Lipids and nanoparticle formulations thereof

Abstract

The present disclosure relates to compounds of Formula I, or a pharmaceutically acceptable salt thereof, pharmaceutical compositions thereof, and methods thereof.

Description

Attorney Docket: 049386-553001WOLIPIDS AND NANOPARTICLE FORMULATIONS THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 748,755, filed January 23, 2025, the contents of which are hereby incorporated by reference in their entirety.FIELD

[0002] The present disclosure relates generally to lipids. For example, the present disclosure relates to synthetic lipids and methods and uses thereof in the formulation and delivery of nucleic acids and therapeutic molecules.BACKGROUND

[0003] The variety of nucleic acid-based therapeutics for targeted delivery creates a challenge for lipid-based delivery vehicles. For example, nucleic acids are structurally diverse in size and type. Examples include DNA used in gene therapy, plasmids, small interfering nucleic acids (siRNA), and microRNA (miRNA) for use in RNA interference (RNAi), antisense molecules, ribozymes, antagomirs, and aptamers.

[0004] The design and use of cationic lipids and ionizable cationic lipids for inclusion in such lipid-based delivery vehicles has shown great advantages. However, use of these lipids can contribute to significant side effects when administered in vivo. One problem that has been observed includes low biodegradability and clearance from target tissues, thus creating an in vivo buildup of the lipid. Another problem is that large amounts of the lipid may cause an adverse immunogenic effect, which can result in discomfort in the subject and a decrease in the therapeutic effect of the active ingredient. A third problem associated with many cationic lipids is a low percentage of effective delivery to the target, thus resulting in a relatively low therapeutic effect or low potency. Moreover, it is not only important that the cationic lipid in the delivery vehicle have a specially tuned pKa so it can formulate with the nucleic acid-based therapeutic agent and protect it from degradation during administration, but be able to release the therapeutic agent once the vehicle has reached its target. With any drug discovery and development paradigms, there needs to be a continuous effort to understand the pharmacology, pharmacokinetics, and toxicology so that drugs with optimum therapeutic index and regulatory approval potential can be initiated into clinical investigations. As room for chemical optimization of nucleic acid is limited, there is a need for optimization of the lipid component of the delivery system.Attorney Docket: 049386-553001WO

[0005] Thus, there is a need in the art for the development of lipids that can meet the special needs of lipid-nucleic acid delivery systems.SUMMARY

[0006] As shown herein, the lipid compounds of the present disclosure are able to form stable lipid formulations such as lipid nanoparticles (LNPs) for use in delivering nucleic acid such as mRNA and siRNA, for therapeutic applications such as protein expression in various species.

[0007] In one aspect, the present disclosure provides a compound of Formula I:IwhereinX1, X2, and X3are each independently O or S,Ra, Rb, and Rcare each independently H, or C1-6alkyl, orRais H or C1-6alkyl, and Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, the heterocyclyl being optionally substituted with one or more C1-3 alkyl,n is 2 to 6,eitherwhereinY1is absent, O, or S,L1is C3-40 alkyl, C3-40 alkenyl, or C3-40 alkynyl,either(a) Y2is absent or C1-4 alkylene, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40Attorney Docket: 049386-553001WOalkynyl, optionally the alkyl, alkenyl, and alkynyl are branched, or(b) Y2is Ci-4 alkylene, optionally substituted with oxo, W is absent, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative; orX2X1X1L2(ii) R1is W and R2iswhereinY1is absent, O, or S,L1is C3-40 alkyl, C3-40 alkenyl, or C3-40 alkynyl,Y2is C1-4 alkylene, W is absent or carbonyl, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative.

[0008] In some embodiments, the compound of Formula I has a structure of Formula I-I:I-Ior a pharmaceutically acceptable salt thereof,whereinX1, X2, and X3are each independently O or S,Ra, Rb, and Rcare each independently H, or C1-6 alkyl, orAttorney Docket: 049386-553001WORais H or C1-6alkyl, and Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, the heterocyclyl being optionally substituted with one or more C1-3alkyl,n is 2 to 6,Y1is absent, O, or S,L1is C3-40alkyl, C3-40alkenyl, or C3-40alkynyl,Y2is absent or C1-4alkylene, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl, optionally the alkyl, alkenyl, and alkynyl are branched, orY2is C1-4 alkylene, optionally substituted with oxo, W is absent, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative.

[0009] In another aspect, the present disclosure provides a lipid composition comprising a nucleic acid and a compound of the present disclosure.

[0010] In another aspect, the present disclosure provides a lipid nanoparticle comprising a compound of the present disclosure. In some embodiments, the compound of the present disclosure self-assembles to form the lipid nanoparticle.

[0011] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure, or a lipid composition of the present disclosure or a lipid nanoparticle of the present disclosure, and a pharmaceutically acceptable excipient.

[0012] In another embodiment, the present disclosure provides a method of treating and / or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition of the present disclosure, a lipid composition of the present disclosure or a lipid nanoparticle of the present disclosure.

[0013] In another embodiment, the present disclosure provides a method of expressing a protein or polypeptide in a target cell, comprising contacting the target cell with a lipid nanoparticle of the present disclosure, or the pharmaceutical composition of the present disclosure.Attorney Docket: 049386-553001WO

[0014] In another embodiment, the present disclosure provides a compound of the present disclosure, or a lipid composition of the present disclosure or a lipid nanoparticle of the present disclosure for use in treating or preventing a disease in a subject in need thereof.

[0015] In another embodiment, the present disclosure provides a use of a compound of the present disclosure, or a lipid composition of the present disclosure or a lipid nanoparticle of the present disclosure for treating and / or preventing a disease in a subject in need thereof.

[0016] In another embodiment, the present disclosure provides a use of a compound of the present disclosure, or a lipid composition of the present disclosure or a lipid nanoparticle of the present disclosure in the preparation of a medicament for treating and / or preventing a disease in a subject in need thereof.

[0017] In another embodiment, the present disclosure provides the compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for use in therapy.BRIEF SUMMARY OF DRAWINGS

[0018] FIG. 1 shows a graph of the percent reduction of FVII plasma protein (as a percentage of base value) at 48 hours after mice are administered intravenously with FVII siRNA encapsulated LNPs at dose levels of 0.01 and 0.03 mg / Kg of FVII siRNA.

[0019] FIG. 2A. shows a bar graph of mouse serum hEPO protein expressions (ng / mL) at 6 hours post intravenous administration of hEPO mRNA in LNPs formulated with DSPC, DMPC, DPPC and DAPC each as helper lipids and L1 as the principal ionizable cationic lipid (iCL) at 0.3 mg / Kg of hEPO mRNA. FIG. 2B. shows a bar graph of hEPO expressions in NHP serum at 0.3 mg / kg dose of hEPO mRNA encapsulated with DSPC, DMPC, DPPC and DAPC each as helper lipids and L1 as the principal iCL (PBS as negative control).

[0020] FIG. 3 shows a bar graph of comparative mouse serum hEPO protein levels 6 h post administration of 0.3 mg / Kg of hEPO mRNA formulated with ionizable lipid L1 in combination with compound 1-2, 1-5, 1-6, 1-7, 1-8 or 1-9. DSPC formulation was used as the comparator. PBS was used as negative control.

[0021] FIG. 4 shows a bar graph of mouse serum hEPO protein expressions (ng / mL) at 6 hours post intravenous administration of hEPO mRNA in LNPs formulated with DSPC, I-2 or I-3 and L1 as the principal iCL at 0.1 or 0.3 mg / Kg of hEPO mRNA. PBS was used as negative control.Attorney Docket: 049386-553001WO

[0022] FIG. 5 shows a bar graph of hEPO expressions in NHP serum at 0.3 mg / kg dose of hEPO mRNA encapsulated with DSPC, I-2 or I-3, and L1 as the principal iCL. PBS was used as negative control.

[0023] FIG. 6A shows a bar graph of relative expression of hEPO in NHPs treated with LNPs formulated with compound I-2 with ionizable lipid L1 (n = 3), 0.3 mg / Kg of hEPO mRNA. FIG.6B shows a bar graph of relative expression of hEPO in NHPs treated with LNPs formulated with compound I-2 with different ionizable lipids (L8, L9) (n = 3), 0.3 mg / Kg of hEPO mRNA. FIG.6C shows a bar graph of relative expression of hEPO in NHPs treated with LNPs formulated with compound I-2 with different ionizable lipids (L10, L11) (n = 3), 0.3 mg / Kg of hEPO mRNA. FIG.6D shows a bar graph of relative expression of hEPO in NHPs treated with LNPs formulated with compound I-2 with different ionizable lipids (L12, L13) (n = 3), 0.3 mg / Kg of hEPO mRNA. FIG.6E shows a bar graph of relative expression of hEPO in NHPs treated with LNPs formulated with compound I-2 with different ionizable lipids (L14, L15) (n = 3), 0.3 mg / Kg of hEPO mRNA. FIG.6F shows a bar graph of relative expression of hEPO in NHPs treated with LNPs formulated with compound I-2 with different ionizable lipids (L16, L17) (n = 3), 0.3 mg / Kg of hEPO mRNA.DETAILED DESCRIPTIONI. General

[0024] The disclosure relates generally to methods and compounds, and pharmaceutically acceptable salts thereof, for intracellular delivery of therapeutic molecules such as nucleic acids. Specifically, the present disclosure relates to compounds of Formula I and pharmaceutically acceptable salts thereof, and compositions thereof. The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.II. Definitions

[0025] As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0026] A dashthat is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -CONH2 is attached through the carbon atom. A dash at the front or end of a chemical group is a matter of convenience; chemical groups can be depictedAttorney Docket: 049386-553001WOwith or without one or more dashes without losing their ordinary meaning. A wavy line drawn through a line in a structure indicates a point of attachment of a group. Unless chemically or structurally required, no directionality is indicated or implied by the order in which a chemical group is written or named.x,-OH

[0027] A squiggly line on a chemical group as shown below, for example, * indicates a point of attachment, i.e., it shows the broken bond by which the group is connected to another described group.

[0028] As used herein, “a compound of the present disclosure” can mean a compound of any of Formula I, or a pharmaceutically acceptable salt thereof. Similarly, the phrase “a compound of Formula (number)” means a compound of that formula and pharmaceutically acceptable salts thereof.

[0029] The prefix “Cu-v” and “Cu-Cv” indicates that the following group has from u to v carbon atoms. For example, “Cns alkyl” and “Ci-Cs alkyl” indicates that the alkyl group has from 1 to 8 carbon atoms.

[0030] “Alkyl” refers to an unbranched or branched saturated hydrocarbon chain. For example, an alkyl group can have 1 to 20 carbon atoms (i.e., C1-C20 alkyl), 1 to 8 carbon atoms (i.e., C1-C8alkyl), 1 to 6 carbon atoms (i.e., C1-C6alkyl), or 1 to 3 carbon atoms (i.e., C1-C3alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1 -propyl f / z-Pr, / / -propyl, -CH2CH2CH3), 2-propyl (z-Pr, z -propyl, -CH(CH3)2), 1 -butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-l -propyl (z-Bu, z -butyl, -CH2CH(CH3)2), 2-butyl f.s-Bu, 5-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, / -butyl, -C(CH3)3), 1 -pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3 -methyl- 1 -butyl (-CH2CH2CH(CH3)2), 2-methyl-l -butyl (-CH2CH(CH3)CH2CH3), 1 -hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2 -pentyl (-CH(CH3)CH2CH(CH3)2), 3 -methyl-3 -pentyl (-C(CH3)(CH2CH3)2), 2 -methyl-3 -pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), and 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, pentadecyl, hexadecyl, heptadecyl and octadecyl.Attorney Docket: 049386-553001WO

[0031] “Alkenyl” refers to an unbranched or branched hydrocarbon chain containing at least two carbon atoms and at least one carbon-carbon double bond. As used herein, alkenyl can have from 2 to 20 carbon atoms (i.e., C2-20 alkenyl), 2 to 8 carbon atoms (i.e., C2-8 alkenyl), 2 to 6 carbon atoms (i.e., C2-6 alkenyl), or 2 to 4 carbon atoms (i.e., C2-4 alkenyl). Alkenyl can include any number of carbons, such as C2, C3, C4, C5, Ce, C7, Cs, C9, C10, C11, C12, C13, C14, C15, Ci6, C17, Cis, C19, C20, or any range therein. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1 -pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl.

[0032] “Alkynyl” refers to an unbranched or branched hydrocarbon chain containing at least one carbon-carbon triple bond. For example, an alkynyl group can have from 2 to 20 carbon atoms (i.e., C2-20 alkynyl), 2 to 8 carbon atoms (i.e., C2-8 alkynyl), 2 to 6 carbon atoms (i.e., C2-6 alkynyl), or 2 to 4 carbon atoms (i.e., C2-4 alkynyl). The term “alkynyl” also includes those groups having one triple bond and one double bond. Examples of C2-6alkynyl include, but are not limited to, ethynyl, prop-l-ynyl, but-l-ynyl, pent-l-ynyl, pent-4-ynyl and penta- 1,4-diynyl.

[0033] “Alkoxy” means a group having the formula -O-alkyl, in which an alkyl group, as defined above, is attached to the parent molecule via an oxygen atom. The alkyl portion of an alkoxy group can have 1 to 20 carbon atoms (i.e., C1-C20 alkoxy), 1 to 12 carbon atoms (i.e., C1-C12 alkoxy), 1 to 8 carbon atoms (i.e., C1-C8alkoxy), 1 to 6 carbon atoms (i.e., C1-C6alkoxy) or 1 to 3 carbon atoms (i.e., C1-C3 alkoxy). Examples of suitable alkoxy groups include, but are not limited to, methoxy (-O-CH3 or -OMe), ethoxy (-OCH2CH3 or -OEt), isopropoxy (-O-CH(CH3)2), t-butoxy (-O-C(CH3)3 or -OtBu) and the like. Other examples of suitable alkoxy groups include, but are not limited to, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like.

[0034] “Alkoxyalkyl” refers an alkoxy group linked to an alkyl group which is linked to the remainder of the compound. Alkoxyalkyl can have any suitable number of carbon, such as from 2 to 6 (C2-6 alkoxyalkyl), 2 to 5 (C2-5 alkoxyalkyl), 2 to 4 (C2-4 alkoxyalkyl), or 2 to 3 (C2-3 alkoxyalkyl). Alkoxy and alkyl are as defined above. Examples of “alkoxyalkyl” include, but are not limited to, methoxymethyl (CH3OCH2-), and methoxyethyl (CH3OCH2CH2).

[0035] “Bridged” means a ring system in which non-adjacent atoms on a ring are connected by a divalent substituent, such as an alkylenyl or hetero alkylenyl group or a single heteroatom.Attorney Docket: 049386-553001WO

[0036] “Hydroxyalkyl” refers to a hydroxy group, -OH, linked to an alkyl group which is linked to the remainder of the compound such that the alkyl group is divalent. Hydroxyalkyl can have any suitable number of carbons, such as from 1 to 8 (C1-8hydroxyalkyl), 1 to 6 (C1-6hydroxyalkyl), 2 to 6 (C2-6 hydroxy alkyl), 2 to 4 (C2-4 hydroxyalkyl), or 2 to 3 (C2-3 hydroxy alkyl). Alkyl is as defined above where the alkyl is divalent.

[0037] “Halo” or “halogen” as used herein refers to fluoro (-F), chloro (-C1), bromo (-Br) and iodo (-1).

