Lipid nanoparticle compositions and uses thereof

Abstract

Provided herein are lipid nanoparticle (LNP) compositions for use in delivering a payload to a cell in the central nervous system (CNS) of a subject. Also provided are a method for treating and / or preventing a CNS disease, kits, pharmaceutical compositions, and other compositions and methods.

Description

LIPID NANOPARTICLE COMPOSITIONS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U. S. Provisional Patent Application No. 63 / 716,236 filed November 4, 2024, and U. S. Provisional Patent Application No. 63 / 803,365 filed May 9, 2025, 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 October 20, 2025, is named 061529-515001WO.xml and is 9,246 bytes in size.BACKGROUND

[0003] There is an unmet need for lipid nanoparticle compositions for specific delivery of polynucleotides to the central nervous system (CNS). The present disclosure provides lipid nanoparticle (LNP) compositions, as well as pharmaceutical compositions, and methods of delivering them to CNS.SUMMARY

[0004] In one aspect, disclosed herein are compounds of Formula I:and pharmaceutically acceptable salts thereof,wherein:RD1is a C1-C4 alkyl;RD2is H or a C1-C4 alkyl;zl and z2 are each independently 1, 2, or 3;z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; andz4 is 0 or 1.

[0005] In some embodiments of the compound of Formula I, RD1is methyl.

[0006] In some embodiments of the compound of Formula I, RD2is methyl. In some embodiments of the compound of Formula I, RD2is H.

[0007] In some embodiments of the compound of Formula I, zl and z2 are each 1. In some embodiments of the compound of Formula I, zl and z2 are each 2.

[0008] In some embodiments of the compound of Formula I, z3 is 6, 7, or 8.

[0009] In some embodiments of the compound of Formula I, z4 is 0. In some embodiments of the compound of Formula I, z4 is 1.

[0010] In some embodiments, the compound of Formula I is:

[0011] In one aspect, disclosed herein are compounds of Formula A:pharmaceutically acceptable salts thereof,wherein:RDlais a Ci-C4alkyl;RD2ais H or a C1-C4 alkyl;zla and z2a are each independently 1, 2, or 3;z3a is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; andz4a is 0 or 1

[0012] In some embodiments of the compound of Formula A, RDlais methyl.

[0013] In some embodiments of the compound of Formula A, RD2ais methyl. In some embodiments of the compound of Formula A, RD2ais H.

[0014] In some embodiments of the compound of Formula A, zla and z2a are each 1. In some embodiments of the compound of Formula A, zla and z2a are each 2.

[0015] In some embodiments of the compound of Formula A, z3a is 6, 7, or 8.

[0016] In some embodiments of the compound of Formula A, z4a is 0. In some embodiments of the compound of Formula A, z4a is 1.

[0017] In some embodiments, the compound of Formula B is:

[0018] In one aspect, disclosed herein are compounds of Formula B:pharmaceutically acceptable salts thereof, whereinXb and Yb are each independently N or CH;zlb, z2b, z3b, z4b are each independently 1, 2, or 3; andz5b is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0019] In some embodiments of the compound of Formula B, zlb, z2b, z3b, and z4b are each 1.

[0020] In some embodiments of the compound of Formula B, z5b is 6, 7, or 8.

[0021] In some embodiments of the compound of Formula B, Xb is N.

[0022] In some embodiments of the compound of Formula B, Yb is N.

[0023] In some embodiments, the compound of Formula B is:

[0024] In another aspect, disclosed herein is a compound selected from:

[0025] In another aspect, disclosed herein are compounds of Formula C:wherein m is an integer from 1 to 9.

[0026] In another aspect, disclosed herein are compounds of Formula D:wherein n is an integer from 1 to 9.

[0027] In another aspect, disclosed herein is a compound selected from:

[0028] In another aspect, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:(i) a first lipid wherein the first lipid is an ionizable cationic lipid,(ii) a second lipid wherein the second lipid is separate from the first lipid, and wherein the second lipid is selected from:compound of Formula I.

[0029] In another aspect, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:(i) a first lipid wherein the first lipid is an ionizable cationic lipid, and(ii) a second lipid wherein the second lipid is separate from the first lipid,wherein the first lipid is a compound of Formula A or a compound of Formula B.

[0030] In another aspect, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:(i) a first lipid wherein the first lipid is an ionizable cationic lipid, and(ii) a second lipid wherein the second lipid is separate from the first lipid,wherein the first lipid is a compound of Formula C.

[0031] In another aspect, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:(i) a first lipid wherein the first lipid is an ionizable cationic lipid, and(ii) a second lipid wherein the second lipid is separate from the first lipid,wherein the first lipid is a compound of Formula D.

[0032] In some embodiments, the second lipid is an ionizable cationic lipid. In some embodiments, the second lipid is a permanently cationic lipid. In some embodiments, the second lipid is a compound selected from Table 7 or Table 8. In some embodiments, the second lipid is selected from:

[0033] In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component further comprises a third lipid, wherein the third lipid is an ionizable cationic lipid or a permanently cationic lipid separate from the first lipid and the second lipid. In some embodiments, the third lipid is a permanently cationic lipid. In some embodiments, the third lipid is l,2-dioleoyl-3-trimethylammonium-propane (14:0 TAP).

[0034] In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component comprises the third lipid in an amount of from about 1 mol% to about 10 mol% (e.g., about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, or about 7 mol%) of the total lipids in the lipid component.

[0035] In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component comprises the first lipid in an amount of from about 10 mol% to about 30 mol% (e.g., from about 15 mol% to about 25 mol%, e.g., about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, or about 22 mol%) of the total lipids in the lipid component.

[0036] In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component comprises the second lipid in an amount of from about 10 mol% to about 30 mol% (e.g., from about 15 mol% to about 25 mol%, e.g., about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, or about 21 mol%) of the total lipids in the lipid component.

[0037] In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component further comprises a phospholipid. In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component comprises the phospholipid in an amount from about 10 mol% to about 30 mol% (e.g., from about 15 mol% to about 25 mol%, e.g., about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, or about 22 mol%) of the total lipids in the lipid component.

[0038] In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component further comprises a PEG lipid. In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component comprises the PEG lipid in an amount of from about 0.5 mol% to about 10 mol% (e.g., from about 0.5 mol% to about 5 mol%, e.g., about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, or about 6 mol%) of the total lipids in the lipid component. In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component comprises the PEG lipid in an amount of about 3.8 mol% of the total lipids in the lipid component.

[0039] In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component further comprises a sterol. In some embodiments, the sterol is cholesterol. In some embodiments of the lipid nanoparticle composition of the disclosure, the lipid component comprises the sterol in an amount of from about 30 mol% to about 50 mol% (e.g., from about 33 mol% to about 43 mol%, e.g., about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, or %, about 42 mol%) of the total lipids in the lipid component.

[0040] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and MO-14-TAP. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and MO-14-TAP in an amount of about 20 mol% of the total lipids in the lipid component.

[0041] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and SL-1. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; andSL-1 in an amount of about 20 mol% of the total lipids in the lipid component.

[0042] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and MO-14-DAP. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and MO-14-DAP in an amount of about 20 mol% of the total lipids in the lipid component.

[0043] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, DEA-14-DAP, and 14:0 TAP. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 33 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component;DEA-14-DAP in an amount of about 20 mol% of the total lipids in the lipid component; and 14:0 TAP in an amount of about 5 mol% of the total lipids in the lipid component.

[0044] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: IL-1, DOPE, cholesterol, PEG-DMG, and ME-14-EPC. For example, insome embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:IL-1 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and ME-14-EPC in an amount of about 20 mol% of the total lipids in the lipid component.

[0045] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and DEA-14-DAP. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and DEA-14-DAP in an amount of about 20 mol% of the total lipids in the lipid component.

[0046] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and DEA-16-DAP. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and DEA-16-DAP in an amount of about 20 mol% of the total lipids in the lipid component.

[0047] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: IL-2, DOPE, cholesterol, PEG-DMG, and Me-14-EPC. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:IL-2 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and Me-14-EPC in an amount of about 20 mol% of the total lipids in the lipid component.

[0048] In some embodiments, a lipid nanoparticle composition of the disclosure further comprises a payload.

[0049] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and MO-14-TAP; and further comprises an mRNA. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and MO-14-TAP in an amount of about 20 mol% of the total lipids in the lipid component; and further comprises mRNA at a lipid:mRNA (weight / weight) ratio of 36: 1.

[0050] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and SL-1; and further comprises an mRNA. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and SL-1 in an amount of about 20 mol% of the total lipids in the lipid component: and further comprises mRNA at a lipid:mRNA (weight / weight) ratio of 36: 1.

[0051] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and MO-14-DAP; and further comprises an mRNA. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and MO-14-DAP in an amount of about 20 mol% of the total lipids in the lipid component; and further comprises mRNA at a lipid:mRNA (weight / weight) ratio of 36: 1.

[0052] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, DEA-14-DAP, and 14:0 TAP; and further comprises an mRNA. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 33 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component;DEA-14-DAP in an amount of about 20 mol% of the total lipids in the lipid component; and 14:0 TAP in an amount of about 5 mol% of the total lipids in the lipid component; and further comprises mRNA at a lipid:mRNA (weight / weight) ratio of 36: 1.

[0053] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: IL-1, DOPE, cholesterol, PEG-DMG, and ME-14-EPC; and further comprises an mRNA. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:IL-1 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and ME-14-EPC in an amount of about 20 mol% of the total lipids in the lipid component; and further comprises mRNA at a lipid:mRNA (weight / weight) ratio of 30: 1.

[0054] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and DEA-14-DAP; and further comprises an mRNA. For example, in some embodiments, disclosed herein is a lipidnanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and DEA-14-DAP in an amount of about 20 mol% of the total lipids in the lipid component; and further comprises mRNA at a lipid:mRNA (weight / weight) ratio of 36:1.

[0055] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: 4A3SC7, DOPE, cholesterol, PEG-DMG, and DEA-16-DAP; and further comprises an mRNA. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and DEA-16-DAP in an amount of about 20 mol% of the total lipids in the lipid component; and further comprises mRNA at a lipid:mRNA (weight / weight) ratio of 36:1.In some aspects, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising: IL-2, DOPE, cholesterol, PEG-DMG, and Me-14-EPC; and further comprises an mRNA. For example, in some embodiments, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising:IL-2 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and Me-14-EPC in an amount of about 20 mol% of the total lipids in the lipid component; and further comprises mRNA at a lipid:mRNA (weight / weight) ratio of 36: 1.

[0056] In some embodiments, the payload comprises a polypeptide or a protein e.g., superoxide dismutase 1 (SOD1), survival of motor neuron 1 (SMN-1), Frataxin, glutamic acid decarboxylase 65 (GAD-65), and glutamic acid decarboxylase 67 (GAD-67), triggering receptorexpressed on myeloid cells 2 (TREM2), tumor necrosis factor (TNF), LGALS3, C-C motif chemokine receptor 7 (CCR7), or glycoprotein nonmetastatic melanoma protein B (GPNMB).

[0057] In some embodiments, the payload comprises a nucleic acid, e.g. 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), or a double stranded RNA (dsRNA). In some embodiments, the payload comprises an mRNA wherein the mRNA encodes a geneediting system or component thereof, e.g., 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), or a guide RNA. In some embodiments, the payload comprises an mRNA wherein the mRNA encodes a protein selected from superoxide dismutase 1 (SOD1), survival of motor neuron 1 (SMN-1), Frataxin, glutamic acid decarboxylase 65 (GAD-65), and glutamic acid decarboxylase 67 (GAD-67). In some embodiments, the payload comprises an mRNA wherein the mRNA encodes a protein selected from myeloid cells 2 (TREM2), tumor necrosis factor (TNF), LGALS3, C-C motif chemokine receptor 7 (CCR7), and glycoprotein nonmetastatic melanoma protein B (GPNMB). In some embodiments, the payload comprises an mRNA, wherein the mRNA encodes purinergic receptor P2Y (P2RY12).

[0058] In some aspects, the disclosure relates to a pharmaceutical composition comprising a lipid nanoparticle composition of the disclosure, and a pharmaceutically acceptable excipient.

[0059] In some aspects, the disclosure relates to a method of treating or preventing a CNS disease or disorder in a subject in need thereof, the method comprising administering an effective amount of the lipid nanoparticle composition or the pharmaceutical composition of the disclosure. In some embodiments, the method comprises selectively delivering the payload to an organ or tissue of the central nervous system of a subject. In some embodiments, the method comprises selectively delivering the payload to a cell of the central nervous system of a subject.

[0060] In some embodiments, the CNS disease is an inflammatory disease of the central nervous system, e.g., multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), neuromyelitis optica (NMO), transverse myelitis or autoimmune encephalitis.

[0061] In some aspects, the disclosure relates to a method of selectively delivering a payload to an organ, tissue, or cell of the central nervous system of a subject in need thereof, the method comprising administering to the subject the lipid nanoparticle composition or the pharmaceutical composition of the disclosure.

[0062] In some aspects, the disclosure relates to a method of delivering a payload to a cell of the central nervous system, comprising contacting the cell of the central nervous system with the lipid nanoparticle composition or the pharmaceutical composition of the disclosure.

[0063] In some embodiments, the payload is an mRNA and wherein the selectively delivering results in expression of a protein encoded by the mRNA in a cell of the central nervous system.

[0064] In some embodiments, wherein the payload is a polynucleotide encoding a gene product, and wherein the selectively delivering results in expression of the gene product in a cell of the central nervous system. In some embodiments, the gene product is functional in the cell of the central nervous system.

[0065] In some embodiments, the payload comprises an mRNA encoding a gene-editing system or component thereof and the selectively delivering results in altered expression of a protein targeted by the gene-editing system in a cell of the central nervous system. In some embodiments, the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 200%, at least 300%, at least 400%, at least 500%, or at least 600% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition. In some embodiments, the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is about 200%, about 300%, about 400%, about 500%, or about 600% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

[0066] In some embodiments, the reference lipid nanoparticle composition comprises a second lipid that does not comprise a compound of Formula (I) but is otherwise identical to the lipid nanoparticle composition administered to the subject. In some embodiments, the reference lipid nanoparticle composition comprises a lipid component comprising a second lipid that does notcomprise M0-14-TAP, M0-14-DAP, DEA-14-DAP, DEA-16-DAP, Me-14-EPC, or SL-1, but is otherwise identical to the lipid nanoparticle composition administered to the subject. In some embodiments, the reference lipid nanoparticle composition comprises a lipid component comprising a first lipid that does not comprise a compound of Formula (A) or (B) but is otherwise identical to the lipid nanoparticle composition administered to the subject. In some embodiments, the reference lipid nanoparticle composition comprises a lipid component comprising a first lipid that does not comprise IL-1 or IL-2 but is otherwise identical to the lipid nanoparticle composition administered to the subject. In some embodiments, the reference lipid nanoparticle composition comprises a lipid component comprising a first lipid that does not comprise 4A3SC7 but is otherwise identical to the lipid nanoparticle composition administered to the subject.

[0067] In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the central nervous system that is at least 500% or at least 1000% of the level of expression of the protein or gene product in a cell from another tissue of the body. In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in a cell of the central nervous system that is from about 500% to about 20000% or from about 1000% to about 20000% of the level of expression of the protein or gene product in a cell from another tissue of the body.

[0068] In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in another cell that is less than 20%, less than 10%, or less than 5% of the level of expression of the protein or gene product in a cell of the central nervous system.

[0069] In some embodiments, the selectively delivering results in a level of expression of the protein or gene product in another cell that is less than 20%, less than 10%, or less than 5% of the total level of expression of the protein or gene product in the subject.

[0070] In some embodiments, the another cell is a lung cell, a spleen cell, or a liver cell. In some embodiments, the another tissue is a lung tissue, a spleen tissue, or a liver tissue.

[0071] In some embodiments, the level of expression is determined by in vivo biofluorescence imaging.

[0072] In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0073] In some embodiments, the cell of the central nervous system is a brain cell. In some embodiments, the cell of the central nervous system is a spinal cord cell.

[0074] In some embodiments, the cell of the central nervous system is a glial cell (e.g., a glial cell of the immune system (microglia), an astrocyte, an oligodendrocyte, or an ependymal cell). In some embodiments, the cell of the central nervous system is a neuron (e.g., neuronal stem cell, a Schwann cell, a glial cell, an oligodendrocyte, an astrocyte, a progenitor cell, a cholinergic cell, a dopaminergic cell, a GABA cell, a glutamatergic cell, and a motor neuron), a choroid plexus cell, or a cell related to blood vessels and coverings (e.g., an endothelial cell).

[0075] In some embodiments, the cell of the central nervous system is a glial cell of the immune system (i.e., the cells of the central nervous system are microglia). In some embodiments, from about 5% to about 30% (e.g., from about 10% to about 30%, e.g., about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 26%) of the cells expressing the protein, polypeptide or gene product are microglia. In some embodiments, about 6%, about 7%, about 8%, about 9% or about 10% of the cells expressing the protein, polypeptide or gene product are microglia.BRIEF DESCRIPTION OF THE DRAWINGS

[0076] 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.

[0077] FIG. 1 shows a FACS scatter plot of cells from Ail4 mice administered composition 6A1 formulated with TdTomato mRNA at a dose of lOmpk. The plot shows 19.9% TdTom+ events found in microglia (CD1 lb+ cells). No microgliosis were observed.

[0078] FIG. 2 shows a graph of normalized percent body weight (BW) change over 24h following administration of composition 6A1 formulated with TdTomato mRNA at doses of 2.5 mpk, 5 mpk, and 10 mpk (n=3).

[0079] FIGs. 3A-3P show single cell sequencing (10X) scatter plots of cells from rats administered composition 6A1. FIG. 3 A shows Cy5 positive cells following administration of a control (15 mM Tris + 10% sucrose). FIG. 3B shows TdTom expression in CD1 lb+ cells following administration of the control. FIG. 3C shows TdTom expression in CD31+ cells following administration of the control. FIG. 3D shows TdTom expression in DoubleNeg cells following administration of the control. FIG. 3E shows Cy5 positive cells following administration of composition 6A1 at a dose of 2.5 mg / kg. FIG. 3F shows TdTom expression in CD1 lb+ cells following administration of composition 6A1 at a dose of 2.5 mg / kg. FIG. 3G shows TdTom expression in CD31+ cells following administration of composition 6A1 at a dose of 2.5 mg / kg. FIG. 3H shows TdTom expression in DoubleNeg cells following administration of composition 6A1 at a dose of 2.5 mg / kg. FIG. 31 shows Cy5 positive cells following administration of 6A1 at a dose of 5 mg / kg. FIG. 3J shows TdTom expression in CD1 lb+ cells following administration of composition 6A1 at a dose of 5 mg / kg. FIG. 3K shows TdTom expression in CD31+ cells following administration of composition 6A1 at a dose of 5 mg / kg. FIG. 3L shows TdTom expression in DoubleNeg cells following administration of composition 6A1 at a dose of 5 mg / kg. FIG. 3M shows Cy5 positive cells following administration of composition 6A1 at a dose of 10 mg / kg. FIG. 3N shows TdTom expression in CD1 lb+ cells following administration of composition 6A1 at a dose of 10 mg / kg. FIG. 30 shows TdTom expression in CD31+ cells following administration of composition 6A1 at a dose of 10 mg / kg. FIG. 3P shows TdTom expression in DoubleNeg cells following administration of composition 6A1 at a dose of 10 mg / kg.

[0080] FIGs. 4A-4G show expression of genes in a single cell sequencing assay of cells harvested from rats administered composition 6A1 a dose of 2.5 mg / kg. FIG. 4B shows expression of Trem2. FIG. 4C shows expression of P2RY12. FIG. 4D shows expression of TNF. FIG. 4E shows expression of Lgals3. FIG. 4F shows expression of Ccr7. FIG. 4G shows expression of Gpnmb.

[0081] FIGs. 5A-5G show expression of genes in a single cell sequencing assay of cells harvested from rats administered composition 6A1 at a dose of 5 mg / kg. FIG. 5A shows the single cell sequencing scatter plot. FIG. 5B shows expression of Trem2. FIG. 5C shows expression of P2RY12. FIG. 5D shows expression of TNF. FIG. 5E shows expression ofLgals3. FIG. 5F shows expression of Ccr7. FIG. 5G shows expression of Gpnmb.

[0082] FIGs. 6A-6G show expression of genes in a single cell sequencing assay of cells harvested from rats administered composition 6A1 at a dose of 10 mg / kg. FIG. 6A shows the single cell sequencing scatter plot. FIG. 6B shows expression of Trem2. FIG. 6C shows expression of P2RY12. FIG. 6D shows expression of TNF. FIG. 6E shows expression of Lgals3. FIG. 6F shows expression of Ccr7. FIG. 6G shows expression of Gpnmb.

[0083] FIG. 7 shows a FACS scatter plot of cells from Ail4 mice administered composition 6 A3 formulated with TdTomato mRNA at a dose of 10 mpk. The plot shows 24.9% TdTom+ events found in microglia (CD1 lb+ cells).

[0084] FIG. 8 shows a graph of normalized percent body weight change over 24h following administration of composition 6A3 formulated with TdTomato mRNA at a dose of 10 mpk. Data for animals 451, 452, and 453 are shown.

