Lipid nanoparticles for extrahepatic delivery

Abstract

Provided herein are lipid nanoparticles composition comprising a lipid component which comprises: (i) a first ionizable cationic lipid, (ii) a PEG-lipid, wherein the PEG-lipid comprises a PEG-Ceramide. Further provided are lipid nanoparticle compositions comprising a lipid component which comprises: (i) a first ionizable cationic lipid, (ii) a PEG-lipid, wherein the PEG-lipid comprises a PEG-Ceramide selected from: N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)5000]} (C8 PEG5000-Ceramide), N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}(C8 PEG2000-Ceramide), N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)750]} (C8 PEG750-Ceramide), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)5000]} (C16 PEG5000-Ceramide), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (C16 PEG200-Ceramide), and N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)750]} (C16 PEG750-Ceramide) (iii) a phospholipid, and (iv) a permanently cationic lipid, an anionic lipid, or a second ionizable cationic lipid separate from the first ionizable cationic lipid.

Description

LIPID NANOPARTICLES FOR EXTRAHEPATIC DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 647,166 filed May 14, 2024, and U.S. Provisional Patent Application No. 63 / 659,796 filed June 13, 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 061529-512001WO.xml and is 48,784 bytes in size.BACKGROUND

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

[0004] In one aspect, the disclosure provides a lipid nanoparticle composition comprising a lipid component which comprises (i) a first ionizable cationic lipid, (ii) a polyethylene glycol (PEG) lipid, wherein the PEG-lipid comprises one or more PEG-Ceramide. In some embodiments, the lipid nanoparticle composition further comprising a phospholipid. In some embodiments, the lipid nanoparticle composition further comprising a permanently cationic lipid, an anionic lipid, or a second ionizable cationic lipid separate from the first ionizable cationic lipid.

[0005] In another aspect, the disclosure provides a lipid nanoparticle composition comprising a lipid component which comprises (i) a first ionizable cationic lipid, (ii) a PEG- lipid, wherein the PEG-lipid comprises one or more PEG-Ceramide selected from: 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-l-{ succinyl [methoxy(poly ethylene glycol)5000]} (C16 PEG5000-ceramide), N-palmitoyl-sphingosine-1-{succinyl [methoxy (poly ethylene glycol)2000]} (C16 PEG200-ceramide), and N-palmitoyl- sphingosine-l-{succinyl[methoxy(polyethylene glycol)750]} (C16 PEG750-ceramide), (iii) a phospholipid, and (iv) a permanently cationic lipid, an anionic lipid, or a second ionizable cationic lipid separate from the first ionizable cationic lipid.

[0006] In some embodiments, the 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 1,2-dipalmitoyl-3-trimethylammonium- propane (16:0 TAP), 1,2-stearoyl-3 -trimethylammonium -propane (18:0 TAP), 1,2- dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC), 1,2-distearoyl-sn-glycero-3- thylphosphocholine (18:0 EPC), and 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). In some embodiments, the permanently cationic lipid or the second ionizable cationic lipid is 16:0 TAP.

[0007] In some embodiments, the composition further comprises an anionic lipid. 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).

[0008] In some embodiments, 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), 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-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. In some embodiments, the phospholipid is selected from 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).

[0009] In another aspect, the disclosure provides a lipid nanoparticle composition comprising a lipid component which comprises (i) a first ionizable cationic lipid, (ii) a PEG- lipid, wherein the PEG-lipid comprises one or more PEG-Ceramide, (iii) a phospholipid, wherein the phospholipid is selected from 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), and (iv) a permanently cationic lipid or a second ionizable cationic lipid separate from the first ionizable cationic lipid, wherein the permanently cationic lipid or a 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.

[0010] In some embodiments, the phospholipid is 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).

[0011] In some embodiments, the PEG-lipid comprises at least one PEG-Ceramide selected from: N-octanoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)5000]} (C8 PEG5000-Ceramide), N-octanoyl-sphingosine-l-{succinyl[methoxy(poly ethylene glycol)2000]}(C8 PEG2000-Ceramide), and N-octanoyl-sphingosine-1- { succinyl [methoxy (poly ethylene glycol)750]} (C8 PEG750-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 PEG-lipid comprises the first PEG-Ceramide and the second PEG-Ceramide in a mol / mol 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. In some embodiments, the first PEG-Ceramide and the second PEG-Ceramide are eachindependently selected from: a PEG750 -Ceramide, a PEG2000-Ceramide, and a PEG5000- Ceramide. In some embodiments, the composition comprises a dual C8-Ceramide comprising a first PEG-Ceramide and the second PEG-Ceramide that are each independently a C8 PEG- Ceramide. 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 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 comprises a dual C8-Ceramide comprising C8 PEG2000-Ceramide and C8 PEG750-Ceramide. In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.1 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the PEG- lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide 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 PEG-lipid comprises C8 PEG2000-Ceramide 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. In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.5 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises C8 PEG2000- Ceramide in an amount of from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises C8 PEG750-Ceramide in an amount of about 0.25 mol %, about 0.5 mol %, about 0.75 mol %, 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 PEG-lipid comprises C8 PEG750-Ceramide in an amount of about 0.75 mol %, about 1.5 mol %, about 2 mol %, or about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises a dual C8-Ceramide comprising C8 PEG2000-Ceramide and C8 PEG750-Ceramide in a total combined amount of about 2 mol % to about 3 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises a dual C8- Ceramide comprising C8 PEG2000-Ceramide in an amount of from about 0.5 mol % to about2.25 mol % of the total lipids in the lipid component, and comprising C8 PEG750-Ceramide in an amount of from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component, and wherein 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.

[0012] In some embodiments, 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 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 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 first ionizable cationic lipid is present in an amount of from about 12 mol % to about 20 mol % of the total lipids in the lipid component. In some embodiments, the first ionizable cationic lipid is present in an amount of from about 14 mol % to about 17 mol % of the total lipids in the lipid component. In some embodiments, the first ionizable cationic lipid is present in an amount of from about 14 mol % to about 16 mol % of the total lipids in the lipid component. In some embodiments, the first ionizable cationic lipid is present in an amount of about 16 mol % of the total lipids in the lipid component. In some embodiments, the first ionizable cationic lipid is present in an amount of about 14 mol % of the total lipids in the lipid component.

[0013] 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 14 mol % of the total lipids in the lipid component. In some embodiments, the phospholipid is present in an amount of about 16 mol % of the total lipids in the lipid component.

[0014] 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 35mol % 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 about 40 mol % of the total lipids in the lipid component. In some embodiments, the permanently cationic lipid or the second ionizable cationic lipid separate from the first ionizable cationic lipid is present in an amount of about 45 mol % of the total lipids in the lipid component.

[0015] 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 from about 2 mol % to about 3 mol % (e.g., about 2% or about 3%) 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 35 mol % to about 50 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 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 from about 2 mol % to about 3 mol % (e.g., about 2% or about 3%) 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.

[0016] In some embodiments, the lipid nanoparticle composition further comprising 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 25 mol % of the total lipids in the lipid component. In some embodiments, the sterol is cholesterol.

[0017] In some embodiments, 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).

[0018] In some embodiments, the first ionizable cationic lipid is a compound selected from Table 4. In some embodiments, the first ionizable cationic lipid is a compound selected from Table 5A. In some embodiments, the first ionizable cationic lipid is a compound selected from Table 5B. In some embodiments, the first ionizable cationic lipid is

[0019] In some embodiments, the lipid nanoparticle composition further comprising a payload. In some embodiments, wherein the payload comprises a polypeptide or a protein. In some embodiments, the polypeptide or the protein is selected from: 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, Polycystin 2, Fibrocystin (or poly ductin), Solute carrier family 3 member 1, Solute carrier family 7 member 9, Paired box gene 9, Myosin VII A, 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.

[0020] 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 regularlyinterspaced 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 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). In some embodiments, the payload comprises an mRNA. In some embodiments, the mRNA encodes a gene-editing system or component thereof.

[0021] 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.

[0022] In some embodiments, the mRNA encodes a protein selected from: 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, Polycystin 2, Fibrocystin (or poly ductin), Solute carrier family 3 member 1, Solute carrier family 7 member 9, Paired box gene 9, Myosin VII A, 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.

[0023] In some embodiments, the payload comprises a guide RNA. In some embodiments, the payload comprises a small interfering RNA (siRNA). In some embodiments, the formulation delivers a payload preferentially in a lung cell, or lung / liver cell.

[0024] In another aspect, the disclosure provides 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 described herein.

[0025] 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.

[0026] In another aspect, the disclosure provides 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 described herein. In some embodiments, the target organ is the lung.

[0027] In some embodiments, the payload is an mRNA and 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 the selectively delivering results in expression of the gene product in a cell of the target organ and optionally the gene product is functional in the cell. In some embodiments, 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.

[0028] In some embodiments, the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is at least 200% of the level of expression of the protein 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 in the cell of the target organ following administration of said lipid nanoparticle composition is at least 300% of the level of expression of the protein 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 in the cell of the target organ following administration of said lipid nanoparticle composition is at least 400% of the level of expression of the protein 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 in the cell of the target organ following administration of said lipid nanoparticle composition is at least 500% of the level of expression of the protein 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 in the cell of the target organ following administration of said lipid nanoparticle composition is at least 600% of the level of expression of the protein 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 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 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 in the cell of the target organ following administration of said lipid nanoparticle composition is about300% of the level of expression of the protein 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 in the cell of the target organ following administration of said lipid nanoparticle composition is about 400% of the level of expression of the protein 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 in the cell of the target organ following administration of said lipid nanoparticle composition is about 500% of the level of expression of the protein 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 in the cell of the target organ following administration of said lipid nanoparticle composition is about 600% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

[0029] In some embodiments, the reference lipid nanoparticle composition comprises a PEG-lipid that does not comprise a PEG-Ceramide, but is otherwise identical to the lipid nanoparticle composition administered to the subject. In some embodiments, the reference lipid nanoparticle composition comprises a phospholipid that is not 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidyl choline (DPPC), or 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), but is otherwise identical to the lipid nanoparticle composition administered to the subject. In some embodiments, the reference lipid nanoparticle composition does not comprise a permanently cationic lipid, an anionic lipid, or a second ionizable cationic lipid separate from the first ionizable cationic lipid, but is otherwise identical to the lipid nanoparticle composition administered to the subject. In some embodiments, the reference lipid nanoparticle composition does not comprise a permanently cationic lipid or a second ionizable cationic lipid separate from the first ionizable cationic lipid, wherein the permanently cationic lipid or a 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, but is otherwise identical to the lipid nanoparticle composition. In some embodiments, the reference lipid nanoparticle compositioncomprises an ionizable cationic lipid having the following structure

[0030] In some embodiments, the reference lipid nanoparticle composition comprises an ionizable cationic lipid having the following structure

[0031] In some embodiments, the reference lipid nanoparticle composition comprises an ionizable cationic lipid having the following structure

[0032] In some embodiments, the reference lipid nanoparticle composition comprises a PEG-lipid selected from PEG-DMG and ALC-0159.

[0033] In some embodiments, the reference lipid nanoparticle composition comprises DSPC.

[0034] In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 500% of the level of expression of the protein in another cell of the body. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 500% of the level of expression of the protein in a liver cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 500% of the level of expression of the protein in a spleen cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 1000% of the level of expression of the protein in another cell of the body. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 1000% of the level of expression of the protein in a liver cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 1000% of the level of expression of the protein in aspleen cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein in another cell of the body. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein in a liver cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein in a spleen cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein in another cell of the body. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein in a liver cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein in a spleen cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a liver cell that is less than 20% of the level of expression of the protein in the target cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a liver cell that is less than 10% of the level of expression of the protein in the target cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a liver cell that is less than 5% of the level of expression of the protein in the target cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a liver cell that is less than 20% of the total level of expression of the protein in the subject. In some embodiments, the selectively delivering results in a level of expression of the protein in a liver cell that is less than 10% of the total level of expression of the protein in the subject. In some embodiments, the selectively delivering results in a level of expression of the protein in a liver cell that is less than 5% of the total level of expression of the protein in the subject. In some embodiments, the selectively delivering does not result in expression of the protein in a liver cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 20% of the level of expression of the protein in the target cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 10% of the level of expression of the protein in the target cell. In some embodiments, the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 5% of the level of expression of the protein in the target cell. In some embodiments, theselectively delivering results in a level of expression of the protein in a spleen cell that is less than 20% of the total level of expression of the protein in the subject. In some embodiments, the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 10% of the total level of expression of the protein in the subject. In some embodiments, the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 5% of the total level of expression of the protein in the subject. In some embodiments, the selectively delivering does not result in expression of the protein in a spleen cell. In some embodiments, the level of expression is determined by in vivo biofluorescence imaging.

