a polyethylene glycol-conjugated lipid (PEG-lipid), an ionizable cationic lipid, and / or a permanently cationic lipid, optionally an ethylphosphocholine.

[0254] In some embodiments the lipid nanoparticle composition further comprises a permanently cationic lipid or a second ionizable cationic lipid separate from the first ionizable cationic lipid. In some embodiments, the permanently cationic lipid or the second ionizable cationic lipid separate from the first ionizable cationic lipid is selected from 16:0 TAP, 18:0 TAP, 16:0 EPC, 18:0 EPC, and DODAP.

[0255] In some embodiments the lipid nanoparticle composition further comprises a permanently cationic lipid or a second ionizable cationic lipid separate from the first ionizable cationic lipid. In some embodiments, the permanently cationic lipid or the second ionizable cationic lipid separate from the first ionizable cationic lipid is selected from 16:0 TAP, 18:0 TAP, 16:0 EPC, 18:0 EPC, and DODAP, and further comprises a compound selected from

[0256] In some embodiments, the lipid nanoparticle composition further comprises an anionic lipid. In some embodiments, the anionic lipid comprises 1,2-dioleoyl-sn-glycero-3- phosphate (18: 1 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), 1,2-dipalmitoyl-sn- glycero-3 -phosphate, (16:0 PA), 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), and 1,2- dilauroyl-sn-glycero-3-phosphate (12:0 PA). In some embodiments, the anionic lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphate (18: 1 PA), 1,2-distearoyl-sn-glycero-3- phosphate (18:0 PA), 1,2-dipalmitoyl-sn-glycero-3 -phosphate, (16:0 PA), 1,2-dimyristoyl-sn- glycero-3 -phosphate (14:0 PA), and 1,2-dilauroyl-sn-glycero-3 -phosphate (12:0 PA).

[0257] In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of from about 10 mol % to about 55 mol % of the total lipids in the lipid component.In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of from about 15 mol % to about 55 mol % of the total lipids in the lipid component. In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of from about 30 mol % to about 55 mol % of the total lipids in the lipid component. In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of from about 35 mol % to about 50 mol % of the total lipids in the lipid component. In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of from about 40 mol % to about 50 mol % of the total lipids in the lipid component. In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of from about 35 mol % to about 45 mol % of the total lipids in the lipid component. In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of from about 10 mol % to about 30 mol % of the total lipids in the lipid component. In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of from about 10 mol % to about 20 mol % of the total lipids in the lipid component. In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of from about 15 mol % to about 20 mol % of the total lipids in the lipid component.

[0258] In some embodiments, the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of about 15, 20, 25, 30, 35, 40, 45 up to about 50 mol % of the total lipids in the lipid component. In some embodiments, the lipid nanoparticle composition comprises a first ionizable cationic lipid, a PEG-lipid, a permanently cationic lipid or second cationic lipid separate from the first ionizable cationic lipid, and a helper lipid. In some embodiments, the PEG-lipid is a PEG-Ceramide. In some embodiments, the helper lipid is a phospholipid. In some embodiments, the lipid nanoparticle composition comprises a first ionizable cationic lipid, a PEG-Ceramide, a permanently cationic lipid or second cationic lipid separate from the first ionizable cationic lipid, and a phospholipid.

[0259] In some embodiments, the lipid nanoparticle composition comprises a first ionizable cationic lipid, a PEG-lipid, a permanently cationic lipid or second cationic lipid separate from the first ionizable cationic lipid, and a helper lipid. In some embodiments, the first ionizable cationic lipid is present in an amount from about 10 mol % up to 20 mol % of the total lipids in the lipid component, the PEG-lipid is present in a total amount of about 3 mol % of the total lipids in the lipid component, the permanently cationic lipid or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of about 40 mol % or about 45 mol % of the total lipids in the lipid component, and the phospholipid is present in an amount of about 10 mol % up to 20 mol % of the total lipids in the lipid component.

[0260] In some embodiments, the lipid nanoparticle composition further comprises a sterol. In some embodiments the sterol is present in an amount of from about 20 mol % to about 50 mol % of the total lipids in the lipid component. In some embodiments the sterol is present in an amount of from about 20 mol % to about 30 mol % of the total lipids in the lipid component. In some embodiments the sterol is present in an amount of about 10, 15, 20, 30, 40, up to about 50 mol % of the total lipids in the lipid component. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is a derivative or analog of cholesterol.

[0261] In some embodiments, the lipid nanoparticle composition comprises a formulation of Table 8. In some embodiments, the lipid nanoparticle composition is a formulation of Table 8. In some embodiments, the lipid nanoparticle composition comprises a formulation of Table 8.

[0262] In some embodiments, the lipid nanoparticle composition comprises formulation B.

[0263] In some embodiments, the lipid nanoparticle composition is formulation B.

[0264] In some embodiments, the lipid nanoparticle composition comprises formulation 2L.

[0265] In some embodiments, the lipid nanoparticle composition is formulation 2L.

[0266] In some embodiments, the lipid nanoparticle composition comprises formulation3F1.

[0267] In some embodiments, the lipid nanoparticle composition is formulation 3F1.

[0268] In some embodiments, the lipid nanoparticle composition comprises formulation2M.

[0269] In some embodiments, the lipid nanoparticle composition is formulation 2M.

[0270] In some embodiments, the lipid nanoparticle composition comprises formulation X.

[0271] In some embodiments, the lipid nanoparticle composition is formulation X.

[0272] In some embodiments, the lipid nanoparticle composition comprises formulation3E4.

[0273] In some embodiments, the lipid nanoparticle composition is formulation 3E4.

[0274] In some embodiments, the lipid nanoparticle composition comprises formulation3E5.

[0275] In some embodiments, the lipid nanoparticle composition is formulation 3E5.

[0276] In some embodiments, the lipid nanoparticle composition comprises formulation3E7.

[0277] In some embodiments, the lipid nanoparticle composition is formulation 3E7.

[0278] In some embodiments, the lipid nanoparticle composition comprises formulationW.

[0279] In some embodiments, the lipid nanoparticle composition is formulation W.

[0280] In some embodiments, the lipid nanoparticle composition comprises formulation2B.

[0281] In some embodiments, the lipid nanoparticle composition is formulation 2B.

[0282] In some embodiments, the lipid nanoparticle composition comprises formulation E.

[0283] In some embodiments, the lipid nanoparticle composition is formulation E.

[0284] In some embodiments, the lipid nanoparticle composition comprises formulationLP01.

[0285] In some embodiments, the lipid nanoparticle composition is formulation LP01.

[0286] In some embodiments, the lipid nanoparticle composition comprises formulation Lipid-V. In some embodiments, the lipid nanoparticle composition is formulation Lipid-V. In some embodiments, in some embodiments, Lipid-V is an ionizable lipid disclosed in W02017 / 004143. In some embodiments, Lipid-V is Compound 9 or Compound 10 of WO20 17 / 004143.

[0287] In some embodiments, the lipid nanoparticle composition comprises formulationLNP-3.

[0288] In some embodiments, the lipid nanoparticle composition is formulation LNP-3.

[0289] In some embodiments, the lipid nanoparticle composition comprises formulationLNP-4.

[0290] In some embodiments, the lipid nanoparticle composition is formulation LNP-4.

[0291] In some embodiments, the payload comprises a polynucleotide, optionally an mRNA, shRNA, or microRNA. In some embodiments, the mRNA encodes a polynucleotide selected from the group shown in Table 9 and / or SEQ ID NOs: 1-6. In some embodiments, themRNA encodes a gene-editing system or component thereof. In some embodiments, the payload comprises a polypeptide or a protein.

[0292] In some embodiments, the ethylphosphocholine is 1,2-dipalmitoyl-sn-glycero-3- ethylphosphocholine (16:0 EPC). In some embodiments, the ionizable cationic lipid is 4A3- SC7. In some embodiments, the helper lipid is 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE).

[0293] In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of about 5 to about 30%, 16:0 EPC at a molar percentage of about 20 to about 50%, DOPE at a molar percentage of about 8 to about 23%, cholesterol at a molar percentage of about 15 to about 46%, and / or DMG-PEG at a molar percentage of about 0.5 to about 10%. In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of about 13 to about 20%, 16:0 EPC at a molar percentage of about 20 to about 40%, DOPE at a molar percentage of about 13 to about 20%, cholesterol at a molar percentage of about 30 to about 40%, and / or DMG-PEG at a molar percentage of about 3 to about 5%. In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of about 16%, 16:0 EPC at a molar percentage of about 30%, DOPE at a molar percentage of about 16%, cholesterol at a molar percentage of about 33%, and / or DMG-PEG at a molar percentage of about 3%. In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of about 14%, 16:0 EPC at a molar percentage of about 40%, DOPE at a molar percentage of about 14%, cholesterol at a molar percentage of about 39%, and / or DMG-PEG at a molar percentage of about 4%.

[0294] In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of 19%, 16:0 EPC at a molar percentage of 20%, DOPE at a molar percentage of 19%, cholesterol at a molar percentage of 38%, and / or DMG-PEG at a molar percentage of 4%. In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of 17%, 16:0 EPC at a molar percentage of 30%, DOPE at a molar percentage of 17%, cholesterol at a molar percentage of 33% and / or DMG-PEG at a molar percentage of 3%. In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of 16%, 16:0 EPC at a molar percentage of 40%, DOPE at a molar percentage of 17%, cholesterol at a molar percentage of 33% and / or DMG-PEG at a molar percentage of 3%. In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of 14%, 16:0 EPC at a molar percentage of 40%, DOPE at a molar percentage of 14%, cholesterol at a molar percentage of 39%, and / or DMG-PEG at a molar percentage of 4%. In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of 14%, 16:0 EPC at a molar percentage of 45%, DOPE at a molar percentage of14%, cholesterol at a molar percentage of 39%, and / or DMG-PEG at a molar percentage of4%. In some embodiments, the composition comprises 4A3-SC7 at a molar percentage of 12%,16:0 EPC at a molar percentage of 50%, DOPE at a molar percentage of 12%, cholesterol at a molar percentage of 24%, and / or DMG-PEG at a molar percentage of 2%.Table 8. Compositions of illustrative hepatic LNP formulations and relevant comparators (italic).

[0295] In some embodiments, a variant of composition B comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, DODAP at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition B may comprise 4A3-SC7 at a molar percentage of 19.05%, DODAP at a molar percentage of 20%, DOPE at a molar percentage of 19.05%, cholesterol at a molar percentage of 38.09%, and / or DMG-PEG at a molar percentage of 3.81%.

[0296] In some embodiments, a variant of composition 2L comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition 2L may comprise 4A3-SC7 at a molar percentage of 23.81%, DOPE at a molar percentage of 23.81%, cholesterol at a molar percentage of 47.62%, and / or DMG-PEG at a molar percentage of 4.76%.

[0297] In some embodiments, a variant of composition 3F1 comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, dendrimer at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition 3F1 may comprise 4A3-SC7 at a molar percentage of 17.86%, dendrimer at a molar percentage of 25%, DOPE at a molar percentage of 17.86%, cholesterol at a molar percentage of 35.71%, and / or DMG-PEG at a molar percentage of 3.57%.

[0298] In some embodiments, a variant of composition 2M comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, dendrimer at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition 2M may comprise 4A3-SC7 at a molar percentage of 14.8%, dendrimer at a molar percentage of 15.6%, DOPE at a molar percentage of 22.2%, cholesterol at a molar percentage of 44.4%, and / or DMG-PEG at a molar percentage of 3%.

[0299] In some embodiments, a variant of composition X comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, DODAP at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition X may comprise 4A3-SC7 at a molar percentage of 14.8%, DODAP at a molar percentage of 15.6%, DOPE at a molar percentage of 22.2%, cholesterol at a molar percentage of 44.4%, and / or DMG-PEG at a molar percentage of 3%.

[0300] In some embodiment, a variant of composition 3E4 comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 14:0 DAP at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition 3E4 may comprise 4A3-SC7 at a molar percentage of 19.05%, 14:0 DAP at a molar percentage of 20%, DOPE at a molar percentage of 19.05%, cholesterol at a molar percentage of 38.09%, and / or DMG-PEG at a molar percentage of 3.81%.

[0301] In some embodiments, a variant of composition 3E5 comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 14:0 DAP at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition 3E5 may comprise 4A3-SC7 at a molar percentage of 19.05%, 14:0 DAP at a molar percentage of 20%, DOPE at a molar percentage of 19.05%, cholesterol at a molar percentage of 38.09%, and / or DMG-PEG at a molar percentage of 3.81%.