[0085] FIGs. 9A-9D show single cell sequencing (10X) scatter plots of cells from rats administered composition 6A3 at a dose of 10 mg / kg. FIG. 9A shows Cy5 positive cells. FIG.9B shows TdTom expression in CD1 lb+ cells. FIG. 9C shows TdTom expression in CD31+ cells. FIG. 9D shows TdTom expression in DoubleNeg cells.

[0086] FIGs. 10A-10G show expression of genes in a single cell sequencing assay of cells harvested from rats administered composition 6A3. FIG. 10A shows the single cell sequencing scatter plot. FIG. 10B shows expression of Trem2. FIG. 10C shows expression ofP2RY12. FIG.10D shows expression of TNF. FIG. 10E shows expression of Lgals3. FIG. 10F shows expression of Ccr7. FIG. 10G shows expression of Gpnmb.

[0087] FIG. 11 shows a FACS scatter plot of cells from Ail4 mice administered composition 5A2 formulated with TdTomato mRNA at a dose of 20 mpk. The plot shows 36.3% TdTom+ events found in microglia (CD1 lb+ cells). No microgliosis observed.

[0088] FIG. 12 shows a FACS scatter plot of cells from Ail4 mice administered composition 5A2 formulated with TdTomato mRNA at a dose of 10 mpk. The plot shows 7.3% TdTom+ events found in microglia (CD1 lb+ cells). No microgliosis observed.

[0089] FIG. 13 shows a graph of normalized percent body weight change following administration of composition 5A2 formulated with TdTomato mRNA at a dose of 10 mpk. Data for animals 551, 552, and 553 are shown.

[0090] FIGs. 14A-14D show single cell sequencing (10X) scatter plots of cells from rats administered composition 6A4 at the high dose. FIG. 14A shows Cy5 positive cells. FIG. 14B shows expression of TdTom in CDllb+ cells. FIG. 14C shows expression of TdTom in CD31+ cells. FIG. 14D shows expression of TdTom in DoubleNeg cells.

[0091] FIGs. 15A-15G show expression of key genes in a single cell sequencing assay of cells harvested from rats administered composition 6A4. FIG. 15A shows the single cell sequencing scatter plot. FIG. 15B shows expression of Trem2. FIG. 15C shows expression of P2RY12. FIG.15D shows expression of TNF. FIG. 15E shows expression of Lgals3. FIG. 15F shows expression of Ccr7. FIG. 15G shows expression of Gpnmb.

[0092] FIG. 16 shows a FACS scatter plot of cells from Ail4 mice administered composition 5A3 formulated with TdTomato mRNA at a dose of lOmpk. The plot shows 11.1% TdTom+ events found in microglia (CD1 lb+ cells). No microgliosis was observed.

[0093] FIG. 17A shows a graph of normalized percent body weight change following administration of composition 5A3 formulated with TdTomato mRNA at a dose of 2.5 mpk. Data for animals 251, 252, and 253 are shown.

[0094] FIG. 17B shows a graph of normalized percent body weight change following administration of composition 5A3 formulated with TdTomato mRNA at a dose of 5 mpk. Data for animals 351, 352, and 353 are shown.

[0095] FIG. 17C shows a graph of normalized percent body weight change following administration of composition 5A3 formulated with TdTomato mRNA at a dose of 10 mpk. Data for animals 451, 452, and 453 are shown.

[0096] FIGs. 18A-18P show single cell sequencing (10X) scatter plots of cells from rats administered composition 5 A3. FIG. 18A shows Cy5 positive cells following administration of a control (15 mM Tris + 10% sucrose). FIG. 18B shows TdTom expression in CD1 lb+ cells following administration of the control. FIG. 18C shows TdTom expression in CD31+ cells following administration of the control. FIG. 18D shows TdTom expression in DoubleNeg cells following administration of the control. FIG. 18E shows Cy5 positive cells following administration of composition 5 A3 at a dose of 2.5 mg / kg. FIG. 18F shows TdTom expression in CD1 lb+ cells following administration of composition 5 A3 at a dose of 2.5 mg / kg. FIG. 18Gshows TdTom expression in CD31 + cells following administration of composition 5A3 at a dose of 2.5 mg / kg. FIG. 18H shows TdTom expression in DoubleNeg cells following administration of composition 5A3 at a dose of 2.5 mg / kg. FIG. 181 shows Cy5 positive cells following administration of composition 5A3 at a dose of 5 mg / kg. FIG. 18 shows TdTom expression in CD1 lb+ cells following administration of composition 5 A3 at a dose of 5 mg / kg. FIG. 18K shows TdTom expression in CD31+ cells following administration of composition 5A3 at a dose of 5 mg / kg. FIG. 18L shows TdTom expression in DoubleNeg cells following administration of composition 5 A3 at a dose of 5 mg / kg. FIG. 18M shows Cy5 positive cells following administration of composition at a dose of 10 mg / kg. FIG. 18N shows TdTom expression in CD1 lb+ cells following administration of composition 5 A3 at a dose of 10 mg / kg. FIG. 180 shows TdTom expression in CD31+ cells following administration of composition 5A3 at a dose of 10 mg / kg. FIG. 18P shows TdTom expression in DoubleNeg cells following administration of composition 5 A3 at a dose of 10 mg / kg.

[0097] FIGs. 19A-19G show expression of key genes in a single cell sequencing assay of cells harvested from rats administered composition 5A3 at a dose of 2.5 mg / kg. FIG. 19A shows the single cell sequencing scatter plot. FIG. 19B shows expression of Trem2. FIG. 19C shows expression of P2RY12. FIG. 19D shows expression of TNF. FIG. 19E shows expression of Lgals3. FIG. 19F shows expression of Ccr7. FIG. 19G shows expression of Gpnmb.

[0098] FIGs. 20A-20G show expression of key genes in a single cell sequencing assay of cells harvested from rats administered composition 5A3 at a dose of 5 mg / kg. FIG. 20A shows the single cell sequencing scatter plot. FIG. 20B shows expression of Trem2. FIG. 20C shows expression of P2RY12. FIG. 20D shows expression of TNF. FIG. 20E shows expression of Lgals3. FIG. 20F shows expression of Ccr7. FIG. 20G shows expression of Gpnmb.

[0099] FIGs. 21A-21G show expression of key genes in a single cell sequencing assay of cells harvested from rats administered composition 5A3 a dose of 10 mg / kg. FIG. 21A shows the single cell sequencing scatter plot. FIG. 21B shows expression of Trem2. FIG. 21C shows expression of P2RY12. FIG. 21D shows expression of TNF. FIG. 21E shows expression of Lgals3. FIG. 21F shows expression of Ccr7. FIG. 21G shows expression of Gpnmb.

[0100] FTGs. 22A-22D show single cell sequencing (10X) scatter plots of cells from rats administered composition 5A1 at the high dose. FIG. 22A shows Cy5 positive cells. FIG. 22B shows CD1 lb+ TdTom. FIG. 22C shows CD31+ TdTom. FIG. 22D shows DoubleNeg TdTom.

[0101] FIGs. 23A-23G show expression of key genes in a single cell sequencing assay of cells harvested from rats administered composition 5A1. FIG. 23 A shows the single cell sequencing scatter plot. FIG. 23B shows expression of Trem2. FIG. 23C shows expression of P2RY12. FIG.23D shows expression of TNF. FIG. 23E shows expression of Lgals3. FIG. 23F shows expression of Ccr7. FIG. 23G shows expression of Gpnmb.

[0102] FIG. 24 shows a FACS scatter plot of cells from Ail4 mice administered composition 6A2 formulated with TdTomato mRNA at a dose of 10 mpk. The plot shows 21.1% TdTom+ events found in microglia (CD1 lb+ cells).

[0103] FIG. 25 shows a graph of the normalized percent body weight (BW) change over 24 h following administration of composition 6A2 formulated with TdTomato mRNA at a dose of 10 mpk. Data for animals 351, 352, and 353 are shown.

[0104] FIG. 26A shows a graph of the normalized percent body weight (BW) change over 24 h following administration of composition 6A2 formulated with TdTomato mRNA at a dose of 10 mpk. Data for animals 251 and 252 are shown.

[0105] FIG. 26B shows a graph of the normalized percent body weight (BW) change over 48 h following administration of composition 6A2 formulated with TdTomato mRNA at a dose of 10 mpk. Data for animals 351 and 352 are shown.

[0106] FIG. 26C shows a graph of the normalized percent body weight (BW) change over 72 h following administration of composition 6A2 formulated with TdTomato mRNA at a dose of 10 mpk. Data for animals 451 and 452 are shown.

[0107] FIGs. 27A-27D show single cell sequencing (10X) scatter plots of cells from rats administered composition 6A2 at the high dose. FIG. 27A shows Cy5 positive cells. FIG. 27B shows expression of TdTom in CD1 lb+ cells. FIG. 27C shows expression of TdTom in CD31 + cells. FIG. 27D shows expression of TdTom in DoubleNeg cells.

[0108] FIGs. 28A-28D show single cell sequencing (10X) scatter plots of cells from rats administered composition 6A2 (24 hour high dose sample; Animal 251). FIG. 28A shows Cy5positive cells. FIG. 28B shows expression of TdTom in CD1 lb+ cells. FIG. 28C shows expression of TdTom in CD31+ cells. FIG. 28D shows expression of TdTom in DoubleNeg cells.

[0109] FIGs. 29A-29D show single cell sequencing (10X) scatter plots of cells from rats administered composition 6A2 (72 hour high dose sample; Animal 452). FIG. 29A shows Cy5 positive cells. FIG. 29B shows expression of TdTom in CDllb+ cells. FIG. 29C shows expression of TdTom in CD31+ cells. FIG. 29D shows expression of TdTom in DoubleNeg cells.

[0110] FIG.30 shows an illustration of an intrathecal (IT) catheter surgery on a rat.

[0111] FIGs. 31A-31G show expression of genes in a single cell sequencing assay of cells harvested from rats administered composition 6A2 rat 24 hours after administration. FIG. 31A shows the single cell sequencing scatter plot. FIG. 3 IB shows expression of Trem2. FIG. 31C shows expression ofP2RY12. FIG. 3 ID shows expression of TNF. FIG. 3 IE shows expression of Lgals3. FIG. 3 IF shows expression of Ccr7. FIG. 31G shows expression of Gpnmb.

[0112] FIGs. 32A-32D show expression of genes in a single cell sequencing assay of cells harvested from rats administered composition 6A2 rat at 24 hours after administration. FIG. 32A shows TdTom expression. FIG. 32B shows expression of TNF. FIG. 32C shows expression of P2RY12 (homeostatic microglia marker). FIG. 32D shows expression of Trem2 (Microglia marker).

[0113] FIGs. 33A-33D show expression of genes in a single cell sequencing assay of cells harvested from rats administered composition 6A2 rat at 72 hours after administration. FIG. 33A shows TdTom expression. FIG. 33B shows expression of TNF. FIG. 33C shows expression of P2RY12 (homeostatic microglia marker). FIG. 33D shows expression of Trem2 (Microglia marker).

[0114] FIG.34 shows a graph of normalized percent body weight (BW) change over 24h in cannulated rats following administration of composition 5A2 formulated with TdTomato mRNA at a dose of 10 mpk. Data for animals 151 Naive, 351, 352, 353, 354, 355, and 356 are shown.

[0115] FIGs. 35A-35D show single cell sequencing (10X) scatter plots of cells from rats administered composition 5A2 demonstrating that 5A2 primarily targets microglia. FIG. 35Ashows the FACS scatter plot. FIG. 35B shows expression of TdTom in CD1 lb+ cells. FIG. 35C shows expression of TdTom in CD31+ cells. FIG. 35D shows expression of TdTom in DoubleNeg cells.

[0116] FIGs. 36A-36D show FACS scatter plots of cells from rats administered composition 5A2, demonstrating that TdTom+ cells label with P2RY12 (a homeostatic microglia marker). This may suggest that microglia that take up 5A2 are not proinflammatory. FIG. 36A shows the FACS scatter plot. 36B shows expression of TdTom in CD1 lb+ T+ cells. FIG. 36C shows expression of TdTom in CDllb+ P2ryl2+T+ cells. FIG. 36D shows expression of TdTom in P2ryl2+T+ cells.

[0117] FIGs. 37A-37D show FACS scatter plots of cells from rats administered composition 5A2, demonstrating that TdTom+ cells label with P2RY12 (a homeostatic microglia marker). This may suggest that microglia that take up 5A2 are not proinflammatory. FIG. 37A shows the FACS scatter plot. 37B shows expression of TdTom in CD1 lb+ T+ cells. FIG. 37C shows expression of TdTom in CDllb+ P2ryl2+T+ cells. FIG. 37D shows expression of TdTom in P2ryl2+T+ cells.

[0118] FIGs. 38A-38D show expression of genes in a single cell sequencing assay of cells harvested from rats administered composition 5A2, at 24 hour take down. FIG. 38A shows TdTom expression. FIG. 38B shows expression of TNF. FIG. 38C shows expression of P2RY12 (homeostatic microglia marker). FIG. 38D shows expression of Trem2 (Microglia marker).

[0119] FIGs. 39A-39B show expression of genes in a single cell sequencing assay of cells harvested from rats intrathecally administered mRNA formulated with composition 6A5 or Control treatment. FIG. 39A shows expression of TdTom mRNA, Trem 2 mRNA (microglial marker), and TNF mRNA in 6A5 treated rats. TNF mRNA is not upregulated relative to control animals. FIG. 39B shows TdTom mRNA expressing cells express P2RY12 at 24 hours and 72 hours post injection. At 72 hours post injection, TdTom mRNA expression decreased while P2RY12 mRNA expression increased.

[0120] FIGs. 40A-40B show the safety, tolerability and selective uptake of composition 6A5 by microglia in non-human primate (NHP) brains after a single intra-cistema magna (ICM) injection. FIG. 40A shows representative FACS plots of brain sections from control brain (top left panel), Img / kg dose (top middle panel), and 3mg / kg dose (top right panel). The bottom plot shows dose-dependent transfection of CDl lb+ cells. FIG. 40B shows expression of tdTomato (top panel), Trem2 (middle panel), and CCR7 (bottom panel) in a single cell assay of cells harvested from NHP brains administered with mRNA formulated with composition 6A5.

[0121] Definitions

[0122] 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.

[0123] 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.

[0124] The singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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 orimprovement 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.

[0132] The terms “decreased”, “decreasing”, “decrease”, “reduced”, “reducing”, “reduce” and the like, are used herein generally to mean a decrease or reduction by a statistically significant amount. In some aspects, “decreased” or “reduced” means a reduction by at least 10% as compared to a reference level, for example a decrease or reduction by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease or reduction (e.g., absent level or non-detectable level as compared to a reference level), or any decrease or reduction between 10-100% as compared to a reference level.

[0133] 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.

[0134] 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 “LNPcomposition” are used herein interchangeably. Lipid nanoparticles can have one or more lipids with at least one dimension on the order of nanometers (e.g., 1-1000 nm). Generally, lipid nanoparticle compositions for delivery are composed of one or more lipids, such as, but not limited to, a synthetic ionizable or cationic lipid, a phospholipid, a structural lipid (e.g., a sterol), and a polyethylene glycol (PEG) lipid. These compositions may also include other lipids. In various embodiments, the lipid nanoparticle composition comprises five components: (i) an ionizable cationic lipid; (ii) a phospholipid; (iii) a steroid or steroid derivative; and (iv) a polymer-conjugated lipid; and (v) an additional cationic ionizable cationic lipid, a permanently cationic lipid or an anionic lipid (“SORT lipid”) separate from said ionizable cationic lipid. In some embodiments, at least one therapeutic agent (e.g., mRNA) can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation of other undesirable effects induced by the biological mechanism of a target subject, tissue, and / or cell, e.g., an adverse immune response. In some embodiments, lipid nanoparticles comprise at least one therapeutic agent (e.g., mRNA) that is either organized within inverse lipid micelles and encased within a lipid monolayer envelop or intercalated between adjacent lipid bilayers. In some embodiments, the morphology of lipid nanoparticles is not like a traditional liposome, which are characterized by a lipid bilayer surrounding an aqueous core. In some embodiments, lipid nanoparticles are substantially non-toxic. In some embodiments, the therapeutic agent (e.g., mRNA) is resistant in aqueous solution to degradation by intracellular or intercellular enzymes by virtue of the lipid nanoparticle.

[0135] 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 organspecific targeting of the LNP (for example as described in Cheng et al. Nat. Nanotechnol.15:313-320 (2020); Wang et al. Nat. Protoc. 18(1):265-291; and US 11,766,408 and US11,229,609, the entire contents of each of which is incorporated herein by reference). A selected SORT lipid provides accurate and specific delivery of the cargo from a rationally-designed LNP based, in part, on the biophysical properties of the selected SORT lipid and its prevalence in the LNP. In some cases, specificity is modulated by a LNP’s surface's acid dissociation constant (pKa), which may be affected by the proportion of charged and uncharged ionizable cationic 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, e.g., Table 7 and Table 8. 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.

[0136] As used herein, the term “ionizable cationic lipid” refers to lipid and lipid-like molecules having at least one pKa in the range of about 4.5-8, such that, without being bound by theory, they may facilitate release of LNP payloads upon uptake into the endosomal compartment of a cell. The ionizable cationic lipid may maintain a neutral charge in pH above the pKa of the lipid; it becomes positively charged in a pH lower than its pKa which facilitates membrane fusion and subsequent cytosolic release of an LNP. Illustrative ionizable cationic lipids have one or more nitrogen atoms having a pKa in the range of about 4.5-8, such are tertiary amine groups.

[0137] 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 (positively charged without pKa or with a pKa greater than 8). 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. PNAS USA.118(52):e2109256118 (2021).

[0138] 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.

[0139] 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 groupat 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.

[0140] 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).

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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 tothe 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.

[0145] 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.

[0146] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof.Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro RNA (miRNA), transfer RNA, ribosomal RNA, a ribozyme, an antisense RNA, a guide RNA (gRNA), cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolated RNA, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences. When the polynucleotides are chemically and / or structurally modified the polynucleotides may be referred to as “modified polynucleotides.”

[0147] 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 havingchemically modified bases or sugars, or backbone modifications. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.

[0148] 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.

[0149] As used herein, the term “microRNA” refers to noncoding RNA consisting of about 22 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.

[0150] 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.

[0151] As used herein, the term “gene-editing system” refers to a DNA or RNA editing system that comprises one or more guide RNA elements and one or more RNA-guided endonuclease elements. The guide RNA element comprises a target RNA comprising a nucleotide sequence substantially complementary to a nucleotide sequence at the one or more target genomic regions or a nucleic acid comprising a nucleotide sequence(s) encoding the target RNA. The RNA-guided endonuclease element comprises an endonuclease that is guided or brought to a target genomic region(s) by a guide RNA element or a nucleic acid comprising a nucleotide sequence(s) encoding such endonuclease.

[0152] 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 referencesequence, 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.

[0153] 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, which is the predominant species present in a preparation, is substantially purified.

[0154] 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.

[0155] The terms “subject” refers to a living organism to which any of the compositions as described herein may be administered. The subject may be suffering from or be at risk for a disease or condition that can be treated by administration of pharmaceutical composition as provided herein or by a therapeutic method disclosed herein. Non-limiting examples of subjects include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the subject is a primate, e.g., a human.

[0156] 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 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 effectiveamount” 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.

[0157] 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.

[0158] “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).

[0159] As used herein, the term “delivering” means causing, through chemical or biophysical properties of a composition (e.g., an LNP composition) and / or the payload (e.g., a polynucleotide) of an LNP to pass from a site of administration to a subject to a target organ (e.g., the lung, liver, heart, or spleen), 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, liver, heart, or spleen)by an LNP composition comprises a selected lipid (e.g., SORT lipid) compared to a reference LNP composition in which the selected lipid is replaced by a control lipid.

[0160] “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 ( / .c., 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.

[0161] ‘ ‘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.

[0162] 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 orstarch, 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.

[0163] 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).

[0164] Chemical moi eties referred to as univalent chemical moi eties (e.g., alkyl, aryl, etc.) also encompass structurally permissible multivalent moi eties, as understood by those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g., CH3CH2-), in appropriate circumstances an “alkyl” moiety can also refer to a divalent radical (e.g, -CH2CH2-, which is equivalent to an “alkylene” group). Similarly, under circumstances where a divalent moiety is required, those skilled in the art will understand that the term “aryl” refers to the corresponding divalent arylene group.