[0035] In some embodiments, the disease or disorder is Acute Interstitial Pneumonia (AIP), alpha-1 antitrypsin deficiency, asthma, bronchiectasis, Bronchiolitis obliterans with Organizing Pneumonia (BOOP), bronchitis, Chronic Obstructive Pulmonary Disease (COPD), coronavirus, cystic fibrosis, Desquamative Interstitial Pneumonia (DIP), emphysema, Idiopathic Interstitial Pneumonia (IIP), influenza, Interstitial Lung Disease (ILD), Interstitial Pulmonary Fibrosis (IPF), Legionnaire’s disease, lung cancer, Non-Specific Interstitial Pneumonia (NSIP), pleurisy, pneumonia, Primary Ciliary Dyskinesia (PCD), pulmonary arterial hypertension, pulmonary edema, pulmonary fibrosis, pulmonary hypertension, Respiratory Bronchiolitis-associated Interstitial Lung Disease (RBILD), restrictive lung disease, sarcoidosis, Severe Acute Respiratory Syndrome, and tuberculosis. In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0036] In another aspect, the disclosure provides a method of delivering a payload to a target cell, comprising contacting the target cell with the lipid nanoparticle composition described herein. In another aspect, the disclosure provides 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 described herein. In some embodiments, the target cell is a lung cell. In some embodiments, the target cell is a ciliated lung cell. In some embodiments, the target cell is a goblet lung cell. In some embodiments, the target cell is an epithelial cell.

[0037] In another aspect, the disclosure provides a pharmaceutical composition comprising the lipid nanoparticle composition described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] 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.

[0039] FIG. 1 shows distribution of Lung / Liver ratio by formulation.

[0040] FIGs. 2A-2C show comparison of formulations with top ratios across organs. FIG. 2A shows top 10 median Lung / Liver ratio; 10 median Lung / Spleen ratio; and top 10 median Lung / (Liver+Spleen) ratio. FIG. 2B shows top 10 median Lung / Liver ratio; 10 median Lung / Spleen ratio; and top 10 median Lung / Other ratio. FIG. 2C shows top 10 median Lung / Liver ratio; 10 median Lung / Spleen ratio; and bottom 10 liver fluorescence.

[0041] FIG. 3 shows hierarchically clustered heatmap of top extrahepatic lipid nanoparticles.

[0042] FIG. 4A-4F show rat in vivo imaging (IVIS®) for top 9 formulations for lung. FIG. 4A shows organ in vivo imaging (IVIS®) of Composition 4F, 4R, and 4M. FIG. 4B shows whole body in vivo imaging (IVIS®) of Composition 4F, 4R, and 4M. FIG. 4C shows organ in vivo imaging (IVIS®) of Composition 40, 4N, and 41. FIG. 4D shows whole body in vivo imaging (IVIS®) of Composition 40, 4N, and 41. FIG. 4E shows organ in vivo imaging (IVIS®) of Composition 4L, 4P, and 4Q. FIG. 4F shows whole body in vivo imaging (IVIS®) of Composition 4L, 4P, and 4Q.

[0043] FIG. 5 shows boxplot of extrahepatic ratios across formulations, conditions, and replicates.

[0044] FIGs. 6A-6E show distribution of liver fluorescence by subtypes. FIG. 6A shows distribution of liver fluorescence by dendrimer subtype. FIG. 6B shows distribution of liver fluorescence by SORT subtype. FIG. 6C shows distribution of liver fluorescence by helper lipid subtype. FIG. 6D shows distribution of liver fluorescence by PEG subtype. FIG. 6E shows distribution of liver fluorescence by SORT-PEG subtype.

[0045] FIGs. 7A-7G show distribution of extrahepatic ratios by subtypes. FIG. 7A 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 PEG-subtype. FIG. 7G shows distribution of extrahepatic ratios by C8-Ceramide / 16:0 TAP vs. other.

[0046] FIGs. 8A-8F 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. FIG. 8B shows statistically significant effect sizes of components on logioLiver variable. FIG. 8C shows statistically significant effect sizes of components on logioSpleen variable. FIG. 8D shows statistically significant effect sizes of components on Lung / (Liver+Spleen) ratio variable. FIG. 8E shows statistically significant effect sizes of components on Lung / Spleen ratio variable. FIG. 8F shows statistically significant effect sizes of components on Lung / Liver ratio variable.

[0047] FIG. 9 shows lung / river ratio of lung formulations vs. comparators.

[0048] FIG. 10 shows clustering process and archetype identification.

[0049] FIG. 11 shows a schematic method of mouse injection.

[0050] FIG. 12 shows lung in vivo imaging (IVIS®) of mouse dosed with lipid nanoparticle compositions.

[0051] FIG. 13A to FIG. 13C show TdTom+ events (%) of cells dosed with lipid nanoparticle compositions. FIG. 13A shows TdTom+ events (%) of lung cells dosed with lipid nanoparticle compositions. FIG. 13B shows TdTom+ events (%) of liver cells dosed with lipid nanoparticle compositions. FIG. 13C shows TdTom+ events (%) of spleen cells dosed with lipid nanoparticle compositions.

[0052] FIG. 14A to FIG. 14C show TdTom+ events (%) of lung cells dosed with lipid nanoparticle compositions. FIG. 14A shows TdTom+ events (%) of lung endothelial (CD31+) cells dosed with lipid nanoparticle compositions. FIG. 14B shows TdTom+ events (%) of lung immune (CD45+) cells dosed with lipid nanoparticle compositions. FIG. 14C shows TdTom+ events (%) of lung epithelial (EpCAMT) cells dosed with lipid nanoparticle compositions.DETAILED DESCRIPTION

[0053] 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 lung 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

[0054] 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.

[0055] 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.

[0056] The singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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 least10% 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.

[0065] 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.

[0066] 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 other words, the terms “formulation,” “lipid nanoparticle,” “LNP,” “lipid nanoparticle composition,” or “LNP composition” are used herein interchangeably. Lipid nanoparticle 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 ionizable cationic lipid; (ii) a phospholipid; (iii) a steroid or steroid derivative; (iv) a polymer-conjugated lipid; and (v) a permanently cationic lipid, an anionic lipid, or a second ionizable lipid separate from the first ionizable cationic lipid (also referred to herein as “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 thebiological 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.

[0067] 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; and US 11,766,408 and US 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 an 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 physicochemical characteristics of the LNP. Illustrative SORT lipids include, but are not limited to, permanently cationic lipids, anionic lipids, zwitterionic lipids, and ionizable cationic lipids (See 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.

[0068] 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 cationiclipids have one or more nitrogen atoms having pKa’s in the range of about 4.5-8, such are tertiary amine groups.

[0069] 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 Dilliard et al., Proc Natl Acad Sci U S A 118:e2109256118 (2021).

[0070] 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.

[0071] 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.

[0072] 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 illustrative sterol 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.

[0073] 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).

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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 free RNA in a sample with intact LNPs compared with the total RNA in a sample treated to disrupt the LNPs.

[0078] 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.

[0079] 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 modificationsthereof. 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.”

[0080] 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.

[0081] 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.

[0082] As used herein, the term “microRNA” refers to noncoding RNA consisting of about22 ribonucleotides which regulates gene expression in the post transcriptional stage by silencing messenger RNA by base-pairing with a complementary sequence in its targeted mRNA.

[0083] 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.

[0084] 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 guidedor brought to a target genomic region(s) by a guide RNA element or a nucleic acid comprising a nucleotide sequence(s) encoding such endonuclease.

[0085] 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. 48:443 (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.

[0086] 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.

[0087] 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.

[0088] 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 asprovided 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.

[0089] 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.

[0090] 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 by intramuscular injection, intravenous injection, intraperitoneal injection, inhalation, or any other suitable route.

[0091] “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).

[0092] 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., apolynucleotide) 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 that comprises a selected lipid (e.g., SORT lipid and / or a PEG-Ceramide) compared to a reference LNP composition in which the selected lipid is replaced by a control lipid.

[0093] “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 the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.

[0094] “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.

[0095] 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.

[0096] 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).

[0097]

[0001] 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

[0098] 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 oralkenyl 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.

[0099] 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), Nl-[2-(didodecylamino)ethyl]Nl,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-y l-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)-chole 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) octanoate (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.

[0100] 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. WO2015199952, 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 in 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 US20130225836; the contents of each of which are herein incorporated by reference in their entirety.

[0101] 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

[0102] In some embodiments, the ionizable cationic lipids are dendrimers. 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 1994.) 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 repeating units 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, mediumsized 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).

[0103] 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.

[0104] Modifying the functional groups and / or the chemical properties of the core, repeating units, and the surface or terminating groups, their physical properties can bemodulated. 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.

[0105] 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 not monodisperse 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 GO, Gl, 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.

[0106] 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.

[0107] 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.

[0108] 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 active or 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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 acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0113] 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)

[0114] In some embodiments of the lipid composition, the ionizable cationic lipid comprises at least two C8-C24alkyl groups. In some embodiments, 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, C5-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 X4 are 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.

[0115] 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-

[0116] In some embodiments of the dendrimer of formula (D-I), the terminating group is a structure selected from the structures in Table 1.

[0117] 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.

[0118] 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.

[0119] In some embodiments of the dendrimer of formula (I), the terminating group is represented by the formula:wherein:Y4is alkanediyl(C≤18); andR10is hydrogen.

[0120] 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.

[0121] 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).

[0122] In some embodiments of the dendrimer of formula (D-I), the degradable diacyl is further defined as:

[0123] In some embodiments of the dendrimer of formula (D-I), the linker is further defined aswherein Y1is C1-C8alkanediyl or substituted C1-C12alkanediyl.

[0124] 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).

[0125] 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.

[0126] In some embodiments of the dendrimer of formula (D-I), the dendrimer is selected from the group consisting of:and pharmaceutically acceptable salts thereof.Dendrimers of Formula (X)

[0127] In some embodiments of the lipid composition, the ionizable cationic lipid is a dendrimer of the formulaIn some embodiments, the ionizable cationic lipid is a dendrimer of the formula

[0128] 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 Rlgor -L2-NRleRlf;R3aand R3bare each independently at each occurrence hydrogen or an optionally substituted (e.g., C1-C6, such as C1-C3) alkyl;Rla, Rlb, Rlc, Rld, Rle, Rlf, and Rlg(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 Rlcand Rld; 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 when(c) each diacyl group independently comprises a structural formula* 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, R3e, and 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; and Y1is 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.

[0129] 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 Rlgor -L2-NRleRlf. 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).

[0130] In some embodiments of XCore, Rla, Rlb, Rlc, Rld, Rle, Rlf, and Rlg(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, Rla, Rlb, Rlc, Rld, Rle, Rlf, and Rlg(if present) are each independently at each occurrence a point of connection to a branch, hydrogen. In some embodiments of XCore, Rla, Rlb, Rlc, Rld, Rle, Rlf, and Rlg(if present) are each independently at each occurrence a point of connection to a branch an optionally substituted alkyl (e.g., C1-C12).

[0131] 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 Rlcand Rld. 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 each occurrence 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 Xc ore, 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 Xc ore, 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 Rlcand Rld. In some embodiments of XCore, part of L1form a heterocycloalkyl (e.g., C4-C6heterocycloalkyl) with one of Rlcand Rldand the heterocycloalkyl can contain one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur.

[0132] 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)1-4-(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, and L2are 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)-).

[0133] 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. Insome embodiments of XCore, x1is 0, 3. In some embodiments of XCorex1is 4. In some embodiments of XCore, x1is 5. In some embodiments of XCore, x1is 6.

[0134] 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 someembodiments of XCore, the core comprises a structural formula:(e.g., T 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 assome 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:some embodiments of XCore, the core comprises a structural formulawherein ring A is an optionally substituted aryl or an optionallysubstituted (e.g., C3-C12, such as C3-C5) heteroaryl. In some embodiments of XCore,the core comprises has a structural formula

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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 when

[0139] 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

[0140] In some embodiments of XBranch, g=2, G=1, Z=2, and each branch of the plurality of branches comprises a structural formula

[0141] In some embodiments of XBranch, g=3, G=3, Z=4, and each branch of the plurality of branches comprises a structural formula

[0142] In some embodiments of XBranch, g=4, G=7, Z=8, and each branch of the plurality of branches comprises a structural formula

[0143] In some embodiments, the dendrimers described herein with a generation (g) = 1 has the structure:

[0144] In some embodiments, the dendrimers described herein with a generation (g) = 1 has the structure

[0145] An example formulation of the dendrimers described herein for generations 1-4 is shown in Table 3. The number of diacyl groups, linker groups, and terminating groups can be calculated based on g.Table 3. Formulation of Dendrimer Groups Based on Generation (g)

[0146] 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.

[0147] 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 Xuranch, Y3is independently at each occurrence an optionally substituted arenylene (e.g., C1- C12).

[0148] 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 some embodiments of the diacyl group of XBranch, A1and A2are each independently aOt 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 ofthe 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.

[0149] 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.

[0150] In some embodiments of the diacyl group, the diacyl group independently at each occurrence comprises a structural formula(e.g.,such asand optionally R3c, R3d,R3e, and R3fare each independently at each occurrence hydrogen or C1-C3alkyl.

[0151] 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.

[0152] In some embodiments of the linker group of XBranchif 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 XBranchif present, Y1is independently at each occurrence an optionally substituted alkylene (e.g., C1- C12). In some embodiments of the linker group of XBranchif present, Y1is independently at each occurrence an optionally substituted alkenylene (e.g., C1-C12). In some embodiments of the linker group of XBranchif present, Y1is independently at each occurrence an optionally substituted arenylene (e.g., C1-C12).