[0038] “Haloalkyl” is an alkyl group, as defined above, in which one or more hydrogen atoms of the alkyl group is replaced with a halogen atom. The alkyl portion of a haloalkyl group can have 1 to 20 carbon atoms (i.e., C1-20 haloalkyl), 1 to 12 carbon atoms (i.e., C1-12 haloalkyl), 1 to 8 carbon atoms (i.e., C1-8 haloalkyl), 1 to 6 carbon atoms (i.e., C1-6 haloalkyl) or 1 to 3 carbon atoms (i.e., C1-3 haloalkyl). The alkyl groups can be substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more halogens. Examples of suitable haloalkyl groups include, but are not limited to, -CF3, -CHF2, -CFH2, -CH2CF3, fluorochloromethyl, difluorochloromethyl, 1,1,1 -trifluoroethyl and pentafluoroethyl.

[0039] “Haloalkoxy” refers to an alkoxy group where some or all of the hydrogen atoms are substituted with halogen atoms. As for an alkyl group, haloalkoxy groups can have any suitable number of carbon atoms, such as C1-6. The alkoxy groups can be substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more halogens. When all the hydrogens are replaced with a halogen, for example by fluorine, the compounds are per-substituted, for example, perfluorinated. Haloalkoxy includes, but is not limited to, trifluoromethoxy, 2, 2, 2, -trifluoroethoxy, perfluoroethoxy, etc.

[0040] “Heteroalkyl” refers to an unbranched or branched saturated hydrocarbon chain containing from 1 to 4 heteroatoms.

[0041] “Cycloalkyl” refers to a saturated or partially saturated cyclic alkyl group having a single ring or multiple rings, such as 2, 3, 4 or more, wherein the multiple rings can be fused, bridged, spiro, or any combination thereof. As used herein, cycloalkyl has from 3 to 20 ring carbon atoms (i.e., C3-20 cycloalkyl), 3 to 12 ring carbon atoms (i.e., C3-12 cycloalkyl), 3 to 10 ring carbon atoms (i.e., C3-10 cycloalkyl), 3 to 8 ring carbon atoms (i.e., C3-8 cycloalkyl), or 3 to 6 ring carbon atoms (i.e., C3-6 cycloalkyl). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups also include partially unsaturated ring systems containing one or more double bonds, including fused ring systems with one aromatic ring and one non-aromatic ring, but not fully aromatic ring systems.Attorney Docket: 049386-553001WO

[0042] “Alkyl-cycloalkyl” refers to a radical having an alkyl component and a cycloalkyl component, where the alkyl component links the cycloalkyl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the cycloalkyl component and to the point of attachment. In some instances, the alkyl component can be absent. The alkyl component can include any number of carbons, such as Ci-6, Ci-2, Ci-3, Ci-4, Ci-5, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. The cycloalkyl component is as defined within. Exemplary alkyl-cycloalkyl groups include, but are not limited to, methyl-cyclopropyl, methyl-cyclobutyl, methyl-cyclopentyl and methyl-cyclohexyl.

[0043] The term “fused” refers to a ring system in which two or more rings in the system share a pair of adjacent ring atoms.

[0044] “Spiro” refers to at least two rings are linked together by one common atom. “Spiro” also refers to a ring substituent which is joined by two bonds at the same carbon atom. Examples of spiro groups include, but are not limited to, 1, 1 -di ethylcyclopentane, dimethyl-dioxolane, and 4-benzyl-4-methylpiperidine, wherein the cyclopentane and piperidine, respectively, are the spiro substituents.

[0045] “Heterocycle” or “heterocyclyl” or “heterocycloalkyl” refer to a saturated or unsaturated cyclic alkyl group, with one or more ring heteroatoms independently selected from nitrogen, oxygen, sulfur and silicon. A heterocyclyl can be a single ring or multiple rings, such as 2, 3, 4 or more, wherein the multiple rings can be fused, bridged, spiro, or any combination thereof. As used herein, heterocyclyl has 3 to 20 ring atoms (i.e., 3 to 20 membered heterocyclyl), 3 to 12 ring atoms (i.e., 3 to 12 membered heterocyclyl), 3 to 10 ring atoms (i.e., 3 to 10 membered heterocyclyl), 3 to 8 ring atoms (i.e., 3 to 8 membered heterocyclyl), 4 to 12 ring carbon atoms (i.e., 4 to 12 membered heterocyclyl), 4 to 8 ring atoms (i.e., 4 to 8 membered heterocyclyl), or 4 to 6 ring atoms (i.e., 4 to 6 membered heterocyclyl). Examples of heterocyclyl groups include pyrrolidinyl, piperidinyl, tetrahydropyridinyl, piperazinyl, oxetanyl, dihydropyranyl, dioxolanyl, azetidinyl, and morpholinyl.

[0046] “Alkyl-heterocycloalkyl” refers to a radical having an alkyl component and a heterocycloalkyl component, where the alkyl component links the heterocycloalkyl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the heterocycloalkyl component and to the point of attachment. The alkyl component can include any number of carbons, such as C0-6, C1-2, C1-3, C1-4, C1-5, C1-6, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. In some instances, the alkyl component can be absent. The heterocycloalkyl component is as defined above.Attorney Docket: 049386-553001WO

[0047] “Aryl” means an aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. For example, an aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. Exemplary aryl groups include, but are not limited to, radicals derived from benzene (e.g., phenyl), naphthalene, anthracene, biphenyl, and the like.

[0048] “Alkyl-aryl” refers to a radical having an alkyl component and an aryl component, where the alkyl component links the aryl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the aryl component and to the point of attachment. The alkyl component can include any number of carbons, such as Co-6, C1-2, C1-3, C1-4, C1-5, C1-6, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. In some instances, the alkyl component can be absent. The aryl component is as defined above. Examples of alkyl-aryl groups include, but are not limited to, benzyl and ethyl-benzene.

[0049] “Heteroaryl” refers to an aromatic group, including groups having an aromatic tautomer or resonance structure, having a single ring, multiple rings, or multiple fused rings, with at least one heteroatom in the ring, i.e., one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the nitrogen or sulfur can be oxidized. Thus, the term includes rings having one or more annular O, N, S, S(O), S(O)2, and N-oxide groups. The term includes rings having one or more annular C(O) groups. As used herein, heteroaryl include 5 to 20 ring atoms (i.e., 5- to 20-membered heteroaryl), 5 to 12 ring atoms (i.e., 5- to 12-membered heteroaryl), or 5 to 10 ring atoms (i.e., 5- to 10-membered heteroaryl), and 1 to 5 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and oxidized forms of the heteroatoms. Examples of heteroaryl groups include, but are not limited to, pyridin-2(1H)-one, pyridazin-3(2H)-one, pyrimidin-4(3H)-one, quinolin-2(1H)-one, pyrimidinyl, purinyl, pyridyl, pyridazinyl, benzothiazolyl, and pyrazolyl. Heteroaryl does not encompass or overlap with aryl as defined above.

[0050] “Alkyl-heteroaryl” refers to a radical having an alkyl component and a heteroaryl component, where the alkyl component links the heteroaryl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the heteroaryl component and to the point of attachment. The alkyl component can include any number of carbons, such as Co-6, C1-2, C1-3, C1-4, C1-5, C1-6, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. In some instances, the alkyl component can be absent. The heteroaryl component is as defined within.Attorney Docket: 049386-553001WO

[0051] Provided are also pharmaceutically acceptable salts, hydrates, solvates, tautomeric forms, polymorphs, and prodrugs of the compounds described herein. “Pharmaceutically acceptable” or “physiologically acceptable” refer to compounds, salts, formulations, dosage forms and other materials which are useful in preparing a pharmaceutical composition that is suitable for veterinary or human pharmaceutical use.

[0052] The compounds described herein can be prepared and / or formulated as pharmaceutically acceptable salts or when appropriate as a free base. Pharmaceutically acceptable salts are nontoxic salts of a free base form of a compound that possess the desired pharmacological activity of the free base. These salts can be derived from inorganic or organic acids or bases. For example, a compound that contains a basic nitrogen can be prepared as a pharmaceutically acceptable salt by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne- 1,4-dioates, hexyne- 1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene- 1 -sulfonates, naphthalene -2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, and mandelates. Lists of other suitable pharmaceutically acceptable salts are found in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21stEdition, Lippincott Wiliams and Wilkins, Philadelphia, Pa., 2006.

[0053] Examples of “pharmaceutically acceptable salts” of the compounds disclosed herein also include salts derived from an appropriate base, such as an alkali metal (for example, sodium, potassium), an alkaline earth metal (for example, magnesium), ammonium and NX4+(wherein X is C1–C4alkyl). Also included are base addition salts, such as sodium or potassium salts.

[0054] Provided are also compounds described herein or pharmaceutically acceptable salts, isomers, or a mixture thereof, in which from 1 to n hydrogen atoms attached to a carbon atom can be replaced by a deuterium atom or D, in which n is the number of hydrogen atoms in the molecule. As known in the art, the deuterium atom is a non-radioactive isotope of the hydrogen atom. Such compounds can increase resistance to metabolism, and thus can be useful for increasing the halflife of the compounds described herein or pharmaceutically acceptable salts, isomer, or a mixtureAttorney Docket: 049386-553001WOthereof when administered to a mammal. See, e.g., Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism,” TRENDS PHARMACOL. SCI., 5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogen atoms have been replaced by deuterium.

[0055] Examples of isotopes that can be incorporated into the disclosed compounds also include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as2H,3H,11C,13C,14C,13N,15N,15O,17O,18O,31P,32P,35S,18F,36Cl,123I, and125I, respectively. Substitution with positron emitting isotopes, such as11C,18F,15O and13N, can be useful in Positron Emission Tomography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds of Formulas I and II, can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Examples as set out below using an appropriate isotopically-labeled reagent in place of the nonlabeled reagent previously employed.

[0056] The compounds of the embodiments disclosed herein, or their pharmaceutically acceptable salts can contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. Where compounds are represented in their chiral form, it is understood that the embodiment encompasses, but is not limited to, the specific diastereomerically or enantiomerically enriched form. Where chirality is not specified but is present, it is understood that the embodiment is directed to either the specific diastereomerically or enantiomerically enriched form; or a racemic or scalemic mixture of such compound(s). As used herein, “scalemic mixture” is a mixture of stereoisomers at a ratio other than 1:1.Attorney Docket: 049386-553001WO

[0057] “Racemates” refers to a mixture of enantiomers. The mixture can comprise equal or unequal amounts of each enantiomer.

[0058] “Stereoisomer” and “stereoisomers” refer to compounds that differ in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds can exist in stereoisomeric form if they possess one or more asymmetric centers or a double bond with asymmetric substitution and, therefore, can be produced as individual stereoisomers or as mixtures. Unless otherwise indicated, the description is intended to include individual stereoisomers as well as mixtures. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see, e.g., Chapter 4 of ADVANCED ORGANIC CHEMISTRY, 4th ed., J. March, John Wiley & Sons, New York, 1992).

[0059] A “subject” or “patient” is meant to describe a human or vertebrate animal including a dog, cat, pocket pet, marmoset, horse, cow, pig, sheep, goat, elephant, giraffe, chicken, lion, monkey, owl, rat, squirrel, slender loris, and mouse. A “pocket pet” refers to a group of vertebrate animals capable of fitting into a commodious coat pocket such as, for example, hamsters, chinchillas, ferrets, rats, guinea pigs, gerbils, rabbits and sugar gliders.

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. A dash at the front or end of a chemical group is a matter of convenience; chemical groups can be depicted with or without one or more dashes without losing their ordinary meaning. A wavy line drawn through a line in a structure indicates a point of attachment of a group. A dashed line indicates an optional bond. Unless chemically or structurally required, no directionality is indicated or implied by the order in which a chemical group is written or the point at which it is attached to the remainder of the molecule. For instance, the group “-SO2CH2-” is equivalent to “-CH2SO2-” and both can be connected in either direction. A prefix such as “Cu-v”, “Cu-Cv” or “(Cu-Cv)” indicates that the following group has from u to v carbon atoms. For example, “C1-6 alkyl” and “Ci-Ce alkyl” both indicate that the alkyl group has from 1 to 6 carbon atoms.

[0061] Unless otherwise specified, the carbon atoms of the compounds of Formula I, are intended to have a valence of four. If in some chemical structure representations, carbon atoms do not have a sufficient number of variables attached to produce a valence of four, the remaining carbon substituents needed to provide a valence of four should be assumed to be hydrogen.

[0062] “Treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. Beneficial or desired clinical results may include one or more of theAttorney Docket: 049386-553001WOfollowing: (a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and / or diminishing the extent of the disease or condition); (b) slowing or arresting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition, and / or preventing or delaying the spread (e.g., metastasis) of the disease or condition); and / or (c) relieving the disease, that is, causing the regression of clinical symptoms (e.g., ameliorating the disease state, providing partial or total remission of the disease or condition, enhancing effect of another medication, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival.

[0063] The term “therapeutically effective amount,” as used herein, is the amount of compound disclosed herein present in a formulation described herein that is needed to provide a desired level of drug in the secretions and tissues of the airways and lungs, or alternatively, in the bloodstream of a subject to be treated to give an anticipated physiological response or desired biological effect when such a formulation is administered by the chosen route of administration. The precise amount will depend upon numerous factors, for example the particular compound disclosed herein, the specific activity of the formulation, the delivery device employed, the physical characteristics of the formulation, its intended use, as well as subject considerations such as severity of the disease state, subject cooperation, etc., and can readily be determined by one skilled in the art based upon the information provided herein.

[0064] “Administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject. The administration can be carried out according to a schedule specifying frequency of administration, dose for administration, and other factors.

[0065] “Co-administration” as used herein refers to administration of unit dosages of the compounds disclosed herein before or after administration of unit dosages of one or more additional therapeutic agents, for example, administration of the compound disclosed herein within seconds, minutes, or hours of the administration of one or more additional therapeutic agents. For example, in some embodiments, a unit dose of a compound of the present disclosure is administered first, followed within seconds or minutes by administration of a unit dose of one or more additional therapeutic agents. Alternatively, in other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed by administration of a unit dose of a compound of the present disclosure within seconds or minutes. In some embodiments, a unitAttorney Docket: 049386-553001WOdose of a compound of the present disclosure is administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of one or more additional therapeutic agents. In other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of a compound of the present disclosure. Co-administration of a compound disclosed herein with one or more additional therapeutic agents generally refers to simultaneous or sequential administration of a compound disclosed herein and one or more additional therapeutic agents, such that therapeutically effective amounts of each agent are present in the body of the patient.

[0066] “Disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein. The disease may be an autoimmune, inflammatory, cancer, infectious (e.g., a viral infection), metabolic, developmental, cardiovascular, liver, intestinal, endocrine, neurological, or other disease. In some embodiments, the disease is cancer (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma).

[0067] The term “adjacent carbons” and “adjacent atoms” as used herein refers to consecutive H H H H H H43.234.T 5 fl carbon atoms that are directly attached to each other. For example, in H H H H, Ci and C2 are adjacent carbons, C2 and C3 are adjacent carbons, C3 and C4 are adjacent carbons, and C4and C5 are adjacent carbons. Similarly, in3Ci and C2 are adjacent carbons, C2 and C3 are adjacent carbons, C3 and C4 are adjacent carbons, and C4 and C5 are adjacent carbons, C5 and Ce are adjacent carbons and Ce and Ci are adjacent carbons.