[0165] As used herein, “Alkyl” refers to optionally substituted, straight and branched chain aliphatic groups having from 1 to 30 carbon atoms. For example, “Ci, C2, C3, C4, C5 or Cg alkyl”, “Ci-C6alkyl”, “alkyl(C<6)”, or “alkyl(Cl-C6)”, is intended to include Ci, C2, C3, C4, C5or C6straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5 or C& branched saturated aliphatic hydrocarbon groups. Examples of alkyl include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, or n-hexyl. In some embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g, Ci-Cg for straight chain, C3-Cg for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms.Analogously, for example “Cis, C19, C20, C21, C22, C23 or C24 alkyl”, “Cis-C24alkyl”, “alkyl(C<24)”, or “alkyl(C8-C24)” is intended to include Cis, C19, C20, C21, C22, C23 or C4straight chain (linear) saturated aliphatic hydrocarbon groups and Cis, C19, C20, C21, C22, C23 orC24 branched saturated aliphatic hydrocarbon groups. Examples of “C18-C24 alkyl” include octadecyl, nonadecyl, didecyl, henicosyl, docosyl, tricosyl, tetracosyl, 5-butylpentadecanyl, 4-methyl-5-(pentan-2-yl)hexadecanyl, 7-methylhenicosanyl, 2, 15,15-trimethylhenicosanyl, 8,9-dimethyldocosanyl, 6-ethyl-8-methylnonadecanyl, and 6,7-dimethyl-8-propyltridecanyl.

[0166] “Alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term “alkenyl” includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenyl groups. The term “C2, C3, C4, C5 or C alkenyl,” “C2-C6 alkenyl,” “alkenyl(C<6)”, or “alkenyl(C2-C6)” includes alkenyl groups containing two to six carbon atoms. The term “Cs, C9, C10, Cn, C12, C13, C14, Ci5, Ci6, C17, Cis, C19, C20, C21, C22, C23 or C24 alkenyl,” “Cs-C24 alkenyl,” or “alkenyl(C8-C24)” includes alkenyl groups containing eight to twenty-four carbon atoms. Examples of “Cs-C24 alkenyl” include 2,6-dimethylhept-2-enyl, 2,6-dimethylhept-2-enyl, 2,8-dimethylnon-2-enyl, 2,7-dimethyldec-2-ene, 3-ethyl-8-methylundec-3-ene, and 2,9,9-trimethyltridec-2-ene.

[0167] The term “optionally substituted alkyl” or “optionally substituted alkenyl” refers to an alkyl or alkenyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryl oxy carbonyl oxy, carboxylate, alkyl carbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, aryl carbonyl ami no, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.I. Central nervous system disease

[0168] Central nervous system disease, also known as central nervous system disorders, are neurological disorders that affect the structure or function of the brain or spinal cord that form the central nervous system (CNS). In some embodiments, a CNS disease is Abulia,Achromatopsia, acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Agraphia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, Akinetopsia, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Amnesia, anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartylglucosaminuria, Asperger syndrome, ataxia, ataxia telangiectasia (Louis-Bar syndrome), ataxias and cerebellar or spinocerebellar degeneration, attention deficit- hyperactivity disorder, autism, autonomic dysfunction, Barth syndrome, Batten disease, Becker's myotonia, Behcet's disease, Bell's palsy, Bernhardt-Roth syndrome, Bipolar disorder, Binswanger's disease, Bloch-Sulzberger syndrome, Bradbury-Eggleston syndrome, Brown-Sequard syndrome, bulbospinal muscular atrophy, CADASIL, Canavan's disease, Carpal tunnel syndrome, causalgia, cavernomas, cavernous angioma, central cervical cord syndrome, central cord syndrome, central pontine myelinolysis, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral beriberi, cerebral gigantism, cerebral palsy, cerebro-oculo-facio-skeletal syndrome (COFS), Charles bonnet syndrome, cholesterol ester storage disease, chorea, choreoacanthocytosis, chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic fatigue syndrome, chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, colpocephaly, congenital myasthenia, corticobasal degeneration, cranial arteritis, cree encephalitis, Creutzfeldt-Jakob disease, Cushing's syndrome, cystinosis, cytomegalic inclusion body disease, Dancing eyes-dancing feet syndrome, Dandy -Walker syndrome, Dan on disease, Dawson disease, De Morsier's syndrome, Dej erine-Klumpke palsy, dementia, dentate cerebellar ataxia, dentatorubral atrophy, Dermatillomania, dermatomyositis, developmental dyspraxia, Devic's syndrome, diffuse sclerosis, Down syndrome, Duchenne muscular dystrophy, dysautonomia, dysgraphia, dyslexia, dysphagia, dyspraxia, dyssynergia cerebellaris myoclonica, dyssynergia cerebellaris progressiva, Epilepsy (such as, e.g, Amish infantile epilepsy syndrome (AIES), benign familial infantile seizures (BFIS), benign familial neonatal seizures (BFNS), childhood absence epilepsy (CAE), childhood-onset epileptic encephalopathy (COEE), Dravet syndrome (DS), early infantile epileptic encephalopathy (EIEE), Exploding head syndrome, familial adult myoclonic epilepsy(FAME), familial febrile seizures (FFS), familial focal epilepsy with variable foci (FFEVF), familial infantile myoclonic epilepsy (FIME), familial temporal lobe epilepsy (FTLE), focal epilepsy and speech disorder (FESD) with or without mental retardation, generalized epilepsy and paroxysmal dyskinesia (GEPD), generalized epilepsy with febrile seizures plus (GEFS+), idiopathic generalized epilepsy (IGE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), myoclonic-atonic epilepsy (MAE), nocturnal frontal lobe epilepsy (NFLE), progressive myoclonic epilepsy (PME), pyridoxamine 5'-phosphate oxidase deficiency (PNPOD), pyridoxine-dependent epilepsy (EPD) and severe myoclonic epilepsy of infancy (SMEI)), Fabry disease, Fahr's syndrome, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralyses, familial spastic paralysis, Farber's disease, Fetal alcohol syndrome, fibromuscular dysplasia, Fisher syndrome, floppy infant syndrome, Foville's syndrome, Friedreich's ataxia, frontotemporal dementia, fucosidosis, galactosialidosis, Gaucher disease, generalized gangliosidosis, Gerstmann's syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 gangliosidosis, GM2 gangliosidosis (Tay-Sachs disease), Gray matter heterotopia, Guillain-Barre syndrome, Hallervorden-Spatz disease, hemicrania continua, hemiplegia alterans, hereditary spastic paraplegia, heredopathia atactica polyneuritiformis, Herpes zoster, Hirayama disease, Holmes-Adie syndrome, holoprosencephaly, Hughes syndrome, Huntington's disease, hydranencephaly, hydromyelia, hypercortisolism, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, iniencephaly, Isaac's syndrome, Isodicentric 15, Joubert syndrome, Karak syndrome, Keams-Sayre syndrome, Kennedy's disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Kliiver-Bucy syndrome, Korsakoff s amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, lateral medullary syndrome, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, Levine-Critchley syndrome, Lewy body dementia, lipoid proteinosis, lissencephaly, locked-in syndrome, Lou Gehrig's disease, lupus, Lyme disease, Machado-Joseph disease, macrencephaly, alpha-mannosidosis, beta-mannosidosis, Melkersson-Rosenthal syndrome, Menkes disease, meralgia paresthetica, metachromatic leukodystrophy, microcephaly,Miller Fisher syndrome, Misophonia, Moebius syndrome, Moyamoya disease, mucopolysaccharidosis type I-H (Hurler syndrome), mucopolysaccharidosis type I-H / S (Hurler-Scheie syndrome), mucopolysaccharidosis type IS (Scheie syndrome), mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type III-A (Sanfdippo syndrome A), mucopolysaccharidosis type III-B (Sanfilippo syndrome B), mucopolysaccharidosis type III-C (Sanfilippo syndrome C), mucopolysaccharidosis type III-D (Sanfilippo syndrome D), mucopolysaccharidosis type IV-B (Morquio syndrome B), mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), mucopolysaccharidosis type VII (Sly syndrome), mucopolysaccharidosis type IX (Natowicz syndrome), multiple sclerosis, muscular dystrophy, myasthenia gravis, myelinoclastic diffuse sclerosis, narcolepsy, neuroacanthocytosis, neurofibromatosis, neuroleptic malignant syndrome, neurosarcoidosis, Niemann-Pick disease, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus myoclonus, O'Sullivan-McLeod syndrome, Otosclerosis, Overuse syndrome, pantothenate kinase- associated neurodegeneration, paraneoplastic syndromes, paresthesia, Parkinson's disease, paroxysmal choreoath etosis, paroxysmal hemicrania, Parry -Romberg syndrome, Pelizaeus-Merzbacher disease, Pena Shokeir II syndrome, periventricular leukomalacia, phytanic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pompe disease, post-polio syndrome, posterior cortical atrophy, Prader-Willi syndrome, primary dentatum atrophy, primary lateral sclerosis, primary progressive aphasia, prion diseases, progressive hemifacial atrophy, progressive locomotor ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, Quadriplegia, Rabies, Ramsay Hunt syndrome I, Ramsay Hunt syndrome II, Rasmussen's encephalitis, Refsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhoff disease, Schilder's disease, Seitelberger disease, Shingles, Shy-Drager syndrome, Sjogren's syndrome, spasticity, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, striatonigral degeneration, Sturge-Weber syndrome, tardive dyskinesia, tauopathy, Tay-Sachs disease, Tetanus, thoracic outlet syndrome, thyrotoxic myopathy, tic douloureux, Todd's paralysis, trigeminal neuralgia, tropical spastic paraparesis, Troyer syndrome, Unverricht-Lundborg disease, vascular dementia, Vertigo, Von Economo's disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's disease, Wallenberg's syndrome, Werdnig-Hoffman disease, Wernicke-Korsakoff syndrome, West syndrome,Whipple's disease, Williams syndrome, Wilson disease, Wolman's disease, X-linked spinal and bulbar muscular atrophy, Y-linked hearing impairment, Zellweger syndrome, multiple sclerosis atrophy, Lewis body dementia (LBD), or Angelman syndrome.Alzheimer’s disease

[0169] Alzheimer’s disease is the most common type of dementia. It is a progressive disease beginning with mild memory loss and possibly leading to loss of the ability to carry on a conversation and respond to the environment. It involves parts of the brain that control thought, memory, and language.Amyotrophic lateral sclerosis (ALS)

[0170] Amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease, is a neurological disease that affects motor neurons. ALS causes loss of muscle control.Huntington’s disease

[0171] Huntington’s disease is an inherited disorder that causes the progressive degeneration of nerve cells in the brain. Huntington’s disease causes changes in the central area of the brain, which affect movement, mood and thinking skills.Parkinson’s disease

[0172] Parkinson’s disease is a brain disorder that causes unintended or uncontrollable movements, such as shaking, stiffness, and difficulty with balance and coordination. Lewy bodies, unusual clumps of the protein a-synuclein, is observed in patient’s brain cells.Spinal muscular atrophy (SMA)

[0173] Spinal muscular atrophy (SMA) is a motor neuron disease involving the loss of motor neurons in the spinal cord. SMA is a genetic disease affecting the central nervous system, peripheral nervous system, and voluntary muscle movement (skeletal muscle).II. Lipid Nanoparticle Compositions

[0174] The present disclosure contemplates LNP compositions useful in the delivery of a payload, for example a polypeptide or polynucleotide encoding a polypeptide to a host cell. TheLNPs are formulated to target different host cells in vitro or in vivo and the payload is then released in the host cell.

[0175] In one aspect, the disclosure provides a lipid nanoparticle (LNP) composition comprising a payload, an ionizable cationic lipid, a selective organ targeting (SORT) lipid, and / or a helper lipid, a sterol, and / or a polyethylene glycol-conjugated lipid (PEG-lipid), wherein the composition delivers the payload to a cell in the central nervous system (CNS) of a subject.Payloads

[0176] The present disclosure contemplates delivery of various payloads useful in the treatment of a CNS disease. Payloads comprise therapeutic polypeptides or polynucleotides encoding polypeptides. For example, the payload may be a polynucleotide encoding a gene related to CNS disease, or a polynucleotide encoding a gene editor for editing a gene related to CNS disease.

[0177] In some embodiments, genes involved in CNS diseases include, but are not limited to, 3R tau, 4R tau, AARS, ABCD1, ACOX1, ADGRV1, ADRA2B, AGA, AGER, ALDH7A1, ALG13, ALS2, ANG, ANXA11, APP, ARHGEF9, ARSA, ARSB, ARV1, ASAHI, ASP A, ATN1, ATP10A, ATP13A2, ATXN1, ATXN2, ATXN3, BAX, BCL-2, BDNF, BICD2, C9orf72, CACNA1A, CACNA1H, CACNB4, CASR, CCNF, CDKL5, CERS1, CFAP410, CHCHD10, CHD2, CHMP2B, CHRNA2, CHRNA4, CHRNA7, CHRNB2, CLCN2a, CLN1, CLN2, CLN3, CLN5, CLN6, CLN8, CNTN2, CPA6, CSTB, CTNS, CTSA, CTSD, DAO, DCTN1, DEPDC5, DMD, DNAJB2, DNM1, DOCK7, DRD2, DYNC1H1, EEF1A2, EFHC1, EGLN1, EPHA4, EPM2A, ERBB4, FGF12, FIG4, FRRS1L, FTL, FUCA1, FUS, FXN, GAA, GABRA1, GABRB1, GABRB3, GABRD, GABRG2, GAL, GALC, GALNS, GBA, GFAP, GLA, GLB1, GLE1, GLT8D1, GNAO1, GNS, GOSR2, GPR98, GRIA1, GRIA2, GRIK1, GRIN1, GRIN2A, GRIN2B, GRIN2D, GSTM1, GUF1, GUSB, HCN1, HGSNAT, HNRNPA1, HTT, HYAL1, IDS, IDUA, IGHMBP2, IL-1, IT15, ITPA, JPH3, KCNA2, KCNB1, KCNC1, KCNMA1, KCNQ2, KCNQ3, KCNT1, KCTD7, LAL, LAMP2, LGI1, LMNB2, LRRK2, MAN2B1, MAN2B2, MAN2C1, MANBA, MATR3, MBD5, MFSD8, NAGA, NAGLU, NECAP1, NEFH, NEK1, NEU1, NHLRC1, NPC1, NPC2, NR4A2, NTRK2, OCA2, OPTN, PARK2, PARK7, PCDH19, PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX11B, PEX12, PEX13, PEX14, PEX16, PEX19, PEX26, PFN1, PINK1, PLCB1, PNPO, PON1, PON2, PON3, PPARGC1A, PRDM8, PRICKLE1, PRKN, PRNP, PRPH, PRRT2, PSAP, S100β, SCARB2, SCN1A, SCN1B,SCN2A, SCN8A, SCN9A, SCN9Ab, SETX, SGSH, SIGMAR1, SIK1, SKP1, SLC1A1, SLC1A2, SLC2A1, SLC6A1, SLC9A6, SLC12A5, SLC13A5, SLC25A12, SLC25A22, SLC17A5, SMN1, SMPD1, SNCA, SNRPN, SOD1, SPG11, SPTAN1, SQSTM1, ST3GAL3, ST3GAL5, STX1B, STXBP1, SYP, SYT1, SZT2, TAF15, TARDBP, TBC1D24, TBCE, TBK1, TBP, TITF-1, TREM2, UBA5, UBE1, UBE3A, UBQLN2, UCH-L1, UNC13A, VAPB, VCP, VPS35, WWOX, and XBPE

[0178] In some embodiments, the payload comprises a polynucleotide, optionally an mRNA, shRNA, or microRNA. In some embodiments, the mRNA encodes a polypeptide or protein selected from the group shown in Table 1, Table 2, and / or SEQ ID NOs: 1-7. In some embodiments, the mRNA encodes a gene-editing system or a component thereof. In some embodiments, the payload comprises a polypeptide or a protein.Polypeptides

[0179] In some embodiments, the polypeptide comprises a peptide or protein that restores the function of a defective protein in a subject.Table 1. Illustrative sequence of genes related to CNS disease

[0180] In some embodiments, the SOD1 polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the SMN-1 polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 2. In some embodiments, the Frataxin polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 3. In some embodiments, the Frataxin polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the Frataxin polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 5. In some embodiments, the GAD-65 polypeptide comprises a polypeptide sequence atleast 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 6. In some embodiments, the GAD-67 polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 7.Polynucleotides

[0181] 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.

[0182] 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 P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.

[0183] In addition, it should be clear that the present disclosure is not limited to the specific polynucleotides disclosed herein. The present disclosure is not limited in scope to any particular source, sequence, or type of polynucleotides, however, as one of ordinary skill in the art could readily identify related homologs in various other sources of the polynucleotides including polynucleotides from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species). It is contemplated that the polynucleotides used in the present disclosure can comprise a sequence based upon a naturally-occurring sequence. Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotide sequence of the naturally-occurring sequence. In some embodiments, the polynucleotide is a complementary sequence to a naturally occurring sequence, or complementary to at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and 100%.Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated herein.

[0184] 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.

[0185] 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 non-coding RNA (IncRNA), a messenger ribonucleic acid (mRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, deoxyribonucleic acid (DNA), a double stranded deoxyribonucleic acid (dsDNA), a single stranded deoxyribonucleic acid (ssDNA), a single stranded ribonucleic acid (ssRNA), a or double stranded ribonucleic acid (dsRNA). In some embodiments, the polynucleotide encodes at least one of the therapeutic agents (or prophylactic agent) described herein.

[0186] In some embodiments, the polynucleotide is greater than 30 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, greater than 200 nucleotides, greater than 300 nucleotides, greater than 400 nucleotides, greater than 500 nucleotides, greater than 600 nucleotides, greater than 700 nucleotides, greater than 800 nucleotides, greater than 900 nucleotides, greater than 1000 nucleotides, greater than 1500 nucleotides, greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides in length.

[0187] 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.

[0188] In some embodiments, the polynucleotide comprises about 50 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 100 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 100000nucleotides. 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.

[0189] 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.

[0190] In some embodiments, the mRNA encodes a gene or a portion of a gene related to CNS disease shown in Table 2.Table 2. Illustrative genes related to CNS

[0191] In someone embodiments, the CNS disease is Alzheimer's disease and the at least one transgene comprises a mRNA comprises cDNA of a gene selected from the group comprising or consisting of 3R tau, 4R tau, AGER, APP, BAX, BCL-2, CHRNA7, DRD2, GFAP, GRIA1, GRIA2, GRIK1, GRIN1, IL-1, SLC1A1, SYP, and SYT1.

[0192] In someone embodiments, the CNS disease is amyotrophic lateral sclerosis (ALS) and the at least one transgene comprises a mRNA comprises cDNA of a gene selected from the group comprising or consisting of ALS2, ANG, ANXA11, ATXN2, C9orf72, CHMP2B, CFAP410, CHCHD10, CCNF, DAO, DCTN1, EPHA4, ERBB4, FIG4, FUS, GLE, GLT8D1, HNRNPA1, MATR3, NEFH, NEK1, OPTN, PFN1, PON1, PON2, PON3, PPARGC1A, PRPH, SETX, SIGMAR1, SMN1, SOD1, SPG11, SQSTM1, TAF15, TARDBP, TBK1, TREM2, UBQLN2, UNC13A, VAPB and VCP.

[0193] In onesome embodiments, the CNS disease is Huntington's disease and the at least one transgene comprises a mRNA comprises cDNA of a gene selected from the group comprising orconsisting of ATN1, ATXN1, ATXN2, ATXN3, FTL, HTT, IT15, JPH3, PRNP, SLC2A3, TBP, TITF-1 and XBP1.

[0194] In someone embodiments, the CNS disease is Parkinson's disease and the at least one transgene comprises a mRNA comprises cDNA of a gene selected from the group comprising or consisting of ATP13A2, BDNF, EGLN1, GBA, GSTM1, LRRK2, NR4A2, NTRK2, PARK2, PARK7, PINK1, PRKN, S1O60, SKP1, SNCA, VPS35 andUCH-Ll.Modified polynucleotides

[0195] In some embodiments, the polynucleotide comprises one or more modifications selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2 -thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2- thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 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-acetyl cytidine, 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-l-methyl-pseudoisocytidine, 4-thio-l -methyl -1-deaza-pseudoisocytidine, 1 -methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 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-m ethyladenosine, 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-m ethoxy -guanosine, 1 -methylguanosine, N2- methylguanosine, N2, N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl -6-thio-guanosine, and N2, N2-dimethyl-6-thio-guanosine, and combinations thereof.

[0196] In some embodiments, a polynucleotide of the disclosure comprises a modified pyrimidine, such as a modified uridine. In some cases a uridine analogue is selected from pseudouridine ( ), 1 -methylpseudouridine (m'P), 2-thiouridine (s2U), 5-methyluridine (m’U), 5-methoxyuridine (mo5U), 4-thiouridine (s4U), 5 -bromouridine (Br5U), 2'0-methyluridine (U2'm), 2'-amino-2'-deoxyuridine ( 'NFh), 2'-azido-2'-deoxyuridine (U2'Ns), and 2'-fluoro-2'-deoxyuridine (U2'F).Modification in untranslated regions

[0197] 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 down-regulation 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 the 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 usedto increase expression of a polynucleotide in a liver. Likewise, use of 5' UTR from muscle proteins (M oD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CDllb, 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.

[0198] Other non-UTR sequences can be incorporated into the 5' (or 3' UTR) UTRs of the polynucleotides of the present disclosure. The 5' and / or 3' UTRs can provide stability and / or translation efficiency of polynucleotides. For example, introns or portions of intron sequences can be incorporated into the flanking regions of a polynucleotide. Incorporation of intronic sequences can also increase the rate of translation of the polynucleotide.