[0302] In some embodiments, a variant of composition 3E7 comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 14:0 DAP at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition 3E7 may comprise 4A3-SC7 at a molar percentage of 19.04%, 14:0 DAP at a molar percentage of 20%, DOPE at a molar percentage of 19.05%, cholesterol at a molar percentage of 38.72%, and / or DMG-PEG at a molar percentage of 3.2%.

[0303] In some embodiments, a variant of composition W comprises D-Lin-MC3-DMA at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition W may comprise D-Lin-MC3-DMA at a molar percentage of 50%, DOPE at a molar percentage of 19.05%, cholesterol at a molar percentage of 38.5%, and / or DMG-PEG at a molar percentage of 1.5%.

[0304] In some embodiments, a variant of composition 2B comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 EPC at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition 2B may comprise 4A3-SC7 at a molar percentage of 16.67%, 16:0 EPC at a molar percentage of 30%, DOPE at a molar percentage of 19.05%, cholesterol at a molar percentage of 33.3%, and / or DMG-PEG at a molar percentage of 3.33%.

[0305] In some embodiments, a variant of composition E comprises 5A2-SC8 at a molar percentage of about 10 to about 50%, DOTAP at a molar percentage of about 10 to about 50%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 60%, and / or DMG-PEG at a molar percentage of about 0.1 to about 10%. Composition E may comprise 5A2-SC8 at a molar percentage of 11.9%, DOTAP at a molar percentage of 50%, DOPE at a molar percentage of 19.05%, cholesterol at a molar percentage of 23.82%, and / or DMG-PEG at a molar percentage of 2.38%.

[0306] As disclosed in the Examples below, lipid nanoparticle compositions of the disclosure (e.g., lipid nanoparticle compositions comprising a dendrimer of 4A3-SC7, a SORT lipid of DODAP or 14: DAP, DMG PEG, and DOPE) were identified as top formulations for liver and other organs. Also, as disclosed in the Examples, MC3 dendrimer (Onpattro) had a very strong liver-tropic effect, as does the 14:0 DAP SORT. Further, as shown in the Examples below, in some embodiments, lipid nanoparticle compositions of the disclosure have a high liver / spleen ratio compared to other formulations, e.g., Formulation W. Moreover, in some embodiments, lipid nanoparticle compositions of the disclosure have a high liver / (lung+spleen) ratio compared to other formulations, e.g., Formulation W. Without wishing to be bound by theory, avoiding unwanted delivery of the nanoparticles of the disclosure, comprising payloads, to organs other than the target organ (e.g., the liver) lowers the potential for toxic effects in those other organs. For example, unwanted delivery to the spleen of an mRNA expressing a polypeptide intended to have a treatment effect in the liver, may lead to expression of the polypeptide in cells of the spleen, where it may have an effect other than the intended effect.

[0307] Without wishing to be bound by theory, formulating LNPs with: an ionizable lipid (e.g., D-Lin-MC3-DMA or 4A3-SC7) and 14:0 DAP SORT, DODAP, DOTAP, 2A11SC7, or 2A9-Cit may increase the selectivity of the LNP for targeting the liver.Payloads

[0308] The present disclosure contemplates delivery of various payloads useful in the treatment of a liver disease. Payloads comprise therapeutic polypeptides or polynucleotides encoding polypeptides. For example, the payload may be a polynucleotide encoding a gene related to liver disease, or a polynucleotide encoding a gene editor for editing a gene related to liver disease.

[0309] In some embodiments, lipid nanoparticle compositions described herein further comprise a payload. In some embodiments, the payload comprises a polypeptide or a protein.In some embodiments, the payload comprises a small interfering RNA (siRNA). In some embodiments, the payload comprises an mRNA. In some embodiments, the mRNA encodes a gene editing system of component thereof. In some embodiments the gene editing system of component thereof comprises a cluster regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), and a guide RNA.

[0310] In some embodiments, the disclosure provides polypeptides comprising one or more therapeutic proteins. Therapeutic proteins comprise, but are not limited to cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anti-coagulants, blood factors, bone morphogenic proteins, immunoglobulins, or enzymes. Some non-limiting examples of particular therapeutic proteins include Erythropoietin (EPO), Granulocyte colonystimulating factor (G-CSF), Alpha-galactosidase A, Alpha-L-iduronidase, Thyrotropin a, N- acetylgalactosamine-4-sulfatase (rhASB), Domase alfa, Tissue plasminogen activator (tPA), Glucocerebrosidase, Interferon (IF) b-la, Interferon b-lb, Interferon gamma, Interferon alpha, TNF-alpha, IL-1 through IL-36, Human growth hormone (rHGH), Human insulin (BHI), Human chorionic gonadotropin a, Darbepoetin a, Follicle-stimulating hormone (FSH), and Factor VIII. In some embodiments, the cytokine is a proinflammatory cytokine. In some embodiments, the cytokine is IL-1β, MIP-1α, MCP-1, TNF-α, G-CSG, or a related cytokine thereof.

[0311] In some embodiments, the polypeptide comprises a peptide or protein that restores the function of a defective protein in a subject. For example, the polynucleotide encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein, Dynein axonemal heavy chain 5, Dynein axonemal heavy chain 11, Bone morphogenetic protein receptor type 2, Fumarylacetoacetate hydrolase, Phenylalanine hydroxylase, Alpha-L-iduronidase, Collagen type IV alpha 3 chain, Collagen type IV alpha 4 chain, Collagen type IV alpha 5 chain, Poly cystin 1, Poly cystin 2, Fibrocystin (or poly ductin), Solute carrier family 3 member 1, Solute carrier family 7 member 9, Paired box gene 9, Myosin VIIA, Cadherin related 23, Usherin, Clarin 1, Gap junction beta-2 protein, Gap junction beta-6 protein, Rhodopsin, dystrophia myotonica protein kinase , Dystrophin, Sodium voltage-gated channel alpha subunit 1, Sodium voltage-gated channel beta subunit 1, Coagulation factor VIII, Coagulation factor IX ,N-glycanase 1, Palmitoyl-protein thioesterase 1, Tripeptidyl peptidase 1, Kvl 1.1 (alpha subunit of potassium ion channel), Palmitoyl-protein thioesterase 1, ATM serine / threonine kinase, or Fibrillin 1.Polynucleotides

[0312] In some embodiments, the lipid composition described herein comprises one or more polynucleotides. In some embodiments, the polynucleotides encode for one or more polypeptides described herein.

[0313] Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-α-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.

[0314] In addition, it should be clear that the present disclosure is not limited to the specific polynucleotides disclosed herein. The present disclosure is not limited in scope to any particular source, sequence, or type of polynucleotides, however, as one of ordinary skill in the art could readily identify related homologs in various other sources of the polynucleotides including polynucleotides from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species). It is contemplated that the polynucleotides used in the present disclosure can comprise a sequence based upon a naturally-occurring sequence. Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotide sequence of the naturally-occurring sequence. In some embodiments, the polynucleotide is a complementary sequence to a naturally occurring sequence, or complementary to at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and 100%. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated herein.

[0315] In some embodiments, the polynucleotide used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In some embodiments, the polynucleotide comprises complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as “mini-genes”. The term “cDNA” is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processedRNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy.

[0316] In some embodiments, the polynucleotide comprises one or more segments comprising a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro-ribonucleic acid (miRNA), a primary micro-ribonucleic acid (pri-miRNA), a long noncoding RNA (IncRNA), a messenger ribonucleic acid (mRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, deoxyribonucleic acid (DNA), a double stranded deoxyribonucleic acid (dsDNA), a single stranded deoxyribonucleic acid (ssDNA), a single stranded ribonucleic acid (ssRNA), a or double stranded ribonucleic acid (dsRNA). In some embodiments, the polynucleotide encodes at least one of the therapeutic agents (or prophylactic agents) described herein.

[0317] In some embodiments, the polynucleotide is greater than 30 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, greater than 200 nucleotides, greater than 300 nucleotides, greater than 400 nucleotides, greater than 500 nucleotides, greater than 600 nucleotides, greater than 700 nucleotides, greater than 800 nucleotides, greater than 900 nucleotides, greater than 1000 nucleotides, greater than 1500 nucleotides, greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500 nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides in length.

[0318] In some embodiments, the mRNA is about 50 nucleotides in length. In some embodiments, the mRNA molecule is about 100 nucleotides in length. In some embodiments, the mRNA molecule is about 200 nucleotides in length. In some embodiments, the mRNA molecule is about 300 nucleotides in length. In some embodiments, the mRNA molecule is about 400 nucleotides in length. In some embodiments, the mRNA molecule is about 500 nucleotides in length. In some embodiments, the mRNA molecule is about 600 nucleotides in length. In some embodiments, the mRNA molecule is about 700 nucleotides in length. In some embodiments, the mRNA molecule is about 800 nucleotides in length. In some embodiments, the mRNA molecule is about 900 nucleotides in length. In some embodiments, the mRNA molecule is about 1000 nucleotides in length. In some embodiments, the mRNA molecule is about 2000 nucleotides in length. In some embodiments, the mRNA molecule is about 3000nucleotides in length. In some embodiments, the mRNA molecule is about 4000 nucleotides in length. In some embodiments, the mRNA molecule is about 5000 nucleotides in length.

[0319] In some embodiments, the polynucleotide comprises about 50 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 100 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 2000 nucleotides.

[0320] In some embodiments, the LNP composition comprises mRNA at a lipid:mRNA(weight / weight) ratio is between 5: 1 and 40: 1. In some embodiments, the LNP comprises mRNA at a lipid:mRNA (weight / weight) ratio between 10:1 and 40: 1, between 15: 1 and 40: 1, between 20: 1 and 40: 1, between 25: 1 and 40: 1, between 30: 1 and 40:1, between 35: 1 and 40: 1, between 20: 1 and 35: 1, between 25: 1 and 35: 1, between 30: 1 and 35:1, between 20: 1 and 30: 1, between 25: 1 and 30: 1, between 20: 1 and 25: 1, between 25: 1 and 30:1, between 25: 1 and 35: 1, between 20: 1 and 36: 1, between 25:1 and 36: 1, between 5: 1 and 45: 1, between 20: 1 and 40:1, between 25: 1 and 40:1, between 35: 1 and 40: 1, or between 30: 1 and 40: 1. In someembodiments, the LNP comprises mRNA at a lipid:mRNA ratio of 30: 1. In some embodiments, the LNP comprises mRNA at a lipid:mRNA ratio of 40: 1.

[0321] In some embodiments, the mRNA encodes a gene or a portion of a gene related to liver disease shown in Table 9. Exemplary sequences of genes related to liver diseases are shown in Table 10.

[0322] It is understood that T is T in DNA and T is U in RNA polynucleotide sequences.Table 9. Exemplary genes related to liver diseasesTable 10. Exemplary sequences of genes related to liver diseases

[0323] In some embodiments, the mRNA encoding ASL comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding SERPINA1 (gene that encodes AAT) comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 2. In some embodiments, the mRNA encoding SLC25A13 comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 3. In some embodiments, the mRNA encoding HBA1 comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding ALDOB comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 5. In some embodiments, the mRNA encoding CFTR comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 6.

[0324] In some embodiments, the mRNA encodes a protein selected from the group consisting of ALDOB, GBE1, FAH, ATP7B, ASL, SLC25A13, LIPA, SERPONA1, CFTR, HFE.

[0325] In some embodiments, the mRNA encodes a protein selected from the group consisting of alpha- 1 -antitrypsin (A1AT), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose- 6-phosphatase (G6Pase), glucose-6-phosphate translocase (G6PT), lysosomal glucocerebrosidase (GB), Niemann-Pick Cl protein (NPC1), Niemann -Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor IX, galactose- 1 -phosphate uridylyltransferase, galactokinase, UDP -galactose 4-epimerase, transthyretin, phenylalanine hydroxylase (PAH),homogentisate 1,2-di oxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL), argininosuccinate synthetase (AS SI), P-type ATPase protein FIC-1, alpha-galactosidase A, acid ceramidase, acid α-L-fucosidase, acid β-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid α-mannosidase, β-mannosidase, arylsulfatase B, arylsulfatase A, N- acetylgalactosamine-6-sulfate sulfatase, acid β-galactosidase, acid α-glucosidase, β- hexosaminidase B, heparan-N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:α- glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alpha-N- acetylgalactosaminidase, sialidase, β-glucuronidase, β-hexosaminidase A. In some embodiments, the polynucleotide is a DNA, such as, for example, a DNA encoding a functional protein associated with a protein deficiency disease (e.g., a protein selected from the proteins listed above).