[0153] 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 ofthe terminating group of XBranch, each terminating group is optionally substituted alkenylthiol (e.g., C1-C18, such as C4-C18).

[0154] 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.

[0155] 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 asC4-C6N-heterocycloalkyl (e.g., N-pyrrol i di ny IN-piperidinylN-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 di- alkylamino)), 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 theterminating 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 structures

[0156] In some embodiments of XCore, the core comprises a structural formula selected, and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a branch of the plurality of branches.

[0157] In some embodiments of the terminating group of XBranch, each terminating group is independently a structure selected from the structure in Table 1. In some embodiments, the dendrimers described herein can comprise a terminating group or pharmaceutically acceptable salt, or thereof selected in Table 1.Table 1. Example terminating groups / peripheries structures

[0158] 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 in Xbranchor 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.

[0159] 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

[0160] 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 is 1; 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.

[0161] 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-Ci2alkyl).

[0162] 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 :

[0163] 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 :

[0164] Additional ionizable cationic lipids that can be used in the compositions and methods of the present application include those ionizable cationic lipids as described in International Patent Publication W02010144740, WO2013149140, WO2016118725, WO2016118724, WO2013063468, WO2016205691, WO2015184256, W02016004202, 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

[0165] 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%.

[0166] 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%.

[0167] 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%.

[0168] 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%.

[0169] In some embodiments, of the lipid nanoparticle composition of the disclosure the first ionizable cationic lipid is a lipid selected from Table 4. In some embodiments, of the lipid nanoparticle composition of the disclosure, 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.

[0170] In some embodiments, the first ionizable cationic lipid comprises:

[0171] In some embodiments, the first ionizable cationic lipid isSelective organ targeting (SORT) lipid

[0172] The lipid composition may further include an anionic lipid, second ionizable cationic lipid separate from the first 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 anionic lipid, second ionizable cationic lipid separate from the first ionizable cationic lipid, or permanently cationic lipid is also referred to herein as a “SORT lipid.”

[0173] 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.

[0174] 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 ammonium group. 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.

[0175] 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-R9 is a group of C8-C24; and A2is a monovalent anion.

[0176] 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).

[0177] In some embodiments of the lipid composition disclosed herein, the lipid composition further comprises a second ionizable cationic lipid (e.g., comprising one or more hydrophobic components and an ionizable group, e.g., a tertiary amino group) separate from the first ionizable cationic lipid. The ionizable positively charged moiety may be positively charged at a physiological pH. One ionizable group that may be used in the ionizable cationic lipid is a tertiary ammine group. In some embodiments of the lipid compositions disclosed herein, the second ionizable cationic 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.

[0178] 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).

[0179] 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).

[0180] In some embodiments of the lipid composition disclosed herein, the second 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 Xis a counterion. 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.

[0181] In some embodiments of the lipid composition disclosed herein, the permanently cationic lipid has a structural formula:, wherein R1and R2are each independently an optionally substituted C6-C24alkyl, or an optionally substituted C6-C24alkenyl.

[0182] In some embodiments of the lipid compositions, the permanently cationic lipid has a structural formula:

[0183] In some embodiments of the lipid composition disclosed herein, the anionic lipid, second ionizable cationic lipid separate from the first ionizable cationic lipid, or permanently cationic 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

[0184] 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.

[0185] 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; and X“ is a monovalent anion.

[0186] 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).

[0187] 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-C6alkyl; X“ is a monovalent anion.

[0188] 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 1,2-dioleoyl-3- trimethyl ammonium -propane (18: 1 DOTAP) (e.g., chloride salt).

[0189] 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; and X2is a monovalent anion.

[0190] 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).

[0191] In some embodiments of the lipid compositions, lipid composition further comprises:1,2-dioleoyl-sn-glycero-3 -phosphate (18: 1

[0192] In some embodiments of the lipid compositions, the additional lipid is selected from the lipids set forth in Table 6.Table 6. Example second ionizable cationic lipid separate from the first ionizable cationic lipid or permanently cationic lipid (e.g., SORT lipids)Table 7. Example anionic lipid (e.g., SORT lipids)

[0193] In some embodiments of the lipid composition of the present application, the anionic lipid, second ionizable cationic lipid separate from the first ionizable cationic lipid, or permanently cationic lipid is present in the composition at a molar percentage from about 5% to about 50%.

[0194] In some embodiments of the lipid composition of the present application, the anionic lipid, second ionizable cationic lipid separate from the first ionizable cationic lipid, or permanently cationic 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%.

[0195] In some embodiments of the lipid composition of the present application, the anionic lipid, second ionizable cationic lipid separate from the first ionizable cationic lipid, or permanently cationic 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% toabout 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%.

[0196] In some embodiments of the lipid composition of the present application, the anionic lipid, second ionizable cationic lipid separate from the first ionizable cationic lipid, or permanently cationic 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

[0197] 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 moiety may 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.

[0198] 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 are 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 cellularrecognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0199] 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.

[0200] 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), 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.

[0201] 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).

[0202] 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.

[0203] 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).

[0204] 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).

[0205] 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).

[0206] 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).

[0207] 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).

[0208] 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.

[0209] 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%.

[0210] 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%.

[0211] 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%.

[0212] 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%.

[0213] 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

[0214] 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 some embodiments 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.

[0215] 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.

[0216] 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%.

[0217] 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%.

[0218] 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 about20% to about 25%, from about 25% to about 50%, from about 25% to about 40%, from about25% to about 30%, from about 30% to about 50%, from about 30% to about 40%, from about30% to about 35%, from about 35% to about 50%, from about 35% to about 45%, from about35% to about 40%, from about 40% to about 50%, from about 40% to about 45%, or from about 45% to about 50%.

[0219] 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 molar percentage 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-conjugated lipid (PEG-lipid)

[0220] 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.

[0221] 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.

[0222] 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 bemodified 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.

[0223] 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-gl y cerol .

[0224] 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.

[0225] 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 anamount 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.

[0226] 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

[0227] 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 12 PEG-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.

[0228] In some embodiments, the PEG-Ceramide comnprises 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 ethylene glycol)2000]} (C16 PEG200-Ceramide), or N-palmitoyl-sphingosine-1-{ succinyl [methoxy(poly ethylene glycol)750]} (C16 PEG750-Ceramide),

[0229] 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(poly ethylene glycol)2000]}(C8 PEG2000-Ceramide), N-octanoyl-sphingosine-l-{succinyl[methoxy(poly ethylene 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).

[0230] In some embodiments, the PEG-Ceramide is C8 PEG750-Ceramide. In some embodiments, the PEG-Ceramide is C8 PEG2000-Ceramide.

[0231] 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 secondPEG-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.

[0232] 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.

[0233] 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.

[0234] 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 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 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 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 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 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 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 ofabout 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.

[0235] In some embodiments, the C8 PEG2000 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 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 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 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 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 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 some embodiments, 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.

[0236] In some embodiments, the C8 PEG5000 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 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 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 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 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 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] In some embodiments of the lipid composition of the present application, the PEG- lipid has a structural formula:, wherein: R12and R13are each independently 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. R12 and R13 are each independently alkyl(c<4-20). In someembodiments, 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.

[0242] 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 ns are 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 about 50. In some embodiments, n2is from 5 to 23. In some embodiments, n2is 11 to about 17. In some embodiments, ns is from 5 to 23. In some embodiments, ns is 11 to about 17.

[0243] 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%.

[0244] 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%.

[0245] 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%.

[0246] 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 someembodiments 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

[0247] In one aspect, the disclosure provides a lipid nanoparticle composition for delivering a payload to a cell in the lung 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.

[0248] 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.

[0249] 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.

[0250] 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).

[0251] 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 cationiclipid 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.

[0252] 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.

[0253] 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 from about 2mol % to about 3 mol % (e.g., about 2% or about 3%) 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.

[0254] 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.

[0255] 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: 1-11. In some embodiments, the mRNA encodes a gene-editing system or component thereof. In some embodiments, the payload comprises a polypeptide or a protein.

[0256] 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).

[0257] 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%.

[0258] 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 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 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 extrahepatic LNP formulations and relevant comparators (italic).

[0259] In some embodiments, a variant of composition 4F comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DOPE at a molar percentage of about 10 to about 30%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1 to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1 to about 10%. Composition 4F may comprise 4A3-SC7 at a molar percentage of 14%, 16:0 TAP at a molar percentage of 45%, DOPE at a molar percentage of 14%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 0.5%, and / or C8 PEG750-Ceramide at a molar percentage of 1.5%.

[0260] In some embodiments, a variant of composition 4R comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DMPE at a molar percentage of about 10 to about 60%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1 to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1 to about 10%. Composition 4R may comprise 4A3-SC7 at a molar percentage of 16%, 16:0 TAP at a molar percentage of 40%, DMPE at a molar percentage of 16%, cholesterol at a molar percentage of25%, C8 PEG2000-Ceramide at a molar percentage of 0.75%, and / or C8 PEG750 -Ceramide at a molar percentage of 2.25%.

[0261] In some embodiments, a variant of composition 4M comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DMPC at a molar percentage of about 10 to about 60%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1 to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1 to about 10%. Composition 4M may comprise 4A3-SC7 at a molar percentage of 16%, 16:0 TAP at a molar percentage of 40%, DMPC at a molar percentage of 16%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 1.5%, and / or C8 PEG750-Ceramide at a molar percentage of 1.5%.

[0262] In some embodiments, a variant of composition 40 comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DMPE at a molar percentage of about 10 to about 60%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1 to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1 to about 10%. Composition 40 may comprise 4A3-SC7 at a molar percentage of 16%, 16:0 TAP at a molar percentage of 40%, DMPE at a molar percentage of 16%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 1.0%, and / or C8 PEG750-Ceramide at a molar percentage of 2.0%.

[0263] In some embodiments, a variant of composition 4N comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DPPC at a molar percentage of about 10 to about 60%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1 to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1 to about 10%. Composition 4N may comprise 4A3-SC7 at a molar percentage of 16%, 16:0 TAP at a molar percentage of 40%, DPPC at a molar percentage of 16%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 1.5%, and / or C8 PEG750-Ceramide at a molar percentage of 1.5%.

[0264] In some embodiments, a variant of composition 41 comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DMPC at a molar percentage of about 10 to about 60%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1 to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1 to about 10%.Composition 41 may comprise 4A3-SC7 at a molar percentage of 16%, 16:0 TAP at a molar percentage of 40%, DMPC at a molar percentage of 16%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 2.25%, and / or C8 PEG750 -Ceramide at a molar percentage of 0.75%.

[0265] In some embodiments, a variant of composition 4L comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DSPE at a molar percentage of about 10 to about 60%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1 to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1 to about 10%. Composition 4L may comprise 4A3-SC7 at a molar percentage of 16%, 16:0 TAP at a molar percentage of 40%, DSPE at a molar percentage of 16%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 2.25%, and / or C8 PEG750 -Ceramide at a molar percentage of 0.75%. In some embodiments, a variant of composition 4L has lipid / mRNA ratio of about 30.

[0266] In some embodiments, a variant of composition 4P comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DSPE at a molar percentage of about 10 to about 60%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1 to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1 to about 10%. Ccomposition 4P may comprise 4A3-SC7 at a molar percentage of 16%, 16:0 TAP at a molar percentage of 40%, DSPE at a molar percentage of 16%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 1.5%, and / or C8 PEG750-Ceramide at a molar percentage of 1.5%.

[0267] In some embodiments, a variant of composition 4Q comprises 4A3-SC7 at a molar percentage of about 10 to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DMPC at a molar percentage of about 10 to about 60%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1 to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1 to about 10%. Composition 4Q may comprise 4A3-SC7 at a molar percentage of 16%, 16:0 TAP at a molar percentage of 40%, DMPC at a molar percentage of 16%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 0.75%, and / or C8 PEG750 -Ceramide at a molar percentage of 2.25%.

[0268] In some embodiments, a variant of composition 4V comprises 4A3-SC7 at a molar percentage of about 10% to about 50%, 16:0 TAP at a molar percentage of about 10% to about60%, DOPE at a molar percentage of about 10% to about 60%, cholesterol at a molar percentage of about 10% to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1% to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1% to about 10%. Composition 4V may comprise 4A3-SC7 at a molar percentage of 16.5%, 16:0 TAP at a molar percentage of 40%, DOPE at a molar percentage of 16%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 0.5%, and / or C8 PEG750- Ceramide at a molar percentage of 1.5%. In some embodiments, a variant of composition 4V has lipid / mRNA ratio of about 30.

[0269] In some embodiments, a variant of composition 4H comprises 4A3-SC7 at a molar percentage of about 10% to about 50%, 16:0 TAP at a molar percentage of about 10 to about 60%, DOPE at a molar percentage of about 10% to about 60%, cholesterol at a molar percentage of about 10 to about 50%, C8 PEG2000-Ceramide at a molar percentage of about 0.1% to about 10%, and / or C8 PEG750-Ceramide at a molar percentage of about 0.1% to about 10%. Composition 4H may comprise 4A3-SC7 at a molar percentage of 16%, 16:0 TAP at a molar percentage of 40%, DOPE at a molar percentage of 16%, cholesterol at a molar percentage of 25%, C8 PEG2000-Ceramide at a molar percentage of 0.75%, and / or C8 PEG750-Ceramide at a molar percentage of 2.25%. In some embodiments, a variant of composition 4H has lipid / mRNA ratio of about 30.