[0199] 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 messenger RNA (mRNA), whereas members of the ELAV family, such as HuR, may increase the stability of mRNA. HuR may bind to AREs of all 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 tothe relevant protein and assaying protein produced at 6 hours, 12 hours, 24 hours, 48 hours, and 7 days post-transfection.

[0200] In some embodiments, a polynucleotide such as a nucleic acid construct, a vector, a polyribonucleotide, or compositions of the disclosure can comprise an engineered 5' cap structure, or a 5 '-cap can be added to a polynucleotide intracellularly. The 5 'cap structure of an mRNA can be involved in binding to the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature pseudo-circular mRNA species. The 5 'cap structure can also be involved in nuclear export, increases in mRNA stability, and in assisting the removal of 5' proximal introns during mRNA splicing.

[0201] In some embodiments, a polynucleotide such as a nucleic acid construct, a vector, or a polynucleotide can be 5 '-end capped generating a 5 '-GpppN-3 ' -triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule. The cap-structure can comprise a modified or unmodified 7-m ethylguanosine linked to the first nucleotide via a 5 '-5 ' triphosphate bridge. This 5'-guanylate cap can then be methylated to generate an N 7-m ethyl -guanyl ate residue (Cap-0 structure). The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5'end of the mRNA may optionally also be 2'-O-methylated (Cap-1 structure). 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a polynucleotide molecule, such as an mRNA molecule, for degradation. In some cases, a cap can comprise further modifications, including the methylation of the 2' hydroxy-groups of the first 2 ribose sugars of the 5' end of the mRNA. For instance, a eukaryotic cap-1 has a methylated 2'-hydroxy group on the first ribose sugar, while a cap-2 has methylated 2 '-hydroxy groups on the first two ribose sugars. The 5' cap can be chemically similar to the 3 ' end of an RNA molecule (the 5 ' carbon of the cap ribose is bonded, and the free 3'-hydroxyls on both 5'- and 3 '- ends of the capped transcripts. Such double modification can provide significant resistance to 5' exonucleases. Non-limiting examples of 5 ' cap structures that can be used with a polynucleotide include, but are not limited to, m7G(5')ppp(5')N (Cap-0), m7G(5')ppp(5')NlmpNp (Cap-1), and m7G(5')-ppp(5 ')NlmpN2mp (Cap-2).

[0202] Modifications to the modified mRNA of the present disclosure may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life whilefacilitating 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-m ethyl -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.

[0203] 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.

[0204] 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 basepairs, 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.

[0205] 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

[0206] 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 agene 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 base-editing protein encoding mRNA and one or more guide RNAs. In some embodiments, the LNPs can comprise at least one nucleic acid suitable for a genome editing technique, such as a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a guide RNA (gRNA), and a DNA repair template. In some embodiments, CRISPR nucleases can have altered activity, for example, modifying the nuclease so that it can be a nickase instead of making double-strand cuts or so that it can bind the sequence specified by the guide RNA but has no enzymatic activity. In some embodiments, the base editing protein can be a fusion protein comprising a deaminase domain and a sequence-specific DNA binding domain, such as an inactive CRISPR nuclease.Gene Editing Methods

[0207] 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.

[0208] In some embodiments, the gene editing can be targeted editing. Targeted editing can be achieved either through a nuclease-independent approach or through a nuclease-dependent approach.

[0209] 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.

[0210] 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.

[0211] 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 doublestrand 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).

[0212] 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 (c.g, endonucleases). Such nuclease-dependent targeted editing can also utilize DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which can occur in response to DSBs. In some embodiments, DNA repair by NHEJ can lead to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which can result in targeted integration of the exogenous genetic material. In some embodiments, a nuclease of the nuclease-dependent targeted editing can include, but not limited to, CRISPR-Cas9, CRISPR-Casl2 (Cpfl), CRISPR-Casl3, C2c2, C2c6, NgAgo, and / or TALEN.

[0213] Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell (e.., to knock out a gene in a cell) are well-known techniques. See for example, Bauer etal., 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.

[0214] In some embodiments, targeted gene editing can be achieved via dual integrase cassette exchange (DICE) system utilizing phiC31 and Bxbl integrases.CRISPR-Ccis9 Gene Editing System

[0215] 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., Cnrr Opin Microbiol 37:67 -78 (2017).

[0216] 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).

[0217] 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.

[0218] 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 thePAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end).

[0219] 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.

[0220] 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, the Cas9 enzyme originates from Streptococcus pyogenes, although other Cas9 homologs can also be used. In some embodiments, the Cas9 enzyme can be wild-type 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).

[0221] 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(1 l):722-36 (2015); Shmakov et al., Mol Cell 60:385-397 (2015)).

[0222] 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.

[0223] 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.

[0224] 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 5. 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, N863A, H983A, and D986A (based on the S. pyogenes Cas9 nuclease).

[0225] 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 someembodiments, 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.

[0226] A Type I CRISPR / Cas system can utilize a large effector complex known as Cascade (CRISPR-associated complex for antiviral defense) for target binding and interference. The Cascade complex can contain multiple Cas proteins, including Cas3, which can be responsible for the destruction of the target DNA. A Type II CRISPR / Cas system, particularly the CRISPR-Cas9 system, can utilize a single Cas9 protein, guided by a synthetic guide RNA (sgRNA), to introduce double-strand breaks in target DNA for subsequent repair or modification. A Type III CRISPR / Cas system can utilize a Csm (CRISPR-Cas subtype multiprotein) or Cmr (CRISPR-Cas subtype ribonucleoprotein) complex for interference. Type III CRISPR / Cas system can target RNA molecules in addition to DNA. A Type V CRISPR / Cas system, including Cpfl (also known as Casl2) and C2c2 (also known as Casl3), can utilize a single effector protein to perform interference. A Type VI CRISPR / Cas system can utilize a single Cas protein, such as C2c2 (also known as Casl3), to target and cleave RNA molecules, making it useful for RNA editing and manipulation.

[0227] Guide RNAs (gRNAs): The CRISPR technology can involve 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.

[0228] 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.

[0229] 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).

[0230] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) can be a doublestranded 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.

[0231] In some embodiments, the genome-targeting nucleic acid (e.g, gRNA) can be a singlemolecule 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.

[0232] A spacer sequence in a gRNA is a sequence (c.g, a 20-nucleotide sequence) that can define the target sequence (e.g., a DNA target sequence, 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.

[0233] The target sequence is in a target gene (c.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 double-stranded 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 spacersequence 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.

[0234] 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.

[0235] In some embodiments, the target nucleic acid sequence can have about 20 nucleotides in length. In some embodiments, the target nucleic acid can have less than about 20 nucleotides in length. In some embodiments, the target nucleic acid can have more than about 20 nucleotides in length. In some embodiments, the target nucleic acid can have at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid can have at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence can have 20 bases immediately 5’ of the first nucleotide of the PAM. For example, in a sequence comprising 5'-NNNNNNNNNNNNNNNNNNNNNRG-3', the target nucleic acid can be the sequence that corresponds to the Ns, wherein N can be any nucleotide, and the underlined NRG sequence can be the S. pyogenes PAM.

[0236] 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.

[0237] 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.

[0238] 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 1 -30 nucleotides at the 5’ end of the sgRNA sequence.

[0239] 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 ofRNAs, e.g., modifications that can enhance stability, reduce the likelihood or degree of innate immune response, and / or enhance other attributes, as described in the art. In some embodiments, non-natural modified nucleobases can be introduced into any of the gRNAs during synthesis or post-synthesis. In some embodiments, modifications can be on internucleoside linkages, purine or pyrimidine bases, or sugar. In some embodiments, a modification can be introduced at the terminal of a gRNA with chemical synthesis or with a polymerase enzyme.

[0240] 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.

[0241] 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.

[0242] 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 the 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.

[0243] In some embodiments, the indel frequency (editing frequency) of a particular CRISPR / Cas nuclease system, comprising one or more specific gRNAs, can be determined using a TIDE analysis, which can be used to identify highly efficient gRNA molecules for editing a target gene. In some embodiments, a highly efficient gRNA can yield a gene editing frequency of higher than 80%. For example, a gRNA can be considered to be highly efficient if it can yield a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.Other Gene Editing Methods

[0244] 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 activatorlike effector nucleases (TALEN), restriction endonucleases, meganucleases homing endonucleases, or the like.

[0245] 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 domainnot 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.

[0246] 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.

[0247] Additional examples of targeted nucleases suitable for use can include, but not limited to, Bxbl, phiC31, PhiBTl, and Wp / 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 Bxb l integrase can recognize a specific 48 base-pair sequence within the attachment sites. The phiC31 nuclease, also known as the phiC31 integrase, is derived from the bacteriophage phiC31. The phiC31 nuclease can catalyze site-specific recombination between two specific DNA sequences, referred to as attB (attachment site in bacteriophage) and attP (attachment site in the phage). The phiC31 nuclease can promote integration of a DNA fragment flanked by attB and attP into the genome in cells of target organ. The phiBTl nuclease can integrate into a different attachment site than phiC31. The Wp / SPBc / TP901-l nuclease, also known as bacteriophage P2Bxbl Cre nuclease, is a site-specific recombination enzyme derived from the temperate bacteriophage P2.Lipids

[0248] The present disclosure contemplates LNP compositions comprising a polynucleotide and a mixture of lipids for delivery to a host cell. Exemplary lipids contemplated for the LNP compositions described herein comprise ionizable cationic lipids, selective organ targeting lipids, helper lipids, structural lipids and polyethylene glycol-conjugated lipids (PEG-lipids).Ionizable cationic lipids

[0249] In some embodiments, the lipid composition comprises an ionizable cationic lipid. In some embodiments, the ionizable cationic lipids contains 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-C24 alkyl or alkenyl carbon groups. These lipid groups may be attached through an ester linkage or may be further added through a Michael addition to a sulfur atom. In some embodiments, these compounds may be a dendrimer, a dendron, a polymer, or a combination thereof.

[0250] A lipid nanoparticle composition 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), N 1 -[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]-dioxolane (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-di oleyloxy -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)-cholest-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-6,1-diyl)bis(2-hexyldecanoate (ALC-0315), or heptadecan-9-yl 8-((2-hydroxy ethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM-102). In addition to these, an ionizable cationic lipid may also be a lipid including a cyclic amine group.

[0251] 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, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373, WO2015199952, W02015074085, 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.

[0252] 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.

[0253] 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. In some embodiments,the ionizable cationic lipid exhibits low toxicity in vitro. In some embodiments, the ionizable cationic lipid exhibits low toxicity in vivo. In some embodiments, the ionizable cationic lipid exhibits liver-specific delivery in vivo.

[0254] 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.

[0255] 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.

[0256] 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-C24 alkyl or alkenyl groups.

[0257] In some embodiments, an ionizable cationic lipid comprises between 2 and 6 hydrophobic tails, often alkyl or alkenyl such as C6-C24 alkyl or alkenyl groups, but may have at least 1, at least 2, at least 3, at least 4, at least 5, or more than 6 tails.Compounds of Formula (D-I)

[0258] In some embodiments of the lipid composition, the ionizable cationic lipid comprises at least two C8-C24 alkyl groups. In some embodiments, the ionizable cationic lipid is 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:Xi is amino or C1-C12 alkylamino, Ci-C 12 dialkylamino, C3-C12 heterocycloalkyl, C5-C12 heteroaryl, or a substituted version thereof;Ri is amino, hydroxy, mercapto, C1-C12 alkylamino, or C1-C12 dialkylamino, or a substituted version of either of these groups; anda is 1, 2, 3, 4, 5, or 6; orthe core has the formula:X2 •4 R2^HID)wherein:X2is N(R5)y;R5 is hydrogen, Ci-Cis alkyl, or substituted Ci-Cis alkyl; andy is 0, 1, or 2, provided that the sum of y and z is 3;R2 is amino, hydroxy, mercapto, C1-C12 alkylamino, or C1-C12 dialkylamino, or a substituted version of either of these groups;b is 1, 2, 3, 4, 5, or 6; andz is 1, 2, or 3; provided that the sum of z and y is 3; orthe core has the formula:R3^X3^R4V / c Ud (D-IV)wherein:X3 is -NRe- wherein Re is hydrogen, Ci-Cs alkyl, or Ci-Cs substituted alkyl, -O-, or Ci-Cs alkylaminodiyl, Ci-Cs alkoxydiyl, Ce-Cs arenediyl, Cs-Cs heteroarenediyl, C3-C8 heterocycloalkanediyl, or a substituted version of any of these groups;R3 and R4 are each independently amino, hydroxy, mercapto, C1-C12 alkylamino, or C1-C12 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 Rf are each independently hydrogen, Ci-Ce alkyl, or substituted Ci-Ce alkyl;c and d are each independently 1, 2, 3, 4, 5, or 6; or the core is Ci-Cis alkylamine, C1-C36 dialkylamine, C3-C12 heterocycloalkane, 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:Ai and A2are each independently -O-, -S-, or -NRa- wherein:Ra is hydrogen, Ci-Ce alkyl, or substituted Ci-Ce alkyl;Y3 is C1-C12 alkanediyl, C1-C12 alkenediyl, C6-C12 arenediyl, or a substituted version of any of these groups; or a group of the formula:wherein:X3 and X4 are C1-C12 alkanediyl, C2-C12 alkenediyl, C6-C12 arenediyl, or a substituted version of any of these groups;Ys is a covalent bond, C1-C12 alkanediyl, C1-C12 alkenediyl, C6-C12 arenediyl, or a substituted version of any of these groups; andR9 is Ci-Cs alkyl or substituted Ci-Cs alkyl;the linker group has the formula:wherein:Yi is C1-C12 alkanediyl, C1-C12 alkenediyl, C6-C12 arenediyl, 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; andthe terminating group has the formula:wherein:Y4 is alkanediyl or an Ci-Cis alkanediyl wherein one or more of the hydrogen atoms on the Ci-Cis alkanediyl has been replaced with -OH, -F, -Cl, -Br, I, SH, OCH3, OCH2CH3, SCH3, or OC(O)CH3;RIO is hydrogen, carboxy, hydroxy,Ce-Ci2aryl, C1-C12 alkylamino, Ci-C 12 dialkylamino, C3-Ci2Y-heterocycloalkyl, -C(O)N(Rn)- Ci-C6alkanediyl- C3-C12 heterocycloalkyl, -C(O)- C1-C12 alkylamino, -C(O)- C1-C12 dialkylamino, or -C(O)- C3-C12 V- heterocycloalkyl, wherein:R11 is hydrogen, Ci-Ce alkyl, or substituted Ci-Ce alkyl;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.

[0259] In some embodiments, the terminating group is further defined by the formula:wherein:Y4 is Ci -Cig alkanediyl; andRio is hydrogen. In some embodiments, Ai and A2are each independently ~O~ or -NRa-

[0260] In some embodiments of the dendrimer of formula (D-I), the terminating group is a structure selected from the structures in Table 4.

[0261] In some embodiments of the dendrimer of formula (D-I), the core is further defined by the formula:X 4 / R2^H2l(D.in)wherein:X2is N(R5)y;Rs is hydrogen or Ci-Cs alkyl, or substituted Ci-Cis alkyl; andy is 0, 1, or 2, provided that the sum of y and z is 3;R2 is amino, hydroxy, or mercapto, or C1-C12 alkylamino, C1-C12 dialkylamino, or a substituted version of either of these groups;b is 1, 2, 3, 4, 5, or 6; andz is 1, 2, 3; provided that the sum of z and y is 3.

[0262] In some embodiments of the dendrimer of formula (D-I), the core is further defined by the formula:wherein:X3 is -NRe-, wherein Ro is hydrogen, Ci-Cg alkyl, or substituted Ci-Cg alkyl, -Q-, or Ci- Cg alkylaminodiyl, Ci-Cg alkoxydiyl, Ci-Cg arenediyl, Ci-Cg heteroarenediyl, Ci- Cg heterocycloalkanediyl, or a substituted version of any of these groups;R3 and R4 are each independently amino, hydroxy, or mercapto, or C1-C12 alkylamino, dialkylamino, or a substituted version of either of these groups; or a group of thewherein:e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3;Rc, Rd, and Rf are each independently hydrogen, Ci-Ce alkyl, or substituted Ci-C6alkyl;c and d are each independently 1, 2, 3, 4, 5, or 6.

[0263] In some embodiments of the dendrimer of formula (I), the terminating group is represented by the formula:wherein:Y4 is alkanediyl(c<is); andRio is hydrogen.

[0264] 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.

[0265] In some embodiments of the dendrimer of formula (D-I), the core comprises a structural formula set forth in Table 3 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).

[0266] In some embodiments of the dendrimer of formula (D-I), the degradable diacyl is further defined as:wherein Yi is Ci-Cs alkanediyl or substituted C1-C12 alkanediyl.

[0268] In some embodiments, in the core of formula (D-IV), Re is H. In some embodiments, in the core of formula (D-IV), Re is Ci-Cs alkyl. In some embodiments, in the core of formula (D-IV), Re is substituted alkyl (e.g, alkyl substituted with -NH2, alkyl substituted with -NHCH3, or alkyl substituted with -NHCH2CH3).

[0269] In s ome 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.

[0270] In some embodiments, of the compound of the disclosure, the core of Formula D-II, D-III, or D-IV has a structure of Table 3 and the terminating group of formula D-VII has astructure of Table 4. In each of the structures of Tables 3 and 4, -"" denotes a point of oattachment to the following structure: wherein the core of Table 3 and the terminating group of Table 4 are attached at opposite ends of the structure.Table 3. Example core structuresTable 4. Example terminating group structures

[0271] In some embodiments, the ionizable cationic lipid is a compound of Formula A:pharmaceutically acceptable salt thereof, wherein:RDlais a C1-C4 alkyl;RD2ais H or a C1-C4 alkyl;zla and z2a are each independently 1, 2, or 3;z3a is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; andz4a is 0 or 1

[0272] In some embodiments of the compound of Formula A, RD1ais methyl.

[0273] In some embodiments of the compound of Formula A, RD2ais methyl. In some embodiments of the compound of Formula A, RD2ais H.

[0274] In some embodiments of the compound of Formula A, zla and z2a are each 1. In some embodiments of the compound of Formula A, zla and z2a are each 2.

[0275] In some embodiments of the compound of Formula A, z3a is 6, 7, or 8.

[0276] In some embodiments of the compound of Formula A, z4a is 0. In some embodiments of the compound of Formula A, z4a is 1.

[0277] In some embodiments, the ionizable cationic lipid is a compound of Formula B:pharmaceutically acceptable salt thereof, whereinXb and Yb are each independently N or CH;zlb, z2b, z3b, z4b are each independently 1, 2, or 3; andz5b is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0278] In some embodiments of the compound of Formula B, zlb, z2b, z3b, and z4b are each 1.

[0279] In some embodiments of the compound of Formula B, z5b is 6, 7, or 8.

[0280] In some embodiments of the compound of Formula B, Xb is N.

[0281] In some embodiments of the compound of Formula B, Yb is N.

[0282] In some embodiments, the ionizable cationic lipid is selected from those set forth in Table 5 and pharmaceutically acceptable salts thereof.Table 5. Example ionizable cationic lipid compoundsAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOAttorney Docket No. 061529-515001WOOther Ionizable cationic lipids

[0283] In some embodiments of the lipid composition, the ionizable cationic lipid comprises a structural formula (D-P):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-C18 alkyl, C3-C18 alkenyl 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-C18 alkyl or C3-C18 alkenyl having one C=C double bond; andwherein one or more of the nitrogen atoms indicated in formula (D-P) may be protonated to provide an ionizable cationic lipid.

[0284] In some embodiments of the ionizable cationic lipid of formula (D-P), a is 1. In some embodiments of the ionizable cationic lipid of formula (D-P), b is 2. In some embodiments ofthe 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, R and R6are each OHindependently H or. In some embodiments of the ionizable cationic lipid of formula OH(D-I’), R1, R2, R3, R4, R\ and R6are each independently H or. In some embodiments of the ionizable cationic lipid of formula (D-I’), R7is C3-C18 alkyl (e.g., Ce-Cn alkyl).

[0285] In some embodiments, the ionizable cationic lipid of formula (D-I’) is 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-ll,25-diol:

[0286] In some embodiments, the ionizable cationic lipid of formula (D-T) is (117?, 257?)- 13,16, 20-tris((?)-2-hy droxy dodecyl)- 13,16,20,23 -tetraazapentatricontane- 11,25-diol:

[0287] Additional ionizable cationic lipids that can be used in the compositions and methods described herein 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 WO2015095340, which are incorporated herein by reference for all purposes. Examples of those ionizable cationic lipids include but are not limited to those as shown in Table 6.Table 6. Example Ionizable Cationic Lipids

[0288] In some embodiments, the ionizable cationic lipid is a dendrimer lipid. In some embodiments, the ionizable cationic lipid is a dendrimer lipid according to Formula (I) or Formula (X). In some embodiments, the ionizable cationic lipid is 4A3-SC7. In some embodiments, the ionizable cationic lipid is 5A2-SC8. In some embodiments, the ionizable cationic lipid is present in the composition at a molar percentage from about 10% to about 35%.