[0326] Polynucleotide sequences can be optimized for expression in various cells and tissues by adjusting codon usage. Codon usage optimization is known in the art, for example at world wide web owpgenomes.urv.es / OPTIMIZER / . In some embodiments, the codon usage of the polynucleotide is optimized for expression in a cell, for example a human cell.Modified polynucleotides

[0327] In some embodiments, the polynucleotide comprises one or more modifications selected from the group consisting of pyridin-4-one ribonucleoside, 5 -aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl- uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl- pseudouridine, 4-thio-l-methyl-pseudouridine, 2- thio-l-methyl-pseudouridine, 1 -methyl- 1- deaza-pseudouridine, 2 -thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5 -hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2 -thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy -pseudoisocytidine, 4-methoxy-l-methyl- pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8 -azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2 -methylthio-adenine, and 2 -methoxy-adenine, inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8 -aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6- thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- m ethylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine, and combinations thereof.

[0328] In some embodiments, a polynucleotide of the disclosure comprises a modified pyrimidine, such as a modified uridine. In some cases, a uridine analogue is selected from pseudouridine (Ψ), 1 -methylpseudouridine (m'P), 2 -thiouridine (s2U), 5-methyluridine (m5U), 5-methoxyuridine (mo5U), 4-thiouridine (s4U), 5 -bromouridine (Br5U), 2'0-methyluridine (U2'm), 2'-amino-2'-deoxyuridine (U2'NH2), 2'-azido-2'-deoxyuridine (U2'N3), and 2'-fluoro- 2'-deoxyuridine (U2'F).Modification in untranslated regions

[0329] In some embodiments, a polynucleotide such as a nucleic acid construct, a vector, or a polyribonucleotide of the disclosure can comprise one or more untranslated regions. An untranslated region can comprise any number of modified or unmodified nucleotides. Untranslated regions (UTRs) of a gene are transcribed but not translated into a polypeptide. In some cases, an untranslated sequence can increase the stability of the polynucleotide and the efficiency of translation. The regulatory features of a UTR can be incorporated into the modified mRNA molecules of the present disclosure, for instance, to increase the stability of the molecule. The specific features can also be incorporated to ensure controlled downregulation of the transcript in case they are misdirected to undesired organ sites. Some 5' UTRs play roles in translation initiation. A 5' UTR can comprise a Kozak sequence which is involved in the process by which the ribosome initiates translation of many genes. Kozak sequences can have the consensus GCC(R)CCAUGG, where R is a purine (adenine or guanine) that is locatedthree bases upstream of the start codon (AUG). 5' UTRs may form secondary structures which are involved in binding of translation elongation factor. In some cases, one can increase the stability and protein production of the polynucleotide molecule of the disclosure, by engineering the features typically found in abundantly expressed genes of specific target organs. For example, introduction of 5'UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, can be used to increase expression of a polynucleotide in a liver. Likewise, use of 5' UTR from muscle proteins (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie- 1, CD36), for myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CD1 lb, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A / B / C / D) can be used to increase expression of a polynucleotide in a desired cell or tissue.

[0330] Other non-UTR sequences can be incorporated into the 5' (or 3' UTR) UTRs of the polynucleotides of the present disclosure. The 5' and / or 3' UTRs can provide stability and / or translation efficiency of polynucleotides. For example, introns or portions of intron sequences can be incorporated into the flanking regions of a polynucleotide. Incorporation of intronic sequences can also increase the rate of translation of the polynucleotide.

[0331] In some embodiments, 3' UTRs may have stretches of Adenosines and Uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into classes: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and T F-α. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif c-Jun and Myogenin are two well-studied examples of this class. Proteins binding to the AREs may destabilize the messengerRNA (mRNA), whereas members of the ELAV family, such as HuR, may increase the stability of mRNA. HuR may bind to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of polynucleotide molecules can lead to HuR binding and thus, stabilization of the message in vivo. Engineering of 3' UTR AU rich elements (AREs) can be used to modulate the stability of a polynucleotide. One or more copies of an ARE can be engineered into a polynucleotide to modulate the stability of a polynucleotide. AREs can be identified, removed, or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conductedin relevant cell lines, using polynucleotides and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE- engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hours, 12 hours, 24 hours, 48 hours, and 7 days post-transfection.

[0332] In some embodiments, a polynucleotide such as a nucleic acid construct, a vector, a polyribonucleotide, or compositions of the disclosure can comprise an engineered 5' cap structure, or a 5'-cap can be added to a polynucleotide intracellularly. The 5'-cap structure of an mRNA can be involved in binding to the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature pseudo-circular mRNA species. The 5'-cap structure can also be involved in nuclear export, increases in mRNA stability, and in assisting the removal of 5' proximal introns during mRNA splicing.

[0333] In some embodiments, a polynucleotide such as a nucleic acid construct, a vector, or a polynucleotide can be 5'-end capped generating a 5'-GpppN-3' -triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. The cap-structure can comprise a modified or unmodified 7- methylguanosine linked to the first nucleotide via a 5'-5' triphosphate bridge. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue (Cap-0 structure). The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5'-end of the mRNA may optionally also be 2'-O-methylated (Cap-1 structure). 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a polynucleotide molecule, such as an mRNA molecule, for degradation. In some cases, a cap can comprise further modifications, including the methylation of the 2' hydroxy -groups of the first 2 ribose sugars of the 5' end of the mRNA. For instance, a eukaryotic cap-1 has a methylated 2'-hydroxy group on the first ribose sugar, while a cap-2 has methylated 2 '-hydroxy groups on the first two ribose sugars. The 5' cap can be chemically similar to the 3' end of an RNA molecule (the 5' carbon of the cap ribose is bonded, and the free 3'-hydroxyls on both 5'- and 3'- ends of the capped transcripts. Such double modification can provide significant resistance to 5' exonucleases. Non-limiting examples of 5' cap structures that can be used with a polynucleotide include, but are not limited to, m7G(5')ppp(5')N (Cap-0), m7G(5')ppp(5')N1mpNp (Cap-1), and m7G(5')- ppp(5')N1mpN2mp (Cap-2).

[0334] Modifications to the modified mRNA of the present disclosure may generate a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life while facilitating efficient translation. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' triphosphate linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with guanosine a-thiophosphate nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap. Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides. Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugars of 5'-terminal and / or 5'-anteterminal nucleotides of the mRNA on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap structures can be used to generate the 5'-cap of a polynucleotide.

[0335] The modified mRNA may be capped post-transcriptionally. According to the present disclosure, 5' terminal caps may include endogenous caps or cap analogues. According to the present disclosure, a 5' terminal cap may comprise a guanine analogue. Useful guanine analogues include, but are not limited to, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine.

[0336] In some embodiments, an untranslated region can comprise any number of nucleotides. An untranslated region can comprise a length of about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs, about 500 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, about 2000 to about 3000 bases or base pairs, about 3000 to about 4000 bases or base pairs, about 4000 to about 5000 bases or base pairs, about 5000 to about 6000 bases or base pairs, about 6000 to about 7000 bases or base pairs, about 7000 to about 8000 bases or base pairs, about 8000 to about 9000 bases or base pairs, or about 9000 to about 10000 bases or base pairs in length. An untranslated region can comprise a length of for example, at least 1 base or base pair, 2 bases or base pairs, 3 bases or base pairs, 4 bases or base pairs, 5 bases or base pairs, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs, 9 bases or base pairs, 10 bases or base pairs, 20 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs, 100 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs, 7000bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or 10000 bases or base pairs in length.

[0337] In some embodiments, a polynucleotide of the disclosure can comprise a poly A sequence. A poly A sequence (e.g., poly A tail) can comprise any number of nucleotides. A poly A sequence can comprise a length of about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs, about 500 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, about 2000 to about 3000 bases or base pairs, about 3000 to about 4000 bases or base pairs, about 4000 to about 5000 bases or base pairs, about 5000 to about 6000 bases or base pairs, about 6000 to about 7000 bases or base pairs, about 7000 to about 8000 bases or base pairs, about 8000 to about 9000 bases or base pairs, or about 9000 to about 10000 bases or base pairs in length. In some examples, a poly A sequence is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in length. A poly A sequence can comprise a length of for example, at least 1 base or base pair, 2 bases or base pairs, 3 bases or base pairs, 4 bases or base pairs, 5 bases or base pairs, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs, 9 bases or base pairs, 10 bases or base pairs, 20 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs, 100 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs, 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or 10000 bases or base pairs in length. A poly A sequence can comprise a length of at most 100 bases or base pairs, 90 bases or base pairs, 80 bases or base pairs, 70 bases or base pairs, 60 bases or base pairs, 50 bases or base pairs, 40 bases or base pairs, 30 bases or base pairs, 20 bases or base pairs, 10 bases or base pairs, or 5 bases or base pairs.Gene Editing Payload

[0338] The LNPs of the present disclosure can comprise one or more components for gene editing, such as, but not limited to, a guide RNA, a tracrRNA, a sgRNA, an mRNA encoding a gene or base editing protein, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease (e.g., Cas9), a DNA template for gene editing, or a combination thereof.In some embodiments, the payload of the LNPs can be suitable for a genome editing technique. In some embodiments, the genome editing technique can be CRISPR or TALEN. In some embodiments, the LNPs can comprise one or more mRNAs, which can encode a gene editing or base editing protein. In some embodiments, the LNPs can comprises both a gene- or baseediting protein encoding mRNA and one or more guide RNAs. In some embodiments, the LNPs can comprise at least one nucleic acid suitable for a genome editing technique, such as a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a guide RNA (gRNA), and a DNA repair template. In some embodiments, CRISPR nucleases can have altered activity, for example, modifying the nuclease so that it can be a nickase instead of making double-strand cuts or so that it can bind the sequence specified by the guide RNA but has no enzymatic activity. In some embodiments, the base editing protein can be a fusion protein comprising a deaminase domain and a sequence-specific DNA binding domain, such as an inactive CRISPR nuclease.Gene Editing Methods

[0339] The presently described LNPs or pharmaceutical composition can comprise a payload of any conventional gene editing methods. In some embodiments, gene editing components can be selectively delivered to the cells of target organ. In some embodiments, the target organ can be liver. In some embodiments, the cells of target organ can be liver cells. In some embodiments, the cells can be ciliated cells, goblet cells, secretory cells, club cells, basal cells or ionocytes.

[0340] In some embodiments, the gene editing can be targeted editing. Targeted editing can be achieved either through a nuclease-independent approach or through a nucleasedependent approach.

[0341] The nuclease-independent targeted editing, such as base-editing and / or prime editing, can involve precise modifications to DNA sequences without creating double-strand breaks. Homologous recombination can be guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the cells of target organ.

[0342] Base editing can allow for the conversion of one DNA base pair into another at a specific target site. In some embodiments, the nuclease can be a fusion of a deaminase enzyme to a modified Cas9 protein (dCas9) or other engineered Cas variants. In some embodiments, base editing can change C (cytosine) to T (thymine) or A (adenine) to G (guanine) in theendogenous DNA. A guide RNA can be designed to target the specific genomic location of interest in the cells of target organ.

[0343] Prime editing can allow for more complex and precise DNA modifications, including insertions, deletions, and all 12 possible base-to-base conversions (A, C, G, T) without double-strand breaks. A prime editing guide RNA, which can consist of a guide sequence and a template for the desired edit, can be designed. The prime editor protein (PE2), which can combine a reverse transcriptase and a Cas9 variant, can be guided to the target site by the prime editing guide RNA. The Cas9 variant can generate a single-strand break (nick) in the DNA. The reverse transcriptase then can use the prime editing guide RNA’s template sequence to copy the desired changes into the nicked strand of DNA. Subsequently, the cellular repair machinery of the cells of target organ can repair the nick, incorporating the edited sequence, via homology-directed repair (HDR).