[0068] The term “double bond” as used herein refers to the formation of an additional single bond between two adjacent atoms that are already connected by a single bond, thus forming a double H H H H H H34.4 34.234bond. For example, in H H H H, Ci and C2 are adjacent carbons, C2 and C3 are adjacent carbons, C3 and C4 are adjacent carbons, and C4 and C5 are adjacent carbons, such that two hydrogens, or R groups, on adjacent atoms are combined with the atoms to which they are attached and the bond linking the adjacent atoms to form a double bond as shown in the following:Attorney Docket: 049386-553001WOH H H H HX 4 x H H H HH H H HSimilarly, in3Ci and C2 are adjacent carbons, C2 and C3 are adjacent carbons, C3 and C4 are adjacent carbons, C4 and C5 are adjacent carbons, C5 and Ce are adjacent carbons, and Ce and Ci are adjacent carbons, such that two hydrogens, or R groups, on adjacent atoms are combined with the atoms to which they are attached and the bond linking the adjacent atoms to form a double bond as shown in the following:

[0069] The term “non-adjacent carbons” and “non-adjacent atoms” as used herein refers to non-consecutive carbons atoms that are not directly attached to each other. For example, in H H H H H H I X 5>4^ 3 > 2< 3 l4AH H H H, and C3 are non-adjacent carbons, Ci and C4 are non-adjacent carbons, C2 and C4 are non-adjacent carbons, Ci and C5 are non-adjacent carbons, C2 and C5 are non-adjacent2 carbons, and C3 and C5 are non-adjacent carbons, among others. Similarly, in3Ci and C3 are non-adjacent carbons, Ci and C4 are non-adjacent carbons, C2 and C4 are non-adjacent carbons, Ci and C5 are non-adjacent carbons, C2 and C5 are non-adjacent carbons, C3 and C5 are non-adjacent carbons, C2 and Ce are non-adjacent carbons, C3 and Ce are non-adjacent carbons, and C4 and Ce are non-adjacent carbons.

[0070] “Solvate” as used herein refers to the result of the interaction of a solvent and a compound. Solvates of salts of the compounds described herein are also provided. Hydrates of the compounds described herein are also provided.

[0071] “Prodrug” as used herein refers to a derivative of a drug that upon administration to the human body is converted to the parent drug according to some chemical or enzymatic pathway.

[0072] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes, but is not limited to, any and all solvents, dispersion media, coatings,Attorney Docket: 049386-553001WOantibacterial and antifungal agents, isotonic and absorption delaying agents, and combinations thereof. The use of pharmaceutically acceptable carriers and pharmaceutically acceptable excipients for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic formulations is contemplated. Supplementary active ingredients can also be incorporated into the formulations. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.III. CompoundsCompound of Formula I

[0073] Disclosed herein are, among other things, compounds of Formula I.

[0074] In one aspect, the present disclosure provides a compound of Formula I:X O OR’Oc +whereinX1, X2, and X3are each independently O or S,Ra, Rb, and Rcare each independently H, or C1-6alkyl, orRais H or C1-6alkyl, and Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, the heterocyclyl being optionally substituted with one or more C1-3 alkyl,n is 2 to 6,eitherwhereinY1is absent, O, or S,L1is C3-40 alkyl, C3-40 alkenyl, or C3-40 alkynyl,eitherAttorney Docket: 049386-553001WO(a) Y2is absent or C1-4 alkylene, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl, optionally the alkyl, alkenyl, and alkynyl are branched, or(b) Y2is C1-4 alkylene, optionally substituted with oxo, W is absent, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative; orX1(ii) R1is w'Land R2i A^Y1'Lwhereinis absent, O, or S,L1is C3-40 alkyl, C3-40 alkenyl, or C3-40 alkynyl,Y2is C1-4 alkylene, W is absent or carbonyl, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative.

[0075] In some embodiments, the compound of Formula I has a structure of Formula I-I:I-Ior a pharmaceutically acceptable salt thereof,whereinX1, X2, and X3are each independently O or S,Ra, Rb, and Rcare each independently H, or C1-6 alkyl, orAttorney Docket: 049386-553001WORais H or C1-6alkyl, and Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, the heterocyclyl being optionally substituted with one or more C1-3alkyl,n is 2 to 6,Y1is absent, O, or S,L1is C3-40alkyl, C3-40alkenyl, or C3-40alkynyl,Y2is absent or C1-4alkylene, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl, optionally the alkyl, alkenyl, and alkynyl are branched, orY2is C1-4 alkylene, optionally substituted with oxo, W is absent, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative.

[0076] In some embodiments, X1, X2, and X3are each independently O.

[0077] In some embodiments, Rais H. In some embodiments, Rband Rcare each independently C1-6 alkyl. In some embodiments, Rais H, and Rband Rcare each independently C1-6 alkyl. In some embodiments, Rais C1-6 alkyl. In some embodiments, Ra, Rband Rcare each independently Ci -6 alkyl.

[0078] In some embodiments, Ra, Rb, and Rcare each independently C1-3 alkyl. In some embodiments, Ra, Rb, and Rcare each independently methyl.

[0079] In some embodiments, Rais C1-6 alkyl, and Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, optionally substituted with one or more C1-3 alkyl. In some embodiments, when Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, Rais C1-3 alkyl. In some embodiments, when Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, Rais methyl.

[0080] In some embodiments, Rband Rctaken together with the N they are attached to form a piperazinyl. Optionally, the piperazinyl is substituted with one or more C1-3 alkyl.

[0081] In some embodiments, n is 2 to 4. In some embodiments, n is 2.Attorney Docket: 049386-553001WO

[0082] In some embodiments, Y1is absent. In some embodiments, Y1is O or S.

[0083] In some embodiments, L1 is C6-25 alkyl, C6-25 alkenyl, or C6-25 alkynyl. In some embodiments, L1 is C6-25 alkyl. In some embodiments, L1 is C10-20 alkyl. In some embodiments, L1 is C6-25 alkenyl. In some embodiments, L1 is C6-25 alkynyl.

[0084] In some embodiments, L1is branched. In some embodiments, L1is unbranched.

[0085] In some embodiments, Y2is absent or C1-4 alkylene, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl.

[0086] In some embodiments, Y2is C1-4 alkylene, W is O or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl.

[0087] In some embodiments, Y2is absent, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl, optionally W is absent.

[0088] In some embodiments, W is O or S. In some embodiments, W is O. In some embodiments, W is absent.

[0089] In some embodiments, L2 is selected from C6-25 alkyl, C6-25 alkenyl, and C6-25 alkynyl. In some embodiments, L2 is selected from C10-20 alkyl, C10-20 alkenyl, and C10-20 alkynyl. In some embodiments, L2is C10-20 alkyl.

[0090] In some embodiments, L2 is C6-25 alkyl.

[0091] In some embodiments, L2is branched. In some embodiments, L2is unbranched.

[0092] In some embodiments, Y2is C1-4 alkylene, W is absent, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative. It is contemplated that the cholesterol derivative can be any suitable cholesterol known in the art with an O atom available for attachment to the rest of the compound of Formula I. For example, a suitable cholesterol derivative can be selected from Beta-sitosterol, stigmasterol, lanosterol, thiocholesterol, stigmastanol, campesterol, fucosterol, brassicasterol and ergosterol

[0093] In some embodiments, Y2is C1-2 alkylene, W is absent, and, L2is cholesterol attached to Y2via an O atom of the cholesterol.Compound of Formula IA

[0094] In some embodiments, the compound of formula I is of Formula IAAttorney Docket: 049386-553001WOIA,or a pharmaceutically acceptable salt thereof.

[0095] In some embodiments, n is 2 to 4. In some embodiments, n is 2.

[0096] In some embodiments, Y2is absent and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl.

[0097] In some embodiments, L2is C3-40 alkyl. In some embodiments, L2 is C6-25 alkyl. In some embodiments, L2is C10-20 alkyl.

[0098] In some embodiments, W is absent or O. In some embodiments, W is absent. In some embodiment, W is O.

[0099] In some embodiments, Y2is C1-4 alkylene and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or cholesterol derivative. The cholesterol derivative is as defined herein.

[0100] In some embodiments, Y2is C1-3 alkylene and L2is cholesterol.

[0101] In some embodiments, Y2is an alkylene substituted with one oxo, such as a C1-4 alkylene or C1-3 alkylene substituted with an oxo. In this case, L2can be attached to Y2via an ester linkage. For example, the cholesterol or the cholesterol derivative can be attached to the rest of the compound of Formula IA via an ester linkage or a thioester linkage with Y2.Compound of Formula IB and IC

[0102] In some embodiments, the compound of formula I is of Formula IBAttorney Docket: 049386-553001WOor a pharmaceutically acceptable salt thereof.

[0103] In some embodiments, the compound of formula I is of Formula ICIC,or a pharmaceutically acceptable salt thereof.

[0104] In some embodiments, n is 2 to 4. In some embodiments, n is 2.

[0105] In some embodiments, L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl. In some embodiments, L2 is C6-25 alkyl. In some embodiments, L2is C10-20 alkyl. In some embodiments, L2is branched. In some embodiments, L2is unbranched.Compound of Formula ID

[0106] In some embodiments, the compound of formula I is of Formula IDAttorney Docket: 049386-553001WOor a pharmaceutically acceptable salt thereof,wherein m is 1 to 3.

[0107] In some embodiments, L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative. The cholesterol derivative is as defined herein.

[0108] In some embodiments, m is 1 or 2. In some embodiments., m is 1.

[0109] For compounds of Formulae IA, to ID, in some embodiments, Ra, Rb, and Rcare each independently C1-3 alkyl. Optionally, Ra, Rb, and Rcare each independently methyl.

[0110] In some embodiments, L1 is C6-25 alkyl, C6-25 alkenyl, or C6-25 alkynyl. In some embodiments, L1 is C6-25 alkyl. In some embodiments, L1 is C10 to 20 alkyl. In some embodiments, L1 is C6-25 alkenyl. In some embodiments, L1 is C10-20 alkenyl.

[0111] In some embodiments, L1is branched. In some embodiments, L1is unbranched.Exemplary Compounds of Formula I

[0112] In some embodiments, the compound of Formula I is selected from the compounds of Table A, the compounds of Table B, and the compounds of Table C, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula I-I is selected from the compounds of Table A, the compounds of Table B, and the compounds of Table C, or a pharmaceutically acceptable salt thereof.

[0113] In some embodiments, the compound of Formula I is selected from the compounds of Table A or a pharmaceutically acceptable salt thereof.Table A - Exemplary Compounds of the Present DisclosureooO' O'O / O'Attorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WO

[0114] In some embodiments, the compound of Formula I is selected fromAttorney Docket: 049386-553001WO

[0115] In some embodiments, the compound of Formula I is selected fromAttorney Docket: 049386-553001WO1 z ——yd QO O 73T °°

[0116] In some embodiments, Y2is C1-4 alkylene, W is absent, and L2is cholesterol or cholesterol derivative attached to Y2via an O of the cholesterol or via an O or S atom of the cholesterol derivative. In some embodiments, L2is a cholesterol derivative. For example, the compound of the present disclosure comprising a cholesterol derivative can be selected from the compounds of Table B and Table C below or a pharmaceutically acceptable salt thereof.Table B - Exemplary compounds comprising cholesterol derivativesCompound number RD1-2-1. f H / Ixj. f TA T A1-2-2• jT H / , | T AT AAttorney Docket: 049386-553001WO-2-3-2-44 ^^ - 26iTii" -~\ 7 \1- ^X \ / \IHI ' -1- i ^ Aii' — '-2-5f H-2-6-2-7-2-8\ l] ^ 1 1hAttorney Docket: 049386-553001WOxx4u L ^ —4^ ^ - \ 7\ / \ \ 7 \x—friiT Xui Xz—4 - —-5-1J3 / 7V^-^-5-2h c\ U ^|||| - - I 1 \ \H, f T fi *T\ Q O=p a: ti-5-3r j ' T b o Y\z —\-5-4-5-5Attorney Docket: 049386-553001WO-5-6I XHX / _ I T A | A-5-7Ji (ni•'■4 (iilTu‘* - ^^iiH" ' —i A Au ' — \ / \1—p-5-8\ / ( Illl -.-5-9 'T— / \4S 111111 -*^^ao o a:Vo o ''z —\-6-1i XHJX / Ix<. x X1^ J-6-2i Xh / <- | T A T AyxA-A>Attorney Docket: 049386-553001WO-6-3-6-44 ^^ - 26iTii" -~\ 7 \1- ^X \ / \IHI ' -1- i ^ Aii' — '-6-5f H-6-6-6-7-6-8\ l] ^ 1 1hAttorney Docket: 049386-553001WOxx4u L ^ —4^ ^ - \ 7\ / \ \ 7 \z x—friiT Xui X —4 - —-7-1J3 / 7V^-^-7-2h c\ U ^|||| - - I 1 \ \H, f T fi *T\ o a=0? o o-7-3- j ' T „zb°0.^) dzz —\-7-4-7-5Attorney Docket: 049386-553001WO-7-6\z—00,' ■o I XHX / 9 o _ I T A | A -7-7Y oo=Ji (ni•'■ 4 (iilTu‘* - ^ ^ \ / \iiH" ' —1— i A Au ' —p-7-8(j\ / ( Illl -.-7-9 'T— / \4S 111111 -*^^-8-1i XHX-8-2i XHs, r T A T AAttorney Docket: 049386-553001WO-8-3-8-44 ^^ - 26iTii" -~\ 7 \1- ^X \ / \IHI ' -1- i ^ Aii' — '-8-5f H-8-6-8-7-8-8\ l] ^ 1 1hAttorney Docket: 049386-553001WOxx4u L ^ —4^ - ^ \ 7\ / \ \ 7 \z x—friiT Xui X —4 - —-12-1J3 / 7V^-^-12-2h c\ U ^|||| - - I 1 \ \H, f T fi *T\ O Q=P or o-12-3r j ' T Vb o'z —\-12-4-12-5Attorney Docket: 049386-553001WO1-12-6i XHX / _ I T A | A1-12-7Ji (ni•'■4 (iilTu‘* - ^^ \ / \i \iH" ' —1—i A Au ' —p1-12-8\ / ( Illl -.1-12-9 □ 'T— / \oe 4S 111111 -*^^\ in oZ=)O Krb oxfa°Table C – Exemplary compounds comprising cholesterol derivatives\Compound RDREnumber1-13-1 _ 0— 'X / Az'X / A / '' — I XHJL / '._T JLAttorney Docket: 049386-553001WO -13-2 0 I 1HA-13-3 0J"\ Illl' \ - \4 \z:- —411101 ii ' -^^-13-4 0 I fH<• f T A T f 4 ^^ - / gZX / Az 7 \ \1—6ii" — '-13-5 0ZX / X / X / X / A / A / A / X / X / ^Q-X £1 JLH, f T A T A-13-6 0-13-7 0 — 7 / 7 / 7 / 77 — ZV — AQ-XAttorney Docket: 049386-553001WO-13-8 0\ l[ ^■ f H / xc jT TA J A-13-9 0\ / r^-9-1 0 — - - I 1H1 L^r t-9-2 0 — AAA\ / A^o-\ ^l AllH ' - \ 7 \ \ 7 \1—1—A>7^-9-3 0-9-4 o-9-5 0I 1H / c r T A T AAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WO-14-3 0?1" c-14-4 o / / < / VX / V^X / X^Q-X 4 \ ' 1- —41111 ii -^^-14-5 0XX / XX^^ / X / XAQX1 XHy XXxT\ 7 \1—friiiTu *-14-6 07^i XHXT / . f TATA-14-7 0-14-8 0 - - - - ^X-Q'X \ l / ^i XHy-XUPAttorney Docket: 049386-553001WO-14-9 0 / VX / ^VA / T^Q-X \ IT^<5^ _-15-1 o IXHJL / \. f TA T A-15-2 o1 1H, f TA T fi-15-3 o 2 (ITH■■■ - \ 7 \1—7^-15-4 o-15-5I 1H / < r TAT AAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WO-16-3 o?1" c-16-4 o / / < V^z\ / ^ / =W^vzV^o-\ 4 \ ' 1- —41111 ii -^^-16-5 o1 XHy XXJxfriiiTu * \ 7 \1—-16-6 o7^i XHXT / . f TATA-16-7 o-16-8 o \ l / ^i XHy-XUPAttorney Docket: 049386-553001WO-16-9 o \ IT^<5^ _-17-1 0 —ZX / X— I XHJL / \. f TA T A-17-2 01 1H, f TA T fi-17-3 o 2 (ITH■■■ - / x / x / =x^=x / x / x / x^oX \ 7 \1—7^-17-4 o / x / x / =x^=x / x / x / x^oX-17-5 0 = = TI 1H / < r TAT AAttorney Docket: 049386-553001WO

[0117] Also falling within the scope herein are the in vivo metabolic products of the compounds described herein. Such products may result for example from the oxidation, reduction, hydrolysis, amidation, esterification and the like of the administered compound, primarily due to enzymatic processes. Accordingly, included are novel and unobvious compounds produced by a process comprising contacting a compound with a mammal for a period of time sufficient to yield a metabolic product thereof. Such products typically are identified by preparing a radiolabelled (e.g.,14C or3H) compound, administering it parenterally in a detectable dose (e.g., greater than about 0.5 mg / kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples. These products are easily isolated since they are labeled (others are isolated by the use of antibodies capable of binding epitopes surviving in the metabolite). The metabolite structures are determined in conventionalAttorney Docket: 049386-553001WOfashion, e.g., by MS or NMR analysis. In general, analysis of metabolites is done in the same way as conventional drug metabolism studies. The conversion products, so long as they are not otherwise found in vivo, are useful in diagnostic assays for therapeutic dosing of the compounds even if they possess no activity of their own.IV. Pharmaceutical Compositions, Lipid Compositions, Lipid Nanoparticles

[0118] Also disclosed herein are pharmaceutical compositions comprising a pharmaceutically effective amount of a compound of the present disclosure (e.g., a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. Also provided herein is a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0119] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.