[0289] In some embodiments of the lipid composition, 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%.

[0290] In some embodiments of the lipid composition, 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% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 10% to about 25%.

[0291] In some embodiments of the lipid composition, 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, 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%.Selective organ targeting (SORI) lipids

[0292] The lipid component of the lipid compositions disclosed herein includes a second (i.e., (“additional”) separate from the first lipid wherein the second lipid is an ionizable cationic lipid, or permanently cationic lipid. In some embodiments of the lipid composition, the lipid (e.g., nanoparticle) composition is preferentially delivered to a target organ.

[0293] In some embodiments, the second comprises 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 second (e.g., SORT lipid) comprises a positive charge upon delivery of a polynucleotide to a cell. In some embodiments the positively charged moiety is quaternary amine or quaternary ammonium ion. In some embodiments, the second (e.g., SORT lipid) comprises, or is otherwise complexed to or interacting with, a counterion.

[0294] In some embodiments, the second is a permanently cationic lipid (i.e., comprising one or more hydrophobic components and a permanently cationic group). The permanently cationic lipid may contain a group which has a positive charge regardless of the pH. One permanently cationic group that may be used in the permanently cationic lipid is a quaternary ammoniumgroup. The permanently cationic lipid may comprise a structural formula:wherein:Yi, Y2, or Y3 are each independently XiC(O)Ri or X2N+R3R4Rs;provided at least one of Yi, Y2, and Y3 is X2N R3R4R5;Ri is C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 alkenyl, C1-C24 substituted alkenyl; Xi is O or NRa, wherein Rais hydrogen, C1-C4 alkyl, or C1-C4 substituted alkyl;X2 is Ci-Cg alkanediyl or Ci-Ce substituted alkanediyl;R3, R4, and Rs are each independently C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 alkenyl, C1-C24 substituted alkenyl; andAi is an anion with a charge equal to the number of X2N R R4R5 groups in the compound.

[0295] In some embodiments, the permanently cationic second (e.g., SORT lipid) has aR°'N. A2Rstructural formula:8(S-II), wherein:R6-R9 are each independently C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 alkenyl, Ci-C24 substituted alkenyl; provided at least one of R6-R9 is a group of C8-C24; andA2 is a monovalent anion.

[0296] In some embodiments, the permanently cationic lipids is l,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), l,2-dipalmitoyl-sn-glycero-3 -ethylphosphocholine (16:0 EPC), l,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), l,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), l,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), l,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), l,2-dioleoyl-3-trimethylammonium-propane (18:1 TAP, DOT AP), or l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).

[0297] In some embodiments, the SORT (additional) lipid is an ionizable cationic lipid (e.g., comprising one or more hydrophobic components and an ionizable group, e.g., a tertiary aminogroup). 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 (e.g., second(e.g., SORT lipid)) has a structural formula:wherein:Ri and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; andR3 and R3' are each independently Ci-Ce alkyl or substituted Ci-Ce alkyl.

[0298] In some embodiments of formula (S-I’a) Ri and R2 are each independently C8-C24 alkenyl (e.g., hexadecane, heptadecene, or octadecene). In some embodiments of formula (S-I’a), R3 and R3' are each independently Ci-Ce alkyl e.g., methyl or ethyl). In some embodiments of formula (S-I’a) Ri and R2 are each independently C8-C24 alkenyl, (e.g., hexadecane, heptadecene, or octadecene) and R3 and R3' are each independently Ci-Ce alkyl (e.g., methyl or ethyl).

[0299] In some embodiments, the ionizable cationic lipids is l,2-distearoyl-3-dimethylammonium-propane (18:0 DAP), l,2-dipalmitoyl-3 -dimethylammonium -propane (16:0 DAP), l,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), l,2-dioleoyl-3-dimethylammonium-propane (18:1 DAP, DODAP), or l,2-dioleyloxy-3 -dimethylaminopropane (DODMA).

[0300] In some embodiments, the additional ionizable cationic lipid or permanently cationic lipid comprises a head group of a particular structure. In some embodiments, the second (e.g., second e.g., SORT lipid)) comprises a headgroup having a structural formula:, wherein L is a linker; Z+is positively charged moiety and X’ is 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.

[0301] In some embodiments, the SORT (additional ionizable cationic lipid or permanentlycationic) lipid has a structural formula:, wherein R1and R2are each independently an optionally substituted C6-C24 alkyl, or an optionally substituted C6-C24 alkenyl.

[0302] In some embodiments, the second (e.g., second (e.g, SORT lipid)) has a structuralformula:, wherein R1and R2are each independently an optionally substituted C6-C24 alkyl, or an optionally substituted C6-C24 alkenyl, and R’, R”, and R”’ are an optionally substituted C1-C4 alkyl.

[0303] In some embodiments, the second (e.g., second e.g., SORT 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 Ci-Ce alkyl

[0304] In some embodiments, the second (e.g., additional (e.g., SORT lipid)) has a structuralformula:wherein:Ri and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group;R3, R3', and R3" are each independently Ci-Ce alkyl or substituted C1-C6 alkyl;R4 is Ci-Ce alkyl or substituted Ci-Ce alkyl; andX is a monovalent anion.

[0305] In some embodiments, the second (e.g., additional (e.g., SORT lipid)) is a phosphatidylcholine (e.g., 14:0 EPC). In some embodiments, the phosphatidylcholine compoundis further defined as:wherein:Ri and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group;R3, R3', and R3" are each independently Ci-Ce alkyl or substituted C1-C6 alkyl; and X is a monovalent anion.

[0306] In some embodiments, the second (e.g., additional (e.g., SORT lipid)) is a phosphocholine lipid. In some embodiments, the second (e.g., additional (e.g., SORT lipid)) is an ethylphosphocholine. The ethylphosphocholine may be, by way of example, without being limited to, l,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), l,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), l,2-dipalmitoyl-sn-glycero-3 -ethylphosphocholine (16:0 EPC), 1,2-dimyristoyl-sn-glycero-3 -ethylphosphocholine (14:0 EPC), l,2-dilauroyl-sn-glycero-3 -ethylphosphocholine (12:0 EPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -ethylphosphocholine (16:0-18:0 EPC).

[0307] In some embodiments, the lipid has a structural formula:wherein:Ri and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group;R3, R3', and R3" are each independently Ci-Ce alkyl or substituted Ci-Ce alkyl; X is a monovalent anion.

[0308] By way of example, and without being limited thereto, the second (e.g., additional (e.g., SORT lipid)) of the structural formula of the immediately preceding paragraph is l,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt).

[0309] In some embodiments, the second (e.g, additional (.g., SORT lipid)) has a structuralformula:wherein:R4 and 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(ci c8), alkenyl(C2-cs), or a substituted version of either group; andX2 is a monovalent anion.

[0310] By way of example, and without being limited thereto, the second (e.g., additional e.g., SORT lipid)) of the structural formula of the immediately preceding paragraph is dimethyldioctadecylammonium (DDAB).

[0311] In some embodiments, the second lipid (e.g., additional (e.g., SORT lipid)) isl,2-dioleoyl-sn-glycero-3 -phosphate (18:1 PA).

[0312] In any of the foregoing embodiments, X or X2 is selected from F, Cl", Br, and I".

[0313] In some embodiments, the additional lipid is a compound of Formula and a compound of formula I:wherein:RD1is a C1-C4 alkyl;RD2is H or a C1-C4 alkyl;zl and z2 are each independently 1, 2, or 3;z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; andz4 is 0 or 1.

[0314] In some embodiments of the compound of Formula I, RD1is methyl.

[0315] In some embodiments of the compound of Formula I, RD2is methyl. In some embodiments of the compound of Formula I, RD2is H.

[0316] In some embodiments of the compound of Formula I, zl and z2 are each 1. In some embodiments of the compound of Formula I, zl and z2 are each 2.

[0317] In some embodiments of the compound of Formula I, z3 is 6, 7, or 8.

[0318] In some embodiments of the compound of Formula I, z4 is 0. In some embodiments of the compound of Formula I, z4 is 1.

[0319] In some embodiments, the second is selected from the lipids set forth in Table 7 and Table 8Table 7. Example Second Lipids (e.g., additional lipids (e.g., SORT lipids))X' is a counterion (e.g., Cl", Br", etc.)Table 8. Example Second Lipids (e.g., additional lipids (e.g., SORT lipids))

[0320] In some embodiments, the permanently cationic lipid is selected from 1,2-dilauroyl-sn-glycero-3 -ethylphosphocholine (12:0 EPC), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), l,2-dipalmitoyl-sn-glycero-3 -ethylphosphocholine (16:0 EPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), l,2-dioleoyl-sn-glycero-3 -ethylphosphocholine (18:1 EPC), l-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), l,2-dimyristoyl-3-trimethylammonium-propane(14:0 TAP), l,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), l,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), l,2-dioleoyl-3-trimethylammonium-propane (18:1 TAP, DOT AP), or 1,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA). In some embodiments, the ethylphosphocholine is l,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC) or 2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC).

[0321] In some embodiments, the second lipid (e.g., additional lipid, e.g. SORT lipid) is 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), l,2-dipalmitoyl-sn-glycero-3 -ethylphosphocholine (16:0 EPC), 1.2-distearoyl-sn-glycero-3 -ethylphosphocholine (18:0 EPC), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:0 EPC), l,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC), Dimethyldioctadecylammonium (18:0 DDAB), l,2-dimyristoyl-3 -trimethylammonium -propane(14:0 TAP), l,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), l,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), l,2-dioleoyl-3-trimethylammonium-propane (18:1 TAP, DOTAP), or l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-sn-glycero-3 -phosphate (18:1 PA), l,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), 1.2-dipalmitoyl-sn-glycero-3 -phosphate (16:0 PA), l,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), or l,2-dilauroyl-sn-glycero-3 -phosphate (12:0 PA).

[0322] In some embodiments, the second lipid (e.g., additional lipid, e.g. SORT lipid) selected from the group shown in Table 7 and Table 8. In some embodiments, the second lipid (e.g., additional lipid, e.g. SORT lipid) is present in the composition at a molar percentage from about 5% to about 65%. In some embodiments, the second lipid (e.g., additional lipid, e.g. SORT lipid) is present in the composition at a molar percentage from about 5% to about 35%.

[0323] In some embodiments of the lipid composition, the second lipid (e.g., additional lipid, e.g. SORT lipid) is present in the composition at a molar percentage about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.

[0324] In some embodiments of the lipid composition, the second lipid (e.g., additional lipid, e.g. SORT lipid) is present in the composition at a molar percentage from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 5% to about 20%, from about 5% to about 10%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 20% to about 25%.

[0325] In some embodiments of the lipid composition, the second lipid (e.g., additional lipid, e.g. SORT lipid) is present at a molar percentage of at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, or at least (about) 55%. In some embodiments of the lipid composition, 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%.Phospholipids

[0326] Phospholipids, as defined herein, are any 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 morelipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. 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, alphalinolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0327] Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.., a dye).

[0328] Phospholipids useful or potentially useful in the compositions and methods may be selected from: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3 -phosphocholine (C16 LysoPC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), 1,2-diphytanoyl-sn-glycero-3 -phosphocholine (4ME 16:0 PC), l,2-diphytanoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) (4ME 16:0 PG), l,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, and l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin.

[0329] 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 di stearoylphosphatidyl choline 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.

[0330] In some embodiments, the phospholipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC).

[0331] In some embodiments, the phospholipid is present in the composition at a molar percentage from about 5% to about 35%.

[0332] In some embodiments of the lipid composition, the phospholipid is present in the composition at a molar percentage from about 7.5% to about 30%.

[0333] In some embodiments of the lipid composition, the phospholipid 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%.

[0334] In some embodiments of the lipid composition, the phospholipid is present in the composition at a molar percentage 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 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%,from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 10% to about 25%.

[0335] In some embodiments of the lipid composition, the phospholipid 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, 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%.Structural lipids

[0336] 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, 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 afused cyclopentyl ring as shown in the formula: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 furtherdefined as:some embodiments, the steroid or steroid derivative is a cholestane or cholestane derivative. In a cholestane, the ring structure is further defined by theformula:described above, a cholestane derivative includes 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.

[0337] Sterols useful in the compositions and methods described herein may be selected from: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.

[0338] In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is sitosterol. In some embodiments, the cholesterol is present in the composition at a molar percentage from about 20% to about 50%.

[0339] In some embodiments of the lipid composition, 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%.

[0340] In some embodiments of the lipid composition, the sterol is present in the composition at a molar percentage from about 10% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 20% to about 25%, from about 25% to about 50%, from about 25% to about 40%, from about 25% to about 30%, from about 30% to about 50%, from about 30% to about 40%, from about 30% to about 35%, from about 35% to about 50%, from about 35% to about 45%, from about 35% to about 40%, from about 40% to about 50%, from about 40% to about 45%, or from about 45% to about 50%.

[0341] In some embodiments of the lipid composition, 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, theionizable 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 lipids (PEG-lipids)

[0342] 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 IntT Pat. Pub. No. WO2012099755 and U. S. Pat. Pub No. 2014 / 0200257.

[0343] 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.

[0344] In one embodiment, PEG-lipids can be PEG-lipids described in IntT Pat. Pub. No.WO 2012 / 099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG-lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG-lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” is a PEG-lipid having one or more hydroxyl ( — OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes 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.

[0345] In some embodiments of the lipid composition, 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-C24 long chain alkyl or alkenyl group or a C6-C24 fatty acid group attached to a linker group with a PEG chain. Some non-limiting examples of a PEG-lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified l,2-diacyloxypropan-3 -amines, PEG modified diacylglycerolsand dialkylglycerols. In some embodiments, PEG modified diastearoylphosphatidylethanolamine or PEG modified dimyristoyl- n-glycerol. 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, to about 15,000. Some non-limiting examples of lipids that may be used 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.

[0346] In some embodiments of the lipid composition, the PEG-lipid has a structural formula:, wherein: R12 and R13 are each independently alkyl(cs24), alkenyl(cs24), or a substituted version of either of these groups; Re is hydrogen, alkyl(c≤8), or substituted alkyl(c≤8); and x is 1-250. In some embodiments, Reis alkyl(c s) such as methyl. R12 and R13 are each independently alkyl(c 4-20). In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG-lipid is 1,2-dimyristoyl- s / z-glycerol, methoxypolyethylene glycol.

[0347] In some embodiments of the lipid composition, the PEG-lipid has a structural formula:, wherein: m is an integer between 1 and 100 and n2 and ns are each independently selected from an integer between 1 and 29. In some embodiments, m is 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 derivabletherein. In some embodiments, is from about 30 to about 50. In some embodiments, n2 is from 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, m is from 5 to 23. In some embodiments, ns is 11 to about 17.

[0348] In some embodiments of the lipid composition, the PEG-lipid is present in the composition at a molar percentage from about 0.5% to about 10%.

[0349] In some embodiments of the lipid composition, the PEG-lipid is present in the composition at a molar percentage of about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0350] In some embodiments of the lipid composition, 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%.

[0351] In some embodiments of the lipid composition, the PEG-lipid is present at a molar percentage of at least (about) 0.5%, at least (about) 1%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, or at least (about) 3.5%. In some embodiments of the lipid composition, 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%.

[0352] In some embodiments, the LNP composition comprises mRNA at a lipid:mRNA (weight / weight) ratio between 5:1 and 40:1. In some embodiments, the LNP composition 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, or between 5:1 and 45:1.

[0353] In some embodiments, the composition is capable of delivering mRNA to a cell in the central nervous system (CNS) of a subject in an amount effective to increase expression and / or function of a gene encoded by the mRNA. In some embodiments, the cell is an endothelial cell. In some embodiments, the cell is a pericyte. In some embodiments, the cell is a smooth muscle cell (SMA).III. Pharmaceutical Compositions

[0354] The disclosure also provides pharmaceutical compositions for the LNP compositions described herein. Such pharmaceutical compositions can be used for generating an immune response against an infectious disease in a subject. The pharmaceutical compositions of the disclosure may include a pharmaceutically acceptable carrier. A thorough discussion of such carriers is available in Chapter 30 of Remington: The Science and Practice of Pharmacy (23rded., 2021).

[0355] In some embodiments, the pharmaceutical composition comprises Tris buffer, optionally at a pH from 6-9. In some embodiments, the pharmaceutical composition comprises sucrose, optionally at 5-15%. In some embodiments, the pharmaceutical composition comprises citrate buffer, optionally at a pH 4-6. In some embodiments, the pharmaceutical composition comprises 15mM Tris buffer, optionally at a pH from 6-9, and / or 5-15% sucrose. In some embodiments, the composition comprises lOmM citrate buffer, optionally at a pH from 4-6.

[0356] In some embodiments, the pharmaceutical compositions include one or more of a poloxamer (e.g., Poloxamer 188) polyethylene glycol (“PEG”), sucrose, and a buffer, wherein the buffer comprises a citrate buffer, an acetate buffer, or a Tris buffer.

[0357] In some embodiments, the pharmaceutical composition comprises a citrate buffer. For example, the citrate buffer is at a pH from 4 to 8. In some embodiments, the buffer is an acetate buffer and has a pH from 4 to 8. In some embodiments, the pharmaceutical composition comprises a Tris buffer, and the Tris buffer has a pH from 4 to 8.

[0358] In some embodiments, the pharmaceutical composition comprises sucrose. In some embodiments, the sucrose is at a concentration from 1% to 15% w / v, 5% to 15% w / v, 1% to 10% w / v, or 5% to 10% w / v.

[0359] In some embodiments, the pharmaceutical composition can also include excipients and / or additives. Examples of these are surfactants, stabilizers, complexing agents, antioxidants, or preservatives which prolong the duration of use of the finished pharmaceutical formulation, flavorings, vitamins, or other additives known in the art. Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof. In some embodiments, preservatives include, but are not limited to, those that protect the solution from contamination with pathogenic particles, including benzalkonium chloride or benzoic acid, or benzoates such as sodium benzoate. Antioxidants include, but are not limited to, vitamins, provitamins, ascorbic acid, vitamin E, salts or esters thereof.

[0360] Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fdlers, 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 pharmaceutical excipients are useful in the present disclosure.

[0361] In some embodiments, one or more tonicity agents may be added to provide the desired ionic strength. Tonicity agents for use herein include those which display no or only negligible pharmacological activity after administration. Both inorganic and organic tonicity adjusting agents may be used.

[0362] In another aspect, the disclosure provides a pharmaceutical composition comprising the composition described herein and a pharmaceutically acceptable excipient and / or diluent. In another aspect, the disclosure provides a kit comprising the composition described herein.IV. Methods of Administration

[0363] In another aspect, the disclosure provides methods of administration for the LNP composition, or the pharmaceutical composition described herein.

[0364] In some embodiments, an LNP composition or pharmaceutical composition described herein is administered intravenously (I. V.). In some embodiments, an LNP composition or pharmaceutical composition described herein is administered intrathecally (LT.). In some embodiments an LNP composition or pharmaceutical composition described herein is administered intramuscularly (I. M.). In some embodiments, an LNP composition or pharmaceutical composition described herein is administered intradermally (ID ). In some embodiments, an LNP composition or pharmaceutical composition described herein is administered by intra-cistema magna (ICM) delivery. In some embodiments an LNP composition or pharmaceutical composition described herein is administered intranasally. In some embodiments, an LNP composition or pharmaceutical composition described herein is administered by inhalation, e.g, of a nebulized pharmaceutical composition or LNP composition. In some embodiments, an LNP composition or pharmaceutical composition described herein is administered via injection.

[0365] In some embodiments, an LNP composition or pharmaceutical composition described herein is administered ata dose of 1 mg per kg of brain weight. In some embodiments, an LNP composition or pharmaceutical composition described herein is administered at a dose of 2 mg per kg of brain weight. In some embodiments, an LNP composition or pharmaceutical composition described herein is administered at a dose of 3 mg per kg of brain weight.

[0366] In some embodiments, the microglial transfection efficiency is about 5% to 10%.Microglial targeting and biodistribution can be validated by single cell RNA sequencing, RNA in situ hybridization, or immunohistochemistry. In some embodiments, the microglial transfection efficiency is about 7% to 15%. In some embodiments, the microglial transfection efficiency is about 12% to 20%.

[0367] In some embodiments, the administration may be given pre-diagnosis. In some embodiments, the administration may be given post-diagnosis.

[0368] In some embodiments, the administration is a single administration. In some embodiments, the administration is a multiple administration. In some embodiments, the multiple administrations occur three times a day, twice a day, once a day, every other day, every third day, weekly, biweekly, every three weeks, every four weeks, or monthly.V. Methods of Use

[0369] In another aspect, the disclosure provides a method for treating and / or preventing a CNS disease in a subject in need thereof, wherein the method comprises administering the composition described herein to the subject by intrathecal injection.

[0370] In some embodiments of the method of the present disclosure, the composition comprises a payload wherein the payload is a messenger RNA (mRNA) and wherein the method results in delivery of the payload to the CNS in an amount effective to increase expression and / or function of a gene encoded by the mRNA.

[0371] In some embodiments, the method results in expression of a polypeptide in a CNS of the subject. In some embodiments, the method results in expression of the polypeptide in the CNS of the subject between 1 and 72 hours after administration of the composition to the subject.