[0344] The nuclease-dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare- cutting nucleases (e.g., endonucleases). Such nuclease-dependent targeted editing can also utilize DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which can occur in response to DSBs. In some embodiments, DNA repair by NHEJ can lead to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which can result in targeted integration of the exogenous genetic material. In some embodiments, a nuclease of the nuclease-dependent targeted editing can include, but not limited to, CRISPR- Cas9, CRISPR-Casl2 (Cpf1), CRISPR-Cas13, C2c2, C2c6, NgAgo, and / or TALEN.

[0345] Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell (e.g., to knock out a gene in a cell) are well-known techniques. See for example, Bauer et al., J Vis Exp 95:e52118 (2015). Available endonucleases capable of introducing specific and targeted DSBs can include, but not limited to, ZFN, TALEN, and CRISPR / Cas9.

[0346] In some embodiments, targeted gene editing can be achieved via dual integrase cassette exchange (DICE) system utilizing phiC31 and Bxbl integrases.CRISPR-Cas9 Gene Editing System

[0347] The CRISPR-Cas9 system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA-targeting platform used for gene editing. It can rely on the DNA nuclease Cas9, and two noncoding RNAs, crisprRNA (crRNA) and transactivating RNA (tracrRNA), to target the cleavage of DNA. CRISPR is a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) with similarity to foreign DNA previously exposed to the cell, for example, by viruses that have infected or attacked the prokaryote. These fragments of DNA can be used by the prokaryote to detect and destroy similar foreign DNA upon re-introduction, for example, from similar viruses during subsequent attacks. Transcription of the CRISPR locus can result in the formation of an RNA molecule comprising the spacer sequence, which can associate with and target Cas (CRISPR-associated) proteins able to recognize and cut the foreign, exogenous DNA. Numerous types and classes of CRISPR / Cas systems have been described in e.g., Koonin et al., Curr Opin Microbiol 37:67-78 (2017).

[0348] CrRNA can drive sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with about 20 nucleotide sequence in the target DNA. Changing the sequence of the 5’ 20 nucleotides in the crRNA can allow targeting of the CRISPR-Cas9 complex to specific loci. The CRISPR-Cas9 complex can only bind DNA sequences that contain a sequence match to the first 20 nucleotides of the crRNA, if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM).

[0349] TracrRNA can hybridize with the 3’ end of crRNA to form an RNA-duplex structure that can be bound by the Cas9 endonuclease to form the catalytically active CRISPR- Cas9 complex, which can then cleave the target DNA.

[0350] Once the CRISPR-Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end).

[0351] After binding of CRISPR-Cas9 complex to DNA at a specific target site and formation of the site-specific DSB, cells can use two main DNA repair pathways to repair the DSB: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is a repair mechanism that is highly active in the majority of cell types, including non-dividing cells. NHEJ can be error-prone and can often result in the removal or addition of between oneand several hundred nucleotides at the site of the DSB, though such modifications can typically be less than 20 nucleotides. The resulting insertions and deletions (indels) can disrupt coding or noncoding regions of genes. Alternatively, HDR can use a long stretch of homologous donor DNA, provided endogenously or exogenously, to repair the DSB with high fidelity. HDR is active only in dividing cells and can occur at a relatively low frequency in most cell types.

[0352] CRISPR Endonuclease: In some embodiments, Cas9 endonuclease can be used in a CRISPR method for genetically engineering cells of the target organ of the LNPs described herein. In some embodiments, Cas9 enzyme can be from Streptococcus pyogenes, although other Cas9 homologs can also be used. In some embodiments, the Cas9 enzyme can be wildtype Cas9. In some embodiments, the Cas9 enzyme can be a modified version of Cas9 (e.g., evolved versions of Cas9, or Cas9 orthologues or variants). In some embodiments, Cas9 can be substituted with another RNA-guided endonuclease, such as Cpfl (class II CRISPR / Cas system).

[0353] In some embodiments, the CRISPR / Cas system can comprise components derived from a Type-I, Type-II, or Type-Ill system. In some embodiments, the CRISPR / Cas system can comprise components derived from Class 1 and Class 2 CRISPR / Cas systems, having Types I to V or Types II, V, and VI, respectively (Makarova et al., Nat Rev Microbiol 13:722- 736 (2015); Shmakov et al., Mol Cell 60:385-397 (2015)).

[0354] Class 2 CRISPR / Cas systems can have single protein effectors. Cas proteins of Types II, V, and VI can be single-protein, RNA-guided endonucleases, herein called Class 2 Cas nucleases. Class 2 Cas nucleases can include, for example, but not limited to, Cas9, Cpfl, C2cl, C2c2, and C2c3 proteins. The Cpfl nuclease is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0355] In some embodiments, the Cas nuclease can be from a Type-II CRISPR / Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease can be from a Class 2 CRISPR / Cas system (a single-protein Cas nuclease, such as a Cas9 protein or a Cpfl protein). The Cas9 and Cpfl family of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, which is further explained infra.

[0356] In some embodiments, a Cas nuclease can comprise more than one nuclease domain. In some embodiments, a Cas9 nuclease can comprise at least one RuvC-like nuclease domain (e.g., Cpfl) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease can introduce a DSB in the target sequence. In some embodiments, the Cas9 nuclease can be modified to contain only one functional nucleasedomain. For example, the Cas9 nuclease can be modified such that one of the nuclease domains can be mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease can be modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease can be modified to contain no functional HNH-like nuclease domain. In some embodiments in which only one of the nuclease domains can be functional, the Cas9 nuclease can be a nickase that can introduce a single-stranded break (nick) into the target sequence. In some embodiments, a conserved amino acid within a Cas9 nuclease domain can be substituted to reduce or alter a nuclease activity. In some embodiments, the Cas nuclease nickase can comprise an amino acid substitution in the RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain can include D10A (based on the S. pyogenes Cas9 nuclease). In some embodiments, the nickase can comprise an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain can include, but not limited to, E762A, H840A, N863 A, H983 A, and D986A (based on the S. pyogenes Cas9 nuclease).

[0357] In some embodiments, the Cas nuclease can be from a Type-I CRISPR / Cas system. In some embodiments, the Cas nuclease can be a component of the Cascade complex of a Type- I CRISPR / Cas system. For example, the Cas nuclease can be a Cas3 nuclease. In some embodiments, the Cas nuclease can be derived from a Type-Ill CRISPR / Cas system. In some embodiments, the Cas nuclease can be derived from Type-IV CRISPR / Cas system. In some embodiments, the Cas nuclease can be derived from a Type-V CRISPR / Cas system. In some embodiments, the Cas nuclease can be derived from a Type-VI CRISPR / Cas system.

[0358] A Type I CRISPR / Cas system can utilize a large effector complex known as Cascade (CRISPR-associated complex for antiviral defense) for target binding and interference. The Cascade complex can contain multiple Cas proteins, including Cas3, which can be responsible for the destruction of the target DNA. A Type II CRISPR / Cas system, particularly the CRISPR-Cas9 system, can utilize a single Cas9 protein, guided by a synthetic guide RNA (sgRNA), to introduce double-strand breaks in target DNA for subsequent repair or modification. A Type III CRISPR / Cas system can utilize a Csm (CRISPR-Cas subtype multiprotein) or Cmr (CRISPR-Cas subtype ribonucleoprotein) complex for interference. Type III CRISPR / Cas system can target RNA molecules in addition to DNA. A Type V CRISPR / Cas system, including Cpfl (also known as Casl2) and C2c2 (also known as Casl3), can utilize a single effector protein to perform interference. A Type VI CRISPR / Cas system can utilize a single Cas protein, such as C2c2 (also known as Cast 3), to target and cleave RNA molecules, making it useful for RNA editing and manipulation.

[0359] Guide RNAs (gRNAs): The CRISPR technology can involves the use of a genometargeting nucleic acid that can direct one or more endonucleases to a specific target sequence within a target gene for gene editing at the specific target sequence. The genome-targeting nucleic acid can be an RNA. A genome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. A guide RNA can comprise at least one spacer sequence that can hybridize to a target nucleic acid sequence within a target gene for editing, and a CRISPR repeat sequence.

[0360] In Type II systems, the gRNA can also comprise a second RNA called the tracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence and tracrRNA sequence can hybridize to each other to form a duplex. In the Type V gRNA, the crRNA can form a duplex. In both systems, the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide can form a complex. In some embodiments, the genome-targeting nucleic acid can provide target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid can thus direct the activity of the site-directed polypeptide.

[0361] As is understood by the person of ordinary skill in the art, each guide RNA can be designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science 337:816-821 (2012); Deltcheva et al., Nature 471:602-607 (2011).

[0362] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) can be a double-stranded guide RNA, comprising two strands of RNA molecules. The first strand can comprise in the 5’ to 3’ direction, an optional spacer extension sequence, a spacer sequence, and a minimum CRISPR repeat sequence. The second strand can comprise a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3 ’ tracrRNA sequence, and an optional tracrRNA extension sequence.

[0363] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) can be a single-molecule guide RNA (sgRNA). sgRNA in a Type II system can comprise, in the 5’ to 3’ direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that can contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins. A single-molecule guide RNA in a Type V system can comprise, in the 5’ to 3’ direction, a minimum CRISPR repeat sequence and a spacer sequence.

[0364] A spacer sequence in a gRNA is a sequence (e.g., a 20-nucleotide sequence) that can define the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target gene of interest (e.g. , DNAI1 or CFTR). In some embodiments, the spacer sequence can range from 15 to 30 nucleotides. For example, the spacer sequence can contain 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence can contain 20 nucleotides.

[0365] The target sequence is in a target gene (e.g., DNAI1 or CFTR) that can be adjacent to a PAM sequence and can be the sequence to be modified by an RNA-guided nuclease (e.g., Cas9). The target sequence is on the PAM strand in a target nucleic acid, which is a doublestranded molecule containing the PAM strand and a complementary non-PAM strand. One of skill in the art recognizes that the gRNA spacer sequence can hybridize to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest. Thus, the gRNA spacer sequence can be the RNA equivalent of the target sequence. The spacer of a gRNA can interact with a target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer thus can vary depending on the target sequence of the target nucleic acid of interest.

[0366] In a CRISPR / Cas system, the spacer sequence can be designed to hybridize to a region of the target nucleic acid that is located 5 ’ of a PAM recognizable by a Cas9 enzyme used in the system. The spacer can perfectly match the target sequence or can have mismatches. Each Cas9 enzyme can have a particular PAM sequence that it can recognize in a target DNA. For example, S. pyogenes can recognize in a target nucleic acid a PAM that comprises the sequence 5’-NRG-3’, where R can comprise either A or G, where N can be any nucleotide and N can be immediately 3’ of the target nucleic acid sequence targeted by the spacer sequence.

[0367] In some embodiments, the target nucleic acid sequence can have about 20 nucleotides in length. In some embodiments, the target nucleic acid can have less than about 20 nucleotides in length. In some embodiments, the target nucleic acid can have more than about 20 nucleotides in length. In some embodiments, the target nucleic acid can have at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid can have at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence can have 20 bases immediately 5’ of the first nucleotide of the PAM. For example, in a sequence comprising 5'-NNNNNNNNNNNNNNNNNNNNNRG-3', the target nucleic acid can be the sequence that corresponds to the Ns, wherein N can be any nucleotide, and the underlined NRG sequence can be the S. pyogenes PAM.

[0368] The guide RNA can target any sequence of interest via the spacer sequence in the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene can be 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch.

[0369] The length of the spacer sequence in gRNAs can depend on the CRISPR / Cas9 system and components used for editing any of the target genes (e.g., DNAI1 or CFTR). For example, different Cas9 proteins from different bacterial species can have varying optimal spacer sequence lengths. Accordingly, the spacer sequence can have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the spacer sequence can have 18-24 nucleotides in length. In some embodiments, the targeting sequence can have 19-21 nucleotides in length. In some embodiments, the spacer sequence can comprise 20 nucleotides in length.

[0370] In some embodiments, the gRNA can be an sgRNA, which can comprise a 20- nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, the sgRNA can comprise a less than 20 nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, the sgRNA can comprise a more than 20 nucleotide spacer sequence at the 5 ’ end of the sgRNA sequence. In some embodiments, the sgRNA can comprise a variable length spacer sequence with about 17-30 nucleotides at the 5’ end of the sgRNA sequence.