[0270] In some embodiments, a variant of composition 5C comprises 5A2-SC8 at a molar percentage of about 10% to about 50%, DOTAP at a molar percentage of about 10% to about 60%, DOPE at a molar percentage of about 10 to about 60%, cholesterol at a molar percentage of about 10% to about 50%, and / or DMG-PEG at a molar percentage of about 0.1% to about 10%. Composition 5C may comprise 5A2-SC8 at a molar percentage of 21.6%, DOTAP at a molar percentage of 40%, DOPE at a molar percentage of 12%, cholesterol at a molar percentage of 24%, and / or DMG-PEG at a molar percentage of 2.4%. In some embodiments, a variant of composition 5C has lipid / mRNA ratio of about 20.

[0271] As described in the Examples below, linear regression demonstrated that the Dual C8-Ceramide PEG, Helper DMPE / DMPC, and SORT 16:0 / 18:0 TAP formulation provided strong extrahepatic LNP targeting. And 16:0 TAP provided the highest beta value in loglO Lung. 14:0 DAP provided the highest beta value in loglO Liver. 18:TAP provided the highest beta value in loglO Spleen.

[0272] In one aspect, the disclosure provides a lipid nanoparticle composition comprising a lipid component which comprises (i) a first ionizable cationic lipid, (ii) a PEG-lipid, wherein the PEG-lipid comprises one or more PEG-Ceramide.

[0273] In some embodiments, the lipid nanoparticle composition further comprising a phospholipid.

[0274] In some embodiments, the lipid nanoparticle composition further comprising a permanently cationic lipid, an anionic lipid, or a second ionizable cationic lipid separate from the first ionizable cationic lipid.

[0275] In another aspect, the disclosure provides a lipid nanoparticle composition comprising a lipid component which comprises (i) a first ionizable cationic lipid, (ii) a PEG- lipid, wherein the PEG-lipid comprises one or more PEG-Ceramide selected from: 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-l-{ succinyl [methoxy(poly ethylene glycol)5000]} (C16 PEG5000-Ceramide), N-palmitoyl-sphingosine-1-{ succinyl [methoxy (poly ethylene glycol)2000]} (C16 PEG200-Ceramide), and N-palmitoyl- sphingosine-l-{succinyl[methoxy(polyethylene glycol)750]} (C16 PEG750-Ceramide), (iii) a phospholipid, and (iv) a permanently cationic lipid, an anionic lipid, or a second ionizable cationic lipid separate from the first ionizable cationic lipid.

[0276] In some embodiments, the 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. In some embodiments, the permanently cationic lipid or the second ionizable cationic lipid is 16:0 TAP.

[0277] In some embodiments, the composition further comprises an anionic lipid. 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).

[0278] In some embodiments, 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), 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-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. In some embodiments, the phospholipid is selected from 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).

[0279] In another aspect, the disclosure provides a lipid nanoparticle composition comprising a lipid component which comprises (i) a first ionizable cationic lipid, (ii) a PEG- lipid, wherein the PEG-lipid comprises one or more PEG-Ceramide, (iii) a phospholipid, wherein the phospholipid is selected from 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), and (iv) a permanently cationic lipid or a second ionizable cationic lipid separate from the first ionizable cationic lipid, wherein the permanently cationic lipid or a 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.

[0280] In some embodiments, the phospholipid is 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).

[0281] In some embodiments, the PEG-lipid comprises at least one PEG-Ceramide selected from: N-octanoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)5000]} (C8 PEG5000-Ceramide), N-octanoyl-sphingosine-l-{succinyl[methoxy(poly ethylene glycol)2000]}(C8 PEG2000-Ceramide), and N-octanoyl-sphingosine-1- { succinyl [methoxy (poly ethylene glycol)750]} (C8 PEG750-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 PEG-lipid comprises the first PEG-Ceramide and the second PEG-Ceramide in a mol / mol 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. 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 composition comprises a dual C8-Ceramide comprising a first PEG-Ceramide and the second PEG-Ceramide that are each independently a C8 PEG- Ceramide. In some embodiments, the lipid component comprises the PEG-lipid 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 lipid component comprises the PEG-lipid in an amount of from about 2 mol % to about 4 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the PEG-lipid in an amount of about 3 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises a dual C8-Ceramide comprising C8 PEG2000-Ceramide and C8 PEG750-Ceramide. In some embodimentsthe PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.1 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises C8 PEG2000- Ceramide 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 PEG-lipid comprises C8 PEG2000-Ceramide 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. In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.5 mol % to about 2.5 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of fromabout 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises C8 PEG750-Ceramide in an amount of about 0.25 mol %, about 0.5 mol %, about 0.75 mol %, 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 PEG-lipid comprises C8 PEG750-Ceramide in an amount of about 0.75 mol %, about 1.5 mol %, about 2 mol %, or about 2.25 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises a dual C8-Ceramide comprising C8 PEG2000-Ceramide and C8 PEG750-Ceramide in a total combined amount of about 2 mol % to about 3 mol % of the total lipids in the lipid component. In some embodiments, the PEG-lipid comprises a dual C8-Ceramide comprising C8 PEG2000- Ceramide in an amount of from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component, and comprising C8 PEG750-Ceramide in an amount of from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component, and wherein 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.

[0282] 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.

[0283] In some embodiments, the lipid component comprises the phospholipid in an amount of from about 5 mol % to about 25 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the phospholipid in an amount of fromabout 10 mol % to about 20 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the phospholipid in an amount of about 14 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the phospholipid in an amount of about 16 mol % of the total lipids in the lipid component.

[0284] In some embodiments, the lipid component comprises the permanently cationic lipid, the lipid component comprises the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 15 mol % to about 55 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the permanently cationic lipid, the lipid component comprises the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 30 mol % to about 55 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the permanently cationic lipid, the lipid component comprises the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 35 mol % to about 50 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the permanently cationic lipid, the lipid component comprises the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 40 mol % to about 50 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the permanently cationic lipid, the lipid component comprises the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 35 mol % to about 45 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the permanently cationic lipid, the lipid component comprises the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of about 40 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the permanently cationic lipid or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of about 45 mol % of the total lipids in the lipid component.

[0285] In some embodiments, the lipid component comprises the first ionizable cationic lipid in an amount from about 10 mol % up to 20 mol % of the total lipids in the lipid component; the PEG-lipid in a total amount from about 2 mol % to about 3 mol % (e.g., about 2% or about 3%) 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 in an amountof about 35 mol % to about 50 mol % of the total lipids in the lipid component; and the phospholipid in an amount of about 10 mol % up to 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 from about 10 mol % up to 20 mol % of the total lipids in the lipid component; the PEG-lipid in a total amount from about 2 mol % to about 3 mol % (e.g., about 2% or about 3%) 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 in an amount of about 40 mol % or about 45 mol % of the total lipids in the lipid component; and the phospholipid in an amount of about 10 mol % up to 20 mol % of the total lipids in the lipid component.

[0286] In some embodiments, the lipid component comprises the lipid nanoparticle composition further comprising a sterol. In some embodiments, the lipid component comprises the sterol in an amount of from about 20 mol % to about 50 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the sterol in an amount of from about 20 mol % to about 30 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the sterol in an amount of about 25 mol % of the total lipids in the lipid component. In some embodiments, the lipid component comprises the sterol is cholesterol.

[0287] In some embodiments, the lipid component comprises the phospholipid is 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the lipid component comprises the phospholipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). In some embodiments, the lipid component comprises the phospholipid is 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments, the lipid component comprises the phospholipid is dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the lipid component comprises the phospholipid is 1,2-distearoyl-sn-glycero-3- phosphorylethanolamine (DSPE).

[0288] In some embodiments, the first ionizable cationic lipid is a compound selected from Table 4. In some embodiments, the first ionizable cationic lipid is a compound selected from Table 5A. In some embodiments, the first ionizable cationic lipid is a compound selected fromTable 5B. In some embodiments, the first ionizable cationic lipid isLung diseases

[0289] Cystic Fibrosis: Cystic fibrosis is a progressive, autosomal recessive 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. The CFTR protein functions as a channel that transports chloride ions across the membrane of cells and is required to regulate the components of mucus, sweat, saliva, tears, and digestive enzymes.

[0290] Disease-causing mutations in the CFTR protein cause dysfunction of its channel activity resulting in abnormal transport of chloride ions, sodium ions, and bicarbonate across the epithelium, leading to the thick, viscous secretions in the lung, pancreas, and other organs, including sweat glands and tissues of the male reproductive tract, characteristic of cystic fibrosis disease. Almost 2,000 variants have been cataloged in the Cystic Fibrosis Mutation database, including variants that cause substitution of a single amino acid, nonsense mutations, frameshifts, mis-splicing variants, or affect the promoter region. The most common cause of cystic fibrosis is the gene variant F508del, which is defined by a deletion of three base pairs that cause the loss of the amino acid phenylalanine at position 508. The loss of phenylalanine causes incorrect folding of the CFTR protein and subsequent degradation. Overall, the variants affect the amount of CFTR protein available in the apical cell membrane, reducing the activity and efficiency of the ion channel.

[0291] The decrease or loss of function of CFTR protein leads to multiorgan dysfunction and a shortened life expectancy. Most cystic fibrosis patients develop severe, chronic lung disease related to airway obstruction partly due to increased levels of sulfated mucins, inflammation, and recurrent infections that are eventually lethal, with the median predicted survival age in the US being 40.7 years. Abnormally viscous secretions in the lung airways cause obstructions that lead to inflammation, tissue damage, frequent respiratory infections,and organ failure. The lungs of individuals with cystic fibrosis are colonized and infected by bacteria from an early age. This leads to chronic airway infection and inflammation, progressing to bronchiectasis, gas trapping, hypoxemia, and hypercarbia. In the initial stage, common bacteria such as Staphylococcus aureus and Hemophilus influenzae colonize and infect the lungs. Eventually, Pseudomonas aeruginosa (and sometimes Burkholderia cepacia) dominates. By 18 years of age, 80% of patients with classic CF harbor P. aeruginosa, and 3.5% harbor B. cepacia. Once within the lungs, these bacteria adapt to the environment and develop resistance to commonly used antibiotics. Cystic fibrosis is the most frequent lethal genetic disease in the white population.

[0292] Several treatments for cystic fibrosis have been developed since the first causal CFTR mutation was identified in 1989. Gene-targeting therapies exist which aim to augment the function of mutant CFTR or restore CFTR function by correcting the mutations. Other therapies are focused on improving mucociliary clearance and preventing infections. Therapies comprised of a group of small molecules that aim to modulate and restore mutant CFTR function are known as CFTR modulators. The compounds elexacaftor-tezacaftor-ivacaftor enhance the activity of mutant CFTR, and patients with the F508del variant taking this combination have improved lung function from 0.2% in the placebo group to 13.6%. However, these modulators are ineffective for CFTR mutations that lead to premature stop codons or splicing defects. For example, patients with mutations that cause premature stop codons (R553X, G542X, and W1282X) or with a splice-site mutation (3849 + 10 kb C>T) cannot benefit from CFTR modulators. Using gene therapy and gene editing approaches to treat patients with nonsense and splicing CFTR mutations can be a viable alternative. See e.g., Ong et al., JAMA 329: 1859-1871 (2023); Cutting G., Nat Rev Genet 16:45-56 (2015); Esposito et al., Life (Basel) 13: 1835 (2023); Hisert et al., Lancet Respir Med 11:916-931 (2023); McHugh et al., PloS One 13:e0199573 (2018); and Krishnamurthy et al., Nucleic Acids Res. 49: 10558- 10572 (2021), which are incorporated by reference herein in its entirety.

[0293] In some embodiments, a patient in need of treatment has received or is concurrently receiving other lung disease medications. For example, a patient in need of treatment may be receiving lumacaftor / ivacaftor combination drug (ORKAMBI®) or may have been on this treatment for at least 28 days prior to commencement of the treatment according to the present disclosure. Other cystic fibrosis medications may include, but are not limited to, routine inhaled therapies directed at airway clearance and management of respiratory infections, such as bronchodilators, rhDNase (PULMOZYME (Dornase alfa)), hypertonic saline, antibiotics, andsteroids; and other routine CF -related therapies such as systemic antibiotics, pancreatic enzymes, multivitamins, and diabetes and liver medications.

[0294] Gene editing has been revolutionizing the medical field by creating permanent therapies across a wide spectrum of diseases. Direct delivery of gene editors to target cells using synthetic nanoparticle- or virus-based systems could facilitate efficient and precise gene editing directly within the patient's body, bypassing the complexities, risks, and costs associated with ex vivo procedures. However, treating genetic lung diseases with gene editing constitutes a large unmet medical. And genome editing approaches for the treatment of cystic fibrosis have been hindered by delivery challenges to protect genome editors from degradation, to achieve cell targeted delivery, and to minimize off-target effects while overcoming significant physiological barriers including mucus, macrophages, and endothelial tissues. See, e.g., Cheng et al., Nat Nanotechnol 15:313-320 (2020); Jinek et al., Science 337: 816-821 (2012); Gaudelli et al., Nature 551:464-471 (2017); and Porto et al., Nat Rev Drug Discov 19:839-859 (2020). The present disclosure addresses, at least, this unmet need and provide compositions and methods for treating cystic fibrosis.