[0372] In some embodiments of the method of the present disclosure, the CNS disease is Abulia, Achromatopsia, acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Agraphia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, Akinetopsia, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Amnesia, anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartylglucosaminuria, Asperger syndrome, ataxia, ataxia telangiectasia (Louis-Bar syndrome), ataxias and cerebellar or spinocerebellar degeneration, attention deficit- hyperactivity disorder, autism, autonomic dysfunction, Barth syndrome, Batten disease, Becker's myotonia, Behcet's disease, Bell's palsy, Bernhardt-Roth syndrome, Bipolar disorder, Binswanger's disease, Bloch-Sulzberger syndrome, Bradbury-Eggleston syndrome, Brown-Sequard syndrome, bulbospinal muscular atrophy, CADASIL, Canavan's disease, Carpal tunnel syndrome, causalgia, cavernomas, cavernous angioma, central cervical cord syndrome, central cord syndrome, central pontine myelinolysis, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral beriberi, cerebral gigantism, cerebral palsy, cerebro-oculo-facio-skeletal syndrome (COFS), Charles bonnet syndrome, cholesterol ester storage disease, chorea, choreoacanthocytosis, chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic fatigue syndrome, chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, colpocephaly, congenital myasthenia, corticobasal degeneration, cranial arteritis, cree encephalitis, Creutzfeldt-lakob disease, Cushing's syndrome, cystinosis, cytomegalic inclusion body disease, Dancing eyes-dancing feet syndrome, Dandy-Walker syndrome, Danon disease, Dawson disease, De Morsier's syndrome, Dejerine-Klumpke palsy, dementia, dentate cerebellar ataxia, dentatorubral atrophy, Dermatillomania, dermatomyositis, developmental dyspraxia, Devic's syndrome, diffuse sclerosis, Down syndrome, Duchenne muscular dystrophy, dysautonomia, dysgraphia, dyslexia, dysphagia, dyspraxia, dyssynergia cerebellaris myoclonica, dyssynergia cerebellaris progressiva, Epilepsy (such as, e.g., Amish infantile epilepsy syndrome (AIES), benign familial infantile seizures (BFIS), benign familial neonatal seizures (BFNS), childhood absence epilepsy (CAE), childhood-onset epileptic encephalopathy (COEE), Dravet syndrome (DS), early infantile epileptic encephalopathy (EIEE), Exploding head syndrome, familial adult myoclonic epilepsy (FAME), familial febrile seizures (FFS), familial focal epilepsy with variable foci (FFEVF), familial infantile myoclonic epilepsy (FIME), familial temporal lobe epilepsy (FTLE), focal epilepsy and speech disorder (FESD) with or without mental retardation, generalized epilepsy and paroxysmal dyskinesia (GEPD), generalized epilepsy with febrile seizures plus (GEFS+), idiopathic generalized epilepsy (IGE), juvenile absence epilepsy (IAE), juvenile myoclonic epilepsy (JME), myoclonic-atonic epilepsy (MAE), nocturnal frontal lobe epilepsy (NFLE), progressive myoclonic epilepsy (PME), pyridoxamine 5'-phosphate oxidase deficiency (PNPOD), pyridoxine-dependent epilepsy (EPD) and severe myoclonic epilepsy of infancy (SMEI)), Fabry disease, Fahr's syndrome, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralyses, familial spastic paralysis, Farber's disease, Fetal alcohol syndrome, fibromuscular dysplasia, Fisher syndrome, floppy infant syndrome, Foville's syndrome, Friedreich's ataxia, frontotemporal dementia, fucosidosis,galactosialidosis, Gaucher disease, generalized gangliosidosis, Gerstmann's syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 gangliosidosis, GM2 gangliosidosis (Tay-Sachs disease), Gray matter heterotopia, Guillain-Barre syndrome, Hallervorden-Spatz disease, hemicrania continua, hemiplegia alterans, hereditary spastic paraplegia, heredopathia atactica polyneuritiformis, Herpes zoster, Hirayama disease, Holmes-Adie syndrome, holoprosencephaly, Hughes syndrome, Huntington's disease, hydranencephaly, hydromyelia, hypercortisolism, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, iniencephaly, Isaac's syndrome, Isodicentric 15, Joubert syndrome, Karak syndrome, Keams-Sayre syndrome, Kennedy's disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Kliiver-Bucy syndrome, Korsakoff s amnesic syndrome, Krabbe disease, Kugelberg- Welander disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, lateral medullary syndrome, Leigh's disease, Lennox -Gastaut syndrome, Lesch-Nyhan syndrome, Levine-Critchley syndrome, Lewy body dementia, lipoid proteinosis, lissencephaly, locked-in syndrome, Lou Gehrig's disease, lupus, Lyme disease, Machado-Joseph disease, macrencephaly, alpha-mannosidosis, beta-mannosidosis, Melkersson-Rosenthal syndrome, Menkes disease, meralgia paresthetica, metachromatic leukodystrophy, microcephaly, Miller Fisher syndrome, Misophonia, Moebius syndrome, Moyamoya disease, mucopolysaccharidosis type LH (Hurler syndrome), mucopolysaccharidosis type LH / S (Hurler-Scheie syndrome), mucopolysaccharidosis type IS (Scheie syndrome), mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type IILA (Sanfilippo syndrome A), mucopolysaccharidosis type III-B (Sanfilippo syndrome B), mucopolysaccharidosis type III-C (Sanfilippo syndrome C), mucopolysaccharidosis type III-D (Sanfilippo syndrome D), mucopolysaccharidosis type IV-B (Morquio syndrome B), mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), mucopolysaccharidosis type VII (Sly syndrome), mucopolysaccharidosis type IX (Natowicz syndrome), multiple sclerosis, muscular dystrophy, myasthenia gravis, myelinoclastic diffuse sclerosis, narcolepsy, neuroacanthocytosis, neurofibromatosis, neuroleptic malignant syndrome, neurosarcoidosis, Niemann-Pick disease, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus myoclonus, O'Sullivan-McLeodsyndrome, Otosclerosis, Overuse syndrome, pantothenate kinase- associated neurodegeneration, paraneoplastic syndromes, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal hemicrania, Parry-Romberg syndrome, Pelizaeus-Merzbacher disease, Pena Shokeir II syndrome, periventricular leukomalacia, phytanic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pompe disease, post-polio syndrome, posterior cortical atrophy, Prader-Willi syndrome, primary dentatum atrophy, primary lateral sclerosis, primary progressive aphasia, prion diseases, progressive hemifacial atrophy, progressive locomotor ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, Quadriplegia, Rabies, Ramsay Hunt syndrome I, Ramsay Hunt syndrome II, Rasmussen's encephalitis, Refsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhoff disease, Schilder's disease, Seitelberger disease, Shingles, Shy-Drager syndrome, Sjogren's syndrome, spasticity, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, striatonigral degeneration, Sturge-Weber syndrome, tardive dyskinesia, tauopathy, Tay-Sachs disease, Tetanus, thoracic outlet syndrome, thyrotoxic myopathy, tic douloureux, Todd's paralysis, trigeminal neuralgia, tropical spastic paraparesis, Troyer syndrome, Unverricht-Lundborg disease, vascular dementia, Vertigo, Von Economo's disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's disease, Wallenberg's syndrome, Werdnig-Hoffman disease, Wernicke-Korsakoff syndrome, West syndrome, Whipple's disease, Williams syndrome, Wilson disease, Wolman's disease, X-linked spinal and bulbar muscular atrophy, Y-linked hearing impairment, Zellweger syndrome, multiple sclerosis atrophy, Lewis body dementia (LBD), or Angelman syndrome.

[0373] In another aspect, the disclosure provides a method of delivering a payload to a cell in a CNS of a subject, wherein the method comprises administering to the subject, by intrathecal injection, the composition described herein.

[0374] In another aspect, the disclosure provides use of the composition described herein for treatment of a CNS disease. In another aspect, the disclosure provides use of the composition described herein for treatment of a CNS disease by intrathecal administration.

[0375] In another aspect, the disclosure provides the composition described herein or the pharmaceutical composition described herein for use in the treatment of a CNS disease in a subject in need thereof.

[0376] In some embodiments of the use or composition-for-use, the CNS disease is acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartylglucosaminuria, Asperger syndrome, ataxia, ataxia telangiectasia (Louis-Bar syndrome), ataxias and cerebellar or spinocerebellar degeneration, attention deficit- hyperactivity disorder, autism, autonomic dysfunction, Barth syndrome, Batten disease, Becker's myotonia, Behcet's disease, Bell's palsy, Bernhardt-Roth syndrome, Binswanger's disease, Bloch-Sulzberger syndrome, Bradbury-Eggleston syndrome, Brown-Sequard syndrome, bulbospinal muscular atrophy, CADASIL, Canavan's disease, causalgia, cavernomas, cavernous angioma, central cervical cord syndrome, central cord syndrome, central pontine myelinolysis, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral beriberi, cerebral gigantism, cerebral palsy, cerebro-oculo-facio-skeletal syndrome (COFS), cholesterol ester storage disease, chorea, choreoacanthocytosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, colpocephaly, congenital myasthenia, corticobasal degeneration, cranial arteritis, cree encephalitis, Creutzfeldt- Jakob disease, Cushing's syndrome, cystinosis, cytomegalic inclusion body disease, dancing eyes-dancing feet syndrome, Dandy-Walker syndrome, Danon disease, Dawson disease, De Morsier's syndrome, Dejerine-Klumpke palsy, dementia, dentate cerebellar ataxia, dentatorubral atrophy, dermatomyositis, developmental dyspraxia, Devic's syndrome, diffuse sclerosis, dysautonomia, dysgraphia, dyslexia, dysphagia, dyspraxia, dyssynergia cerebellaris myoclonica, dyssynergia cerebellaris progressiva, epilepsy (such as, e.g., Amish infantile epilepsy syndrome [AIES], benign familial infantile seizures [BFIS], benign familial neonatal seizures [BFNS], childhood absence epilepsy [CAE], childhood-onset epilepticencephalopathy [COEE], Dravet syndrome [DS], early infantile epileptic encephalopathy [EIEE], familial adult myoclonic epilepsy [FAME], familial febrile seizures [FFS], familial focal epilepsy with variable foci [FFEVF], familial infantile myoclonic epilepsy [FEME], familial temporal lobe epilepsy [FTLE], focal epilepsy and speech disorder [FESD] with or without mental retardation, generalized epilepsy and paroxysmal dyskinesia [GEPD], generalized epilepsy with febrile seizures plus [GEFS+], idiopathic generalized epilepsy [IGE], juvenile absence epilepsy [JAE], juvenile myoclonic epilepsy [JME], myoclonic-atonic epilepsy [MAE], nocturnal frontal lobe epilepsy [NFLE], progressive myoclonic epilepsy [PME], pyridoxamine 5'-phosphate oxidase deficiency [PNPOD], pyridoxine-dependent epilepsy [EPD] and severe myoclonic epilepsy of infancy [SMEI]), Fabry disease, Fahr's syndrome, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralyses, familial spastic paralysis, Farber's disease, fibromuscular dysplasia, Fisher syndrome, floppy infant syndrome, Friedreich's ataxia, frontotemporal dementia, fucosidosis, galactosialidosis, Gaucher disease, generalized gangliosidosis, Gerstmann's syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 gangliosidosis, GM2 gangliosidosis (Tay-Sachs disease), Guillain-Barre syndrome, Hallervorden-Spatz disease, hemicrania continua, hemiplegia alterans, hereditary spastic paraplegia, heredopathia atactica polyneuritiformis, Holmes- Adie syndrome, holoprosencephaly, Hughes syndrome, Huntington's disease, hydranencephaly, hydromyelia, hypercorti soli sm, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, iniencephaly, Isaac's syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy's disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Kliiver-Bucy syndrome, Korsakoff s amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, lateral medullary syndrome, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, Levine-Critchley syndrome, Lewy body dementia, lipoid proteinosis, lissencephaly, locked-in syndrome, Lou Gehrig's disease, lupus, Lyme disease, Machado-Joseph disease, macrencephaly, alpha-mannosidosis, beta-mannosidosis, Melkersson-Rosenthal syndrome, Menkes disease, meralgia paresthetica, metachromatic leukodystrophy, microcephaly,Miller Fisher syndrome, Moebius syndrome, mucopolysaccharidosis type I-H (Hurler syndrome), mucopolysaccharidosis type I-H / S (Hurl er- Scheie syndrome), mucopolysaccharidosis type IS (Scheie syndrome), mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type III-A (Sanfilippo syndrome A), mucopolysaccharidosis type III-B (Sanfilippo syndrome B), mucopolysaccharidosis type III-C (Sanfilippo syndrome C), mucopolysaccharidosis type III-D (Sanfilippo syndrome D), mucopolysaccharidosis type IV-B (Morquio syndrome B), mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), mucopolysaccharidosis type VII (Sly syndrome), mucopolysaccharidosis type IX (Natowicz syndrome), multiple sclerosis, muscular dystrophy, myasthenia gravis, myelinoclastic diffuse sclerosis, narcolepsy, neuroacanthocytosis, neurofibromatosis, neuroleptic malignant syndrome, neurosarcoidosis, Niemann-Pick disease, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus myoclonus, O'Sullivan-McLeod syndrome, pantothenate kinase- associated neurodegeneration, paraneoplastic syndromes, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal hemicrania, Parry-Romberg syndrome, Pelizaeus-Merzbacher disease, Pena Shokeir II syndrome, periventricular leukomalacia, phytanic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pompe disease, post-polio syndrome, posterior cortical atrophy, primary dentatum atrophy, primary lateral sclerosis, primary progressive aphasia, prion diseases, progressive hemifacial atrophy, progressive locomotor ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, Ramsay Hunt syndrome I, Ramsay Hunt syndrome II, Rasmussen's encephalitis, Refsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhoff disease, Schilder's disease, Seitelberger disease, Shy-Drager syndrome, Sjogren's syndrome, spasticity, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, striatonigral degeneration, Sturge-Weber syndrome, tardive dyskinesia, tauopathy, Tay-Sachs disease, thoracic outlet syndrome, thyrotoxic myopathy, tic douloureux, Todd's paralysis, trigeminal neuralgia, tropical spastic paraparesis, Troyer syndrome, vascular dementia, Von Economo's disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's disease, Wallenberg's syndrome, Werdnig-Hoffman disease, Wernicke-Korsakoff syndrome, West syndrome, Whipple's disease, Williams syndrome, Wilson disease, Wolman's disease, X-linkedspinal and bulbar muscular atrophy, Zellweger syndrome, multiple sclerosis atrophy, Lewis body dementia (LBD), or Angelman syndrome.

[0377] In another aspect, the disclosure provides a composition or method as described herein.EXAMPLES

[0378] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.Example 1. Methods

[0379] Preparation of lipid nanoparticles: For formation of LNP compositions, mRNA was dissolved in lx PBS or citrate buffer (lOmM, pH 4.0), and mixed rapidly into ethanol containing the lipids.[03801LNP characterization; The different LNP compositions were characterized by size, polydispersity index (PDI), and zeta-potential as assessed by Dynamic Light Scattering (DLS, Malvern, 173° Scattering angle). DLS measures the scattering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (at or around the mean particle diameter) in a population, with a perfectly uniform population having a PDI of zero. The encapsulation efficacy (EE %) was tested using RiboGreen RNA Assay (Zhao et al., 2016).

[0381] Tissue Dissociation: Neural tissue (e.g., from brains) from the tested animals was collected and dissociated into single cell suspensions using the MACS™ Adult Brain Dissociation Kit (Miltenyi Biotec) or the MACS™ Multi Tissue Dissociation Kit (Miltenyi Biotec), via a combination of mechanical dissociation and enzymatic degradation of the extracellular matrix.

[0382] FACS: For fluorescence-activated cell sorting (FACS) analysis, the cells acquired via the tissue dissociation described above, were incubated with fluorescently labeled antibodies in eBioscience™ Flow Cytometry Staining Buffer or PBS with 0.02-0.05% Bovine serum albumin (BSA) for 10 minutes at room temperature. For some experiments, cells were stained withappropriate antibodies for analysis (species specific). These included CD1 lb (added 1:250), CD31 (added 1:50), and CD45 (added 1:250), and CD3 (added 1:250).

[0383] Single-cell RNA sequencing (scRNA): Cells acquired via the tissue dissociation described above were resuspended in FACS sorting buffer / Nuclease free water and loaded into either a 10X Connect Machine or master mix was added when using Chromium X using the 5’ Gene Expression Single Cell Kit from 10X Genomics. Cell types were classified using machine learning algorithm trained on Human Lung Atlas data(https: / / www.science.org / doi / 10.! 126 / science.abl5197).Example 2: Formulation Screening Using Rodent Studies

[0384] The compositions in Table 9 were screened via rodent studies with Td Tom or Cre mRNA as a cargo. A 1 OX scRNA assay was used to evaluate expression of genes including Trem2, P2RY12. TNF, Lgals3, Ccr7, and Gpnmb. Fluorescence-activated cell sorting (FACS) provided for quantification of TdTom+ events found in microglia (CD1 lb+ cells). Clinical observations included body weight monitoring, which allowed for calculation of normalized percent body weight change.Table 9. Exemplary LNP Compositions

[0385] Lipid nanoparticle characterization data is summarized in Table 10.Table 10. Lipid Nanoparticle Composition Characterization DataMouse Model

[0386] Ai 14 mice were administered ere mRNA or TdTom mRNA formulated in lipid nanoparticle compositions of the disclosure. Details of the compositions used in the Ail4 mouse model are summarized in Table 11. Study details (e.g. dosage) and clinical observations, including body weight (BW) change, are summarized in Tables 12A-12B.[0387JFIG. 1 illustrates TdTom+ events as high as 20% found in microglia (CD1 lb+ cells) for composition 6A1. No microgliosis was observed.[0388JFIG. 7 illustrates TdTom+ events as high as 25% found in microglia (CD1 lb+ cells) for composition 6A3.[0389JFIG. 11 and FIG. 12 illustrate TdTom+ events as high as 36% found in microglia (CD1 lb+ cells) at 20 mpk and 7.3% TdTom+ events at lOmpk for composition 5A2. No microgliosis observed.[0390JFIG. 16 illustrates TdTom+ events as high as 11% found in microglia (CD1 lb+ cells) for composition 5 A3. No microgliosis observed.

[0391] FIG. 24 illustrates TdTom+ events as high as 21% found in microglia (CD1 lb+ cells) for composition 6A2.Table 11. Formulation Details for Ail4 Mouse ModelTable 12A. Ail4 Mice Model Experimental Details and ResultsNSO: Non-Significant OutcomeTable 12B. Ail4 Mice Model Experimental Details and Results (Continued)Rat model

[0392] Rats were intrathecally administered with a lipid nanoparticle composition of the disclosure formulated with tdTomato mRNA. Details of the LNP formulations used in the rat model are summarized in Table 9 above, and Table 13 below. Study details and clinical observations, including body weight change, are summarized in Tables 14A-14D.Table 13. LNP Formulation Details (Rat Model)Table 14A. Rat Model Experimental Details and ResultsTable 14B. Rat Model Experimental Details and Results (Continued)Table 14C. Rat Model Experimental Details and Results (5A1 + TdTomato mRNA)* trypan blue in spinal canal but leakage found around the neck areaTable 14D. Rat Model Experimental Details and Results (6A2 Tdt mRNA)[0393JFACS data is shown in FIGs.3A-3P, 9A-9D, 14A-14D, 18A-18P, 22A-22D, and 27A- 27D

[0394] 10X scRNA seq data are shown in FIGs. 4A-4G, 5A-5G, 6A-6G, 10A-10G, 15A-15G, 19A-19G, 20A-20G, 21A-21G, 23A-23G, and 39A-39B

[0395] Without wishing to be bound by theory, in some formulations, ScRNA-seq in rats indicated initial microglial activation post-LNP uptake, followed by a return to a homeostatic state suggesting that the changes are transient.

[0396] Further clinical observations are described below.Composition 6A1

[0397] Catheter Patency: All catheters were good with no leakage found during necropsy.

[0398] Body Weight Loss: All groups had a loss of 10%+ (see FIG. 2).

[0399] Clinical observations: black porphryin staining around eyes and nose, hunched posture, hypoactive, sunken eyes at 24 hours; clinical observation was observed in animals starting at 2 hours post-dose. All groups had similar body weight loss and clinical observations (no clear dose response).

[0400] Necropsy Observations: Animals 251 and 353 had pink blotches on the brain postperfusion.Composition 6A3

[0401] Catheter Patency: Animal 451: trypan blue in thoracic region but leakage found in the neck area. Animal 452: n / a (line accidentally cut during necropsy). Animal 453: good catheter no leakage.

[0402] Body Weight Loss: Animal 453 with the good catheter had 14% body weight loss.Animals 451 & 452 with unclear catheter patency had around 7% loss (see e.g., FIG. 8).