[0120] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0121] In some embodiments, the present disclosure provides a lipid composition comprising a nucleic acid and a compound of the present disclosure or a pharmaceutically acceptable salt or solvate thereof.

[0122] In some embodiments, the nucleic acid is RNA or DNA. In some embodiments, the nucleic acid is selected from an siRNA, an mRNA, a self-replicating RNA, a DNA plasmid, and an antisense oligonucleotide. In some embodiments, the nucleic acid is an siRNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a selfreplicating RNA. In some embodiments, the nucleic acid is a DNA plasmid. In some embodiments, the nucleic acid is an antisense oligonucleotide. In some embodiment, the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a protein. In some embodiments, the protein is an enzyme, an antibody, an antigen, a receptor, or a transporter. In some embodiments, the protein is a gene-editing enzyme. In some embodiments, the protein is an enzyme. In some embodiments, the protein is an antibody. In some embodiments, the protein is an antigen. In some embodiments, the protein is a receptor. In some embodiments, the proteinAttorney Docket: 049386-553001WOis a transporter. For example, the gene-editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease.

[0123] The nucleic acid may be encapsulated or complexed within the lipid portion of the composition, thereby protecting the nucleic acid from nuclease degradation. In some embodiments, the nucleic acid is substantially fully encapsulated within the lipid portion of the lipid formulation, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the nucleic acid in the lipid formulation is not substantially degraded after exposure of the particle to a nuclease at 37 °C for at least 20, 30, 45, or 60 minutes. In certain other instances, the nucleic acid in the lipid formulation is not substantially degraded after incubation of the formulation in serum at 37 °C for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the nucleic acid is complexed with the lipid portion of the formulation.

[0124] In the context of nucleic acids, full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid. Encapsulation is determined by adding the dye to a lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E = (10 - I) / I0, where I and 10 refer to the fluorescence intensities before and after the addition of detergent.

[0125] In some embodiments, the lipid composition (e.g. lipid nanoparticles) comprise a nucleic acid that is fully encapsulated within the lipid portion of the formulation, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction thereof orAttorney Docket: 049386-553001WOrange therein) of the particles have the nucleic acid encapsulated therein. The amount may be any value or subvalue within the recited ranges, including endpoints.

[0126] It can be appreciated that the lipid composition can exist in different structural forms. For example, the lipid composition comprises liposomes, lipoplexes, or lipid nanoparticles. In some embodiments, the lipid composition comprises or is lipid nanoparticles.

[0127] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the lipid nanoparticle of the present disclosure, and a pharmaceutically acceptable excipient.

[0128] In some embodiments, the pharmaceutical composition is a lyophilized composition.

[0129] Therapies based on the intracellular delivery of nucleic acids to target cells face both extracellular and intracellular barriers. Indeed, naked nucleic acid materials cannot be easily systemically administered due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, phagocyte uptake and their ability in activating the immune response, all features that preclude their clinical development. When exogenous nucleic acid material (e.g., mRNA) enters the human biological system, it is recognized by the reticuloendothelial system (RES) as foreign pathogens and cleared from blood circulation before having the chance to encounter target cells within or outside the vascular system. It has been reported that the half-life of naked nucleic acid in the blood stream is around several minutes (Kawabata K, Takakura Y, Hashida M Pharm Res. 1995 Jun; 12(6):825-30). Chemical modification and a proper delivery method can reduce uptake by the RES and protect nucleic acids from degradation by ubiquitous nucleases, which increase stability and efficacy of nucleic acidbased therapies. In addition, RNAs or DNAs are anionic hydrophilic polymers that are not favorable for uptake by cells, which are also anionic at the surface. The success of nucleic acidbased therapies thus depends largely on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells and obtain sufficient levels of expression in vivo with minimal toxicity.

[0130] Moreover, upon internalization into a target cell, nucleic acid delivery vectors are challenged by intracellular barriers, including endosome entrapment, lysosomal degradation, nucleic acid unpacking from vectors, translocation across the nuclear membrane (for DNA), and release at the cytoplasm (for RNA). Successful nucleic acid-based therapy thus depends upon the ability of the vector to deliver the nucleic acids to the target sites inside of the cells in order to obtain sufficient levels of a desired activity such as expression of a gene.Attorney Docket: 049386-553001WO

[0131] While several gene therapies have been able to successfully utilize a viral delivery vector (e.g., AAV), lipid-based formulations have been increasingly recognized as one of the most promising delivery systems for RNA and other nucleic acid compounds due to their biocompatibility and their ease of large-scale production. One of the most significant advances in lipid-based nucleic acid therapies happened in August 2018 when Patisiran (ALN-TTR02) was the first siRNA therapeutic approved by the Food and Drug Administration (FDA) and by the European Commission (EC). ALN-TTR02 is an siRNA formulation based upon the so-called Stable Nucleic Acid Lipid Particle (SNALP) transfecting technology. Despite the success of Patisiran, the delivery of nucleic acid therapeutics, including mRNA, via lipid formulations is still undergoing development. The use of mRNA in lipid delivery vehicles quickly rose to prominence as a result of the COVID-19 pandemic with several vaccines delivering mRNA encoding the spike protein of COVID-19 showing strong protective capabilities. Such lipid-based mRNA vaccines include Pfizer and BioNtech’s BNT162b2 and Moderna’s mRNA-1273, which have received emergency use authorization around the world.

[0132] Some art-recognized lipid-formulated delivery vehicles for nucleic acid therapeutics include, according to various embodiments, polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, multivesicular liposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, micelles, and emulsions. These lipid formulations can vary in their structure and composition, and as can be expected in a rapidly evolving field, several different terms have been used in the art to describe a single type of delivery vehicle. At the same time, the terms for lipid formulations have varied as to their intended meaning throughout the scientific literature, and this inconsistent use has caused confusion as to the exact meaning of several terms for lipid formulations. Among the several potential lipid formulations, liposomes, cationic liposomes, and lipid nanoparticles are specifically described in detail and defined herein for the purposes of the present disclosure.Liposomes

[0133] Conventional liposomes are vesicles that consist of at least one bilayer and an internal aqueous compartment. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). They generally present as spherical vesicles and can range in size from 20 nm to a few microns. Liposomal formulations can be prepared as a colloidalAttorney Docket: 049386-553001WOdispersion or they can be lyophilized to reduce stability risks and to improve the shelf-life for liposome-based drugs. Methods of preparing liposomal compositions are known in the art and are within the skill of an ordinary artisan.

[0134] Liposomes that have only one bilayer are referred to as being unilamellar, and those having more than one bilayer are referred to as multilamellar. The most common types of liposomes are small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), and multilamellar vesicles (MLV). In contrast to liposomes, lysosomes, micelles, and reversed micelles are composed of monolayers of lipids. Generally, a liposome is thought of as having a single interior compartment, however some formulations can be multivesicular liposomes (MVL), which consist of numerous discontinuous internal aqueous compartments separated by several nonconcentric lipid bilayers.

[0135] Liposomes have long been perceived as drug delivery vehicles because of their superior biocompatibility, given that liposomes are basically analogs of biological membranes, and can be prepared from both natural and synthetic phospholipids (Int. J. Nanomedicine. 2014; 9:1833-1843). In their use as drug delivery vehicles, because a liposome has an aqueous solution core surrounded by a hydrophobic membrane, hydrophilic solutes dissolved in the core cannot readily pass through the bilayer, and hydrophobic compounds will associate with the bilayer. Thus, a liposome can be loaded with hydrophobic and / or hydrophilic molecules. When a liposome is used to carry a nucleic acid such as RNA, the nucleic acid is contained within the liposomal compartment in an aqueous phase.

[0136] Liposomes can be composed of cationic, anionic, and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are liposomes that are made in whole or part from positively charged lipids, or more specifically a lipid that comprises both a cationic group and a lipophilic portion. In addition to the general characteristics profiled above for liposomes, the positively charged moieties of cationic lipids used in cationic liposomes provide several advantages and some unique structural features. For example, the lipophilic portion of the cationic lipid is hydrophobic and thus will direct itself away from the aqueous interior of the liposome and associate with other nonpolar and hydrophobic species. Conversely, the cationic moiety will associate with aqueous media and more importantly with polar molecules and species with which it can complex in the aqueous interior of the cationic liposome. For these reasons, cationic liposomes are increasingly being researched for use in gene therapy due to their favorability towards negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required forAttorney Docket: 049386-553001WOin vivo clinical applications. Cationic lipids suitable for use in cationic liposomes are listed herein below.Lipid Nanoparticles

[0137] In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNP) have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phase in its interior, but rather the lipids from the bilayer or monolayer shell are directly complexed to the internal compound thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles having a relatively uniform dispersion of shape and size. While sources vary on what size qualifies a lipid particle as being a nanoparticle, there is some overlap in agreement that a lipid nanoparticle can have a diameter in the range of from 10 nm to 1000 nm. However, more commonly they are considered to be smaller than 120 nm or even 100 nm.

[0138] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to include an ionizable cationic lipid which can complex to and associate with the negatively charged backbone of the nucleic acid core. Ionizable cationic lipids with apparent pKa values below about 7 have the benefit of providing a cationic lipid for complexing with the nucleic acid’s negatively charged backbone and loading into the lipid nanoparticle at pH values below the pKa of the ionizable lipid where it is positively charged. Then, at physiological pH values, the lipid nanoparticle can adopt a relatively neutral exterior allowing for a significant increase in the circulation half-lives of the particles following IV administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutics, and low levels of cytotoxicity and immunogenicity.

[0139] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely studied as synthetic materials for delivery of nucleic acid medicines. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and characterized by broad size distributions ranging from the submicron scale to a few microns. Lipoplexes, such as the LIPOFECTAMINE® reagent, have found considerable utility for in vitro transfection. However, these first-generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (imparted by the cationic lipid) result in rapid plasma clearance, hemolytic and other toxicities, as well as immune system activation.Attorney Docket: 049386-553001WO

[0140] In some embodiments, the present disclosure provides a lipid nanoparticle comprising a plurality of lipids, wherein each lipid is independently a compound of the present disclosure or a pharmaceutically acceptable salt or solvate thereof. For example, the plurality of lipids can selfassemble to form the lipid nanoparticle. In another aspect, the present disclosure provides a lipid nanoparticle comprising a compound of Formula I of the present disclosure or a pharmaceutically acceptable salt or solvate thereof. For example, the compound of the present disclosure or the salt thereof self-assembles into the lipid nanoparticle. It is understood that a lipid nanoparticle generally comprising an interior and exterior.

[0141] The lipid nanoparticle can be a range of sizes as measured by for example average diameter. For example, the average particle size of the lipid nanoparticle is less than about 1000 nm, less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 90 nm. For example, the average particle size of the lipid nanoparticle is more than about 10 nm, more than about 20 nm, more than about 30 nm, more than about 40 nm, more than about 50 nm, more than about 60 nm, more than about 80 nm, more than about 100 nm, more than about 150 nm, or more than about 200 nm. In some embodiments, the average particle size of the lipid nanoparticle is less than about 100 nm. In some embodiments, the average particle size of the lipid nanoparticle is about 40 nm to about 100 nm, about 50 nm to about 100 nm, about 60 nm to about 100 nm, about 50 nm to about 95 nm, about 50 nm to about 90 nm, or about 60 nm to about 90 nm. The diameter may be any value or subvalue within the recited ranges, including endpoints.

[0142] In some embodiments, the lipid nanoparticle has a polydispersity index (PDI) of about 0.010 to about 1.10. In embodiments, the PDI is about 0.010 to about 1.05, about 0.010 to about 1.00, about 0.010 to about 0.95, about 0.010 to about 0.90, about 0.010 to about 0.85, about 0.010 to about 0.80, about 0.010 to about 0.75, about 0.010 to about 0.70, about 0.010 to about 0.65, about 0.010 to about 0.60, about 0.010 to about 0.55, about 0.010 to about 0.50, about 0.010 to about 0.45, about 0.010 to about 0.40, about 0.010 to about 0.35, about 0.010 to about 0.30, about 0.010 to about 0.25, about 0.010 to about 0.20, about 0.010 to about 0.15, about 0.010 to about 0.10, about 0.010 to about 0.09, about 0.010 to about 0.08, about 0.010 to about 0.07, about 0.010 to about 0.06, about 0.010 to about 0.05, about 0.010 to about 0.04, about 0.010 to about 0.03, about 0.010 to about 0.02, about 0.010 to about 0.019, about 0.010 to about 0.018, about 0.010 to about 0.017, about 0.010 to about 0.016, about 0.010 to about 0.015, about 0.010 to about 0.014, about 0.010 to about 0.013, about 0.010 to about 0.012, about 0.010 to about 0.011 (or any ranges therein.) The amount may be any value or subvalue within the recited ranges, including endpoints.Attorney Docket: 049386-553001WO

[0143] In some embodiments, the lipid nanoparticle is substantially non-toxic. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease.

[0144] In some embodiments, the lipid nanoparticle further comprises a nucleic acid. For example, the nucleic acid can be encapsulated in the interior. In some embodiments, the nucleic acid is as defined herein.Additional Components

[0145] It is contemplated that the pharmaceutical composition, lipid composition and lipid nanoparticle of the present disclosure can further comprise other components. Exemplary additional components can be one or more of other lipids, cholesterol, polyethylene glycol (PEG)-lipid conjugate as described herein. The other lipids can be helper lipids and ionizable cationic lipids as described herein. It is also contemplated that the pharmaceutical composition, lipid composition and lipid nanoparticle of the present disclosure can further comprise other therapeutic agents.