[0371] In some embodiments, the gRNAs can comprise unmodified ribonucleic acid. In some embodiments, the gRNAs can comprise modified ribonucleic acid. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic generation of RNAs, e.g., modifications that can enhance stability, reduce the likelihood or degree of innate immune response, and / or enhance other attributes, as described in the art. In some embodiments, non-natural modified nucleobases can be introduced into any of the gRNAs during synthesis or post-synthesis. In some embodiments, modifications can be on internucleoside linkages, purine or pyrimidine bases, or sugar. In some embodiments, a modification can be introduced at the terminal of a gRNA with chemical synthesis or with a polymerase enzyme.

[0372] In some embodiments, more than one guide RNAs can be used with a CRISPR / Cas nuclease system. Each guide RNA can contain a different targeting sequence, such that the CRISPR / Cas system can cleave more than one target nucleic acid. In some embodiments, one or more guide RNAs can have the same or differing properties, such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA can be used, each guide RNA can be encoded on the same or on different vectors. The promoters used to drive expression of the more than one guide RNA can be the same or different.

[0373] In some embodiments, enzymatic or chemical ligation methods can be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, and the like.

[0374] In some embodiments, the CRISPR / Cas nuclease system can contain multiple gRNAs, for example, 2, 3, or 4 gRNAs. Such multiple gRNAs can target different sites in a same target gene. Alternatively, the multiple gRNAs can target different genes. In some embodiments, the guide RNA(s) and the Cas protein can form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex. The guide RNAs can guide the Cas protein to a target sequence(s) on one or more target genes (e.g., DNAI1 and CFTR), where the Cas protein can cleave the target gene at the target site. In some embodiments, the CRISPR / Cas complex can be a Cpfl / guide RNA complex. In some embodiments, the CRISPR complex can be a Type-II CRISPR / Cas9 complex. In some embodiments, the Cas protein can be a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex can be a Cas9 / guide RNA complex.

[0375] In some embodiments, the indel frequency (editing frequency) of a particular CRISPR / Cas nuclease system, comprising one or more specific gRNAs, can be determined using a TIDE analysis, which can be used to identify highly efficient gRNA molecules for editing a target gene. In some embodiments, a highly efficient gRNA can yield a gene editing frequency of higher than 80%. For example, a gRNA can be considered to be highly efficient if it can yield a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.Other Gene Editing Methods

[0376] Besides the CRISPR system disclosed herein, additional gene editing systems as known in the art can also be used as a payload of the LNPs described herein. In some embodiments, the additional gene editing system can comprise zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), restriction endonucleases, meganucleases homing endonucleases, or the like.

[0377] ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which can be a polypeptide domain that can bind DNA in a sequencespecific manner through one or more zinc fingers. A zinc finger can be a domain of about 30 amino acids within the zinc finger binding domain whose structure can be stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain can be a domain not occurring in nature whose design / composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. A selected zinc finger domain can be a domain not found in nature whose production can result primarily from an empirical process such as phage display, interaction trap or hybrid selection. In some embodiments, a ZFN can be a fusion of the FokI nuclease with a zinc finger DNA binding domain.

[0378] A TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. A “transcription activator-like effector DNA binding domain,” “TAL effector DNA binding domain,” or “TALE DNA binding domain” is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins can be secreted by plant pathogens of the genus Xanthomonas during infection. These proteins can enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains. TAL effector DNA binding domain specificity can depend on an effector-variable number of imperfect 34 amino acid repeats, which can comprise polymorphisms at select repeat positions called repeat variable diresidues (RVD). In some embodiments, a TALEN can be a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain.

[0379] Additional examples of targeted nucleases suitable for use can include, but not limited to, Bxbl, phiC31, PhiBTl, and Wβ / SPBc / TP901-l, whether used individually or in combination. The Bxbl nuclease, also known as the Bxbl integrase, is a site-specific recombinase enzyme derived from the mycobacteriophase Bxbl . The Bxbl integrase can catalyze site-specific recombination between two specific DNA sequences, referred to as attachment (att) sites. The Bxbl integrase can recognize a specific 48 base-pair sequence within the attachment sites. The phiC31 nuclease, also known as the phiC31 integrase, is derived from the bacteriophage phiC31. The phiC31 nuclease can catalyze site-specific recombination between two specific DNA sequences, referred to as attB (attachment site in bacteriophage)and attP (attachment site in the phage). The phiC31 nuclease can promote integration of a DNA fragment flanked by attB and attP into the genome in cells of target organ. The phiBTl nuclease can integrate into a different attachment site than phiC31. The Wβ / SPBc / TP901-1 nuclease, also known as bacteriophage P2 Bxb1 Cre nuclease, is a site-specific recombination enzyme derived from the temperate bacteriophage P2.

[0380] In some embodiments, the polynucleotide encodes a gene-editing system or component thereof. In some embodiments, the gene-editing system selected from the group consisting of alpha- 1 -antitrypsin (A1AT), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose- 6-phosphatase (G6Pase), glucose-6-phosphate translocase, lysosomal glucocerebrosidase (GB), Niemann-Pick Cl protein (NPC1), Niemann -Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor IX, galactose- 1 -phosphate uridylyltransf erase, galactokinase, UDP -galactose 4-epimerase, transthyretin, phenylalanine hydroxylase (PAH), homogentisate 1,2-di oxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL), argininosuccinate synthetase (ASS1), P-type ATPase protein FIC-1, alpha-galactosidase A, acid ceramidase, acid α-L-fucosidase, acid β-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid α-mannosidase, β-mannosidase, arylsulfatase B, arylsulfatase A, N- acetylgalactosamine-6-sulfate sulfatase, acid β-galactosidase, acid α-glucosidase, β- hexosaminidase B, heparan-N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:α- glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alpha-N- acetylgalactosaminidase, sialidase, β-glucuronidase, β-hexosaminidase A. In some embodiments, the polynucleotide is a DNA, such as, for example, a DNA encoding a functional protein associated with a protein deficiency disease (e.g., a protein selected from the proteins listed above).Pharmaceutical composition

[0381] The disclosure also provides pharmaceutical compositions comprising the LNP composition described herein and a pharmaceutically acceptable excipient and / or diluent. Suchcompositions can be used for the treatment of a liver disease as described herein in a patient or subject. The pharmaceutical compositions of the disclosure may include a pharmaceutically acceptable carrier, and a thorough discussion of such carriers is available in Chapter 30 of Remington: The Science and Practice of Pharmacy (23rded., 2021).

[0382] In some embodiments, the composition comprises Tris buffer, optionally at a pH from 6-9. In some embodiments, the composition comprises sucrose, optionally at 5-15%. In some embodiments, the composition comprises citrate buffer, optionally at a pH 4-6. In some embodiments, the composition comprises 15 mM Tris buffer, optionally at a pH from 6-9, and / or 5-15% sucrose. In some embodiments, the composition comprises 10 mM citrate buffer, optionally at a pH from 4-6.

[0383] In some embodiments, the pharmaceutical compositions include one or more of a poloxamer (e.g., Poloxamer 188) polyethylene glycol (“PEG”), sucrose, and a buffer, wherein the buffer comprises a citrate buffer, an acetate buffer, or a Tris buffer.

[0384] In some embodiments, the composition comprises a citrate buffer. For example, the citrate buffer is at a pH from 4 to 8. In some embodiments, the buffer is an acetate buffer and has a pH from 4 to 8. In another embodiments, the composition comprises a Tris buffer, and the Tris buffer has a pH from 4 to 8.

[0385] In some embodiments, the composition comprises sucrose. In some embodiments, the sucrose is at a concentration from 1% to 15% w / v, 5% to 15% w / v, 1% to 10% w / v, or 5% to 10% w / v.

[0386] In some embodiments, pharmaceutical compositions can also include excipients and / or additives. Examples of these are surfactants, stabilizers, complexing agents, antioxidants, or preservatives which prolong the duration of use of the finished pharmaceutical formulation, flavorings, vitamins, or other additives known in the art. Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof. In some embodiments, preservatives include, but are not limited to, those that protect the solution from contamination with pathogenic particles, including benzalkonium chloride or benzoic acid, or benzoates such as sodium benzoate. Antioxidants include, but are not limited to, vitamins, provitamins, ascorbic acid, vitamin E, salts, or esters thereof.

[0387] In some embodiments, one or more tonicity agents may be added to provide the desired ionic strength. Tonicity agents for use herein include those which display no or only negligible pharmacological activity after administration. Both inorganic and organic tonicity adjusting agents may be used.Method of treatment

[0388] In some embodiments, the LNP compositions and pharmaceutical compositions described herein can be employed to treat or prevent a liver disease or disorder, including but not limited to a disease or disorder from the following: Glycogen Storage Disease Type IV, Hereditary Fructose Intolerance, Wilson Disease, Type I Tyrosinemia, Hereditary Hemochromatosis, Alpha- 1 Antitrypsin Deficiency, Cystic fibrosis.

[0389] In some embodiments, the LNP compositions can be employed to treat Glycogen Storage Disease Type IV. In some embodiments, the LNP compositions can be employed to treat Hereditary Fructose Intolerance. In some embodiments, the LNP compositions can be employed to treat Wilson Disease. In some embodiments, the LNP compositions can be employed to treat Type I Tyrosinemia. In some embodiments, the LNP compositions can be employed to treat Hereditary Hemochromatosis. In some embodiments, the LNP compositions can be employed to treat Alpha- 1 Antitrypsin Deficiency. In some embodiments, the LNP compositions can be employed to treat Cystic fibrosis.

[0390] In some embodiments, the LNP composition can be delivered to liver, wherein the composition delivers a payload preferentially in a liver cell. In some embodiments, the LNP composition can be delivered to liver, wherein the composition delivers a payload preferentially to both a liver cell and a lung cell.

[0391] In another aspect, the disclosure provides a method for treating and / or preventing a liver disease in a subject in need thereof, wherein the method comprises administering the composition described herein to the subject by intravenous injection.

[0392] In some embodiments, the payload is a messenger RNA (mRNA) and the method results in delivery of the payload to the liver in an amount effective to increase expression and / or function of a gene encoded by the mRNA.

[0393] In some embodiments, the method results in expression of a polypeptide in a liver of the subject, wherein the expression is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increased compared to the polypeptide expression prior to delivery.

[0394] In some embodiments, the method results in expression of the polypeptide in the liver of the subject between 10 min and 24 hours after administration of the composition to the subject.

[0395] In another aspect, the disclosure provides a method of delivering a payload to a cell in a liver of a subject, wherein the method comprising administering to the subject, by intravenous injection, the LNP composition described herein.

[0396] In another aspect, the disclosure provides a kit including the composition described herein. In another aspect, the disclosure provides use of the LNP composition described herein for treatment of a liver disease by intravenous injection.Liver diseases[1] Glycogen Storage Disease Type IV is an autosomal recessive disease due to mutations in the gene encoding the glycogen branching enzyme (GBE1) that catalyzes the alpha 1, bond of the first glucose in the side chains of glycogen. The altered glycogen branching reduces its solubility, thus impairing the osmotic pressure within the hepatocyte. Hereditary Fructose Intolerance is an autosomal recessive disease due to the deficiency of fructose 1 -phosphate aldolase (aldolase B) involved in the metabolism of fructose- 1 -phosphate into dihydroxyacetone phosphate and D-glyceraldehyde. Wilson Disease is an autosomal recessive disorder which depends on mutations in the gene encoding the ATP7B Cu translocase. ATP7B, mainly expressed by the hepatocyte, regulates the levels of copper in the liver. When the activity of ATP7B is reduced, copper accumulates within the hepatocyte. Furthermore, ATP7B modulates the synthesis of ceruloplsmin. Type I Tyrosinemia is an autosomal recessive disease which is the most severe form of genetic tyrosinemia and is the only one that causes a severe liver involvement. Type I tyrosinemia is due to the altered activity of fumarylacetoacetate hydrolase, which causes the elevation of plasma and urine succinylacetone and high plasma concentration of tyrosine, methionine, and phenylalanine. Hereditary Hemochromatosis is an autosomal recessive disease characterized by iron overload that my cause liver cirrhosis, cardiomyopathy, diabetes, and arthritis. Molecular analysis in HFE gene confirm hereditary hemochromatosis. Homozygous patients for Cys22Tyr have a higher risk for iron overload. Alpha-1 Antitrypsin Deficiency is an autosomal recessive disease due to mutations in the SERPINA1 gene which encodes the serine protease inhibitor AAT. The protein is mainly synthesized by liver cells, and inhibits proinflammatory proteases. The liver damage is due to the accumulation of AAT mutant polymers and not to the lack of circulating AAT. Cystic fibrosis is a progressive, genetic disease that affects the lungs, pancreas, liver, kidneys, and other organs. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the CFTR protein to become dysfunctional, resulting in thick and sticky mucus that blocks airways and leads to lung damage and traps germs and makes infections more likely.Method of administration

[0397] In another aspect, the disclosure provides a method of delivering a payload to a cell in a liver of a subject, wherein the method comprising administering to the subject, by intravenous injection, the composition disclosed herein. In another aspect, the disclosure provides a kit comprising the composition disclosed herein.