[0295] Primary ciliary dyskinesis (PCD): Primary ciliary dyskinesia is a disorder characterized by chronic respiratory tract infections. Mutations in genes which provide instructions for making proteins that form the inner structure of cilia and produce the force needed for cilia to bend because the disease and it results in defective cilia that move abnormally or are unable to move. Mutations in the DNAI1 and DNAH5 gene account for up to 30 percent of all cases of PCD.

[0296] Chronic obstructive pulmonary disease (COPD): Chronic obstructive pulmonary disease is a chronic inflammatory lung disease that causes obstructed airflow from the lungs. In the vast majority of people with COPD, the lung damage that leads to COPD is caused by long-term cigarette smoking. In about 1% of people with COPD, the disease results from a genetic disorder that causes low levels of a protein called alpha- 1 -antitrypsin (AAT). AAT is made in the liver and secreted into the bloodstream to help protect the lungs. Alpha- 1- antitrypsin deficiency can cause liver disease, lung disease, or both.

[0297] As disclosed in the Examples below, lipid nanoparticle compositions of the disclosure (e.g., lipid nanoparticle compositions comprising a PEG-Ceramide, and it particular, two separate PEG-Ceramides, were identified as top formulations targeting the lung. 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, e.g., lipid nanoparticles comprising a PEG-Ceramide, and it particular, two separate PEG-Ceramides, have a high lung / spleen, lung / (spleen + liver), and lung / (other organs) ratio compared formulations not comprising a PEG-Ceramide. See, e.g., Formulations 5 A, 5B, X, B, W, and 2J, which comprise PEG-DMG as the PEG-lipid. 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 lung) 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 lung, may lead to expression of the polypeptide in cells of the spleen, where it may have an effect other than the intended effect.Payloads

[0298] The present disclosure contemplates delivery of various payloads useful in the treatment of a lung disease. Payloads comprise therapeutic polypeptides or polynucleotides encoding polypeptides. For example, the payload may be a polynucleotide encoding a gene related to lung disease, or a polynucleotide encoding a gene editor for editing a gene related to lung disease.

[0299] 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 thereofcomprises 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.Polypeptides

[0300] 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), Dornase alfa, Tissue plasminogen activator (TP A)Activase, 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.

[0301] 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, Polycystin 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 l,Kvl 1.1 (alpha subunit of potassium ion channel), Palmitoyl -protein thioesterase 1, ATM serine / threonine kinase, or Fibrillin 1.Polynucleotides

[0302] 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.

[0303] 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.

[0304] 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 polynucleotidesincluding 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.

[0305] 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-processed RNA 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.

[0306] 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 agent (or prophylactic agent) described herein.

[0307] In some embodiments, the polynucleotide is greater than 30 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, greater than 200 nucleotides, greater than300 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.

[0308] 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 3000 nucleotides 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.

[0309] 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 200nucleotides. 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.

[0310] 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 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 some embodiments, 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.

[0311] In some embodiments, the mRNA encodes a gene or a portion of a gene related to lung disease shown in Table 9 or Table 10.

[0312] It is understood that T is T in DNA and T is U in RNA polynucleotide sequences.Table 9. Examples of genes related to lung diseasesTable 10. Example sequences of genes related to lung diseases

[0313] 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: 1. In some embodiments, the mRNA encoding DNAI1 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 DNAH5 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 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: 4. In some embodiments, the mRNA encoding ARMC4 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 DNAAF1 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. In some embodiments, the mRNA encoding DNAAF2 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: 7. In some embodiments, the mRNA encoding DNAAF4 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: 8. In some embodiments, the mRNA encoding ZMYND10 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: 9. In some embodiments, the mRNA encoding CCDC39 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: 10. In some embodiments, the mRNA encoding CCDC40 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: 11.

[0314] 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

[0315] 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 -taurinom ethyluridine, 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine, 1 -taurinom ethyl-4-thio-uri dine, 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- 1-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- methylguanosine, 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.

[0316] 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 frompseudouridine (Ψ), 1 -methylpseudouridine (m1P), 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

[0317] 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 located three 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 an 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.

[0318] 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 sequencescan be incorporated into the flanking regions of a polynucleotide. Incorporation of intronic sequences can also increase the rate of translation of the polynucleotide.

[0319] 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-a. 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 conducted in 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.

[0320] In some embodiments, a polynucleotides 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.

[0321] In some embodiments, a polynucleotides 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 ofthe 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, an 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')NlmpNp (Cap-1), and m7G(5')-ppp(5 ')NlmpN2mp (Cap-2).

[0322] 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.

[0323] 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, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine.

[0324] 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, 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or 10000 bases or base pairs in length.

[0325] In some embodiments, a polynucleotide of the disclosure can comprise a polyA sequence. A polyA sequence (e.g., polyA tail) can comprise any number of nucleotides. A polyA 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 polyA sequence is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in length. A polyA 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 polyA 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

[0326] 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 tracr RNA, 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.

[0327] In some embodiments, the LNP comprises a gene editing system, wherein the gene editing system comprises a gRNA and the mRNA of the base editor. In some embodiments, the gRNA and the mRNA of the base editor are present at a molar ratio that is 1 : 1. In someembodiments, the gRNA and the mRNA of the base editor are present at a molar ratio that is not 1:1. In some embodiments, the gRNA and the mRNA of the base editor are present at a molar or weight ratio less than 1:1. In some embodiments, the gRNA and the mRNA of the base editor are present at a molar or weight ratio of at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1 :24, 1 :25, 1 :26, 1 :27, 1 :28, 1 :29, or 1 :30. In some embodiments, the gRNA and the mRNA of the base editor are present at a molar or weight ratio of at least about 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. In some embodiments, the gRNA and the mRNA of the base editor are present at a molar or weight ratio of about 1:1, 1:2, 1:3, 1 :4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30, or a range between any two of the foregoing values.

[0328] In some embodiments, the mRNA of the base editor and the gRNA are present at a molar or weight ratio that is not 1:1. In some embodiments, the mRNA of the base editor and the gRNA are present at a molar or weight ratio less than 1 : 1. In some embodiments, the mRNA of the base editor and the gRNA are present at a molar or weight ratio of at most about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30. In some embodiments, the mRNA of the base editor and the gRNA are present at a molar or weight ratio of at least about 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1. In some embodiments, the mRNA of the base editor and the gRNA are present at a molar or weight ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30, or a range between any two of the foregoing values.Gene Editing Methods

[0329] 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 lungs. In some embodiments, the cells of target organ can be lung cells. In some embodiments, the cells can be ciliated cells, goblet cells, secretory cells, club cells, basal cells or ionocytes.

[0330] 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.

[0331] 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.

[0332] 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 the endogenous DNA. A guide RNA can be designed to target the specific genomic location of interest in the cells of target organ.

[0333] 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).

[0334] 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, anuclease of the nuclease-dependent targeted editing can include, but not limited to, CRISPR- Cas9, CRISPR-Cas 12 (Cpfl), CRISPR-Casl3, C2c2, C2c6, NgAgo, and / or TALEN.

[0335] 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.

[0336] In some embodiments, targeted gene editing can be achieved via dual integrase cassette exchange (DICE) system utilizing phiC31 and Bxb1 integrases.CRISPR-Cas proteins

[0337] As used herein, the term “Cas protein” or “CRISPR-Cas protein” refers to a full- length Cas protein obtained from nature, a recombinant Cas protein having a sequence that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods, i.e., (i) possession of nucleic-acid binding of the Cas protein to a target DNA, (ii) ability to create double-strand breaks in the target DNA sequence; and / or (iii) ability to nick the target DNA sequence on one strand. The Cas proteins contemplated herein comprise CRISPR Cas9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (dCas9) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (c.g, engineered or recombinant), and may include a Cas9 equivalent from any type of CRISPR system (e.g., type II, V, VI), including Cpfl (a type-V CRISPR-Cas systems), C2cl (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al., Science 353:aaf5573 (2016), the contents of which are incorporated herein by reference.

[0338] In some aspects, the disclosure provides base editors comprising one or more adenosine deaminase variants disclosed herein and a CRISPR-Cas protein. In some embodiments, the CRISPR-Cas protein comprises a Cas homolog. The CRISPR-Cas protein may be selected from any CRISPR associated protein, including but not limited to a Cas9, a Cas9n, a dCas9, a CasX, a CasY, a C2cl, a C2c2, a C2c3, a GeoCas9, a CjCas9, a Cas 12a, a Casl2b, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Casl4, a Csn2, an xCas9, an SpCas9-NG, an SpCas9-NG-CP1041, an SpCas9-NG-VRQR, an LbCasl2a, an AsCasl2a, a Cas9-KKH, a circularly permuted Cas9, an Argonaute (Ago) domain, aSmacCas9, a Spy-macCas9, an SpCas9-VRQR, an SpCas9-NRRH, an SpaCas9-NRTH, an SpCas9-NRCH. Other non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. In certain embodiments, the CRISPR-Cas protein comprises or is a Cas9 protein or a Casl2a protein derived from S. pyogenes or S. aureus.

[0339] In some embodiments, the CRISPR-Cas protein comprises a nuclease dead Cas9 (dCas9) protein, a Cas9 nickase (nCas9) protein, or a nuclease active Cas9 protein. As used herein, the term “dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9. The term dCas9 is not meant to be particularly limiting and may be referred to as a “dCas9 or equivalent.” Any suitable mutation which inactivates both Cas9 endonucleases may be used to form the dCas9.

[0340] In other embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. In other embodiments, Cas9 variants having mutations may result in the full or partial inactivation of the endogenous Cas9 nuclease activity (e.g., dCas9 or nCas9, respectively). In some embodiments, variants or homologues of Cas9 (e.g., variants of Cas9 from Streptococcus pyogenes) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the sequence of wild type Cas9 from Streptococcus pyogenes.

[0341] In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9

[0342] In some embodiments, the disclosure also may utilize Cas9 fragments which retain their functionality and which are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0343] In some embodiments, the CRISPR-Cas protein of any of the disclosed base editors comprises a dead S. pyogenes Cas9 (dSpCas9). In some embodiments, the CRISPR-Cas protein of any of the disclosed base editors comprises a dead Lachnospiraceae bacterium Casl2a (dLbCasl2a).

[0344] In some embodiments, the disclosed base editors may comprise a CRISPR-Cas protein that comprises a nickase. In some embodiments, the base editors described herein comprise a Cas9 nickase. The term “Cas9 nickase” or “nCas9” refers to a variant of Cas9 which is capable of introducing a single-strand break in a double strand DNA molecule target. In some embodiments, the Cas9 nickase comprises only a single functioning nuclease domain. The wild type Cas9 (e.g., the canonical SpCas9) comprises two separate nuclease domains, namely, the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). In one embodiment, the Cas9 nickase comprises a mutation in the RuvC domain which inactivates the RuvC nuclease activity.

[0345] The catalytically impaired Cas9 protein can be, but is not limited to NRRH, NRTH, NRCH, xCas9, SpCas9-NG, SpCas9, SpG, SpRY, SauriCas9, SaCas9, Nme2Cas9, VRER- SpCas9, and VQRSpCas9. In some embodiments, the catalytically impaired Cas9 protein is SpCas9-NG.

[0346] In some embodiments, the Cas protein may be complexed with a guide polynucleotide.

[0347] Exemplary CRISPR-Cas proteins include but are not limited to S. pyogenes Cas9 nickase (SpCas9n) and S. aureus Cas9 nickase (SaCas9n). Additional exemplary CRISPR-Cas proteins include S. aureus Cas9-KKH (SaCas9-KKH), LbCasl2a, enAsCasl2a (an engineered AsCasl2a recently reported by Kleinstiver et al., Nat Biotechnol 33: 1293-1298 (2015)), SpCas9-NG, SpCas9-VRQR, SpCas9-NG-CP1041, SpCas9-NG-VRQR, SpCas9-NRCH, CP1028-SpCas9, and CP1041-SpCas9. In some embodiments, the CRISPR-Cas protein comprises a Cas9 nickase (nCas9) protein. In some embodiments, the CRISPR-Cas protein comprises an SpCas9n protein. In certain embodiments, the CRISPR-Cas protein of any of the disclosed base editors is a SaCas9n. In certain embodiments, the CRISPR-Cas protein of any of the disclosed base editors is an SpCas9-NRCH. In certain embodiments, the CRISPR-Casprotein of any of the disclosed base editors is an LbCasl2a, e.g., a catalytically inactive or "dead" LbCasl2a. In certain embodiments, the CRISPR-Cas protein of any of the disclosed base editors is an AsCasl2a, e.g., an enAsCasl2a. In certain embodiments, the CRISPR-Cas protein of any of the disclosed base editors is a circular permuted variant of SpCas9, e.g., a CP1028 SpCas9 or a CP1041 SpCas9. In certain embodiments, the CRISPR-Cas protein of any of the disclosed base editors is an evolved SpCas9, e.g., an SpCas9-NG. In certain embodiments, the CRISPR-Cas protein of any of the disclosed base editors is an SpCas9-NG- CP1041. In certain embodiments, the CRISPR-Cas protein of any of the disclosed based editors is SpCas9-NG-VRQR.