[0403] Clinical observations: Animal 453 was moribund at 24 hour post-dose; clinical observations included bleeding out of eyes, black porphyrin staining around eyes and nose, hypoactive, and ruffled fur at 24 hours. Animals 451 & 452 also had black porphyrin staining around eyes and nose, ruffled fur, lethargic. Clinical observations observed starting at 3 hours post-dose.Composition 6A4

[0404] Catheter patency: Animal 551: trypan blue in thoracic region but leakage found in the neck area. Animal 552: good catheter no leakage. Animal 553: good catheter no leakage.

[0405] Body Weight Loss: Animal 551 with the unclear catheter patency had 11% body weight loss. Animals 552 & 553 with good catheter patency had around 8-10% loss, (see e.g., FIG. 13).

[0406] Clinical Observations: Animal 551: Mild porphyrin staining around eyes, black porphyrin staining around nose, mild lethargic, ruffled fur, sunken eyes. Animal 552 & 553: Mild porphyrin staining around eyes and nose, mild sunken eyes, ruffled fur, mild lethargy. Clinical observations observed starting at 3 hours post-dose.

[0407] Necropsy Observations: Animal 552 had a pink blotch on the cerebellum post-perfusion.Composition 5A3

[0408] Catheter patency: Group 2: good catheter no leakage. Animal 351 bad catheter with subcutaneous leakage. Animals 352 & 353: good catheter no leakage. Animal 451: good catheter no leakage. Animals 452 & 453: bad catheter with subcutaneous leakage.

[0409] Body weight loss: Group 2 with good catheter patency had 5-7% body weight loss.Animals 352 & 353 with good catheter patency had 4-7% loss. Animal 451 with good catheter patency had about 13% body weight loss. Animals with bad catheter patency had minimal body weight loss to up to 3% body weight gain (see e.g., FIGs. 17A-17C).

[0410] Clinical observations: Animals 251 and 252 ruffled fur and active in home-cage. Animal 253 mild porphyrin staining around eyes and active in home-cage. Animal 351 bright, alert, and active. Animal 352 ruffled fur, moderate-severe porphyrin staining around eyes. Animal 353ruffled fur, moderate porphyrin staining around eyes, slight shaking. Group 4 ruffled fur; mild porphyrin staining around eyes.Composition 5A1

[0411] Catheter patency: Animal 255: bad catheter with leakage. Animal 252: unclear catheter patency with leakage found in the neck area, but trypan blue in thoracic spinal space. Animal 253: bad catheter with subcutaneous leakage. Remaining animals: good catheters no leaks.

[0412] Body weight loss (24 hr): 3 mpk cohort: range of 0.3-7% body weight loss and one animal with 0.5% body weight gain. 7.5 mpk cohort: range of 2-8% body weight loss. 10 mpk cohorts: average 8.3% body weight loss (excluding animals that failed catheter patency); Animal 252 had the highest body weight loss of 12%.

[0413] Clinical Observations (24 hr): 3 mpk cohort: ruffled fur, mild porphyrin staining around eyes, active in home cage. 7.5 mpk cohort: mild dehydration. 10 mpk cohorts: ruffled fur, hypoactive, black porphyrin staining around eyes and nose, lethargic.Composition 6A2

[0414] Catheter patency: Animal 351: bad catheter with subcutaneous leakage. Animal 352 & 353: good catheter no leakage. Animal 251: good catheter with trypan blue in cervical region. Animal 252: trypan blue found in thoracic region but not in cervical spinal space. Animal 351: bad catheter with trypan blue found in insertion site. Animal 352: trypan blue found in thoracic region but not in cervical spinal space. Animal 451: bad catheter with subcutaneous leakage. Animal 452: good catheter with trypan blue in cervical region.

[0415] Body Weight Loss: Animal 351 with the failed catheter patency had 1% body weight gain. Animals 352 & 353 with unclear catheter patency had around 10% loss, (see e.g., FIG.25). Animal 251 with good catheter 4% body weight loss at 24 hrs. Animal 452 with good catheter 2% body weight loss at 24 hr, 5% loss at 48 hr, and 4% at 72 hr. Animals 252 and 352 with unclear patency had minimal body weight loss. Animal 351 with a bad catheter had less than %1 BW loss over 24 hr and 48 hr. Animal 451 gained up to 6% body weight at 72 hr. (see e.g., FIGs.26A-26C).

[0416] Clinical Observations: Animal 351: bright, alert, and active. Animal 352: mild porphyrin staining around left eye and nose, ruffled fur, active. Animal 353: black porphyrin stainingaround eyes and nose, ruffled fur, active in cage. Clinical observations observed starting at 3 hours post-dose.

[0417] Clinical Observations (24 hour): Animal 251: ruffled fur, bright, alert, and active. Animal 252: ruffled fur, bright, alert, and active. Animal 351: bright, alert, and active. Animal 352: mild sunken eyes, hypoactive, responsive to stimuli. Animal 451: mild hypoactivity. Animal 452: sunken eyes, ruffled fur; hypoactivity, lethargic, black porphyrin staining around eyes, responsive to stimuli. At 48 hr post-dose, animal dragging left hindleg / paw; clinical observations remained to end of study.

[0418] Necropsy Observations: Animal 251 had pink blotch on cerebellum and both lobes postperfusion.Additional observations

[0419] Dexamethasone pretreatment reduced perivascular macrophage reporter expression by >80% without affecting microglial transfection levels. Increasing lipid to mRNA ratio did not change the tolerability profde but increased microglial reporter expression. No adverse cytokine production was observed following treatment (see FIGs.39A-39B).Summary:

[0420] Various formulations were screened for CNS cell tropism. Readouts were measured in 3 categories: Tolerability at lOmg / kg; Uptake by microglia by FACS; Uptake by non-microglial cells. The results are summarized in Table 15A-15B.Table 15A. Summary of CNS Cell Tropism Screening using Exemplary LNPsTable 15B. Summary of CNS Cell Tropism Screening using Exemplary LNPshydroxydodecyl)amino)butyl)piperazine-2, 5-dione (cKK-E12) as the ionizable lipid.** Body weight (BW) score in Rats were measured at 24 h post dose and percent BW change from pre-dose baseline was calculated. Percent BW change was categorized and scored as follows: 0% change = score 0, (-) 0.1-4% = score 1, (-) 5-9% = score 2, (-) 10-14%=score 3.Example 3: Non-human Primate (NHP) Biodistribution and Tolerability Analysis

[0421] Macaques (n=3) received a single intra-cistema magna (ICM) injection of a traditional 4-component LNP control (including DLin-MC3-DMA as the first lipid and no second lipid), or Composition 6A5 (Size = 85.4mm, PDI=0.179, 95.9% EE, 1.29mg / ml) in a buffer solution at doses of 1 mg per kg of brain weight or 3 mg per kg of brain weight of each subject (corresponding to a 0.3 mL volume at concentrations of 0.233 mg / mL or 0.70 mg / mL and total dosage of,07mg or 0.210 mg). A control group was also included in the study. Brain sections from different regions were analyzed at 24h post dosing (p.d.) via FACS, single-cell RNA sequencing (scRNA-seq), RNA in situ hybridization (RISH), and immunohistochemistry (IHC)to confirm microglial targeting and biodistribution. Cytokine levels from CSF samples were measured at 4h and 24h p.d.

[0422] Tissue Dissociation; Neural tissue (e.g., from brains) from the tested NHPs was collected from each subject and dissociated into single cell suspensions using the MACS™ Adult Brain Dissociation Kit (Miltenyi Biotec) or the MACS™ Multi Tissue Dissociation Kit (Miltenyi Biotec), via a combination of mechanical dissociation and enzymatic degradation of the extracellular matrix.[04231 Single-cell RNA screening (scRNA analysis): A 10X scRNA assay was used to evaluate expression of genes including tdTom+, Trem2, CTSD, and Gpnmb. Cells acquired via the tissue dissociation described above were resuspended in FACS sorting buffer / Nuclease free water and loaded into either a 10X Connect Machine or master mix was added when using Chromium X using the 5’ Gene Expression Single Cell Kit from 10X Genomics. Cell types were classified using machine learning algorithm trained on Human Lung Atlas data (see C. Dominguez Conde et al., Science, 376, eabl5197 (2022)).FACS: Fluorescence-activated cell sorting (FACS) provided for quantification of TdTom+ events found in microglia (CD1 lb+ cells). For fluorescence-activated cell sorting (FACS) analysis, the cells acquired via the tissue dissociation described above, were incubated with fluorescently labeled antibodies in eBioscience™ Flow Cytometry Staining Buffer or PBS with 0.02-0.05% Bovine serum albumin (BSA) for 10 minutes at room temperature. For some experiments, cells were stained with appropriate antibodies for analysis (species specific). These included CDllb (added 1:250), CD31 (added 1:50), and CD45 (added 1:250), and CD3 (added 1:250).RISH: RNA in situ hybridization was performed on the Leica Biosystems BOND RX Research Advanced Staining System using the RNAscope™ LS Multiplex Fluorescent Reagent Kit (Advanced Cell Diagnostics, Inc., Newark, CA). First, brain tissues fixed in 10% NBF for 24h at room temperature were processed to formalin fixed paraffin embedded blocks and 5pm sections from each sample collected onto Fisherbrand™ SuperFrost™ Plus microscope glass slides (Fisher Sci catalog no 12-550-15). The tissue sections were pretreated with heat and protease prior to hybridization with the target oligo probes Preamplifier and amplifier was then hybridized sequentially, followed by TSA-fluorophore reaction. Each channel reacts with aunique fluorophore. Following staining completion, slides were removed from the Leica Bond Rx and coverslips were mounted with ProLong™ Gold Antifade Mountant (Invitrogen cat# P36930). Images were collected at 40X objective using 3DHistech Pannoramic SCAN II digital slide scanner. Each sample was quality controlled for RNA integrity with a RNAscope™ probe specific to POLR2A / PPIB / UBC RNA and for background with a probe specific to bacterial dapB RNA. Specific RNA staining signal was identified as fluorescent, punctate dots. Samples were counterstained with D PI for nuclear staining and slides were mounted using Prolong Gold antifade mountant (Invitrogen cat# P36930).

[0424] Both Composition 6A5 doses were well tolerated, and all animals recovered without any observable clinical symptoms. FACS analysis of coronal brain sections revealed high levels of tdTomato positive cells co-localizing with microglial cells (CD1 lb+). See FIG. 40A. Microglial transfection efficiency was dose-dependent, with 5% at low dose (Img / kg of brain weight) and 23% at high dose (3mg / kg). No uptake was detected in endothelial cells (CD31+). ScRNA-seq identified transfected microglial subtypes, with no evidence of inflammatory responses (see FIG.40B) or peripheral immune cell infiltration. RISH confirmed exclusive microglial targeting.ABBREVIATIONSLNP lipid nanoparticlePBS phosphate-buff ered salineFBS Fetal bovine serumPFA ParaformaldehydeMAV Meninges and associated vasculatureIVIS in vivo imaging systemDOTAP l,2-Dioleoyl-3 -trimethylammonium propaneDOPE dioleoylphosphatidylethanolamineELISA enzyme-linked immunoassayINCORPORATION BY REFERENCE

[0425] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0426] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. The scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWhat is claimed is:

1. A lipid nanoparticle composition comprising a lipid component comprising:(i) a first lipid wherein the first lipid is an ionizable cationic lipid, and(ii) a second lipid wherein the second lipid is separate from the first lipid, wherein the second lipid is a compound of formula I:pharmaceutically acceptable salt thereof,wherein:RD1is a C1-C4 alkyl;RD2is H or a C1-C4 alkyl;zl and z2 are each independently 1, 2, or 3;z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; andz4 is 0 or 1.

2. The lipid nanoparticle composition of claim 1, wherein in the compound of Formula I, RD1is methyl.

3. The lipid nanoparticle composition of claim 1 or 2, wherein in the compound of Formula I, RD2is methyl.

4. The lipid nanoparticle composition of claim 1 or 2, wherein in the compound of Formula I, RD2is H.

5. The lipid nanoparticle composition of any one of claims 1-4, wherein in the compound of Formula I, zl and z2 are each 1.

6. The lipid nanoparticle composition of any one of claims 1-4, wherein in the compound of Formula I, zl and z2 are each 2.

7. The lipid nanoparticle composition of any one of claims 1-6, wherein in the compound of Formula I, z3 is 6, 7, or 8.

8. The lipid nanoparticle composition of any one of claims 1-7, wherein in the compound of Formula I, z4 is 0.

9. The lipid nanoparticle composition of any one of claims 1-7, wherein in the compound of Formula I, z4 is 1.

10. The lipid nanoparticle composition of claim 1, wherein the compound of Formula I is:

11. A lipid nanoparticle composition comprising a lipid component comprising:(i) a first lipid wherein the first lipid is an ionizable cationic lipid, and(ii) a second lipid wherein the second lipid is separate from the first lipid,wherein the second lipid is a compound of Formula C:wherein m is an integer from 1 to 10.

12. The lipid nanoparticle composition of claim 11, wherein m is an integer from 3 to 8.

13. A lipid nanoparticle composition comprising a lipid component comprising:(i) a first lipid wherein the first lipid is an ionizable cationic lipid, and(ii) a second lipid wherein the second lipid is separate from the first lipid,wherein the second lipid is a compound of Formula D:wherein n is an integer from 1 to 10.

14. The lipid nanoparticle composition of claim 13, wherein n is an integer from 3 to 8.

15. A lipid nanoparticle composition comprising a lipid component comprising:(i) a first lipid wherein the first lipid is an ionizable cationic lipid, and(ii) a second lipid wherein the second lipid is separate from the first lipid,wherein the second lipid selected from:

16. The lipid nanoparticle composition of any one of claims 1-15, wherein the ionizable cationic lipid is a compound of Formula A:pharmaceutically acceptable salt thereof,wherein:RDlais a C1-C4 alkyl;RD2ais H or a C1-C4 alkyl;zla and z2a are each independently 1, 2, or 3;z3a is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; andz4a is 0 or 1; ora compound of Formula B:pharmaceutically acceptable salt thereof,whereinXb and Yb are each independently N or CH;zlb, z2b, z3b, z4b are each independently 1, 2, or 3; andz5b is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

17. The lipid nanoparticle composition of any one of claims 1-15, wherein the first lipid is a compound selected from Table 5.

18. The lipid nanoparticle composition of any one of claims 1-15, wherein the first lipid is a compound selected from Table 6.

19. The lipid nanoparticle composition of any one of claims 1-15, wherein the first lipid is a compound selected from:

20. A lipid nanoparticle composition comprising a lipid component comprising:(i) a first lipid wherein the first lipid is an ionizable cationic lipid, and(ii) a second lipid wherein the second lipid is separate from the first lipid, wherein the first lipid is:a compound of Formula A:pharmaceutically acceptable salt thereof,wherein:RDlais a C1-C4 alkyl;RD2ais H or a C1-C4 alkyl;zla and z2a are each independently 1, 2, or 3;z3a is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; andz4a is 0 or 1; ora compound of Formula B:pharmaceutically acceptable salt thereof,whereinXb and Yb are each independently N or CH;zlb, z2b, z3b, z4b are each independently 1, 2, or 3; andz5b is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

21. The lipid nanoparticle composition of claim 16 or 20, wherein in the compound of Formula A, RDlais methyl.

22. The lipid nanoparticle composition of claim 16, 20, or 21, wherein in the compound of Formula A, RD2ais methyl.

23. The lipid nanoparticle composition of claim 16, 20, or 21, wherein in the compound of Formula A, RD2ais H.

24. The lipid nanoparticle composition of any one of claims 16 and 20-23, wherein in the compound of Formula A, zla and z2a are each 1.

25. The lipid nanoparticle composition of any one of claims 16 and 20-23, wherein in the compound of Formula A, zla and z2a are each 2.

26. The lipid nanoparticle composition of any one of claims 16 and 20-25, wherein in the compound of Formula A, z3a is 6, 7, or 8.

27. The lipid nanoparticle composition of any one of claims 16 and 20-26, wherein in the compound of Formula A, z4a is 0.

28. The lipid nanoparticle composition of any one of claims 16 and 20-26, wherein in the compound of Formula A, z4a is 1.

29. The lipid nanoparticle composition of claim 16 or 20, wherein in the compound of Formula B, zlb, z2b, z3b, and z4b are each 1.

30. The lipid nanoparticle composition claim 16, 20, or 29, wherein in the compound of Formula B, z5b is 6, 7, or 8.

31. The lipid nanoparticle composition of any one of claims 16, 20, and 29-30, wherein in the compound of Formula B, Xb is N.

32. The lipid nanoparticle composition of any one of claims 16, 20, and 29-31, wherein in the compound of Formula B, Yb is N.

33. The lipid nanoparticle composition of any one of claims 20-32, wherein the first lipid is34. The lipid nanoparticle composition of any one of claims 20-33, wherein the second lipid is an ionizable cationic lipid.

35. The lipid nanoparticle composition of any one of claims 20-33, wherein the second lipid is a permanently cationic lipid.

36. The lipid nanoparticle composition of any one of claims 20-33, wherein the second lipid is a compound selected from Table 7 or Table 8.

37. The lipid nanoparticle composition of any one of claims 20-33, wherein the second lipid is selected from:

38. The lipid nanoparticle of any one of claims 1-37, wherein the lipid component further comprises a third lipid, wherein the third lipid is an ionizable cationic lipid or a permanently cationic lipid separate from the first and the second lipid.

39. The lipid nanoparticle composition of claim 38, wherein the third lipid is a permanently cationic lipid.

40. The lipid nanoparticle composition of claim 38, wherein the third lipid is l,2-dioleoyl-3- trimethylammonium-propane (14:0 TAP).

41. The lipid nanoparticle composition of any one of claims 38-40, wherein the lipid component comprises the third lipid in an amount of from about 1 mol% to about 10 mol % of the total lipids in the lipid component.

42. The lipid nanoparticle composition of claim 41, wherein the lipid component comprises the third lipid in an amount of about 5 mol% of the total lipids in the lipid component.

43. The lipid nanoparticle composition of any one of claims 1-42, wherein the lipid component comprises the first lipid in an amount of from about 10 mol% to about 30 mol % of the total lipids in the lipid component.

44. The lipid nanoparticle composition of claim 43, wherein the lipid component comprises the first lipid in an amount of from about 15 mol% to about 25 mol% of the total lipids in the lipid component.

45. The lipid nanoparticle composition of claim 43, wherein the lipid component comprises the first lipid in an amount of about 19 mol% of the total lipids in the lipid component.

46. The lipid nanoparticle composition of any one of claims 1-45, wherein the lipid component comprises the second lipid in an amount of from about 10 mol% to about 30 mol % of the total lipids in the lipid component.

47. The lipid nanoparticle composition of claim 46, wherein the lipid component comprises the second lipid in an amount of from about 15 mol% to about 25 mol % of the total lipids in the lipid component.

48. The lipid nanoparticle composition of claim 46, wherein the lipid component comprises the second lipid in an amount of about 20 mol% of the total lipids in the lipid component.

49. The lipid nanoparticle composition of any one of claims 1-48, wherein the lipid component further comprises a phospholipid.

50. The lipid nanoparticle composition of 49, wherein the phospholipid is selected from 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3- phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3 -phosphorac^ 1 -glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), l,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.

51. The lipid nanoparticle composition of claim 49, wherein the phospholipid is DOPE or DSPC.

52. The lipid nanoparticle composition of any one of claims 49-51, wherein the lipid component comprises the phospholipid in an amount of from about 10 mol% to about 30 mol% of the total lipids in the lipid component.

53. The lipid nanoparticle composition of claim 52, wherein the lipid component comprises the phospholipid in an amount of from about 15 mol% to about 25 mol% of the total lipids in the lipid component.

54. The lipid nanoparticle composition of claim 52, wherein the lipid component comprises the phospholipid in an amount of about 19 mol% of the total lipids in the lipid component.

55. The lipid nanoparticle composition of any one of claims 1-54, wherein the lipid component further comprises a PEG lipid.

56. The lipid nanoparticle composition of claim 55, wherein the PEG lipid is selected from: 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

57. The lipid nanoparticle composition of claim 55, wherein the PEG lipid is 1,2-dimyristoyl-sn- glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG).

58. The lipid nanoparticle composition of any one of claims 55-57, wherein the lipid component comprises the PEG lipid in an amount of from about 0.5 mol% to about 10 mol % of the total lipids in the lipid component.

59. The lipid nanoparticle composition of claim 58, 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.

60. The lipid nanoparticle composition of claim 58, wherein the lipid component comprises the PEG lipid in an amount of about 4 mol% of the total lipids in the lipid component.

61. The lipid nanoparticle composition of claim 58, wherein the lipid component comprises the PEG lipid in an amount of about 3.8 mol% of the total lipids in the lipid component.

62. The lipid nanoparticle composition of any one of claims 1-61, wherein the lipid component further comprises a sterol.

63. The lipid nanoparticle composition of claim 62, wherein the sterol is cholesterol.

64. The lipid nanoparticle composition of claim 62 or 63, wherein the lipid component comprises the sterol in an amount of from about 30 mol% to about 50 mol% of the total lipids in the lipid component.

65. The lipid nanoparticle composition of claim 64, wherein the lipid component comprises the sterol in an amount of from about 33 mol% to about 43 mol% of the total lipids in the lipid component.