[0398] In some embodiments, the administering to the subj ect is done by intravenous (I V.) delivery. In some embodiments, the administering to the subject is done by intrathecal (LT.) delivery. In some embodiments the administering to the subject is done by intramuscular (I.M.) delivery. In some embodiments, the administering to the subject is done by intradermal (I D.) delivery. In some embodiments of the method, the administering to the subject is done by intranasal delivery.

[0399] In some embodiments, the administration is single administration. In some embodiments, the administration is a multiple administration. In some embodiments, the multiple administrations occur three times a day, twice a day, once a day, every other day, every third day, weekly, biweekly, every three weeks, every four weeks, or monthly.EXAMPLESExample 1. Methods of Analysis

[0400] Formulations: 171 formulations were screened in vivo using 8 dendrimers, 13 SORT lipids, 9 phospholipids, 17 PEGylated lipids, and 6 sterols (lanosterol, desmosterol, cholesterol, sitostanol, sitosterol, stigmasterol). Luciferase mRNA cargos were encapsulated within the formulations to measure absolute and relative organ tropism within relevant destination organs.

[0401] Exemplary formulations of LNP compositions are shown in Table 11.Table 11. Compositions of illustrative hepatic LNP formulations and relevant comparators (italic).

[0402] Individual Study Plans: Most individual study plans observed the effects of various conditions (e.g., various buffers) on 1-3 formulations. In vivo imaging (IVIS®) fluorescence readouts (in units of photons / second / square centimeters / steradian [p / s / cm2 / sr]) were taken using the Spectral Instruments IVIS Aura Software, with Region of Interest (ROIs) drawn for each of six organs (Lung, Liver, Spleen, Heart, Kidney 1, and Kidney2).

[0403] Holistic QC: A total of 57 study plans encompassing 1344 rats (Rattus norvegicus), and formulation / condition combinations were considered for the holistic analysis. All data were compiled from GraphPad Prism (.pzfx) files derived from raw IVIS data. Formulation numbers were extracted from the data, and data without formulation numbers (e.g., vehicle controls) were discarded. As radiance is calculated based on RO I total emission, total emissions close to 0 can sometimes cause negative values; organs possessing negative fluorescence values were removed from the analysis. An outlier study (with average fluorescence valuesmuch greater than its counterparts with similar formulations) was also removed. Subjects used in multiple study plans as references were deduplicated. Only rats that were dosed at a concentration of 0.3 mg / kg with a necropsy performed 4 hours post-dose were analyzed, and rats dosed with formulations created with other cargos other than Luciferase (e.g., eGFP, mCherry, and tdTomato) were omitted from this analysis.

[0404] Hepatic candidate statistical selection: Hepatic ratios - Liver / Lung, Liver / Spleen. Liver / (Lung+Spleen) and Liver / Other - were calculated across all subjects in all study plans. Formulations were sorted by the medians of these ratios across subjects dosed with those formulations.

[0405] Component-level Linear Regression: To discover drivers of extrahepatic activity at a lipid component level, feature transformations were applied to the underlying raw data to make it compatible with a linear regression model. The following independent variables were used as inputs to the linear regression:

[0406] Categorical variables: Dendrimer type, SORT type, PEGylated type, Phospholipid type, and Cholesterol type. Categorical variables were made compatible with regression by using one-hot encoding.

[0407] Numerical variables: mol % of each of the types.

[0408] After feature transformation, there were 48 independent variables in the regression.The following dependent variables were used: logic Lung Fluorescence, logic SpleenFluorescence, logic Liver Fluorescence, Liver / Lung Ratio, Liver / Spleen Ratio, Liver / (Lung + Spleen) Ratio. Logic fluorescence values were used as opposed to raw values to conform to the assumptions made by linear regression (which assumes a Gaussian distribution for the dependent variables). Linear regression was run without covariates with the intent to assess associations between independent and dependent variables. P- values were corrected using a Bonferroni multiple hypothesis testing correction at a significance threshold of 0.05.Example 2. IVIS analyses to measure LNP tropism in rodents

[0409] IVIS images were collected to analyze distribution of hepatic ratios of lipid nanoparticle formulations. In Vivo Imaging System (IVIS) is a technique that can be used to read luminescence and fluorescence and determine the distribution of luminescent or fluorescent protein expression in small animals. By measuring the intensity and distribution of protein expression in mice and rats, an LNP formulation’s tropism can be determined.

[0410] LNP compositions of the disclosure comprising firefly luciferase mRNA were intravenously administered to the tail vain of Female Sprague Dawley rats (N=4) at dosages of 0.3 mg / kg. The IV bolus contained the LNP composition in PBS or a mixture of 15 mM Tris buffer with 10% sucrose.

[0411] The rats were sacrificed and perfusion was carried out using routine murine subject protocols, organs were then trimmed to remove surrounding tissues, and then placed on the imaging sheet. Imaging data was collected (AMI HTX In Vivo Imaging System) and then analyzed using IVIS software (Aura Imaging Software by Spectral Instruments Imaging).

[0412] .pzfx (GraphPad Prism) files containing IVIS (in vivo) data were extracted, cleaned, and the values were compiled. Hepatic ratios (Liver / Lung, Liver / Spleen, Liver (Lung+Spleen), Liver / Other (all other organs)) were calculated and sorted by medians of ratios. The Liver / Lung ratio of various formulations are shown in FIG. 1 and FIG. 10.

[0413] Comparison of formulations with top ratios across organs are shown in FIGs. 2A- 2B. Lipid nanoparticles are considered as a “top hepatic LNP” if at least 3 of: (Top 1 Liver / Lung Ratio, Top 1 Liver / Spleen Ratio, Top 1 Liver / (Lung+Spleen) Ratio, Top 1 Liver / Other Ratio). Table 12 shows lipid nanoparticles that fit the criteria.Table 12. Top hepatic LNP formulations from statistical analysis with relevant comparators (italicized).

[0414] Top formulations showed increased hepatic ratios when compared to previous and benchmark formulations, though at lower sample sizes. Hierarchically clustered heatmap of median formulation spread through organs of interest for top hepatic LNP formulations and relevant comparators are shown in FIG. 3. X-axis depicts formulation, and Y-axis depicts organ.

[0415] LNP formulations share a dendrimer of 4A3-SC7, a SORT lipid of DODAP or 14: DAP, DMG PEG, and DOPE. IVIS images for top formulations for liver and organs are shown in FIGs. 4A-4F.Example 3. Distribution of extrahepatic ratios by subtypes

[0416] This example describes distribution of extrahepatic ratios by lipid composition subtypes.

[0417] Boxplot of extrahepatic ratios across formulations, conditions, and replicates are shown in FIG. 5. Distribution of liver fluorescences by subtypes were measured to determine whether LNPs that consistently appear at high ratios in extrahepatic tissues while having a high lung / low liver absolute fluorescence, (FIGs. 6A-6E). FIG. 6B shows that 14:0 DAP has the highest median value of liver fluorescence following by DODAP. Helper subtype showed DSPC and DOPE have the highest liver fluorescence values (FIG. 6C).

[0418] Formulation 4F (14% 4A3-SC7, 45% 16:0 TAP, 25% Cholesterol, 16.0% DOPE,0.5% C8 PEG2000-Cera, 1.5%C8 PEG750-Cera), Formulation 4L (16.0% 4A3-SC7, 40% 16:0 TAP, 25% Cholesterol, 16.0% DSPE, 2.25% C8 PEG2000-Cera, 0.75%C8 PEG750-Cera), and Formulation 4R (16.0% 4A3-SC7, 40% 16:0 TAP, 25% Cholesterol, 16.0% DMPE, 0.75% C8 PEG2000-Cera, 2.25% C8 PEG750-Cera) are the top three formulations for extrahepatic activity.

[0419] Distribution of extrahepatic ratios by subtypes are shown in FIGs. 7A-7F. Lung / Liver Ratio, Lung / Spleen Ratio, Lung / (Liver+Spleen) Ratio, Lung / Other (all other organs) Ratio by dendrimer subtype shows 5A5-SC7 and 4A3-SC7 have the highest median values compared to other dendrimers (FIG. 7 A). Extrahepatic ratios by SORT lipid subtypeshows either 16:0 TAP and 16:0 EPC have the highest values compare to other SORT lipids (FIG. 7B). Extrahepatic ratios by PEG-lipid subtype shows C8 PEG2000-Ceramide / C8 PEG750-Ceramide has the highest values compare to other SORT lipids. (FIGs. 7D-7E). FIG. 7F shows distribution of extrahepatic ratios of formulations comprising C8-Ceramide / 16:0 TAP vs other. The results shows that formulations comprising C8-Ceramide / 16:0 TAP have higher extrahepatic ratios compared to others.Example 4. Linear regression

[0420] This example describes linear regression model results which assess the associations between lipid component variables and relevant extrahepatic ratios and absolute fluorescences.

[0421] Linear Regression aims to find a linear relationship between independent and dependent variables, by quantifying the effect of the independent variables on the dependent variables via a Beta value (and a confidence in that Beta value [Confidence Interval + p-value]). The component variables (i.e., the independent variables) here are either categorical variables (Dendrimer, SORT, Helper, Cholesterol, and PEG subtypes) or numerical variables (Corresponding mol %) and the dependent variables here are Liver / Lung Ratio, Liver / Spleen Ratio, Li ver / (Lung+ Spleen) Ratio, logi Lung, logi Liver, or logi Spleen. Statistically significant effect sizes of each separate component on each dependent variable are shown in FIGs. 8A-8B (point estimates are shown as dots; 95% confidence intervals are shown as bars). A Beta value of 2 indicates that a change in the independent variable by 1 unit will correspond to a change of 2 in the dependent variable. The top results are reproduced in Table 13. The MC3 dendrimer (Onpattro) has a very strong liver-tropic effect, as does the 14:0 DAP SORT. The other two values represented here have negative values, i.e. they actually lead to more lung tropism.Table 13. Top component-level effect sizes on dependent variables from linear regression.

[0422] FIG. 9 shows Formulation 3E4, Formulation 3E5, and Formulation 3E7 have the highest liver / lung ratio compared to Formulation W. Component-level IVIS data reanalysis suggests formulating LNPs with: D-Lin-MC3-DMA Dendrimer, 14:0 DAP SORT.Example 5. IV Liver formulation screening and Analysis

[0423] Orthogonal protein expression in rats

[0424] LNP formulations described herein comprising DNAI1-HA mRNA or DNAI1-HA mRNA were administered intravenously to Sprague Dawley rats. Dosing was varied for different mouse groups 1-15 as shown in Table 14. The rats were then evaluated for DNAI1 expression, cytokine expression, and body weight, as described herein.Table 14. Dosing in rat IV

[0425] Selected cytokine analysis

[0426] Cytokine levels in blood serum of the rats were measured at 4 hours and 24 hours after administration. FIGs. 11A-11E shows the occurrence (pg / mL) of cytokines detected in the IV liver screen using lipid nanoparticle (LNP) compositions described herein.

[0427] FIG. 17 shows a hierarchically clustered heatmap of top cytokines expressed in lipid nanoparticle (LNP) compositions described herein. “Group” refers to the dosing groups identified in Table 14.

[0428] Clinical Chemistry

[0429] Blood samples were collected from rats administered lipid nanoparticle compositions of the disclosure at 4 hours and 24 hours after administration. Liver enzyme levels, triglycerides and total complements (CH50) in the blood serum of the rats were measured by standard methods (e.g., enzyme-linked immunosorbent assay (ELISA)).

[0430] FIGs. 18A-18C show the occurrence (pg / mL) of liver enzymes in the blood serum of Sprague Dawley rat subjects intravenously dosed with lipid nanoparticle (LNP) compositions described herein. FIGs. 18D-18E show levels of triglycerides and total complements (CH50), respectively in the blood serum of Sprague Dawley rat subjects intravenously dosed with lipid nanoparticle (LNP) compositions described herein.