[0348] The nuclease in the compositions described herein may be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR-Cas protein can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence of any one of the genes described herein. For example, the CRISPR enzyme may be directed and cleaved a genomic locus of CFTR.

[0349] The CRISPR-Cas protein may be mutated with respect to a corresponding wildtype enzyme such that the mutated CRISPR-Cas protein lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to- alanine substitution (D10A) in the RuvC catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce non -homologous end-joining (NHEJ) or homology directed repair (HDR). See e.g., WO2022216619, which is incorporated herein by reference in its entirety.CRISPR-Cas9 Gene Editing System

[0350] 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).

[0351] The term “Cas9” or “Cas9 domain” comprises any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. The term Cas9 is not meant to be particularly limiting and may be referred to as a “Cas9 or equivalent.” Additional Cas9 sequences and structures are well known to those of skill in the art (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti et al., Proc Natl Acad Sci U S A 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. " Deltcheva et al., , Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." linek et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference).

[0352] Examples of Cas9 and Cas9 equivalents are provided as follows; however, these specific examples are not meant to be limiting. The base editors of the present disclosure may use any suitable CRISPR-Cas domain, including any suitable Cas9 or Cas9 equivalent.

[0353] 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).

[0354] 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.

[0355] 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).

[0356] 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 one and 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.

[0357] In other embodiments, the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species. In some embodiments, the Cas9 protein is an ortholog comprising a sequence of at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs. In some embodiments, the adenine base editor may include any of the above Cas9 ortholog sequences, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0358] 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).

[0359] In some embodiments, the CRISPR / Cas system can comprise components derived from a Type-I, Type-II, or Type-III 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(11):722-36 (2015); Shmakov et al., Mol Cell 60:385-397 (2015)).

[0360] 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.

[0361] 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.

[0362] 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 nuclease domain. 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).

[0363] 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-III 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.

[0364] A Type I CRISPR / Cas system can utilize a large effector complex known as Cascade (CRISPR-associated complex for antiviral defense) for target binding andinterference. 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.

[0365] 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.

[0366] 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.

[0367] 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).

[0368] 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.

[0369] 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.

[0370] 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.

[0371] 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.

[0372] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR-Cas protein (e.g., a Cas9 or Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP, and Maq (available at maq.sourceforge.net).

[0373] In some embodiments, a guide sequence is above or more than about 5, 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, 75, or more nucleotides in length. In some embodiments, each gRNA comprises a guide sequence of at least 10 contiguous nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that is complementary to a target sequence (or off target site).

[0374] In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a base editor to a target sequence may be assessed by any suitable assay. For example, the components of a base editor, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of a base editor disclosed herein, followed by an assessment of preferential cleavage within the target sequence. Similarly, cleavage of a target polynucleotide sequence may be evaluated in situ by providing the target sequence, components of a base editor, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.

[0375] 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.

[0376] 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 someembodiments, 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.

[0377] 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.

[0378] 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.

[0379] 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.

[0380] 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 someembodiments, 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.

[0381] 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.

[0382] 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.

[0383] 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.

[0384] 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%.

[0385] In some embodiments, a guide RNA (gRNA) comprising a sequence that is complementary to a target sequence (e.g. CFTR gene) and is used with RNA-guided nucleases,e.g., Cas, to induce a DNA break at the target site or target position. Methods for designing gRNAs and exemplary targeting domains can include those described in, e.g., WO2015 / 161276, W02017 / 193107, WO2017 / 093969, US2016 / 272999 and US2015 / 056705, the contents of which are incorporated by reference. Methods for introducing a genetic disruption at one or more target sites and gRNAs that target the target sites include those described in, e.g., WO2015 / 161276, W02015 / 070083, WO2019 / 070541, WO2019 / 195491, WO20 19 / 195492, WO2019 / 089884, and WO2020 / 223535, the contents of which are incorporated by reference.Other CRISPR-Cas tools

[0386] In addition to nucleases that cleave target sequences, there are three other classes of CRISPR-Cas tools: base editors, transposases, and prime editors. These four classes can mediate different types of genomic edits, including conversion, deletion, or insertion of nucleic acids. These tools can use a nuclease dead Cas or deactivated Cas (dCas) system. The nuclease domains are mutated to abolish cleavage activity and obtain a dCas, such as for Cas9, where two point mutations are introduced to attain dCas9 (Xu, Y., & Li, Z. (2020). CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Computational and Structural Biotechnology Journal., 18, 2401-15, which is incorporated by reference herein in its entirety). Importantly, the DNA binding activity of dCas9 is not affected. Fusing dCas systems with other effector domains can further extend CRISPR-Cas applications as catalytically inactive Cas nucleases are useful programmable proteins that localize the fused proteins to the target regions. For example, the CRISPR-dCas9 system fused to transcriptional activators (CRISPRa) or repressors (CRISPRi) can be used to activate or inhibit the transcription of target genes, respectively (Xu, Y., & Li, Z. (2020). CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Computational and Structural Biotechnology Journal., 18, 2401-15, which is incorporated by reference herein in its entirety). Furthermore, the development of the prime editing system, a versatile fusion between Cas9, a reverse transcriptase, and a prime editing gRNA (pegRNA), can mediate insertions, deletions, and all 12 types of base substitutions without double-strand breaks or donor templates (Anzalone et al., Nature 576: 149-157 (2019), which is incorporated by reference herein in its entirety).Base editors

[0387] In some embodiments, the methods provided herein comprises the administration of an LNP, wherein the LNP comprises a nucleic acid encoding a base editor. Base editors comprise fusions between impaired Cas enzymes which are unable to create double stranded breaks (DSBs), and a base-modification enzyme that modifies single-stranded nucleic acids only. Base editing can precisely convert one nucleic acid or base pair into another in genomic DNA or cellular RNA without double-strand breaks, DNA repair templates, or relying on repair mechanisms. Two classes of DNA base editors can convert a base pair to another: cytosine base editors (CBEs) can convert a C-G base pair into a T-A base pair, and adenine base editors (ABEs) can convert an A-T base pair to a G-C base pair. These editors can be used to perform all possible transition mutations. However, while base editors avoid the generation of indels, due to target sequence requirements, the base editing window is more restricted. Of note, cytosine to guanine base editors (CBGEs) have recently also been under development (Kurt et al., Nat Biotechnol 39:41-46 (2021), which is incorporated by reference herein in its entirety).

[0388] In some embodiments, the gene editing system comprises a base editor and a guide RNA (gRNA). In some embodiments, the base editor is an adenine base editor (ABE). In other embodiments, the base editors is a cytosine base editor (CBE). An ABE comprises a tRNA adenosine deaminase (TadA) protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence. A CBE comprises a cytidine deaminase protein, or a variant thereof, fused to a catalytically impaired Cas protein capable of binding to a specific nucleotide sequence. In some embodiments, the present disclosure provides base editors having adenosine deaminase domains that are mutated (e.g., evolved to have mutations) that enable the deaminase domain to have improved activity when used with Cas homologs (e.g., homologs other than SpCas9). Accordingly, the present disclosure provides variants of adenosine deaminases (e.g., variants of TadA-7.10). One example of an adenosine deaminase variant is TadA-8e, which contains eight additional mutations relative to the TadA-7.10 deaminase domain (where TadA-7.10 contains the mutations W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N in the ecTadA sequence). TadA-8e is broadly compatible with diverse Cas9 or Casl2 homologs, and exhibits improved editing efficiencies when paired with previously incompatible Cas9 or Casl2 homologs. For instance, disclosed adenosine deaminase variants such as TadA-8e exhibit higher editing efficiencies when paired in a base editor with certain Cas9 variants, such as circularly permuted variants CP 1041 and CP 1028, than exhibited by theTadA-7.10 deaminase. In some embodiments, the adenosine deaminase is TadA-7.10. In some embodiments, the adenosine deaminase is TadA-8e.

[0389] In some embodiments, any of the disclosed base editors are capable of deaminating adenosine in a nucleic acid sequence (e.g., DNA or RNA). In various embodiments, the adenosine deaminases of the base editors hydrolytically deaminate a targeted adenosine in a nucleic acid of interest to an inosine, which is read as a guanosine (G) by DNA polymerase enzymes. In some embodiments, the base editor is an ABE. In another embodiment, the base editor is ABE8e. In other embodiments, the base editor is a CBE. In some embodiments, the gene editing system comprises a ABE and a guide RNA (gRNA). In some embodiments, the gene editing system comprises a CBE and a guide RNA (gRNA).Cytosine base editors (CBEs)

[0390] In some embodiments, the base editor is a cytosine base editor. The first generation of CBEs were made up of a cytidine deaminase enzyme, such as an Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 1 (APOBEC1), fused to the amino terminus of a catalytically impaired Cas, which can only edit single- stranded DNA. The Cas protein can either be a catalytically inactive dCas, or a partially inactive Cas nickase (nCas), which includes mutations that only allow the enzyme to nick the non-edited strand (Porto et al., Nat Rev Drug Discov 19: 839-859 (2020); Huang et al., Nat Protoc 16: 1089-1128 (2021), which are incorporated by reference herein in its entirety). The use of nCas9 promotes repair of the non-edited strand using the deaminated strand as template, which increases editing efficiency (Huang et al., Nat Protoc 16: 1089-1128 (2021), which is incorporated by reference herein in its entirety). Improved versions also include uracil glycosylase inhibitor (UGI) in the CBE fusion complex to improve editing efficiency. UGI inhibits uracil DNA glycosylase (UNG), an enzyme which eliminates uracil bases through the base-excision repair (BER) pathway.

[0391] The CBE base editing process begins with sgRNA directing the Cas protein to the target locus. Cas binding to the target denatures the DNA duplex to generate a ssDNA R-loop formation that exposes a region of DNA with target cytosines that the cytidine deaminase enzyme can deaminate. CBEs convert a C-G base pair to a T-A base pair by deaminating the target cytosine to generate uracil, which will be read as a thymine by polymerases (Porto et al., Nat Rev Drug Discov 19: 839-859 (2020), which is incorporated by reference herein in its entirety). Further fusion proteins have been developed as cytosine base-editors to improve base-editing efficiency, modify the editing window, and reduce indel formation during base-editing (Kim et al., Nat Biotechnol 35:371-376 (2017), which is incorporated by reference herein in its entirety).Adenine base editors (ABEs)

[0392] Adenine base editors can deaminate an adenosine that leads to a point mutation from adenine (A) to guanine (G). The adenine base editors can comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein including any naturally occurring variant, mutant, or otherwise engineered version of Cas9 that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenesis process.

[0393] As methylated cytosines undergo high rates of spontaneous cytosine deamination, and almost half of the pathogenic point mutations identified can be corrected by a base pair conversion from an A-T base pair to a G-C base pair, ABEs are highly relevant in the context of correcting disease-causing mutations. ABEs contain a catalytically impaired Cas protein, either a dCas, with no endonuclease activity, or nCas, which yield single-stranded breaks, fused to a DNA modifying enzyme, Escherichia coli tRNA adenosine deaminase (ecTadA). As ssDNA adenosine deaminase enzymes are not naturally occurring, Tad A required extensive engineering and development through the directed mutagenesis to produce the first generation of ABE. Similar to CBEs, sgRNA guides the Cas domain to the intended target locus, which exposes a stretch of ssDNA in an R-loop for editing. TadA deaminates an adenine’s exocyclic amine to yield inosine, which is read as guanine by polymerases, converting A-T base pairs to G-C base pairs (Porto et la., Nat Rev Drug Discov 19:839-859 (2020), which is incorporated by reference herein in its entirety).

[0394] In contrast to CBEs, which are compatible with a variety of Cas homologs, ABEs are more restricted. Optimization and protein engineering of ABEs have been performed to improve editing efficiency and expand the targeting range. For example, a previous version of ABE, ABE7.10, was compatible with limited Cas9 enzymes and exhibited lower DNA editing efficiency than CBEs. Through directed evolution, the adenosine deaminase enzyme of ABE7.10, TadA-7.10, was evolved to include 8 additional mutations, yielding TadA-8e, which allowed for greater compatibility with more Cas9 and Cas 12a homologs, higher deamination rates, and overall improved DNA editing efficiency. The base editor variant ABE8e contains an ecTadA-8e fused to a Streptococcus pyogenes Cas9 nickase (SpCas9n). ABE8e has a broader base-editing window than ABE7.10, resulting in an editing window that is on par with that of CBEs (Richter et al., Nat Biotechnol 38):883-891 (2020), which is incorporated by reference herein in its entirety).