66. The lipid nanoparticle composition of claim 64, wherein the lipid component comprises the sterol in an amount of about 38 mol% of the total lipids in the lipid component.

67. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7, DOPE, cholesterol, PEG-DMG, and M0-14-TAP.

68. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7, DOPE, cholesterol, PEG-DMG, and SL-1.

69. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7, DOPE, cholesterol, PEG-DMG, and M0-14-DAP.

70. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7, DOPE, cholesterol, PEG-DMG, DEA-14-DAP, and 14:0 TAP.

71. A lipid nanoparticle composition comprising a lipid component comprising:IL-1, DOPE, cholesterol, PEG-DMG, and ME-14-EPC.

72. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7, DOPE, cholesterol, PEG-DMG, and DEA-16-DAP.

73. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7, DOPE, cholesterol, PEG-DMG, and DEA-14-DAP.

74. A lipid nanoparticle composition comprising a lipid component comprising:IL-2, DOPE, cholesterol, PEG-DMG, and Me-14-EPC.

75. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and MO-14-TAP in an amount of about 20 mol% of the total lipids in the lipid component.

76. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and SL-1 in an amount of about 20 mol% of the total lipids in the lipid component.

77. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and MO-14-DAP in an amount of about 20 mol% of the total lipids in the lipid component.

78. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 33 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component;DEA-14-DAP in an amount of about 20 mol% of the total lipids in the lipid component; and 14:0 TAP in an amount of about 5 mol% of the total lipids in the lipid component.

79. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component;DEA-16-DAP in an amount of about 20 mol% of the total lipids in the lipid component80. A lipid nanoparticle composition comprising a lipid component comprising:IL-1 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and ME-14-EPC in an amount of about 20 mol% of the total lipids in the lipid component.

81. A lipid nanoparticle composition comprising a lipid component comprising:4A3SC7 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and DEA-14-DAP in an amount of about 20 mol% of the total lipids in the lipid component.

82. A lipid nanoparticle composition comprising a lipid component comprising:IL-2 in an amount of about 19 mol% of the total lipids in the lipid component;DOPE in an amount of about 19 mol% of the total lipids in the lipid component; cholesterol in an amount of about 38 mol% of the total lipids in the lipid component;PEG-DMG in an amount of about 4 mol% of the total lipids in the lipid component; and Me-14-EPC in an amount of about 20 mol% of the total lipids in the lipid component.

83. The lipid nanoparticle composition of any one of claims 1-82, further comprising a payload.

84. The lipid nanoparticle composition of claim 83, wherein the payload comprises a polypeptide or a protein.

85. The lipid nanoparticle composition of claim 84, wherein the polypeptide or protein is selected from superoxide dismutase 1 (SOD1), survival of motor neuron 1 (SMN-1), Frataxin, glutamic acid decarboxylase 65 (GAD-65), and glutamic acid decarboxylase 67 (GAD-67).

86. The lipid nanoparticle composition of claim 84, wherein the polypeptide or protein is selected from triggering receptor expressed on myeloid cells 2 (TREM2), tumor necrosis factor (TNF), LGALS3, C-C motif chemokine receptor 7 (CCR7), and glycoprotein nonmetastatic melanoma protein B (GPNMB).

87. The lipid nanoparticle composition of claim 83, wherein the payload comprises a nucleic acid.

88. The lipid nanoparticle composition of claim 87, 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).

89. The lipid nanoparticle composition of claim 88, wherein the payload comprises a small interfering RNA (siRNA).

90. The lipid nanoparticle composition of claim 88, wherein the payload comprises an mRNA.

91. The lipid nanoparticle composition of claim 90, wherein the mRNA encodes a gene-editing system or component thereof.

92. The lipid nanoparticle composition of claim 91, 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.

93. The lipid nanoparticle composition of claim 90, wherein the mRNA encodes a protein selected from superoxide dismutase 1 (SOD1), survival of motor neuron 1 (SMN-1), Frataxin, glutamic acid decarboxylase 65 (GAD-65), and glutamic acid decarboxylase 67 (GAD-67).

94. The lipid nanoparticle composition of claim 90, wherein the mRNA encodes a protein selected from myeloid cells 2 (TREM2), tumor necrosis factor (TNF), LGALS3, C-C motif chemokine receptor 7 (CCR7), and glycoprotein nonmetastatic melanoma protein B (GPNMB).

95. The lipid nanoparticle composition of claim 90, wherein the mRNA encodes purinergic receptor P2Y (P2RY12).

96. The lipid nanoparticle composition of claim 88, wherein the payload comprises a guide RNA.

97. A pharmaceutical composition comprising a lipid nanoparticle composition of any one of claims 1-96 and a pharmaceutically acceptable excipient.

98. A method of treating or preventing a CNS 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 83-96 or a pharmaceutical composition of claim 97.

99. The method of claim 98, wherein the method comprises selectively delivering the payload to an organ or tissue of the central nervous system of a subject.

100. The method of claim 98, wherein the method comprises selectively delivering the payload to a cell of the central nervous system of a subject.

101. The method of any one of claims 98-100, wherein the CNS disease is selected from Abulia, Achromatopsia, acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Agraphia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, Akinetopsia, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Amnesia, anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartylglucosaminuria, Asperger syndrome, ataxia, ataxia telangiectasia (Louis-Bar syndrome), ataxias and cerebellar or spinocerebellardegeneration, attention deficit- hyperactivity disorder, autism, autonomic dysfunction, Barth syndrome, Batten disease, Becker's myotonia, Behcet's disease, Bell's palsy, Bernhardt-Roth syndrome, Bipolar disorder, Binswanger's disease, Bloch-Sulzberger syndrome, Bradbury-Eggleston syndrome, Brown-Sequard syndrome, bulbospinal muscular atrophy, CADASIL, Canavan's disease, Carpal tunnel syndrome, causalgia, cavernomas, cavernous angioma, central cervical cord syndrome, central cord syndrome, central pontine myelinolysis, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral beriberi, cerebral gigantism, cerebral palsy, cerebro-oculo-facio-skeletal syndrome (COFS), Charles bonnet syndrome, cholesterol ester storage disease, chorea, choreoacanthocytosis, chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic fatigue syndrome, chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, colpocephaly, congenital myasthenia, corticobasal degeneration, cranial arteritis, cree encephalitis, Creutzfeldt-Jakob disease, Cushing's syndrome, cystinosis, cytomegalic inclusion body disease, Dancing eyes-dancing feet syndrome, Dandy-Walker syndrome, Danon disease, Dawson disease, De Morsi er's syndrome, Dejerine-Klumpke palsy, dementia, dentate cerebellar ataxia, dentatorubral atrophy, Dermatillomania, dermatomyositis, developmental dyspraxia, Devic's syndrome, diffuse sclerosis, Down syndrome, Duchenne muscular dystrophy, dysautonomia, dysgraphia, dyslexia, dysphagia, dyspraxia, dyssynergia cerebellaris myoclonica, dyssynergia cerebellaris progressiva, Epilepsy (such as, e.g., Amish infantile epilepsy syndrome (AIES), benign familial infantile seizures (BFIS), benign familial neonatal seizures (BFNS), childhood absence epilepsy (CAE), childhood-onset epileptic encephalopathy (COEE), Dravet syndrome (DS), early infantile epileptic encephalopathy (EIEE), Exploding head syndrome, familial adult myoclonic epilepsy (FAME), familial febrile seizures (FFS), familial focal epilepsy with variable foci (FFEVF), familial infantile myoclonic epilepsy (FIME), familial temporal lobe epilepsy (FTLE), focal epilepsy and speech disorder (FESD) with or without mental retardation, generalized epilepsy and paroxysmal dyskinesia (GEPD), generalized epilepsy with febrile seizures plus (GEFS+), idiopathic generalized epilepsy (IGE), juvenile absence epilepsy (J AE), juvenile myoclonic epilepsy (JME), myoclonic-atonic epilepsy (MAE), nocturnal frontal lobe epilepsy (NFLE), progressive myoclonic epilepsy (PME), pyridoxamine 5'-phosphate oxidase deficiency (PNPOD), pyridoxine-dependent epilepsy (EPD) and severe myoclonic epilepsy of infancy (SMEI)), Fabry disease, Fahr's syndrome, familial dysautonomia, familial hemangioma, familialidiopathic basal ganglia calcification, familial periodic paralyses, familial spastic paralysis, Farber's disease, Fetal alcohol syndrome, fibromuscular dysplasia, Fisher syndrome, floppy infant syndrome, Foville's syndrome, Friedreich's ataxia, frontotemporal dementia, fucosidosis, galactosialidosis, Gaucher disease, generalized gangliosidosis, Gerstmann's syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 gangliosidosis, GM2 gangliosidosis (Tay-Sachs disease), Gray matter heterotopia, Guillain-Barre syndrome, Hallervorden-Spatz disease, hemicrania continua, hemiplegia alterans, hereditary spastic paraplegia, heredopathia atactica polyneuritiformis, Herpes zoster, Hirayama disease, Holmes-Adie syndrome, holoprosencephaly, Hughes syndrome, Huntington's disease, hydranencephaly, hydromyelia, hypercorti soli sm, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, iniencephaly, Isaac's syndrome, Isodicentric 15, Joubert syndrome, Karak syndrome, Keams-Sayre syndrome, Kennedy's disease, Kinsboume syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Kliiver-Bucy syndrome, Korsakoff s amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, lateral medullary syndrome, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, Levine-Critchley syndrome, Lewy body dementia, lipoid proteinosis, lissencephaly, locked-in syndrome, Lou Gehrig's disease, lupus, Lyme disease, Machado-Joseph disease, macrencephaly, alpha-mannosidosis, beta-mannosidosis, Melkersson-Rosenthal syndrome, Menkes disease, meralgia paresthetica, metachromatic leukodystrophy, microcephaly, Miller Fisher syndrome, Misophonia, Moebius syndrome, Moyamoya disease, mucopolysaccharidosis type I-H (Hurler syndrome), mucopolysaccharidosis type I-H / S (Hurler-Scheie syndrome), mucopolysaccharidosis type IS (Scheie syndrome), mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type IILA (Sanfilippo syndrome A), mucopolysaccharidosis type IILB (Sanfilippo syndrome B), mucopolysaccharidosis type III-C (Sanfilippo syndrome C), mucopolysaccharidosis type III-D (Sanfilippo syndrome D), mucopolysaccharidosis type IV-B (Morquio syndrome B), mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), mucopolysaccharidosis type VII (Sly syndrome), mucopolysaccharidosis type IX (Natowicz syndrome), multiple sclerosis, muscular dystrophy,myasthenia gravis, myelinoclastic diffuse sclerosis, narcolepsy, neuroacanthocytosis, neurofibromatosis, neuroleptic malignant syndrome, neurosarcoidosis, Niemann-Pick disease, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus myoclonus, O'Sullivan-McLeod syndrome, Otosclerosis, Overuse syndrome, pantothenate kinase- associated neurodegeneration, paraneoplastic syndromes, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal hemicrania, Parry-Romberg syndrome, Pelizaeus-Merzbacher disease, Pena Shokeir II syndrome, periventricular leukomalacia, phytanic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pompe disease, post-polio syndrome, posterior cortical atrophy, Prader-Willi syndrome, primary dentatum atrophy, primary lateral sclerosis, primary progressive aphasia, prion diseases, progressive hemifacial atrophy, progressive locomotor ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, Quadriplegia, Rabies, Ramsay Hunt syndrome I, Ramsay Hunt syndrome II, Rasmussen's encephalitis, Refsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhoff disease, Schilder's disease, Seitelberger disease, Shingles, Shy-Drager syndrome, Sjogren's syndrome, spasticity, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, striatonigral degeneration, Sturge-Weber syndrome, tardive dyskinesia, tauopathy, Tay-Sachs disease, Tetanus, thoracic outlet syndrome, thyrotoxic myopathy, tic douloureux, Todd's paralysis, trigeminal neuralgia, tropical spastic paraparesis, Troyer syndrome, Unverricht-Lundborg disease, vascular dementia, Vertigo, Von Economo's disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's disease, Wallenberg's syndrome, Werdnig-Hoffman disease, Wernicke-Korsakoff syndrome, West syndrome, Whipple's disease, Williams syndrome, Wilson disease, Wolman's disease, X-linked spinal and bulbar muscular atrophy, Y-linked hearing impairment, Zellweger syndrome, multiple sclerosis atrophy, Lewis body dementia (LBD), and Angelman syndrome.

102. The method of any one of claims 98-100, wherein the CNS disease is an inflammatory disease of the central nervous system.

103. The method of any one of claims 98-100, wherein the CNS disease is selected from multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), neuromyelitis optica (NMO), transverse myelitis and autoimmune encephalitis.

104. The method of any one of claims 98-103, wherein the method further comprises pre-treating the subject with dexamethasone.

105. A method of selectively delivering a payload to an organ, tissue, or cell of the central nervous system 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 83-96 or a pharmaceutical composition of claim 97.

106. The method of any one of claims 99-104, 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 central nervous system.

107. The method of any one of claims 99-104, wherein the payload is a polynucleotide encoding a gene product and wherein the selectively delivering results in expression of the gene product in a cell of the central nervous system.

108. The method of claim 107, wherein the gene product is functional in the cell of the central nervous system.

109. The method of any one of claims 99-104, 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 central nervous system.

110. The method of any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 200% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

111. The method of any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 300% of the level ofexpression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

112. The method of any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 400% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

113. The method of any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 500% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

114. The method any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is at least 600% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

115. The method of any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is about 200% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

116. The method any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is about 300% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

117. The method of any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is about 400% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

118. The method of any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is about 500% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

119. The method of any one of claims 106-109, wherein the level of expression of the protein or gene product in the cell of the central nervous system following administration of said lipid nanoparticle composition or pharmaceutical composition is about 600% of the level of expression of the protein or gene product in the cell of the central nervous system following administration of a reference lipid nanoparticle composition.

120. The method of any one of claims 106-119, wherein the reference lipid nanoparticle composition comprises a second lipid that does not comprise a compound of Formula (I), but is otherwise identical to the lipid nanoparticle composition administered to the subject.

121. The method of any one of claims 106-119, wherein the reference lipid nanoparticle composition comprises a lipid component comprising a second lipid that does not comprise MO-14-TAP, MO-14-DAP, DEA-14-DAP, DEA-16-DAP, Me-14-EPC, or SL-1, but is otherwise identical to the lipid nanoparticle composition administered to the subject.

122. The method of any one of claims 106-119, wherein the reference lipid nanoparticle composition comprises a lipid component comprising a first lipid that does not comprise a compound of Formula (A) or (B), but is otherwise identical to the lipid nanoparticle composition administered to the subject.

123. The method of any one of claims 106-119, wherein the reference lipid nanoparticle composition comprises a lipid component comprising a first lipid that does not comprise acompound of Formula (C) or (D), but is otherwise identical to the lipid nanoparticle composition administered to the subject.

124. The method of any one of claims 106-119, wherein the reference lipid nanoparticle composition comprises a lipid component comprising a first lipid that does not comprise IL-1 or IL-2, but is otherwise identical to the lipid nanoparticle composition administered to the subject.

125. The method of any one of claims 106-119, wherein the reference lipid nanoparticle composition comprises a lipid component comprising a first lipid that does not comprise 4A3SC7, but is otherwise identical to the lipid nanoparticle composition administered to the subject.

126. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the central nervous system that is at least 500% of the level of expression of the protein or gene product in a cell from another tissue of the body.

127. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the central nervous system that is at least 1000% of the level of expression of the protein or gene product in cell from another tissue of the body.

128. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the central nervous system that is from about 500% to about 20000% of the level of expression of the protein or gene product in another tissue of the body.

129. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product in a cell of the central nervous system that is from about 1000% to about 20000% of the level of expression of the protein or gene product in a cell from another tissue of the body.

130. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product in another cell that is less than 20% of the level of expression of the protein or gene product in a cell of the central nervous system.

131. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product in a in another cell that is less than 10% of the level of expression of the protein or gene product in a cell of the central nervous system.

132. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product in another cell that is less than 5% of the level of expression of the protein or gene product in a cell of the central nervous system.

133. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product in another cell that is less than 20% of the total level of expression of the protein or gene product in the subject.

134. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product another cell that is less than 10% of the total level of expression of the protein or gene product in the subject.

135. The method of any one of claims 98-125, wherein the selectively delivering results in a level of expression of the protein or gene product another cell that is less than 5% of the total level of expression of the protein or gene product in the subject.

136. The method of any one of claims 98-135, wherein the another cell is a lung cell, a spleen cell, or a liver cell.

137. The method of claims 106-136, wherein the level of expression is determined by in vivo biofluorescence imaging.

138. The method of any one of claims 98-137, wherein the subject is a primate.

139. The method of any one of claims 98-137, wherein the subject is a human.

140. A method of delivering a payload to a cell of the central nervous system, comprising contacting the cell of the central nervous system with the lipid nanoparticle composition of any one of claims 83-96 or a pharmaceutical composition of claim 97.

141. The method of any one of claims 98-140, wherein the cell of the central nervous system is a brain cell.

142. The method of any one of claims 98-140, wherein the cell of the central nervous system is a spinal cord cell.

143. The method of any one of claims 98-140, wherein the cell of the central nervous system is a glial cell.

144. The method of any one of claims 98-140, wherein the cell of the central nervous system is a neuron, a choroid plexus cell, or a cell related to blood vessels and coverings.

145. The method of claim 144, wherein the neuron is selected from a neuronal stem cell, a Schwann cell, a glial cell, an oligodendrocyte, an astrocyte, a progenitor cell, a cholinergic cell, a dopaminergic cell, a GABA cell, a glutamatergic cell, and a motor neuron.

146. The method of claim 143 or 145, wherein the glial cell is a glial cell of the immune system (microglia), an astrocyte, an oligodendrocyte, or an ependymal cell.

147. The method of claim 144, wherein the cell related to blood vessels and coverings is an endothelial cell.

148. The method of any one of claims 98-140, wherein from about 5% to about 30% of the cells expressing the protein, polypeptide or gene product are microglia.

149. The method of any one of claims 98-140, wherein from about 10% to about 30% of the cells expressing the protein, polypeptide or gene product are microglia.

150. The method of any one of claims 98-140, wherein about 20% of the cells expressing the protein, polypeptide or gene product are microglia.

151. The method of any one of claims 98-140, wherein about 25% of the cells expressing the protein, polypeptide or gene product are microglia.

152. The method of any one of claims 98-140, wherein about 8% of the cells expressing the protein, polypeptide or gene product are microglia.

153. A compound of formula I:wherein:RD1is a C1-C4 alkyl;RD2is H or a C1-C4 alkyl;zl and z2 are each independently 1, 2, or 3;z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; andz4 is 0 or 1.

154. The compound of claim 153, wherein in the compound of Formula I, RD1is methyl.

155. The compound of claim 153 or 154, wherein in the compound of Formula I, RD2is methyl.

156. The compound of claim 153 or 154, wherein in the compound of Formula I, RD2is H.

157. The compound of any one of claims 153-156, wherein in the compound of Formula I, zl and z2 are each 1.

158. The compound of any one of claims 153-156, wherein in the compound of Formula I, zl and z2 are each 2.

159. The compound of any one of claims 153-158, wherein in the compound of Formula I, z3 is 6, 7, or 8.

160. The compound of any one of claims 153-159, wherein in the compound of Formula I, z4 is 0.

161. The compound of any one of claims 153-159, wherein in the compound of Formula I, z4 is 1.

162. A compound selected from:

163. A compound of Formula A:pharmaceutically acceptable salt thereof,wherein:RDlais a C1-C4 alkyl;RD2ais H;zla and z2a are each independently 1, 2, or 3;z3a is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; andz4a is 0 or 1;164. The compound of claim 163, wherein in the compound of Formula A, RDlais methyl.

165. The compound of claim 163 or 164, wherein in the compound of Formula A, zla and z2a are each 1.

166. The compound of claim 163 or 164, wherein in the compound of Formula A, zla and z2a are each 2.

167. The compound of any one of claims 163-166, wherein in the compound of Formula A, z3a is 6, 7, or 8.

168. The compound of any one of claims 163-167, wherein in the compound of Formula A, z4a is 0.

169. The compound of any one of claims 163-167, wherein in the compound of Formula A, z4a is 1.

170. A compound of Formula B:pharmaceutically acceptable salt thereof,whereinXb and Yb are each independently N or CH;zlb, z2b, z3b, z4b are each independently 1, 2, or 3; andz5b is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

171. The compound of claim 170, wherein in the compound of Formula B, zlb, z2b, z3b, and z4b are each 1.

172. The compound claim 170 or 171, wherein in the compound of Formula B, z5b is 6, 7, or 8.

173. The compound of any one of claims 170-172, wherein in the compound of Formula B, Xb isN.

174. The compound of any one of claims 170-173, wherein in the compound of Formula B, Yb isN.

175. A compound selected from:

176. A compound of Formula C:wherein m is an integer from 1 to 10.

177. The compound of claim 176, wherein m is an integer from 3 to 8.

178. A compound of Formula D:wherein n is an integer from 1 to 10.

179. The compound of claim 178, wherein m is an integer from 3 to 8.

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