[0431] Hematology

[0432] Platelet and neutrophil levels in blood serum of the rats were measured at 4 hours and 24 hours after administration by standard methods (e.g., flow cytometry).

[0433] FIGs. 19A and 19B show the effect intravenously dosed lipid nanoparticle (LNP) compositions described herein on levels of platelets and neutrophils in the blood of Sprague Dawley rat subjects.

[0434] Liver formulation screen and validation

[0435] Rat body weight was measured before administration of LNP compositions of the disclosure, and at 24 hours after dosing. FIGs. 12A-12C shows body weight change in Sprague Dawley rats subjected to selected LNP compositions described herein.

[0436] 24 hours after dosing, lung, liver, and spleen tissues were collected from the rats and analyzed using an automated western blot using single plex assay for DNAI1-HA detected with HA tag antibody (HA-tag). The results are shown in FIG. 14 which shows DNAI1-HA expression and their biodistributions in Sprague Dawley rat subjects following administrationof LNP compositions described herein. FIGs. 15A-15C shows DNAI1-HA expression of LNP compositions described herein and their biodistributions in Sprague Dawley rat subjects shown qualitatively using Western Blots.

[0437] FIGs. 16A-16B shows the reduction of DNAI1-HA protein expression in the lung (top) and spleen (bottom) after 24 hours post-dosing of rat subjects with LNP compositions described herein.

[0438] Stability optimization of 3E5

[0439] Stability optimization of 3E5 was carried out using IVIS analyses. 3E5 was formulated in PBS and another buffer and administered to rats pre- and post-freeze thaw. Particle size, poly dispersity index and encapsulation efficiency for LNP 3E5 under the tested conditions are summarized in Table 15. The particle size, poly dispersity index and encapsulation efficiency for further LNP compositions of the disclosure are summarized in Table 16

[0440] Table 15. Stability optimization data using 3E5

[0441] Table 16. Formulation properties of LNP compositions used for the liver formulation screen validation.

[0442] Formulations of LNP 3E5 (comprising Luc-HA mRNA) in PBS and in another buffer, as described in Table 16, were administered to rats, intravenously at a dose of 0.1 mg / kg. IVIS organ imaging was carried out 4 hours after payload dosing. The Results show in FIG. 13A and FIG. 13B show increased occurrence in the liver compared to the spleen and lung in rat subjects.ABBREVIATIONSLNP lipid nanoparticleNHP non-human primatesRIP A buffer Radioimmunoprecipitation assay bufferBCA assay Bicinchoninic acid assayCV coefficient of varianceHPLC High performance liquid chromatographyDFA Difluoroacetic acidMS mass spectrometryPBS phosphate-buffered salineFBS Fetal bovine serumI VIS in vivo imaging systemINCORPORATION BY REFERENCE

[0443] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0444] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. The scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS1. A lipid nanoparticle composition comprising a lipid component which comprises:(i) a first lipid wherein the first lipid is an ionizable cationic lipid,(ii) a second lipid wherein the second lipid is separate from the first lipid,(iii) a phospholipid, and(iv) a PEG-lipid wherein: the second lipid is a lipid having a structural formula:wherein R1and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group, and R3and R3are each independently C1-C6alkyl or a substituted version thereof.

2. The lipid nanoparticle composition of claim 1, wherein in the compound of formula (S-I’a), R3and R3are each independently unsubstituted C1-C6alkyl.

3. The lipid nanoparticle composition of claim 1, wherein in the compound of formula (S- I’a), R3and R3are each independently methyl, ethyl, or propyl.

4. The lipid nanoparticle composition of claim 1, wherein in the compound of formula (S- I’a), R3and R3are each methyl.

5. The lipid nanoparticle composition of any one of claims 1-4, wherein in the compound of formula (S-I’a), R1and R2are each independently unsubstituted C8-C24alkenyl.

6. The lipid nanoparticle composition of any one of claims 1-4, in the compound of formula (S-I’a), R1and R2are each independently unsubstituted C8-C24alkyl.

7. The lipid nanoparticle composition of claim 1, wherein the second lipid is 1,2-dioleoyl- 3-dimethylammonium-propane (18: 1 DODAP).

8. The lipid nanoparticle composition of claim 1, wherein the second lipid is 1,2- dimyristoyl-3-dimethylammonium-propane (14:0 DAP).

9. A lipid nanoparticle composition comprising a lipid component which comprises:(i) a first lipid wherein the first lipid is an ionizable cationic lipid,(ii) a second lipid wherein the second lipid is separate from the first lipid,(iii) a phospholipid, and(iv) a PEG-lipid wherein: the second lipid is a lipid having a structural formula:wherein RT1aand RT1bare each independently C4-C18alkyl, C4-C18alkenyl, or a substituted version of either group, RC1a, RC1band, RC1care each independently C1-C6alkyl or a substituted version thereof; and xl and x2 are each independently 1, 2, 3, or 4.

10. The lipid nanoparticle of claim 9, wherein in the compound of formula (S-I’b) RT1aand RT1bare each independently unsubstituted C4-C18alkyl or C4-C18alkenyl.

11. The lipid nanoparticle of claim 9, wherein in the compound of formula (S-I’b) RT1aand RT1bare each independently unsubstituted C5-C8alkyl or C6-C20alkenyl.

12. The lipid nanoparticle of any one of claims 9-11, wherein in the compound of formula (S-I’b) RC1a, RC1b, and RC1care each independently unsubstituted C1-C6alkyl.

13. The lipid nanoparticle of any one of claims 9-11, wherein in the compound of formula (S-I’b) RC1a, RC1b, and RC1care each independently methyl.

14. The lipid nanoparticle of any one of claims 9-11, wherein in the compound of formula (S-I’b) RC1a, RC1b, and RC1care each independently ethyl.

15. The lipid nanoparticle of any one of claims 9-14, wherein in the compound of formula (S-I’b) x1and x2 are each 1.

16. The lipid nanoparticle of any one of claims 9-14, wherein in the compound of formula (S-I’b) x1and x2 are each 2.

17. The lipid nanoparticle of claim 9, wherein the compound of formula (S-I’b) is 2A11- SC7, having the following structure:

18. A lipid nanoparticle composition comprising a lipid component which comprises:(i) a first lipid wherein the first lipid is an ionizable cationic lipid,(ii) a second lipid wherein the second lipid is separate from the first lipid,(iii) a phospholipid, and(iv) a PEG-lipid wherein: the second lipid is a lipid having a structural formula:wherein RT2aand RT2bare each independently C4-C18alkyl, C4-C18alkenyl, or a substituted version of either group, RC2aand, RC2bare each independently C1-C6alkyl or a substituted version thereof, and yl is 1, 2, 3, or 4.

19. The lipid nanoparticle of claim 18, wherein in the compound of formula (S-I’c) RT2aand RT2bare each independently unsubstituted C4-C18alkyl or C4-C18alkenyl.

20. The lipid nanoparticle of claim 18, wherein in the compound of formula (S-I’c) RT2aand RT2bare each independently unsubstituted C5-C8alkyl or C6-C20alkenyl.

21. The lipid nanoparticle of any one of claims 18-20, wherein in the compound of formula (S-I’c) RC2aand RC2bare each independently unsubstituted C1-C6alkyl.

22. The lipid nanoparticle of any one of claims 18-20, wherein in the compound of formula (S-I’c) RC2aand RC2bare each independently methyl.

23. The lipid nanoparticle of any one of claims 18-20, wherein in the compound of formula (S-I’c) RC2aand RC2bare each independently ethyl.

24. The lipid nanoparticle of any one of claims 18-23, wherein in the compound of formula (S-I’c) y1is 1.

25. The lipid nanoparticle of any one of claims 18-23, wherein in the compound of formula (S-I’c) y1is 2.

26. The lipid nanoparticle of claim 18, wherein in the compound of formula (S-I’c), RT2aand RT2bare each a group having the following structure: wheredenotes the point of attachment.

27. The lipid nanoparticle of claim 18, wherein the compound of formula (S-I’c) is a 2A9-Cit, having the following structure:

28. The lipid nanoparticle composition of any one of claims 1-27, wherein the phospholipid is selected from 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), 1-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-sn-glycero-3- phosphocholine (OChemsPC), 1 -hexadecyl -sn-glycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2-diarachidonoyl-sn-glycero-3- phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1 ,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3 -phosphorac^ 1 -glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), 1- stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, and lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) sphingomyelin.

29. The lipid nanoparticle composition of any one of claims 1-28, wherein the PEG-lipid is selected from: 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

30. The lipid nanoparticle composition of any one of claims 1-28, wherein the PEG-lipid is 1,2-dimyristoyl-sn-glycero-3 -methoxypoly ethylene glycol -2000 (PEG2000-DMG).

31. The lipid nanoparticle composition of any one of claims 1-30, wherein the PEG-lipid is present in an amount of from about 0.5 mol % to about 5 mol % of the total lipids in the lipid component.

32. The lipid nanoparticle composition of any one of claims 1-30, wherein the PEG-lipid is present in an amount of from about 2 mol % to about 4 mol % of the total lipids in the lipid component.

33. The lipid nanoparticle composition of any one of claims 1-30, wherein the PEG-lipid is present in an amount of about 3 mol % of the total lipids in the lipid component.

34. The lipid nanoparticle composition of any one of claims 1-30, wherein the PEG-lipid is present in an amount of about 4 mol % of the total lipids in the lipid component.

35. The lipid nanoparticle composition of any one of claims 1-35, wherein the first lipid is present in an amount of from about 5 mol % to about 40 mol % of the total lipids in the lipid component.

36. The lipid nanoparticle composition of any one of claims 1-35, wherein the first lipid is present in an amount of from about 5 mol % to about 30 mol % of the total lipids in the lipid component.

37. The lipid nanoparticle composition of any one of claims 1-35, wherein the first lipid is present in an amount of from about 10 mol % to about 20 mol % of the total lipids in the lipid component.

38. The lipid nanoparticle composition of any one of claims 1-35, wherein the first lipid is present in an amount of from about 15 mol % to about 20 mol % of the total lipids in the lipid component.

39. The lipid nanoparticle composition of any one of claims 1-35, wherein the first lipid is present in an amount of from about 40 mol % to about 50 mol % of the total lipids in the lipid component.

40. The lipid nanoparticle composition of any one of claims 1-35, wherein the first lipid is present in an amount of about 20 mol % of the total lipids in the lipid component.

41. The lipid nanoparticle composition of any one of claims 1-35, wherein the first lipid is present in an amount of about 19 mol % of the total lipids in the lipid component.

42. The lipid nanoparticle composition of any one of claims 1-41, wherein the phospholipid is present in an amount of from about 5 mol % to about 25 mol % of the total lipids in the lipid component.

43. The lipid nanoparticle composition of any one of claims 1-41, wherein the phospholipid is present in an amount of from about 10 mol % to about 20 mol % of the total lipids in the lipid component.

44. The lipid nanoparticle composition of any one of claims 1-41, wherein the phospholipid is present in an amount of about 20 mol % of the total lipids in the lipid component.

45. The lipid nanoparticle composition of any one of claims 1-41, wherein the phospholipid is present in an amount of about 19 mol % of the total lipids in the lipid component.

46. The lipid nanoparticle composition of any one of claims 1-45, wherein the second lipid separate from the first ionizable cationic lipid is present in an amount of from about 5 mol % to about 40 mol % of the total lipids in the lipid component.

47. The lipid nanoparticle composition of any one of claims 1-45, wherein the second lipid separate from the first ionizable cationic lipid is present in an amount of from about 10 mol % to about 40 mol % of the total lipids in the lipid component.

48. The lipid nanoparticle composition of any one of claims 1-45, wherein the second lipid separate from the first ionizable cationic lipid is present in an amount of from about 10 mol % to about 30 mol % of the total lipids in the lipid component.

49. The lipid nanoparticle composition of any one of claims 1-45, wherein the second lipid separate from the first ionizable cationic lipid is present in an amount of about 20 mol % of the total lipids in the lipid component.

50. The lipid nanoparticle composition of any one of claims 1-27, wherein: the first lipid is present in an amount from about 10 mol % up to 20 mol % of the total lipids in the lipid component; the PEG-lipid is present in a total amount of about 4 mol % of the total lipids in the lipid component;the second lipid separate from the first ionizable cationic lipid is present in an amount of about 10 mol % to about 30 mol % of the total lipids in the lipid component; and the phospholipid is present in an amount of about 10 mol % up to 20 mol % of the total lipids in the lipid component.