[0395] In certain embodiments, the adenine base editors contemplated herein can include a Cas9 protein that is of smaller molecular weight than the canonical SpCas9 sequence. In some embodiments, the smaller-sized Cas9 variants may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery. The canonical SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. Smaller- sized Cas9 variants can be at least 1300 amino acids, or at least less than 1290 amino acids, or than less than 1280 amino acids, or less than 1270 amino acid, or less than 1260 amino acid, or less than 1250 amino acids, or less than 1240 amino acids, or less than 1230 amino acids, or less than 1220 amino acids, or less than 1210 amino acids, or less than 1200 amino acids, or less than 1190 amino acid, or less than 1180 amino acids, or less than 1170 amino acids, or less than 1160 amino acids, or less than 1150 amino acids, or less than 1140 amino acids, or less than 1130 amino acids, or less than 1120 amino acids, or less than 1110 amino acids, or less than 1100 amino acids, or less than 1050 amino acids, or less than 1000 amino acids, or less than 950 amino acids, or less than 900 amino acids, or less than 850 amino acids, or less than800 amino acids, or less than 750 amino acids, or less than 700 amino acids, or less than 650 amino acids, or less than 600 amino acids, or less than 550 amino acids, or less than 500 amino acids, but at least larger than about 400 amino acids and retaining the required functions of the Cas9 protein.

[0396] In one embodiment, the base editor is ABE 0.1, ABE 0.2, ABE 1.1, ABE 1.2, ABE2.1, ABE 2.2, ABE 2.3, ABE 2.4, ABE 2.5, ABE 2.6, ABE 2.7, ABE 2.8, ABE 2.9, ABE 2.10, ABE 2.11, ABE 2.12, ABE 3.1, ABE 3.2, ABE 3.3, ABE 3.4, ABE 3.5, ABE 3.6, ABE 3.7, ABE 3.8, ABE 4.1, ABE 4.2, ABE 4.3, ABE 5.1, ABE 5.2, ABE 5.3, ABE 5.4, ABE 5.5, ABE 5.6, ABE 5.7, ABE 5.8, ABE 5.9, ABE 5.10, ABE 5.11, ABE 5.12, ABE 5.13, ABE 5.14, ABE6.1, ABE 6.2, ABE 6.3, ABE 6.4, ABE 6.5, ABE 6.6, ABE 7.1, ABE 7.2, ABE 7.3, ABE 7.4, ABE 7.5, ABE 7.6, ABE 7.7, ABE 7.8, ABE 7.9, ABE 7.10, or ABEmax, as described in US 2020 / 0308571, which is hereby incorporated by reference in its entirety. In another embodiment, the base editor is an ABE8 variant. In some embodiments, the base editor is ABE8e.

[0397] Exemplary ABEs include, but are not limited to, ABE7.10 (or ABEmax), ABE8e, SaKKH-ABE8e, NG-ABE8e, ABE-xCas9, SaKKH-ABE7.10, NG-ABE7.10, ABE7.10- VRQR, ABE8e-NRTH, ABE8e-NRRH, ABE8e-NRCH, NG-CP1041-ABE8e, ABE8eCP1041, ABE8e-CP 1028, and ABE8e-VRQR. In certain embodiments, the ABE used in the disclosed methods is an ABE8e or an ABE7.10. ABE8e may be referred to herein as “ABE8” or “ABE8.0.” The ABE8e base editor and variants thereof may comprise anadenosine deaminase domain containing a TadA-8e adenosine deaminase monomer (monomer form) or a TadA-8e adenosine deaminase homodimer or heterodimer (dimer form). ABE8e is further described in Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol. 2020 Jul;38(7):883-891, which is incorporated by reference herein in its entirety. Other ABEs can be used to deaminate a target adenosine in accordance with the disclosure.

[0398] In some embodiments, the adenosine deaminase domain of any of the disclosed base editors comprises a single adenosine deaminase, or a monomer. In some embodiments, the adenosine deaminase domain comprises 2, 3, 4 or 5 adenosine deaminases. In some embodiments, the adenosine deaminase domain comprises two adenosine deaminases, or a dimer. In some embodiments, the deaminase domain comprises a dimer of an engineered (or evolved) deaminase and a wild-type deaminase, such as a wild-type E. coli-derived deaminase. Base editors are further described in International Publication No. WO 2018 / 027078, published August 2, 2018; International Publication No. WO 2019 / 079347 on April 25, 2019; international Application No PCT / US2019 / 033848, filed May 23, 2019, which published as International Publication No. WO 2019 / 226953 on November 28, 2019; U.S. Patent Publication No. 2018 / 0073012, published March 15, 2018, which issued as U.S. Patent No. 10,113,163, on October 30, 2018; U.S. Patent Publication No. 2017 / 0121693, published May 4, 2017, Which issued as U.S. Patent No. 10,167,457 on January 1, 2019; International Publication No. WO 2017 / 070633, published April 27, 2017; U.S. Patent Publication No. 2015 / 0166980, published June 18, 2015; U.S. Patent No. 9,840,699, issued December 12, 2017; and U.S. Patent No. 10,077,453, issued September 18, 2018, and International Patent Application No. PCT / US2020 / 28568, filed April 16, 2020: all of which are incorporated herein by reference in their entireties.Other Gene Editing Methods

[0399] 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.

[0400] 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 sequence-specific 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.

[0401] 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.

[0402] In some embodiments, the gene editing system comprises a TAL-effector nuclease (TALEN). In some embodiments, a “TALE-nuclease” (TALEN) is a fusion protein comprising a nucleic acid binding domain typically derived from a Transcription Activator Like Effector (TALE) and a nuclease catalytic domain that cleaves a nucleic acid target sequence. The catalytic domain comprises a nuclease domain or a domain having endonuclease activity, like for instance I-TevI, ColE7, NucA and Fok-I. In a particular embodiment, the TALE domain can be fused to a meganuclease like for instance I-Crel and I-Onul or functional variant thereof. In some embodiments, the TALEN is a monomeric TALEN. A monomeric TALEN is a TALEN that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered TAL repeats with the catalytic domain of I-TevI described in WO2012138927. TALENs have been described and used for gene targeting and genemodifications (see, e.g., Boch et al., Science 326: 1509-1512 (2009); Moscou and Bogdanove, Science 326: 1501 (2009); Christian et al., Genetics 186:757-761 (2010); Li et al., Nucleic Acids Res 39: 359-372(2011).

[0403] 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...

Claims

CLAIMS1. A lipid nanoparticle composition comprising a lipid component which comprises:(i) a first ionizable cationic lipid,(ii) a PEG-lipid, wherein the PEG-lipid comprises one or more PEG-Ceramide.

2. The lipid nanoparticle composition of claim 1, further comprising: a phospholipid.

3. The lipid nanoparticle composition of claim 1 or 2, further comprising: a permanently cationic lipid, an anionic lipid, or a second ionizable cationic lipid separate from the first ionizable cationic lipid.

4. A lipid nanoparticle composition comprising a lipid component which comprises:(i) a first ionizable cationic lipid,(ii) a PEG-lipid, wherein the PEG-lipid comprises one or more PEG-Ceramide selected from: 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-1-{ succinyl [methoxy (poly ethylene glycol)750]} (C8 PEG750-Ceramide), N-palmitoyl- sphingosine-l-{succinyl[methoxy(polyethylene glycol)5000]} (C16 PEG5000-Ceramide), N- palmitoyl-sphingosine-l-{succinyl[methoxy(poly ethylene glycol)2000]} (C16 PEG200- Ceramide), and N-palmitoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)750]} (C16 PEG750-Ceramide),(iii) a phospholipid, and(iv) a permanently cationic lipid, an anionic lipid, or a second ionizable cationic lipid separate from the first ionizable cationic lipid.

5. The lipid nanoparticle composition of any one of the preceding claims, wherein the composition further comprises a permanently cationic lipid or a second ionizable cationic lipid separate from the first ionizable cationic lipid.

6. The lipid nanoparticle composition of claim 5, wherein 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.

7. The lipid nanoparticle composition of claim 5, wherein the permanently cationic lipid or the second ionizable cationic lipid is 16:0 TAP.

8. The lipid nanoparticle composition of any one of claims 1-4, wherein the composition further comprises an anionic lipid.

9. The lipid nanoparticle composition of claim 8, wherein 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).

10. The lipid nanoparticle composition of any one of claims 2-9, 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), 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-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), 1- stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), 1 -stearoyl -2-oleoyl- phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid,palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE) sphingomyelin.

11. The lipid nanoparticle composition of any one of claims 2-9, wherein the phospholipid is selected from 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) 1,2-dimyristoyl-sn-gly cero- 3 -phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1,2-distearoyl- sn-gly cero-3 -phosphoryl ethanol amine (D SPE) .

12. A lipid nanoparticle composition comprising a lipid component which comprises:(i) a first ionizable cationic lipid,(ii) a PEG-lipid, wherein the PEG-lipid comprises one or more PEG-Ceramide,(iii) a phospholipid, wherein the phospholipid is selected from 1,2-dioleoyl-sn- gly cero-3 -phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-gly cero-3 - phosphoethanolamine (DMPE) 1,2-dimyristoyl-sn-gly cero-3 -phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1,2-distearoyl-sn-glycero-3- phosphorylethanolamine (DSPE), and(iv) a permanently cationic lipid or a second ionizable cationic lipid separate from the first ionizable cationic lipid, wherein the permanently cationic lipid or a 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.

13. The lipid nanoparticle composition of any one of claims 2-12, wherein the phospholipid is 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).

14. The lipid nanoparticle composition of any one of the preceding claims, wherein the PEG-lipid comprises at least one PEG-Ceramide selected from: 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), and N-octanoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)750]} (C8 PEG750- Ceramide).

15. The lipid nanoparticle composition of any one of the preceding claims, wherein 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.

16. The lipid nanoparticle composition of claim 15, wherein the PEG-lipid comprises the first PEG-Ceramide and the second PEG-Ceramide in a mol / mol 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.

17. The lipid nanoparticle composition of claim 15 or 16, wherein the first PEG-Ceramide and the second PEG-Ceramide are each independently selected from: a PEG750-Ceramide, a PEG2000-Ceramide, and a PEG5000-Ceramide.

18. The lipid nanoparticle composition of claim 15 or 16, wherein the lipid component comprises a dual C8-Ceramide comprising a first PEG-Ceramide and the second PEG- Ceramide that are each independently a C8 PEG-Ceramide.

19. The lipid nanoparticle composition of any one of the preceding claims, wherein the lipid component comprises the PEG-lipid in an amount of from about 0.5 mol % to about 5 mol % of the total lipids in the lipid component.

20. The lipid nanoparticle composition of any one of the preceding claims, wherein the lipid component comprises the PEG-lipid in an amount of from about 2 mol % to about 4 mol of the total lipids in the lipid component.

21. The lipid nanoparticle composition of any one of the preceding claims, wherein the lipid component comprises the PEG-lipid in an amount of about 3 mol % of the total lipids in the lipid component.

22. The lipid nanoparticle composition of any one of claims 4-21, wherein the PEG-lipid comprises a dual C8-Ceramide comprising C8 PEG2000-Ceramide and C8 PEG750- Ceramide.

23. The lipid nanoparticle composition of any one of claims 4-22, wherein the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.1 mol % to about 2.5 mol % of the total lipids in the lipid component.

24. The lipid nanoparticle composition of any one of claims 4-23, wherein the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component.

25. The lipid nanoparticle composition of any one of claims 4-24, wherein the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.75 mol % to about 2.25 mol % of the total lipids in the lipid component.

26. The lipid nanoparticle composition of any one of claims 4-25, wherein the PEG-lipid comprises C8 PEG2000-Ceramide 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.

27. The lipid nanoparticle composition of any one of claims 4-26, wherein the PEG-lipid comprises C8 PEG2000-Ceramide 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.

28. The lipid nanoparticle composition of any one of claims 4-27, wherein the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.5 mol % to about 2.5 mol % of the total lipids in the lipid component.

29. The lipid nanoparticle composition of any one of claims 4-28, wherein the PEG-lipid comprises C8 PEG2000-Ceramide in an amount of from about 0.75 mol % to about 2.25 mol of the total lipids in the lipid component.

30. The lipid nanoparticle composition of any one of claims 4-29, wherein the PEG-lipid comprises C8 PEG750-Ceramide in an amount of about 0.25 mol %, about 0.5 mol %, about 0 75 mol %, 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.

31. The lipid nanoparticle composition of any one of claims 4-30, wherein the PEG-lipid comprises C8 PEG750-Ceramide in an amount of about 0.75 mol %, about 1.5 mol %, about 2 mol %, or about 2.25 mol % of the total lipids in the lipid component.

32. The lipid nanoparticle composition of any one of claims 4-31, wherein the PEG-lipid comprises a dual C8-Ceramide comprising C8 PEG2000-Ceramide and C8 PEG750-Ceramide in a total combined amount of about 2 mol % to about 3 mol % of the total lipids in the lipid component.

33. The lipid nanoparticle composition of any one of claims 4-32, wherein the PEG-lipid comprises a dual C8-Ceramide comprising C8 PEG2000-Ceramide in an amount of from about 0.5 mol % to about 2.25 mol % of the total lipids in the lipid component, and comprising C8 PEG750-Ceramide in an amount of from about 0.75 mol % to about 2.25 mol of the total lipids in the lipid component, and wherein 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.