[0146] In some embodiments, the lipid nanoparticle further comprises a helper lipid, which can be referred to as a neutral lipid, a neutral helper lipid, non-cationic lipid, non-cationic helper lipid, anionic lipid, anionic helper lipid, or a zwitterionic lipid. In some situations, lipid formulations, particularly cationic liposomes and lipid nanoparticles have increased cellular uptake if helper lipids are present in the formulation. (Curr. Drug Metab. 2014; 15(9):882-92). For example, some studies have indicated that neutral and zwitterionic lipids such as l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), Di-Oleoyl-Phosphatidyl-Ethanolamine (DOPE) and 1,2-DiStearoyl-sn-glycero-3-PhosphoCholine (DSPC), being more fusogenic (i.e., facilitating fusion) than cationic lipids, can affect the polymorphic features of lipid-nucleic acid complexes, promoting the transition from a lamellar to a hexagonal phase, and thus inducing fusion and a disruption of the cellular membrane. (Nanomedicine (Lond). 2014 Jan; 9(1): 105-20). In addition, the use of helper lipids can help to reduce any potential detrimental effects from using many prevalent cationic lipids such as toxicity and immunogenicity. Non-limiting examples of noncationic lipids suitable for lipid formulations of the present disclosure include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG),Attorney Docket: 049386-553001WOdioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethyl -phosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0147] In some embodiments, the helper lipid is selected from: dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is distearoylphosphatidylcholine (DSPC).

[0148] Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof. One study found that cholesterol increases the spacing of the charges of the lipid layer interfacing with the nucleic acid making the charge distribution match that of the nucleic acid more closely. (J. R. Soc. Interface. 2012 Mar 7; 9(68): 548-561). Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5 a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5 a-cholestanone, and cholesteryl decanoate; and mixtures thereof. In preferred embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.

[0149] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the helper lipid present in the lipid formulation comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid formulation. In yet other embodiments, the helper lipid present in the lipid formulation comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid formulation. In some embodiments, the lipid composition or nanoparticle further comprises cholesterol.Attorney Docket: 049386-553001WO

[0150] Other examples of helper lipids include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkylaryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.

[0151] In some embodiments, the lipid nanoparticle further comprises cholesterol. In some embodiments, the lipid nanoparticle is DSPC free. In some embodiments, the lipid nanoparticle is free of helper lipid.

[0152] In some embodiments, the lipid nanoparticle further comprises a PEG-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG. Optionally, the PEG-lipid conjugate is PEG2000-DMG.

[0153] In some embodiments, the lipid nanoparticle further comprises an ionizable cationic lipid. In some embodiments, the lipid nanoparticle is free from ionizable cationic lipids.

[0154] The lipid composition further includes a cationic lipid suitable for forming a cationic liposome or lipid nanoparticle. Cationic lipids are widely studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake. Generally, cationic lipids are amphiphiles containing a positive hydrophilic head group, two (or more) lipophilic tails, or a steroid portion and a connector between these two domains. Preferably, the cationic lipid carries a net positive charge at about physiological pH. Cationic liposomes can be used in non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA / small hairpin RNA-shRNA. Cationic lipids, such as DOTAP, (l,2-dioleoyl-3- trimethylammoniumpropane) and DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N, N, N-trimethyl- ammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids by electrostatic interaction, providing high in vitro transfection efficiency.

[0155] In the presently disclosed lipid formulations, the cationic lipid may include, for example, N, N-dimethyl-N, N-di-9-cis-octadecenylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride and 1.2-Dioleyloxy-3 -trimethylaminopropane chloride salt), N-(l-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1.2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N, N-dimethylaminopropane (y-Attorney Docket: 049386-553001WODLenDMA), 1,2-Dilinoleylcarbamoyloxy-3 -dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3 -dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -Linoleoyl-2-linoleyloxy— 3 dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA. Cl), 1,2-Dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP. Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N, N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N, N-Dioleylamino)-l,2-propanediol (DOAP), l,2-Dilinoleyloxo-3-(2-N, N- dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N, N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31 -tetraen-19-yl-4-(dimethylamino)butanoate (MC3), 1, 1 '-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethylazanediyl)didodecan-2-ol (C 12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 3-((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yloxy)-N, N-dimethylpropan-l-amine (MC3 Ether), 4-((6Z,9Z,28Z,31 Z)-heptatriaconta-6, 9, 28, 31 -tetraen-19-yloxy)-N, N-dimethylbutan-1 -amine (MC4 Ether), or any combination thereof. Other cationic lipids include, but are not limited to, N, N-distearyl-N, N-dimethylammonium bromide (DDAB), 3P-(N-(N', N'-dimethylaminoethane)- carbamoyl)cholesterol (DC-Chol), N-(l-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N, N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-sn-3 -phosphoethanolamine (DOPE), 1,2-dioleoyl-3 -dimethylammonium propane (DODAP), N-(l,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (XTC). Additionally, commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL), and Lipofectamine (comprising DOSPA and DOPE, available from GIBCO / BRL).

[0156] Exemplary suitable cationic lipids are disclosed in International Publication Nos. WO 2023 / 086514 and WO 2024 / 233750; and U. S. Patent Nos. 10,383,952, 10,980,895, 10,526,284, and 10,961,188, the contents of each of which are incorporated by reference herein. In some embodiments, cationic lipids included in lipid formulations provided herein are ionizable cationic lipids.

[0157] In some embodiments, the ionizable cationic lipid is a compound of Formula A:Attorney Docket: 049386-553001WOO— L6 R4O — L8R2NZ\1(A)wherein:R1and R2are each independently H or C1-6 alkyl; orR1and R2are joined to form a saturated heterocyclic ring, wherein:R1is a linear C1-4 alkylene; andR2is -(CH2)m(X)n-, whereinX is O, S, or NR9, wherein R9is H or C1-6 alkyl;m is 1, 2, 3 or 4, andn is 0 or 1;L1 is a linear C1-6 alkylene optionally substituted with one to three methyl groups; Y is selected from the group consisting of:wherein:each asterisk (*) indicates the atom attached to L2 and L3; and R10is H or C1-6 alkyl;L2 and L3 are each independently a linear C1-8 alkylene;L4, L5, L6, L7, L8 and L9 are each independently absent or -CH2-, provided that:Attorney Docket: 049386-553001WOat least two of L4, L6 and L8 are -CH2-; andat least two of L5, L7 and L9 are -CH2-;R3and R4are each independently H, methyl or ethyl; andR5, R6, R7and R8are each independently selected from the group consisting of:linear Ci-20 alkyl, wherein each said linear Ci-20 alkyl is optionally substituted with one or more substituents selected from the group consisting of:C1-6 alkyl, C1-6 alkoxy and -F, wherein each said C1-6 alkyl substituent is optionally substituted with one or more groups selected from the group consisting of C1-3 alkoxy and -F;C3-8 monocycloalkyl, wherein each said C3-8 monocycloalkyl is optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, C1-6 alkoxy and -F; C7- 12 bicycloalkyl, wherein each said C7-12 bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, C1-6 alkoxy and -F; and Ce-io aryl, wherein each said Ce-io aryl is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, Ci -6 alkoxy and -F;C3-8 monocycloalkyl, wherein each said C3-8 monocycloalkyl is optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, C1-6 alkoxy and -F;C7- 12 bicycloalkyl, wherein each said C7-12 bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, C1-6 alkoxy and -F; andCe-io aryl, wherein each said Ce-io aryl is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, C1-6 alkoxy and -F.

[0158] In some embodiments, the ionizable cationic lipid is a compound of Formula B:Attorney Docket: 049386-553001WOR6O— L6 R4.0R® n — L8L2 R 02— L1— NOL3 \1R5 O — L7O RL X) — L9 R3(B)wherein:R1and R2are each independently H or C1-6 alkyl; orR1and R2are joined to form a saturated heterocyclic ring, wherein:R1is a linear C1-4 alkylene; andR2is -(CH2)m(X)n-, whereinX is O, S, or NR9, wherein R9is H or C1-6 alkyl;m is 1, 2, 3 or 4, andn is 0 or 1;L1 is a linear C1-6 alkylene optionally substituted with one to three methyl groups; Y is selected from the group consisting of:wherein:each asterisk (*) indicates the atom attached to L2 and L3; and R10is H or C1-6 alkyl;L2 and L3 are each independently a linear C1-8 alkylene;L4, L5, L6, L7, L8 and L9 are each independently absent or -CH2-, provided that:Attorney Docket: 049386-553001WOat least two of L4, L6 and L8 are -CH2-; andat least two of L5, L7 and L9 are -CH2-;R3and R4are each independently H, methyl or ethyl; andR5, R6, R7and R8are each independently selected from the group consisting of:linear Ci-20 alkyl, wherein each said linear Ci-20 alkyl is optionally substituted with one or more substituents selected from the group consisting of:C6-10 aryl, wherein each said C6-10 aryl is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more C6-10 aryl that is optionally substituted with one or more C1-6 alkyl; 6-10 membered heteroaryl, wherein each said 6-10 membered heteroaryl is a monocyclic or bicyclic aromatic system optionally substituted with one or more C1-6 alkyl; C6-10 aryl, wherein each said C6-10 aryl is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more C6-10 aryl that is optionally substituted with one or more C1-6 alkyl; and 6-10 membered heteroaryl, wherein each said 6-10 membered heteroaryl is a monocyclic or bicyclic aromatic system optionally substituted with one or more C1-6 alkyl.

[0159] In some embodiments, the ionizable cationic lipid is a compound of Formula C:O(C)whereinRi is a-CH((CH2)nCH3)2, -CH((CH2)nCH3)((CH2)n-iCH3) or -CH((CH2)y(CH=CH)(CH2)mCH3)2, wherein n is 2-8, y is 2-4 and m is 2-4;R2 is a linear C5-C10 alkenyl or a branched C8-C18 alkyl;R3 is a linear C2-C6_alkylene;R4 and R5 are the same or different, each a hydrogen or a linear alkyl of 1 to 6 carbons or a branched alkyl of 3 to 6 carbons;L1 is a linear C1-C8 alkylene;L2 is a linear C1-C8 alkylene; andAttorney Docket: 049386-553001WOX is S;or a pharmaceutically acceptable salt or solvate thereof.

[0160] In some embodiments, the ionizable cationic lipid is a compound of Formula D:O(D) wherein Ri isR2 isL1 and L2 are each independently a linear C1-C7 alkyl;X1 is O or S;R3 is a linear C1-C6 alkyl; andR4 and R5 are each independently a linear C1-C6 alkyl;or a pharmaceutically acceptable salt thereof.

[0161] In some embodiments, the ionizable cationic lipid is a compound selected from Table D, or is a pharmaceutically acceptable salt of a compound selected from Table D.Attorney Docket: 049386-553001WOTable D - Exemplary ionizable cationic lipids.Attorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WOAttorney Docket: 049386-553001WO

[0162] Other suitable cationic lipids are disclosed in International Publication Nos. WO 09 / 086558, WO 09 / 127060, WO 10 / 048536, WO 10 / 054406, WO 10 / 088537, WO 10 / 129709, and WO 2011 / 153493; U. S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803; U. S. Patent No. 8,158,601; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are herein incorporated by reference.

[0163] Other suitable cationic lipids include those having alternative fatty acid groups and other dialkylamino groups, including those, in which the alkyl substituents are different (e.g., N-ethyl-N-methylamino-, and N-propyl-N-ethylamino-). These lipids are part of a subcategory of cationic lipids referred to as amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. In general, amino lipids having less saturated alkyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of filter sterilization. Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of C14to C22may be used. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.Attorney Docket: 049386-553001WO

[0164] In some embodiments, cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. Of course, it will be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the disclosure. In certain embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11. In some embodiments, the ionizable cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of an ionizable cationic lipid is about 6 to about 7.

[0165] Depending on the intended use of the lipid formulation, the proportions of the components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0166] In some embodiments, based on the total amount of lipids in the lipid nanoparticle, the lipid nanoparticle comprises about 5 mol% to 15 mol% of a combined amount of the compound of Formula I of the present disclosure and the helper lipid, about 30 mol% to about 70 mol% of the ionizable cationic lipid, about 20 mol% to about 60 mol% of cholesterol, and about 0.5 mol% to about 5 mol% of a PEG-lipid conjugate

[0167] In some embodiments, based on the total amount of lipids in the lipid nanoparticle, the lipid nanoparticle comprises about 5 mol% to 10 mol% of a combined amount of the compound of Formula I of the present disclosure and the helper lipid, about 40 mol% to about 60 mol% of the ionizable cationic lipid, about 30 mol% to about 50 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate.

[0168] In some embodiments, based on the total amount of lipids in the lipid nanoparticle, the lipid nanoparticle comprises about 5 mol% to 12 mol% of a combined amount of the compound of Formula I of the present disclosure and the helper lipid, about 45 mol% to about 55 mol% of the ionizable cationic lipid, about 35 mol% to about 45 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate.

[0169] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary dependent on the specific formulation, for example, by ± 5 mol%.Attorney Docket: 049386-553001WO

[0170] In some embodiments, lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 50:1 to about 10:1. In some embodiments, the lipid nanoparticle has a total lipid: nucleic acid weight ratio of about 40:1 to about 20:1. In some embodiments, the lipid nanoparticle has a total lipid: nucleic acid weight ratio of about 35:1 to about 25:1. In some embodiments, the lipid nanoparticle has a total lipid: nucleic acid weight ratio of about 32:1 to about 28:1. In some embodiments, the lipid nanoparticle has a total lipid: nucleic acid weight ratio of about 31:1 to about 29:1.

[0171] The pharmaceutical composition can be formulated for therapeutic use, in which case other components such as pharmaceutically acceptable buffers and salts, and stabilizers such as cryoprotectants, can be included.

[0172] In some embodiments, the dosage form of the pharmaceutical compositions described herein can be a liquid suspension of nucleic acid-lipid nanoparticles described herein. In some embodiments, the liquid suspension is in a buffered solution. In some embodiments, the buffered solution comprises a buffer selected from the group consisting of HEPES, MOPS, TES, and TRIS. In some embodiments, the buffer has a pH of about 7.4. In some preferred embodiments, the buffer is HEPES. In some further embodiments, the buffered solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from a sugar and glycerol or a combination of a sugar and glycerol. In some embodiments, the sugar is a dimeric sugar. In some embodiments, the sugar is sucrose. In some preferred embodiments, the buffer comprises HEPES, sucrose, and glycerol at a pH of 7.4. In some embodiments, the suspension is frozen during storage and thawed prior to administration. In some embodiments, the suspension is frozen at a temperature below about -70 °C. In some embodiments, the suspension is diluted with sterile water prior to inhalable administration. In some embodiments, an inhalable administration comprises diluting the suspension with about 1 volume to about 4 volumes of sterile water. In some embodiments, a lyophilized nucleic acid-lipid nanoparticle formulation can be resuspended in a buffer as described herein.

[0173] In some embodiments, the pharmaceutical composition comprises a HEPES buffer at a pH of about 7.4. In some embodiments, the HEPES buffer is at a concentration of about 7 mg / mL to about 15 mg / mL.

[0174] In some embodiments, the pharmaceutical composition further comprises about 2.0 mg / mL to about 4.0 mg / mL of NaCl.Attorney Docket: 049386-553001WO

[0175] In some embodiments, the pharmaceutical composition further comprises one or more cryoprotectants. In some embodiments, the one or more cryoprotectants are selected from sucrose, glycerol, or a combination of sucrose and glycerol. In some embodiments, the cryoprotectant is sucrose.

[0176] In some embodiments, the pharmaceutical composition comprises a combination of sucrose at a concentration of about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of about 50 mg / mL to about 70 mg / mL.

[0177] The compounds disclosed herein can be formulated with conventional carriers and excipients. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. Exemplary excipients include, but are not limited to, those set forth in the “HANDBOOK OF PHARMACEUTICAL EXCIPIENTS” (1986). Excipients can include, for example, ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, and combinations thereof. In some embodiments, the formulation is basic. In some embodiments, the formulation is acidic. In some embodiments, the formulation has a neutral pH. In some embodiments, the pH of the formulations is from 2 to 11 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 6-7, 6-8, 6-9, 6-10, 6-11, 7-8, 7-9, 7-10, 7-11, 8-9, 8-10, 8-11, 9-10, or 9-11).