51. The lipid nanoparticle composition of any one of claims 1-27, wherein: the first lipid is present in an amount from about 10 mol % up to 20 mol % of the total lipids in the lipid component; the PEG-lipid is present in a total amount of about 3 mol % of the total lipids in the lipid component; the second lipid separate from the first ionizable cationic lipid is present in an amount of about 10 mol % or about 30 mol % of the total lipids in the lipid component; and the phospholipid is present in an amount of about 10 mol % up to 20 mol % of the total lipids in the lipid component.

52. The lipid nanoparticle composition of any one of the preceding claims, further comprising a sterol.

53. The lipid nanoparticle composition of claim 52, wherein the sterol is present in an amount of from about 20 mol % to about 50 mol % of the total lipids in the lipid component.

54. The lipid nanoparticle composition of claim 52, wherein the sterol is present in an amount of from about 30 mol % to about 40 mol % of the total lipids in the lipid component.

55. The lipid nanoparticle composition of claim 52, wherein the sterol is present in an amount of about 38 mol % of the total lipids in the lipid component.

56. The lipid nanoparticle composition of any one of claims 52-55, wherein the sterol is selected from lanosterol, desmosterol, cholesterol, sitostanol, sitosterol, stigmasterol, optionally cholesterol.

57. The lipid nanoparticle of any one of claims 1-56, wherein the phospholipid is 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE).

58. The lipid nanoparticle of any one of claims 1-56, wherein the phospholipid is 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE).

59. The lipid nanoparticle of any one of claims 1-56, wherein the phospholipid is 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

60. The lipid nanoparticle of any one of claims 1-56, wherein the phospholipid is dipalmitoylphosphatidylcholine (DPPC).

61. The lipid nanoparticle of any one of claims 1-56, wherein the phospholipid is 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE).

62. The lipid nanoparticle of any one of claims 1-56, wherein the phospholipid is 1,2- di stearoyl -sn-gly cero-3 -phosphocholine (D SPC) .

63. The lipid nanoparticle composition of any one of claims 1-62, wherein the first lipid is a compound selected from Table 4.

64. The lipid nanoparticle composition of any one of claims 1-62, wherein the first lipid is a compound selected from Table 5A.

65. The lipid nanoparticle composition of any one of claims 1-62, wherein the first lipid is a compound selected from Table 5B.

66. The lipid nanoparticle composition of any one of claims 1-62, wherein the first lipid is a compound having the following structure:wherein:RD1is a C1-C4alkyl; z1 and z2 are each independently 1, 2, or 3; and z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

67. The lipid nanoparticle composition of any one of claims 1-62, wherein the first lipid is:

68. The lipid nanoparticle composition of any one of claims 1-67, further comprising a payload.

69. The lipid nanoparticle composition of claim 68, wherein the payload comprises a polypeptide or a protein.

70. The lipid nanoparticle composition of claim 69, wherein the polypeptide or protein is selected from: alpha- 1 -antitrypsin (A1AT), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose- 6-phosphatase (G6Pase), glucose-6-phosphate translocase (G6PT), lysosomal glucocerebrosidase (GB), Niemann-Pick Cl protein (NPC1), Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor IX, galactose- 1 -phosphate uridylyltransferase, galactokinase, UDP-galactose 4-epimerase, transthyretin, phenylalanine hydroxylase (PAH), homogentisate 1,2-di oxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL), argininosuccinate synthetase (ASS1), P-type ATPase protein FIC-1, alpha-galactosidase A, acid ceramidase, acid α-L-fucosidase, acid β-galactosidase, iduronate-2-sulfatase, alpha-L- iduronidase, galactocerebrosidase, acid α-mannosidase, β-mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid β-galactosidase, acid α- glucosidase, β-hexosaminidase B, heparan -N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alpha-N-acetylgalactosaminidase, sialidase, β-glucuronidase, β-hexosaminidase A.

71. The lipid nanoparticle composition of claim 68, wherein the payload comprises a nucleic acid.

72. The lipid nanoparticle composition of claim 71, wherein the nucleic acid is selected from an siRNA, a miRNA, a pri-miRNA, a messenger RNA (mRNA), a cluster regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), a plasmid DNA (pDNA), a transfer RNA (tRNA), an antisense oligonucleotide (ASO), a guide RNA, a double stranded DNA (dsDNA), a single stranded DNA (ssDNA), a single stranded RNA (ssRNA), and a double stranded RNA (dsRNA).

73. The lipid nanoparticle composition of claim 72, wherein the payload comprises a small interfering RNA (siRNA).

74. The lipid nanoparticle composition of claim 72, wherein the payload comprises an mRNA.

75. The lipid nanoparticle composition of claim 74, wherein the mRNA encodes a geneediting system or component thereof.

76. The lipid nanoparticle composition of claim 75, wherein the gene-editing system or component thereof comprises a cluster regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), and a guide RNA.

77. The lipid nanoparticle composition of claim 72, wherein the mRNA encodes a protein selected from: alpha- 1 -antitrypsin (A1AT), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose- 6-phosphatase (G6Pase), glucose-6-phosphate translocase (G6PT), lysosomal glucocerebrosidase (GB), Niemann-Pick Cl protein (NPC1), Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor IX, galactose- 1 -phosphate uridylyltransferase, galactokinase, UDP-galactose 4-epimerase, transthyretin, phenylalanine hydroxylase (PAH), homogentisate 1,2-di oxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL), argininosuccinate synthetase (ASS1), P-type ATPase protein FIC-1, alpha-galactosidase A,acid ceramidase, acid α-L-fucosidase, acid β-galactosidase, iduronate-2-sulfatase, alpha-L- iduronidase, galactocerebrosidase, acid α-mannosidase, β-mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid β-galactosidase, acid α- glucosidase, β-hexosaminidase B, heparan -N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alpha-N-acetylgalactosaminidase, sialidase, β-glucuronidase, β-hexosaminidase A78. The lipid nanoparticle composition of claim 72, wherein the payload comprises a guide RNA.

79. The lipid nanoparticle composition of any one of claims 1-78, wherein the lipid nanoparticle composition is capable of delivering a payload preferentially to a liver cell, or to both a liver cell and a lung cell.

80. The lipid nanoparticle composition of any one of claims 1-78, wherein the lipid nanoparticle composition delivers a payload preferentially to a liver cell, or to both a liver cell and a lung cell.

81. A pharmaceutical composition comprising a lipid nanoparticle composition of any one of claims 1-78 and a pharmaceutically acceptable excipient.

82. A method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of claims 1-80 or a pharmaceutical composition of claim 81.

83. The method of claim 82, wherein the method comprises selectively delivering the payload to a target organ.

84. The method of claim 82, wherein the method comprises selectively delivering the payload to a target cell.

85. A method of selectively delivering a payload to a target organ of a subject in need thereof, the method comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of claims 1-80.

86. The method of any one of claim 83-85, wherein the target organ is the liver.

87. The method of any one of claims 83-86, wherein the payload is an mRNA and wherein the selectively delivering results in expression of a protein encoded by the mRNA in a cell of the target organ.

88. The method of any one of claims 83-86, wherein the payload is a polynucleotide encoding a gene product and wherein the selectively delivering results in expression of the gene product in a cell of the target organ and optionally wherein the gene product is functional in the cell of the target organ.

89. The method of any one of claims 83-86, wherein the payload comprises an mRNA encoding a gene-editing system or component thereof and wherein the selectively delivering results in altered expression of a protein targeted by the gene-editing system in a cell of the target organ.

90. The method of any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 200% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

91. The method of any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 300% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

92. The method of any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 400% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

93. The method of any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 500% of the level ofexpression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

94. The method any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 600% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

95. The method of any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is about 200% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

96. The method any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is about 300% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

97. The method of any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is about 400% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

98. The method of any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is about 500% of the level of expression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

99. The method of any one of claims 87-89, wherein the level of expression of the protein or gene product in the cell of the target organ following administration of said lipid nanoparticle composition or pharmaceutical composition is about 600% of the level ofexpression of the protein or gene product in the cell of the target organ following administration of a reference lipid nanoparticle composition.

100. The method of any one of claims 82-99, wherein the reference lipid nanoparticle composition comprises a second ionizable cationic lipid that does not comprise a 1,2- dimyristoyl-3-dimethylammonium-propane, but is otherwise identical to the lipid nanoparticle composition administered to the subject.

101. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is at least 500% of the level of expression of the protein or gene product in a cell from another tissue of the body.

102. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is at least 500% of the level of expression of the protein or gene product in a cell from lung tissue.

103. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is at least 500% of the level of expression of the protein or gene product in a cell from spleen tissue.

104. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is at least 1000% of the level of expression of the protein or gene product in cell from another tissue of the body.

105. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is at least 1000% of the level of expression of the protein or gene product in a cell from lung tissue.

106. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is at least 1000% of the level of expression of the protein or gene product in a cell from spleen tissue.

107. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein or gene product in another tissue of the body.

108. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein or gene product in a cell from lung tissue.

109. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein or gene product in a cell from spleen tissue.

110. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein or gene product in a cell from another tissue of the body.

111. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein or gene product in a cell from lung tissue.

112. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein or gene product in a cell from spleen tissue.

113. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a lung cell that is less than 20% of the level of expression of the protein or gene product in a cell from the target tissue.

114. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a lung cell that is less than 10% of the level of expression of the protein or gene product in a cell from the target tissue.

115. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a lung cell that is less than 5% of the level of expression of the protein or gene product in a cell from the target tissue.

116. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a lung cell that is less than 20% of the total level of expression of the protein or gene product in the subject.

117. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a lung cell that is less than 10% of the total level of expression of the protein or gene product in the subject.

118. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a lung cell that is less than 5% of the total level of expression of the protein or gene product in the subject.

119. The method of any one of claims 82-100, wherein the selectively delivering does not result in expression of the protein or gene product in a cell from lung tissue.

120. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a spleen cell that is less than 20% of the level of expression of the protein or gene product in cell from the target tissue.

121. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a spleen cell that is less than 10% of the level of expression of the protein or gene product in cell from the target tissue.

122. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a spleen cell that is less than 5% of the level of expression of the protein or gene product in a cell from the target tissue.

123. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a spleen cell that is less than 20% of the total level of expression of the protein or gene product in the subject.

124. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a spleen cell that is less than 10% of the total level of expression of the protein or gene product in the subject.

125. The method of any one of claims 82-100, wherein the selectively delivering results in a level of expression of the protein or gene product in a spleen cell that is less than 5% of the total level of expression of the protein or gene product in the subject.

126. The method of any one of claims 82-100, wherein the selectively delivering does not result in expression of the protein or gene product in a spleen tissue.

127. The method of claims 87-126, wherein the level of expression is determined by in vivo biofluorescence imaging.

128. The method of any one of claim 82-127, wherein the subject is a primate.

129. The method of any one of claim 82-127, wherein the subject is a human.

130. A method of delivering a payload to a target cell, comprising contacting the target cell with the lipid nanoparticle composition of any one of claims 1-80 or the pharmaceutical composition of claim 81.

131. An in vivo method of selectively delivering a payload to a target cell in a subject in need thereof, comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of claims 1-80 or the pharmaceutical composition of claim 81.

132. The method of claim 130 or claim 131, wherein the target cell is a liver cell.

133. The lipid nanoparticle composition of any one of claims 56-80, wherein: the first lipid is present in an amount of about 19 mol % of the total lipids in the lipid component; the PEG-lipid is present in a total amount of about 4 mol % of the total lipids in the lipid component;the second lipid separate from the first ionizable cationic lipid is present in an amount of about 20 mol % of the total lipids in the lipid component; the phospholipid is present in an amount of about 19 mol % of the total lipids in the lipid component; and the cholesterol is present in an amount of about 38 mol % of the total lipids in the lipid component.

134. The lipid nanoparticle composition of any one of claims 56-80, wherein: the first lipid is present in an amount about 19 mol % of the total lipids in the lipid component; the PEG-lipid is present in a total amount of about 3 mol % of the total lipids in the lipid component; the second lipid separate from the first ionizable cationic lipid is present in an amount of about 20 mol % of the total lipids in the lipid component; and the phospholipid is present in an amount of about 19 mol % of the total lipids in the lipid component; and the cholesterol is present in an amount of about 39 mol % of the total lipids in the lipid component.

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