34. The lipid nanoparticle composition of any one of the preceding claims, wherein 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.

35. The lipid nanoparticle composition of any one of the preceding claims, wherein 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.

36. The lipid nanoparticle composition of any one of the preceding claims, wherein 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.

37. The lipid nanoparticle composition of any one of the preceding claims, wherein 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.

38. The lipid nanoparticle composition of any one of the preceding claims, wherein 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.

39. The lipid nanoparticle composition of any one of the preceding claims, wherein 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.

40. The lipid nanoparticle composition of any one of the preceding claims, wherein the lipid component comprises the first ionizable cationic lipid in an amount of about 16 mol % of the total lipids in the lipid component.

41. The lipid nanoparticle composition of any one of claims 1-39, wherein the lipid component comprises the first ionizable cationic lipid in an amount of about 14 mol % of the total lipids in the lipid component.

42. The lipid nanoparticle composition of any one of claims 2-41, wherein the lipid component comprises the phospholipid 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 2-42, wherein the lipid component comprises the phospholipid 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 2-43, wherein the lipid component comprises the phospholipid in an amount of about 14 mol % of the total lipids in the lipid component.

45. The lipid nanoparticle composition of any one of claims 2-44, wherein the lipid component comprises the phospholipid in an amount of about 16 mol % of the total lipids in the lipid component.

46. The lipid nanoparticle composition of any one of claims 3-45, wherein the lipid component comprises the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 15 mol % to about 55 mol % of the total lipids in the lipid component.

47. The lipid nanoparticle composition of any one of claims 3-46, wherein the lipid component comprises the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 30 mol % to about 55 mol % of the total lipids in the lipid component.

48. The lipid nanoparticle composition of any one of claims 3-47, wherein the lipid component comprises the permanently cationic lipid, the anionic lipid, or the secondionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 35 mol % to about 50 mol % of the total lipids in the lipid component.

49. The lipid nanoparticle composition of any one of claims 3-48, wherein the lipid component comprises the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 40 mol % to about 50 mol % of the total lipids in the lipid component.

50. The lipid nanoparticle composition of any one of claims 3-49, wherein the lipid component comprises the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of from about 35 mol % to about 45 mol % of the total lipids in the lipid component.

51. The lipid nanoparticle composition of any one of claims 3-50, wherein the lipid component comprises the permanently cationic lipid, the anionic lipid, or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of about 40 mol % of the total lipids in the lipid component.

52. The lipid nanoparticle composition of any one of claims 3-51, wherein the lipid component comprises the permanently cationic lipid or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of about 45 mol % of the total lipids in the lipid component.

53. The lipid nanoparticle composition of any one of claims 3-52, wherein: the lipid component comprises the first ionizable cationic lipid in an amount from about 10 mol % up to 20 mol % of the total lipids in the lipid component; the lipid component comprises the PEG-lipid in a total amount from about 2 mol % to about 3 mol % of the total lipids in the lipid component; the lipid component comprises the permanently cationic lipid or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of about 35 mol % to about 50 mol % of the total lipids in the lipid component; and the lipid component comprises the phospholipid in an amount of about 10 mol % up to 20 mol % of the total lipids in the lipid component.

54. The lipid nanoparticle composition of any one of claims 3-53, wherein:the lipid component comprises the first ionizable cationic lipid in an amount from about 10 mol % up to 20 mol % of the total lipids in the lipid component; the lipid component comprises the PEG-lipid in a total amount from about 2 mol % to about 3 mol % of the total lipids in the lipid component; the lipid component comprises the permanently cationic lipid or the second ionizable cationic lipid separate from the first ionizable cationic lipid in an amount of about 40 mol % or about 45 mol % of the total lipids in the lipid component; and the lipid component comprises the phospholipid in an amount of about 10 mol % up to 20 mol % of the total lipids in the lipid component.

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

56. The lipid nanoparticle composition of claim 55, wherein the lipid component comprises the sterol in an amount of from about 20 mol % to about 50 mol % of the total lipids in the lipid component.

57. The lipid nanoparticle composition of claim 55, wherein the lipid component comprises the sterol in an amount of from about 20 mol % to about 30 mol % of the total lipids in the lipid component.

58. The lipid nanoparticle composition of claim 55, wherein the lipid component comprises the sterol in an amount of about 25 mol % of the total lipids in the lipid component.

59. The lipid nanoparticle composition of any one of claims 55-58, wherein the sterol is selected from lanosterol, desmosterol, cholesterol, sitostanol, sitosterol, stigmasterol, optionally cholesterol.

60. The lipid nanoparticle of any one of claims 2-59, wherein the phospholipid is 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE).

61. The lipid nanoparticle of any one of claims 2-59, wherein the phospholipid is 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE).

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

63. The lipid nanoparticle of any one of claims 2-59, wherein the phospholipid is dipalmitoylphosphatidylcholine (DPPC).

64. The lipid nanoparticle of any one of claims 2-59, wherein the phospholipid is 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE).

65. The lipid nanoparticle composition of any one of the preceding claims, wherein the first ionizable cationic lipid is a compound selected from Table 4.

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

67. The lipid nanoparticle composition of any one of claims 1-64, wherein the first ionizable cationic lipid is a compound selected from Table 5B.

68. The lipid nanoparticle composition of any one of claims 1-64, wherein the first ionizable cationic lipid is:

69. The lipid nanoparticle composition of any one of the preceding claims, further comprising a payload.

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

71. The lipid nanoparticle composition of claim 70, wherein the polypeptide or the protein is selected from: 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 boxgene 9, Myosin VII A, 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.

72. The lipid nanoparticle composition of claim 69, wherein the payload comprises a nucleic acid.

73. The lipid nanoparticle composition of claim 72, 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).

74. The lipid nanoparticle composition of claim 73, 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 any one of claim 74-76, wherein the mRNA encodes a protein selected from: 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 boxgene 9, Myosin VII A, 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.

78. The lipid nanoparticle composition of any one of claims 73-76, wherein the payload comprises a guide RNA.

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

80. The lipid nanoparticle composition of any one of claims 1-79, wherein the composition delivers a payload preferentially in a lung cell, or lung / liver cell.

81. 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 of any one of claims 69-80.

82. The method of claim 81, wherein the method comprises selectively delivering the payload to a target organ.

83. The method of claim 81, wherein the method comprises selectively delivering the payload to a target cell.

84. 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 69-78.

85. The method of any one of claims 81-84, wherein the target organ is the lung.

86. The method of any one of claims 81-85, 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.

87. The method of any one of claims 81-86, wherein the payload is a polynucleotide encoding a gene product and wherein the selectively delivering results in expression of thegene product in a cell of the target organ and optionally wherein the gene product is functional in the cell.

88. The method of any one of claims 81-87, 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.

89. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is at least 200% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

90. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is at least 300% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

91. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is at least 400% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

92. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is at least 500% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

93. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is at least 600% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

94. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition isabout 200% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

95. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is about 300% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

96. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is about 400% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

97. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is about 500% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

98. The method of claim 86 or 88, wherein the level of expression of the protein in the cell of the target organ following administration of said lipid nanoparticle composition is about 600% of the level of expression of the protein in the cell of the target organ following administration of a reference lipid nanoparticle composition.

99. The method of any one of claims 84-98, wherein the reference lipid nanoparticle composition comprises a PEG-lipid that does not comprise a ceramide PEG, but is otherwise identical to the lipid nanoparticle composition administered to the subject.

100. The method of any one of claims 84-99, wherein the reference lipid nanoparticle composition comprises a phospholipid that is not 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), or 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), but is otherwise identical to the lipid nanoparticle composition administered to the subject.

101. The method of any one of claims 84-100, wherein the reference lipid nanoparticle composition does not comprise a permanently cationic lipid, an anionic lipid, or a secondionizable cationic lipid separate from the first ionizable cationic lipid, but is otherwise identical to the lipid nanoparticle composition administered to the subject.

102. The method of any one of claims 84-101, wherein the reference lipid nanoparticle composition does not comprise a permanently cationic lipid or a second ionizable cationic lipid separate from the first ionizable cationic lipid, wherein the permanently cationic lipid or a 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, but is otherwise identical to the lipid nanoparticle composition.

103. The method of any one of claims 84-102, wherein the reference lipid nanoparticle composition comprises an ionizable cationic lipid having the following structure:

104. The method of any one of claims 84-103, wherein the reference lipid nanoparticle composition comprises an ionizable cationic lipid having the following structure:

105. The method of any one of claims 84-104, wherein the reference lipid nanoparticle composition comprises an ionizable cationic lipid having the following structure:

106. The method of any one of claims 84-105, wherein the reference lipid nanoparticle composition comprises a PEG-lipid selected from107. The method of any one of claims 84-106, wherein the reference lipid nanoparticle composition comprises DSPC.

108. The method of any one of claims 84-107, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 500% of the level of expression of the protein in another cell of the body.

109. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 500% of the level of expression of the protein in a liver cell.

110. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 500% of the level of expression of the protein in a spleen cell.

111. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 1000% of the level of expression of the protein in another cell of the body.

112. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 1000% of the level of expression of the protein in a liver cell.

113. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is at least 1000% of the level of expression of the protein in a spleen cell.

114. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein in another cell of the body.

115. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein in a liver cell.

116. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 500% to about 20000% of the level of expression of the protein in a spleen cell.

117. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein in another cell of the body.

118. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein in a liver cell.

119. The method of any one of claims 84-108, wherein the selectively delivering results in a level of expression of the protein in a cell of the target organ that is from about 1000% to about 20000% of the level of expression of the protein in a spleen cell.

120. The method of any one of claims 84-119, wherein the selectively delivering results in a level of expression of the protein in a liver cell that is less than 20% of the level of expression of the protein in the target cell.

121. The method of any one of claims 84-119, wherein the selectively delivering results in a level of expression of the protein in a liver cell that is less than 10% of the level of expression of the protein in the target cell.

122. The method of any one of claims 84-119, wherein the selectively delivering results in a level of expression of the protein in a liver cell that is less than 5% of the level of expression of the protein in the target cell.

123. The method of any one of claims 84-122, wherein the selectively delivering results in a level of expression of the protein in a liver cell that is less than 20% of the total level of expression of the protein in the subject.

124. The method of any one of claims 84-122, wherein the selectively delivering results in a level of expression of the protein in a liver cell that is less than 10% of the total level of expression of the protein in the subject.

125. The method of any one of claims 84-122, wherein the selectively delivering results in a level of expression of the protein in a liver cell that is less than 5% of the total level of expression of the protein in the subject.

126. The method of any one of claims 84-122, wherein the selectively delivering does not result in expression of the protein in a liver cell.

127. The method of any one of claims 84-126, wherein the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 20% of the level of expression of the protein in the target cell.

128. The method of any one of claims 84-126, wherein the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 10% of the level of expression of the protein in the target cell.

129. The method of any one of claims 84-126, wherein the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 5% of the level of expression of the protein in the target cell.

130. The method of any one of claims 84-129, wherein the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 20% of the total level of expression of the protein in the subject.

131. The method of any one of claims 84-129, wherein the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 10% of the total level of expression of the protein in the subject.

132. The method of any one of claims 84-129, wherein the selectively delivering results in a level of expression of the protein in a spleen cell that is less than 5% of the total level of expression of the protein in the subject.

133. The method of any one of claims 84-129, wherein the selectively delivering does not result in expression of the protein in a spleen cell.

134. The method of claims 84-133, wherein the level of expression is determined by in vivo biofluorescence imaging.

135. The method of any one of claims 84-134, wherein the disease or disorder is Acute Interstitial Pneumonia ( AIP), alpha-1 antitrypsin deficiency, asthma, bronchiectasis, Bronchiolitis obliterans with Organizing Pneumonia (BOOP), bronchitis, Chronic Obstructive Pulmonary Disease (COPD), coronavirus, cystic fibrosis, Desquamative Interstitial Pneumonia (DIP), emphysema, Idiopathic Interstitial Pneumonia (IIP), influenza, Interstitial Lung Disease (ILD), Interstitial Pulmonary Fibrosis (IPF), Legionnaire’s disease, lung cancer, Non-Specific Interstitial Pneumonia (NSIP), pleurisy, pneumonia, Primary Ciliary Dyskinesia (PCD), pulmonary arterial hypertension, pulmonary edema, pulmonary fibrosis, pulmonary hypertension, Respiratory Bronchiolitis-associated Interstitial Lung Disease (RBILD), restrictive lung disease, sarcoidosis, Severe Acute Respiratory Syndrome, and tuberculosis.

136. The method of any one of claim 81-135, wherein the subject is a primate.

137. The method of any one of claim 81-135, wherein the subject is a human.

138. 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.

139. 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.

140. The method of claim 138 or claim 139, wherein the target cell is a lung cell.

141. The method of claim 140, wherein the target cell is a ciliated lung cell.

142. The method of claim 140, wherein the target cell is a goblet lung cell.

143. The method of claim 140, wherein the target cell is an epithelial cell.

144. A pharmaceutical composition comprising the lipid nanoparticle composition of any one of claims 1-80.

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