[0178] In some embodiments, the compounds disclosed herein are administered alone. In some embodiments, the compounds disclosed herein are administered in pharmaceutical compositions. In some embodiments, the pharmaceutical compositions are for veterinary use. In some embodiments, the pharmaceutical compositions are for human use. In some embodiments, the pharmaceutical compositions disclosed herein include at least one additional therapeutic agent. In some embodiments, the pharmaceutical compositions disclosed herein include one or more additional therapeutic agent. In some embodiments, the one or more additional therapeutic agents is independently a chemotherapeutic agent, an immunotherapeutic agent, a hormonal agent, an anti-hormonal agent, a targeted therapy agent, or an anti-angiogenesis agent.

[0179] Pharmaceutical compositions disclosed herein can be in any form suitable for the intended method of administration. The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient (i.e., nucleic acid) with an excipient and / or one or more other accessory ingredients. A pharmaceutical composition in accordance withAttorney Docket: 049386-553001WOthe present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. The pharmaceutical compositions disclosed herein can be presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. Exemplary techniques and formulations can be found, for instance, in REMINGTON’S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, PA). Such methods can include the step of bringing into association a compound disclosed herein with the carrier that constitutes at least accessory ingredients. In general, the formulations can be prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0180] The pharmaceutical compositions can be in the form of a sterile injectable or intravenous preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable or intravenous preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. Among the acceptable vehicles and solvents that can be employed include, but are not limited to, water, Ringer’s solution isotonic sodium chloride solution, and hypertonic sodium chloride solution.

[0181] The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time -release formulation intended for oral administration to humans can contain approximately 1 mg to 2000 mg of active material compounded with an appropriate and convenient amount of carrier material, which can vary from 5% to 95% of the total formulations (weight: weight). For example, a time-release formulation intended for oral administration to humans can contain approximately 1 mg to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material, which can vary from 5% to 95% of the total formulations (weight:weight). The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion can contain from 3 μg to 500 μg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of 30 mL / hr can occur.Attorney Docket: 049386-553001WO

[0182] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions that can include suspending agents and thickening agents.

[0183] The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately before use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit-dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.

[0184] In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present disclosure can include, without limitation, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with a primary DNA construct, or mRNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

[0185] In some embodiments, the formulations described herein can include one or more excipients, each in an amount that together increases the stability of the nucleic acid in the lipid formulation, increases cell transfection by the nucleic acid (e.g., mRNA or siRNA), increases the expression of an encoded protein, and / or alters the release profile of the encoded protein, or increases knockdown of a target native nucleic acid. Further, a nucleic acid may be formulated using self-assembled nucleic acid nanoparticles.

[0186] A dosage form of the composition of this disclosure can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0187] To formulate compositions for pulmonary delivery within the present disclosure, the nucleic acid-lipid formulation can be combined with various pharmaceutically acceptable additives, as well as a base or carrier for dispersion of the nucleic acid-lipid formulation(s). Examples of additives include pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodium chloride, mannitol, sorbitol), adsorptionAttorney Docket: 049386-553001WOinhibitors (e.g., Tween 80), solubility enhancing agents (e.g., cyclodextrins and derivatives thereof), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione). When the composition for mucosal delivery is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w / v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced in the mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and most often 3 / 4 to 1.7.

[0188] The nucleic acid-lipid formulation may be dispersed in a base or vehicle, which may comprise a hydrophilic compound having a capacity to disperse the nucleic acid-lipid formulation and any desired additives. The base may be selected from a wide range of suitable carriers, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or carrier, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer, and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc., can be employed as carriers. Hydrophilic polymers and other carriers can be used alone or in combination and enhanced structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking, and the like. The carrier can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of a selected carrier in this context may result in promotion of absorption of the nucleic acid-lipid formulation.

[0189] The compositions of this disclosure may alternatively contain as pharmaceutically acceptable carriers substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.Attorney Docket: 049386-553001WO

[0190] It should be understood that in addition to the ingredients particularly mentioned above the formulations can include other agents conventional in the art having regard to the type of formulation in question.

[0191] Further provided are veterinary formulations comprising a compound disclosed herein together with a veterinary carrier therefor.

[0192] Veterinary carriers are materials useful for the purpose of administering the formulation and can be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary formulations can be administered orally, parenterally, or by any other desired route.

[0193] Compounds herein are used to provide controlled release pharmaceutical compositions containing as active ingredient one or more of the compounds (“controlled release formulations”) in which the release of the active ingredient can be controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given active ingredient.V. Kits

[0194] Also provided herein are kits that includes a compound of the present disclosure or a pharmaceutically acceptable salt thereof, a pharmaceutical composition of the present disclosure, a lipid composition of the present disclosure, or a lipid nanoparticle of the present disclosure. In some embodiments the kits described herein can comprise a label and / or instructions for use of the compound, the composition, or the lipid nanoparticle in the treatment of a disease or condition in a subject (e.g., human) in need thereof. In some embodiments, the disease or condition is cancer, or immune diseases. In some embodiments, the disease or condition is associated with abnormal expression of a gene such as over expression of a gene.

[0195] In some embodiments, the kit can also comprise one or more additional therapeutic agents and / or instructions for use of additional therapeutic agents in combination with the compound disclosed herein in the treatment of the disease or condition in a subject (e.g., human) in need thereof.

[0196] In some embodiments, the kits provided herein comprise individual dose units of a compound as described herein, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate thereof. Examples of individual dosage units can include pills, tablets, capsules, prefilled syringes or syringe cartridges, IV bags, inhalers, nebulizers etc., each comprising a therapeutically effectiveAttorney Docket: 049386-553001WOamount of the compound in question, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate thereof. In some embodiments, the kit can contain a single dosage unit and in others multiple dosage units are present, such as the number of dosage units required for a specified regimen or period.

[0197] Also provided are articles of manufacture that include a compound disclosed herein, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers or tautomer thereof; and a container. In some embodiments, the container of the article of manufacture is a vial, jar, ampoule, preloaded syringe, blister package, tin, can, bottle, box, an intravenous bag, an inhaler, or a nebulizer.VI. Administration

[0198] One or more of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, a pharmaceutical composition of the present disclosure, a lipid composition of the present disclosure, or a lipid nanoparticle of the present disclosure are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. Preferably, the administration is by parenteral such as intravenously or intramuscularly.

[0199] The compounds of the present disclosure (also referred to herein as the active ingredients), can be administered by any route appropriate to the condition to be treated.

[0200] Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), transdermal, vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the route may vary with for example the condition of the recipient. An advantage of certain compounds disclosed herein is that they are orally bioavailable and can be dosed orally.

[0201] A compound, composition or lipid nanoparticle of the present disclosure may be administered to an individual in accordance with an effective dosing regimen for a desired period of time or duration, such as at least about one month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 12 months or longer. In some embodiments, the compound, composition or lipid nanoparticle is administered on a daily or intermittent schedule for the duration of the individual’s life. In some instances, when the compound, composition orAttorney Docket: 049386-553001WOlipid nanoparticle of the present disclosure is used as a prophylaxis, for example as a vaccine, the compound, composition or lipid nanoparticle may be administered only once, twice, or three times.

[0202] The dosage or dosing frequency of a compound, composition or lipid nanoparticle of the present disclosure may be adjusted over the course of the treatment, based on the judgment of the administering physician.

[0203] The compound may be administered to an individual (e.g., a human) in an effective amount. In some embodiments, the compound is administered once daily.

[0204] According to the present disclosure, a therapeutically effective dose of the provided composition, when administered regularly, may result in an increased nucleic acid activity level in a subject as compared to a baseline activity level before treatment. Typically, the activity level is measured in a biological sample obtained from the subject such as blood, plasma or serum, urine, or solid tissue extracts. The baseline level can be measured immediately before treatment. In some embodiments, administering a pharmaceutical composition described herein results in an increased nucleic acid activity level in a biological sample (e.g., plasma / serum or lung epithelial swab) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level before treatment. In some embodiments, administering the provided composition results in an increased nucleic acid activity level in a biological sample (e.g., plasma / serum or lung epithelial swab) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level before treatment for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days.VII. Methods of Use

[0205] In some embodiments, the present disclosure includes a method treating and / or preventing a disease in a subject in need thereof, comprising administering a therapeutically effective amount to the subject, the compound, the pharmaceutical composition, the lipid composition, or the lipid nanoparticle of the present disclosure.

[0206] In some embodiments, the present application includes a method of expressing a protein or polypeptide in a target cell, comprising contacting the target cell with the compound, theAttorney Docket: 049386-553001WOpharmaceutical composition, the lipid composition, or the lipid nanoparticle of the present disclosure.

[0207] In some embodiments, the present application includes a method of delivering a nucleic acid to a subject in need thereof, comprising encapsulating a therapeutically effective amount of the nucleic acid in the compound, the pharmaceutical composition, the lipid composition, or the lipid nanoparticle of the present disclosure, and administering the compound, the pharmaceutical composition, the lipid composition, or the lipid nanoparticle of the present disclosure to the subject.

[0208] In some embodiments, the protein or polypeptide is an antigen, and expression of the antigen elicits an in vivo immunogenic response.

[0209] The compound, the pharmaceutical composition, the lipid composition, or the lipid nanoparticle of the present disclosure can be administered by any suitable methods known in the field. In some embodiments, the compound, the pharmaceutical composition, the lipid composition, or the lipid nanoparticle of the present disclosure is administered intravenously or intramuscularly. It is understood that for intravenous or intramuscular administration, the compound, the pharmaceutical composition, the lipid composition, or the lipid nanoparticle of the present disclosure can be suitable formulated as described herein or as understood by conventional means known in the field.

[0210] In some embodiments, the lipid formulations may be administered in a local rather than systemic manner. Local delivery can be affected in various ways, depending on the tissue to be targeted. For example, aerosols containing compositions of the present disclosure can be inhaled (for nasal, tracheal, or bronchial delivery).

[0211] In some embodiments, the composition or the lipid nanoparticle of the present disclosure further comprises a nucleic acid. The lipid composition or lipid nanoparticle of the present disclosure can be used as a delivery vehicle for delivering the nucleic acid to a target cell or tissue.

[0212] In some embodiments, the lipid composition or lipid nanoparticle of the present disclosure may be administered to any desired tissue. In some embodiments, the nucleic acid delivered by a lipid formulation or composition of the present disclosure is active in the tissue in which the lipid formulation and / or composition was administered. In some embodiments, the nucleic acid is active in a tissue different from the tissue in which the lipid formulation or composition was administered. Example tissues in which the nucleic acid may be delivered include, but are notAttorney Docket: 049386-553001WOlimited to the lung, trachea, and / or nasal passages, muscle, liver, eye, or the central nervous system.

[0213] In some embodiments, there are provided a methods of treating a disease or disorder in a mammalian subject. A therapeutically effective amount of a compound, a pharmaceutical composition, a lipid composition or a lipid nanoparticle of the present disclosure may be administered to a subject having a disease or disorder associated with expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition. The lipid compositions and lipid nanoparticles described herein can be used in a methods for treating cancer or inflammatory disease. The disease may be one selected from the group consisting of central nervous system disorders, peripheral nervous system disorders, muscle atrophies, muscle dystrophies, immune disorder, cancer, renal disease, fibrotic disease, genetic abnormality, inflammation, and cardiovascular disorder.VIII. Mechanism of Action for Cellular Update of Lipid Composition / Nanoparticle

[0214] Lipid formulations for the intracellular delivery of nucleic acids, particularly liposomes, cationic liposomes, and lipid nanoparticles, are designed for cellular uptake by penetrating target cells through exploitation of the target cells’ endocytic mechanisms where the contents of the lipid delivery vehicle are delivered to the cytosol of the target cell. (Nucleic Acid Therapeutics, 28(3): 146-157, 2018). Specifically, in the case of a nucleic acid-lipid formulations described herein, the lipid formulation enters cells through receptor mediated endocytosis. Prior to endocytosis, functionalized ligands such as a the lipid conjugate of the disclosure at the surface of the lipid delivery vehicle can be shed from the surface, which triggers internalization into the target cell. During endocytosis, some part of the plasma membrane of the cell surrounds the vector and engulfs it into a vesicle that then pinches off from the cell membrane, enters the cytosol and ultimately undergoes the endolysosomal pathway. For ionizable cationic lipid-containing delivery vehicles, the increased acidity as the endosome ages results in a vehicle with a strong positive charge on the surface. Interactions between the delivery vehicle and the endosomal membrane then result in a membrane fusion event that leads to cytosolic delivery of the payload. For mRNA or self-replicating RNA payloads, the cell’s own internal translation processes will then translate the RNA into the encoded protein. The encoded protein can further undergo post -translational processing, including transportation to a targeted organelle or location within the cell.

[0215] By controlling the composition and concentration of the lipid conjugate, one can control the rate at which the lipid conjugate exchanges out of the lipid formulation and, in turn, the rate at which the lipid formulation becomes fusogenic. In addition, other variables including, e.g., pH,Attorney Docket: 049386-553001WOtemperature, or ionic strength, can be used to vary and / or control the rate at which the lipid formulation becomes fusogenic. Other methods which can be used to control the rate at which the lipid formulation becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure. Also, by controlling the composition and concentration of the lipid conjugate, one can control the liposomal or lipid particle size.IX. Compound Preparation

[0216] In some embodiments, the present disclosure provides processes and intermediates useful for preparing the compounds disclosed herein or pharmaceutically acceptable salts thereof.

[0217] Compounds disclosed herein can be purified by any of the means known in the art, including chromatographic means, including but not limited to high-performance liquid chromatography (HPLC), preparative thin layer chromatography, flash column chromatography, ion exchange chromatography, and supercritical fluid chromatography (SFC). Any suitable stationary phase can be used, including but not limited to, normal and reversed phases as well as ionic resins. In some embodiments, the disclosed compounds are purified via silica gel and / or alumina chromatography.

[0218] During any of the processes for preparation of the compounds provided herein, it can be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by means of conventional protecting groups as described in standard works, such as T. W. Greene and P. G. M. Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th ed., Wiley, New York 2006. The protecting groups can be removed at a convenient subsequent stage using methods known from the art.

[0219] Exemplary chemical entities useful in methods of the embodiments will now be described by reference to illustrative synthetic schemes for their general preparation herein and the specific examples that follow. Skilled artisans will recognize that, to obtain the various compounds herein, starting materials can be suitably selected so that the ultimately desired substituents will be carried through the reaction scheme with or without protection as appropriate to yield the desired product. Alternatively, it can be necessary or desirable to employ, in the place of the ultimately desired substituent, a suitable group that can be carried through the reaction scheme and replaced as appropriate with the desired substituent. Furthermore, one of skill in the art will recognize that the transformations shown in the schemes below can be performed in any order that is compatible with the functionality of the particular pendant groups.Attorney Docket: 049386-553001WO

[0220] The methods of the present disclosure generally provide a specific enantiomer or diastereomer as the desired product, although the stereochemistry of the enantiomer or diastereomer was not determined in all cases. When the stereochemistry of the specific stereocenter in the enantiomer or diastereomer is not determined, the compound is drawn without showing any stereochemistry at that specific stereocenter even though the compound can be substantially enantiomerically or disatereomerically pure.

[0221] Compounds disclosed herein can be prepared from commercially available reagents using the synthetic methods and reaction schemes described herein, or using other reagents and conventional methods known to persons of ordinary skill in the art. For instance, representative syntheses of compounds of the present disclosure are described in the schemes below, and the particular examples that follow.X. Lipid Composition / Nanoparticle Preparation

[0222] There are many different methods for the preparation of lipid formulations comprising a nucleic acid. (Curr. Drug Metabol. 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20). The techniques of thin film hydration, double emulsion, reverse phase evaporation, microfluidic preparation, dual asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution, and encapsulation in preformed liposomes are briefly described herein.Thin Film Hydration

[0223] In Thin Film Hydration (TFH) or the Bangham method, the lipids are dissolved in an organic solvent, then evaporated through the use of a rotary evaporator leading to a thin lipid layer formation. After the layer hydration by an aqueous buffer solution containing the compound to be loaded, Multilamellar Vesicles (MLVs) are formed, which can be reduced in size to produce Small or Large Unilamellar vesicles (LUV and SUV) by extrusion through membranes or by the sonication of the starting MLV.Double Emulsion

[0224] Lipid formulations can also be prepared through the Double Emulsion technique, which involves lipids dissolution in a water / organic solvent mixture. The organic solution, containing water droplets, is mixed with an excess of aqueous medium, leading to a water-in-oil-in-water (W / O / W) double emulsion formation. After mechanical vigorous shaking, part of the water droplets collapse, giving Large Unilamellar Vesicles (LUVs).Attorney Docket: 049386-553001WOReverse Phase Evaporation

[0225] The Reverse Phase Evaporation (REV) method also allows one to achieve LUVs loaded with nucleic acid. In this technique a two-phase system is formed by phospholipids dissolution in organic solvents and aqueous buffer. The resulting suspension is then sonicated briefly until the mixture becomes a clear one-phase dispersion. The lipid formulation is achieved after the organic solvent evaporation under reduced pressure. This technique has been used to encapsulate different large and small hydrophilic molecules including nucleic acids.Microfluidic Preparation

[0226] The Microfluidic method, unlike other bulk techniques, gives the possibility of controlling the lipid hydration process. The method can be classified in continuous-flow microfluidic and droplet-based microfluidic, according to the way in which the flow is manipulated. In the microfluidic hydrodynamic focusing (MHF) method, which operates in a continuous flow mode, lipids are dissolved in isopropyl alcohol which is hydrodynamically focused in a microchannel cross junction between two aqueous buffer streams. Vesicles size can be controlled by modulating the flow rates, thus controlling the lipids solution / buffer dilution process. The method can be used for producing oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three-inlet and one-outlet ports.Dual Asymmetric Centrifugation

[0227] Dual Asymmetric Centrifugation (DAC) differs from more common centrifugation as it uses an additional rotation around its own vertical axis. An efficient homogenization is achieved due to the two overlaying movements generated: the sample is pushed outwards, as in a normal centrifuge, and then it is pushed towards the center of the vial due to the additional rotation. By mixing lipids and an NaCl-solution a viscous vesicular phospholipid gel (VPC) is achieved, which is then diluted to obtain a lipid formulation dispersion. The lipid formulation size can be regulated by optimizing DAC speed, lipid concentration and homogenization time.Ethanol Injection

[0228] The Ethanol Injection (El) method can be used for nucleic acid encapsulation. This method provides the rapid injection of an ethanolic solution, in which lipids are dissolved, into an aqueous medium containing nucleic acids to be encapsulated, through the use of a needle. Vesicles are spontaneously formed when the phospholipids are dispersed throughout the medium.Attorney Docket: 049386-553001WODetergent Dialysis

[0229] The Detergent dialysis method can be used to encapsulate nucleic acids. Briefly lipid and plasmid are solubilized in a detergent solution of appropriate ionic strength, after removing the detergent by dialysis, a stabilized lipid formulation is formed. Unencapsulated nucleic acid is then removed by ion-exchange chromatography and empty vesicles by sucrose density gradient centrifugation. The technique is highly sensitive to the cationic lipid content and to the salt concentration of the dialysis buffer, and the method is also difficult to scale.Spontaneous Vesicle Formation by Ethanol Dilution

[0230] Stable lipid formulations can also be produced through the Spontaneous Vesicle Formation by Ethanol Dilution method in which a stepwise or dropwise ethanol dilution provides the instantaneous formation of vesicles loaded with nucleic acid by the controlled addition of lipid dissolved in ethanol to a rapidly mixing aqueous buffer containing the nucleic acid.EXAMPLESI. Abbreviations

[0231] Certain abbreviations and acronyms are used in describing experimental details. Although most of these would be understood by one skilled in the art, Table 1 contains a list of many of these abbreviations and acronyms.Table 1. List of Abbreviations and AcronymsAbbreviation Meaning°C degree(s) Celsiuspg or ug microgram(s)pL or uL microliter(s)pm or um micron(s)pmol or umol micromole(s)aq AqueousBoc tert-butoxycarbonylbr s broad singletCbz Benzyloxycarbonyld DoubletDCM Dichloromethanedd doublet of doubletsddd doublet of doublet of doubletsddt doublet of doublet of tripletsAttorney Docket: 049386-553001WODIPEA N,N-diisopropylethylamineDMAP 4-dimethylaminopyridineDMF DimethylformamideDMP Dess-Martin periodinaneDMSO dimethyl sulfoxidedt doublet of tripletsEt EthylEtOAc ethyl acetateg gram(s)h hour(s)HATU hexafluorophosphate azabenzotriazole tetramethyl uronium HPLC high-performance liquid chromatographyHz HertziPr isopropylIPA or iPrOH isopropyl alcoholJ coupling constantKHMDS potassium bis(trimethylsilyl)amideKOAc potassium acetateLAH lithium aluminum hydrideLCMS liquid chromatography mass spectrometryLDA lithium diisopropylamideLiHMDS lithium bis(trimethylsilyl)amidem multipletM MolarityMe MethylMeCN acetonitrileMeOH methanolmg milligram(s)MHz megahertzmin minute(s)mL milliliter(s)mm millimeter(s)mmol millimole(s)MOMO ormethoxymethylOMOMNaOMe sodium methoxiden-BuLi n-butyllithiumNMR nuclear magnetic resonanceOSEM 2-(trimethylsilyl)ethoxymethoxyOTf trifluoromethanesulfonatePiv PivaloylAttorney Docket: 049386-553001WOqd quartet of doubletsrt room temperatures SingletSFC supercritical fluid chromatographyt TripletTBAF tetrabutylammonium fluorideTBS tert-butyldimethylsilylTBSCl tert-Butyldimethylsilyl chloridetBu tert-butyltd triplet of doubletsTES triethylsilaneTF2O or Tf2O trifluoromethanesulfonic anhydrideTFA trifluoroacetic acidTHF tetrahydrofuranTIPS triisopropylsilylTMS trimethylsilyltt triplet of tripletsv / v volume / volumewt Weightδ parts per million referenced to residual non-deuterated solvent peakII. CompoundsExample 1 Synthesis of Compound 1-1„ I Q O'"N P'O^|^OH a. SOCl2, CH2Cl2, 25ºC to 40ºC, 97% crude; b. i. OH dibutyltin oxide, i-PrOH, 25ºC to reflux ii. cool to 25ºC, Et3N 25%; c. icosanoyl chloride (1-4), Et3N, CHCl3, 39%Attorney Docket: 049386-553001WOScheme 1-12-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)acetyl chloride (1-2)1-2

[0232] 2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)acetic acid (1-1, 25.0g, 56.2 mmol) Chorny, M.; Alferiev, I.S.; Fishbein, I.; Tengood, J.E.; Folchman-Wagner, Z.; Forbes, S.P.; Levy, R.J. Pharm.Res. 2012, 29, 1232-1241 was dissolved in CH2CI2 (125 mL) at room temperature under nitrogen. To this solution was added thionyl chloride (125 mL, 205.0g, 1.72 mol) dropwise over a period of 20 minutes. After the addition was complete the mixture was stirred for 2 hours then the mixture was concentrated in vacuo to give crude 1-2 (25.3g, 54.6 mmol, 97% crude yield) as a white solid.

[0233] ‘H-NMR (300 MHz, CDCI3): 5 5.38 (m, 1H), 4.46 (s, 2H), 3.31 (m, 1H), 2.31 (m, 2H), 1.76-2.08 (5H), 0.65-1.60 (33H), 0.69 (s, 3H).(2R)-3-(2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)acetoxy)-2-hydroxypropyl (2-(trimethylammonio)ethyl) phosphate (1-3)Q O- 9, PC.0,oOH1-3

[0234] A mixture of (R)-2,3-dihydroxypropyl (2-(trimethylammonio)ethyl) phosphate (sn-glycero-3-phosphocholine, 5.00g, 19.41 mmol) and dibutyltin oxide (5.08g, 20.41 mmol) wasAttorney Docket: 049386-553001WOdissolved in isopropanol (200 mL), under nitrogen. After solution was achieved the mixture was heated under reflux for 2 hours, then was cooled to room temperature. To this cooled solution was added Et3N (2.36g, 23.35 mmol), in one portion, followed by the addition of 1-2 (10.8g, assumed 23.31 mmol). The mixture was allowed to stir for 3 hours at room temperature then was filtered and the filtrate was concentrated in vacuo to give the crude reaction mixture. The crude mixture was dissolved in CH2CI2 (150 mL) and silica gel (30g, type: ZCX-100-200 mesh) was added. The solvent was removed in vacuo and the silica gel containing adsorbed 1-3 was loaded atop a silica gel column (150g, type: ZCX-100-200 mesh) which was eluted with a gradient of CH2Cl2: MeOH from 100:0 to 90:10. Qualified fractions were pooled and concentrated in vacuo to afford 1-3 (3.30g, 4.83 mmol, 25%) as a white solid.

[0235] LC-MS: (+ mode) RT 2.251 min, Calcd. For C37H66NO8P 683.45, Found 684.50 (M+H+); ‘H-NMR (300 MHz, CD3OD): d 5.38 (m, 1), 4.12-4.38 (6H), 3.85-4.09 (3H), 3.62-3.69 (2H), 3.23 (s, 9H), 1.81-2.40 (8H), 0.87-1.69 (34H), 0.73 (s, 3H).Icosanoyl chloride (1-4)O1-4

[0236] Icosanoic acid (8.00g, 25.60 mmol) was dissolved in CH2CI2 (50 mL) under nitrogen. To this solution was added thionyl chloride (40 mL, 65.6g, 0.551 mol) dropwise over a period of 25 minutes. The mixture was allowed to stir for 30 minutes after the addition was complete, then was warmed to 70 °C and was stirred for 2 hours. The mixture was concentrated in vacuo to give crude icosanoyl chloride (8.40g, 25.4 mmol, 99% crude yield) as a white solid. ’H-NMR (400 MHz, CDCI3): d2.90 (t, J= 7.3Hz, 2H), 1.74 (m, 2H), 1.20-1.42 (32H), 0.91 (t, J= 6.7Hz, 3H).(2R)-3-(2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)acetoxy)-2-(icosanoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (1-1)Attorney Docket: 049386-553001WO

[0237] To a solution of 1-3 (3.30g, 4.83 mmol) in CHCl3 (75 mL), under nitrogen at room temperature, was added in order Et3N (2.44g, 24.15 mmol) and icosanoyl chloride (8.00g, 24.2 mmol). The mixture was stirred at room temperature under nitrogen for 48 hours, then was concentrated in vacuo to give crude 1 that was purified by Prep-Chiral HPLC (column: CHIRALPAK IG 3x25cm, 5mm, 87475S901G0SCZ-WJ001); Mobile Phase A: hexanes with 0.1% Et3N, Mobile Phase B: EtOH, gradient 15%-15% over 40 minutes. Qualified fractions were combined, concentrated in vacuo and the residue was dissolved in CHCI3 (20 mL). The CHCI3 solution of 1 was washed with water (3 x 20mL) and concentrated in vacuo to provide 1 (0.60g, 0.613 mmol, 12.7%) as a pale yellow, solid. SFC-MS: (+ mode) RT 3.13 min. Calcd. For C57H104NO9P 977.74, Found 979.10 (M+H+); ¹H-NMR (400MHz, CDCl3): δ 5.25-5.40 (2H), 4.10-4.64 (6H), 4.00 (m, 1H), 3.84 (m, 2H), 3.46 (s, 9H), 3.26 (m, 1H), 2.92 (m, 1H), 2.20-2.42 (4H), 1.74-2.10 (5H), 0.85-1.60 (70H), 0.69 (s, 3H); ³¹P-NMR (400MHz, CDCl3): 16.01ppm.Example 2 Synthesis of Compound 1-2a. i. sn-glycero-3-phosphocholine, dibutyltin oxide, i-PrOH, 25 °C to reflux ii. cool to 25°C, Et3N, 82%; b. 2-(cholesteryl-3-yl)oxyacetic acid, DCC, DMAP, CHCl3, 55%Scheme 1-2( / ?)-2-hydroxy-3-(icosanoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (2-1)OHAttorney Docket: 049386-553001WO

[0238] A mixture of (R)-2,3-dihydroxypropyl (2-(trimethylammonio)ethyl) phosphate (sn-glycero-3-phosphocholine, 7.40g, 28.76 mmol) and dibutyltin oxide (7.18g, 28.85 mmol) was dissolved in isopropanol (300 mL), under nitrogen. After solution was achieved the mixture was heated under reflux for 4 hours, then was cooled to room temperature. To this cooled solution was added Et3N (3.49g, 34.53 mmol), in one portion, followed by the addition of 1-4 (10.50g, assumed 31.72 mmol). The mixture was allowed to stir for 30 minutes at room temperature then was heated to 80°C and was allowed to stir for 12 hours. The mixture was cooled to room temperature, then was cast into water (250 mL), filtered and the filtrate was concentrated in vacuo to give the crude reaction mixture. The crude mixture was dissolved in CH₂Cl₂ (250 mL) and silica gel (50g, type: ZCX- 100-200 mesh) was added. The solvent was removed in vacuo and the silica gel containing adsorbed 2-1 was loaded atop a silica gel column (300g, type: ZCX-100-200 mesh) which was eluted with a gradient of CH2Cl2: MeOH from 100:0 to 90:10. Qualified fractions were pooled and concentrated in vacuo to afford 2-1 (13.00g, 23.56 mmol, 82%) as a white solid. LC-MS (+ mode): RT 2.388 min, Calcd. for C28H58NO7P 551.40, Found 552.45; ¹H-NMR (400MHz, CD3OD): δ 4.37 (m, 2H), 4.14 (m, 2H), 3.80-4.02 (3H), 3.68 (m, 2H), 3.30 (s, 9H), 2.34 (m, 2H), 1.61 (m, 2H), 1.22-1.32 (33H), 0.90 (t, J = 6.0Hz, 3H).(2R)-2-(2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)acetoxy)-3-(icosanoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (1-2)1-2

[0239] To a solution of 2-1 (4.59g, 8.32 mmol) and 1-1 (4.40g, 9.89 mmol) in CHCl3 (75 mL), cooled in an ice-water bath under nitrogen, was successively added DCC (4.12g, 19.96 mmol) and DMAP (0.40 g, 3.27 mmol). The mixture was stirred for 30 minutes at 0 °C, warmed to room temperature and was allowed to stir for 12 hours. The mixture was concentrated in vacuo and the residue was dissolved in CH2CI2 (100 mL). The solvent was removed in vacuo and the silica gelAttorney Docket: 049386-553001WOcontaining adsorbed 2 was loaded atop a silica gel column (400g, type: ZCX- 100-200 mesh) which was eluted with a gradient of CH₂Cl₂:MeOH from 100:0 to 80:20. Qualified fractions were pooled and concentrated in vacuo to afford 2 (4.50g, 4.60 mmol, 55%) as a white solid.

[0240] LC-MS (+ mode): RT 3.97 min, Calcd. for C57H104NO9P 977.74, Found 979.1 (M+H+); ‘H-NMR (300MHz, CDCI3): d 5.30-5.50 (2H), 4.30-4.50 (3H), 4.10-4.29 (3H), 4.03 (m, 2H), 3.84 (m, 2H), 3.39 (s, 9H), 2.20-2.46 (4H), 1.80-2.10 (5H), 0.80-1.70 (71H), 0.66 (s, 3H).Example 3 Synthesis of Compound 1-3a. i. LDA, THF, HMPA, -20°C; ii. n-BuBr, -20°C to 25°C 37%; b. SOCI2, CH2CI2, 25°C to 40°C, 89% crude; c. i.sn-glycero-3-phosphocholine, dibutyltin oxide, i-PrOH, 25°C to reflux ii. cool to 25°C, Et3N 70%; d. icosanoic acid, DCC, DMAP, CHCI3, 39%Scheme 1-3(+ / -)-2-Butylheptadecanoic acid (3-2)

[0241] A solution of i-Pr₂NH (16.40g, 0.162 mol) in anhydrous THF (200 mL) was cooled to -20°C under nitrogen, the n-BuLi (2.5 M in hexanes, 65 mL, 0.163 mol) was added over a period of 30 minutes. The mixture was allowed to stir for 30 minutes at -20°C, then a solution ofAttorney Docket: 049386-553001WOheptadecanoic acid (3-1, 20.0g, 74 mmol) in THF (50 mL) was added over 20 minutes. The solution was stirred for 20 minutes (-20°C) and a solution of n-butylbromide (10.1g, 74 mmol) in THF (20 mL) was added dropwise over a period of 10 minutes. The mixture was allowed to stir for 30 minutes after the addition was complete, then was warmed to room temperature and was stirred for 16 hours. The reaction mixture was poured into cold (0°C) 10% aq. HC1 (250 mL) and the solution was stirred for 20 minutes. The solution was warmed to room temperature and was extracted with petroleum ether (2 x 200 mL) and the combined organic phases were washed with brine (250 mL), dried (MgSO₄), filtered, and concentrated in vacuo to give crude 3-2 as a sticky, pale yellow semi-solid. Crude 3-2 was dissolved in CH2CI2 (100 mL) and silica gel (60g, type: ZCX- 100-200 mesh) was added. The solvent was removed in vacuo and the silica gel containing adsorbed 3-2 was loaded atop a silica gel column (300g, type: ZCX-100-200 mesh) which was eluted with a gradient of petroleum ether: EtOAc from 100:0 to 90:10. Qualified fractions were pooled and concentrated in vacuo to afford 3-2 (9.00g, 27.6 mmol, 37%) as a pale yellow solid. ‘H-NMR (400MHz, CDCI3): d 2.38 (m, 1H), 1.64 (m, 2H), 1.51 (m, 2H), 1.20-1.40 (30H), 0.85-0.92 (6H).(+ / -)-2-Butylheptadecanoyl chloride (3-3)3-3

[0242] To a solution of 3-2 (9.00g, 27.6 mmol) in CH2CI2 (45 mL), under nitrogen at room temperature, was added thionyl chloride (45 mL, 73.8g, 0.62 mol) over a period of 15 minutes. The mixture was stirred at room temperature for 30 minutes, then was warmed to 40°C and was stirred for 6 hours. The mixture was cooled to room temperature and then was concentrated in vacuo, the residue was diluted with toluene (50 mL) and the solvent was removed in vacuo, this operation was repeated one more time to give crude 3-3 (8.5g, assumed 24.6 mmol, assumed 89%). ^-NMR (400MHz, CDCI3): d 2.77 (m, 1H), 1.75 (m, 2H), 1,56 (m, 2H), 1.20-1.41 (30H), 0.83-0.94 (6H).(2 / ?)-3-((+ / - 2-Butylheptadecanoyl)oxy)-2-hydroxypropyl (2-(trimethylammonio)ethyl) phosphate (3-4)Attorney Docket: 049386-553001WO3-4

[0243] A mixture of ( / ?)-2,3-clihydroxypropyl (2-(trimethylammonio)ethyl) phosphate (sn-glycero-3-phosphocholine (6.20g, 24.1 mmol) and dibutyltin oxide (6.33g, 25.43 mmol) was dissolved in isopropanol (400 mL), under nitrogen. After solution was achieved the mixture was heated under reflux for 4 hours, then was cooled to room temperature. To this cooled solution was added Et₃N (2.93g, 29 mmol), in one portion, followed by the addition of 3-3 (10.0g, assumed 29.0 mmol). The mixture was allowed to stir for 16 hours at room temperature then was cast into water (100 mL) and the resulting solid was removed by filtration. The solid was dissolved in CH2CI2 (250 mL) and silica gel (60g, type: ZCX-100-200 mesh) was added. The solvent was removed in vacuo and the silica gel containing adsorbed 3-4 was loaded atop a silica gel column (300g, type: ZCX-100-200 mesh) which was eluted with a gradient of CH₂Cl₂:MeOH from 100:0 to 90:10. Qualified fractions were pooled and concentrated in vacuo to afford 3-4 (9.50g, 16.8 mmol, 70%) as a white solid. LC-MS (+ mode): RT 2.295 min, Calcd. For C29H60NO7P: 565.41; Found: 566.52 (M+H+); ^-NMR (300MHz, CD3OD): d 3.80-4.35 (7H), 3.58-3.70 (3H), 3.22 (s, 9H), 2.38 (m, 1H), 1.40-1.70 (4H), 1.19-1.40 (30H), 0.85-0.92 (6H).(2 / ?)-3-((+ / - 2-butylheptadecanoyl)oxy)-2-(icosanoyloxy)propyl (2-(trimethylammonio) ethyl) phosphate (3)3-4

[0244] To a room temperature solution of 3-4 (5.00g, 8.83 mmol) and icosanoic acid (3.31g, 10.59 mmol) in CHCI3 (100 mL), under nitrogen, was added in order DMAP (0.54g, 4.42 mmol) and DCC (5.47g, 26.51 mmol). The reaction mixture was stirred for 72 hours at room temperature, solid materials were removed by filtration through a sintered glass funnel and the filtrate was concentrated in vacuo to give crude 3. Crude 3 was dissolved in CH2CI2 (100 mL) and silica gel (25g, type: ZCX-100-200 mesh) was added. The solvent was removed in vacuo and the silica gel containing adsorbed 3 was loaded atop a silica gel column (150g, type: ZCX-100-200 mesh)Attorney Docket: 049386-553001WOwhich was eluted with a gradient of CH₂Cl₂:MeOH from 100:0 to 90:10, then the column was eluted with CHC13: MeOH: H2O (78:20:2). Qualified fractions were pooled and concentrated in vacuo to afford 3 (3.00g, 3.49 mmol, 39%) as a white solid. LC-MS (+ mode): RT 3.422 min, Calcd. For C49H98NO8P 859.70. Found 861.10; *H-NMR (400MHz, CDCI3): d5.24 (m, 1H), 4.40-4.55 (3H), 4.20 (m, 1H), 3.85-4.08 (4H), 3.42 (s, 9H), 2.25-2.40 (3H), 1.40-1.65 (6H), 1.15-1.40 (62H), 0.86-0.93 (9H);31P-NMR (400MHz, CDCI3): 45.29ppm.Example 4 Synthesis of Compound 1-44-2a. i. Mg, 12, THF, ii. pentanal, 66%; b. i. NaH (4.0 eq), THF, ii. bromoacetic acid, THF, 50%; c. DCC, DMAP, CHC13, 80%Scheme 1-4(+ / -)-icosan-5-ol (4-2)OH4-2

[0245] Dried magnesium turnings (7.40g, 0.304 mol) were covered with dry THF (125 mL), under nitrogen, and solid iodine (0.295g, 1.16 mmol) was added. The mixture was warmed to 40°C and after the color of iodine had disappeared a solution of 1 -bromopentadecane (4-1, 76.0g, 0.257 mol) in THF (150 mL) was added at such a rate that the temperature of the reaction mixture didAttorney Docket: 049386-553001WOnot exceed 55°C. After the addition was complete the temperature of the mixture was allowed to cool to 40°C and was maintained at that temperature for 30 minutes. The Grignard solution was then cooled to 20°C and a solution of pentanal (20.0g, 0.232 mol) in THF (50 mL) was added at such a rate that the temperature of the reaction mixture did not exceed 40°C. The mixture was stirred at 40°C for 18 hours after the addition was complete, then was cooled to 10°C and the reaction was quenched by the addition of 25% aq. NH4CI solution (500 mL). The mixture was filtered through a sintered glass funnel and then was extracted with hexanes (2 x 500 mL). The combined organic phases were dried (Na₂SO₄), filtered, and concentrated in vacuo to give crude 4-2 as a pale yellow, oil. Crude 4-2 was purified by chromatography on a column of silica gel (750g, 60-200 mesh) packed with hexanes-Et2O (99: 1) and eluted with a gradient of hexanes-Et2O (99: 1 to 85: 15). Qualified fractions were combined and concentrated in vacuo to afford 4-2 (46.0g, 0.151 mol, 65%) as a clear, colorless oil. TOF-MS Calcd. for C20H42O 298.32, Found 316.30 (M+NH4+); ^-NMR (400MHZ, CDCI3): d 3.59 (m, 1H), 1.23-1.50 (34H), 0.84-0.95 (6H).2-([+ / -]-icosan-5-yloxy)acetic acid (4-3)4-3

[0246] To NaH (90%, 1.79g, 67.1 mmol), in dry THF (25 mL) under nitrogen at room temperature, was added, dropwise, a solution of 4-2 (5.00g, 16.74 mmol) in THF (25 mL) over a period of 30 minutes. The mixture was allowed to stir for 2 hours at room temperature, then a solution of bromoacetic acid (2.56g, 18.42 mmol) in THF (15 mL) was added dropwise over 15 minutes. The mixture was allowed to stir for 30 minutes after the addition was complete and n-BU4NI (0.31g, 1.00 mmol) was added in 1 portion, followed by dry DMF (10 mL) and the mixture was warmed to 40°C and was stirred for 48 hours. The mixture was cooled to room temperature and MeOH (10 mL) was slowly added, and the mixture was cast into water (100 mL). The solution was extracted with hexanes (2 x 100 mL) and the combined organic phases were washed with water (3 x 75L) and dried (Na₂SO₄). Filtration and concentration in vacuo gave crude 4-3 which was further azeotropically dried with toluene (3 x 40 mL) and purified by chromatography on a column of silica gel (130g, 60-200 mesh), packed with hexanes-EtOAc (95:5) and eluted with a gradient of hexanes-EtOAc (95:5 to 80:20). Qualified fractions were combined and concentrated in vacuo to give 4-3 (3.00g, 8.41 mmol, 50%) as a clear, viscous oil. TOF-MS: Calcd. for C22H44O3Attorney Docket: 049386-553001WO356.33, Found 355.5 (M-H+); ‘H-NMR (400MHZ, CDCI3): d 4.07 (s, 2H), 3.44 (m, 1H), 1.44-1.58 (4H), 1.25-1.40 (30H), 0.84-0.95 (6H).(2 / ?)-2-(2-([+ / -]-icosan-5-yloxy)acetoxy)-3-(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (4), PX / X / xO O'0^0o

[0247] (7?)-2-hydroxy-3-(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (4-4, Avanti Polar Lipids #855775, 2.20g, 4.20 mmol) and 4-3 (1.80g, 5.05 mmol) were dissolved in CHCI3 (50 mL) at room temperature under nitrogen. To this solution was added in order DMAP (0.26g, 2.13 mmol) and DCC (2.60g, 12.60 mmol) and the resulting reaction mixture was stirred at room temperature for 72 hours. Suspended solids were removed by filtration and the filtrate was concentrated in vacuo to afford crude 4 as a sticky semi-solid that was dissolved in a mixture of CHCl₃ / MeOH / ammonium hydroxide (3 mL, 80:20:2) and was purified by medium pressure chromatography over a column of silica gel (60g, 60-200 mesh) using a gradient from CHCl₃ / MeOH / ammonium hydroxide (80:20:2) to CHCL / MeOH / water (80:20:2). Qualified fractions were pooled, and non-qualified fractions containing 4 were concentrated in vacuo and subjected to further chromatography under identical conditions. Qualified fractions from both chromatographic runs were combined and concentrated in vacuo and dried azeotropically by dissolution in i-PrOH / CHCl₃ (1:1, 10 mL) and concentration in vacuo. The azeotropic drying was repeated 3 additional times. The product was then dissolved in cyclohexane / EtOH (96:4), frozen and lyophilized to provide 4 (2.88g, 3.34 mmol, 80%) as a white solid. TOF-MS: Calcd. for C48H96NO9P 861.68, Found 863.0 (M+H+); ^-NMR (400MHz, CDCI3): d 5.27 (m, 1H), 4.30-4.47 (3H), 4.09-4.26 (3H), 3.83 (m, 2H), 3.39 (s, 9H), 3.35 (m, 1H), 2.32 (m, 2H), 1.45-1.70 (8H), 1.23-1.43 (58H), 0.85-0.96 (9H).Attorney Docket: 049386-553001WOExample 5 Synthesis of Compound 1-5a OH(a). DCC, DMAP, CHCL, r.t. 12h, 45%Scheme 1-5(2R)-2-(2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)- 2,3,4,7,8,9,10,ll,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl)oxy)acetoxy)-3-(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (5)5

[0248] To a solution of 4-4 (2.0g, 3.8 mmol) and 2-(((35,,107?,137?,177?)-10,13-dimethyl-17-((7?)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,ll,12,13,14, 15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl)oxy)acetic acid (2.04g, 4.59 mmol) in DCM (50 mL), cooled in an ice-water bath under nitrogen, was added in order DCC (2.3g, 11.4 mmol) and DMAP (0.234g, 1.9 mmol). The mixture was stirred for 30 minutes at 0°C after the additions were complete, then was warmed to room temperature and was allowed to stir for 48 hours. The mixture was concentrated in vacuo and the residue was dissolved in CH2CI2 (100 mL) and 30g silica gel wasAttorney Docket: 049386-553001WOadded. The solvent was removed in vacuo and the silica gel containing adsorbed 5 was loaded atop a silica gel column (250g, type: ZCX-100-200 mesh) which was eluted with a gradient of CH₂Cl₂:MeOH from 100:0 to 80:20 followed by DCM: MeOH: H2O ( v / v / v = 65:25:4). Eluent was concentrated and freeze dried to afford 5 (1.72 g, HPLC = 95.6%) as an off white solid. Yield = 45.41%. LC-MS (+ mode): RT 2.47 min, Calcd. for C55H100NO9P 949.71, Found 950.90 (M+H+); ‘H NMR (400 MHz, Chloro form-t / ) δ 4.31 (s, 3H), 4.22 - 4.07 (m, 3H), 3.98 (t, J= 6.5 Hz, 2H), 3.80 (s, 2H), 3.36 (s, 10H), 2.25 (dt, J= 20.0, 10.3 Hz, 4H), 2.06 - 1.81 (m, 5H), 1.56 (dd, J= 9.6, 4.7 Hz, 3H), 1.47 (ddt, J= 20.7, 10.2, 5.5 Hz, 3H), 1.37 - 1.25 (m, 10H), 1.25 (s, 21H), 1.18 - 1.09 (m, 3H), 1.12 - 0.96 (m, 10H), 0.99 (s, 4H), 0.96 - 0.83 (m, 13H), 0.68 (s, 3H).Example 6 Synthesis of Compound 1-6OH6-1 6-2(a), i. sn-glycero-3-Phosphocholine, dibutyltin oxide, i-PrOH, 25°C to reflux ii. cool to 25°C, Et₃N (b). DCC, DMAP, CHCI3, r.t. 12h, 45%.Scheme 1-6(R)-2-hydroxy-3-(tetradecanoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (6-2)OH6-2Attorney Docket: 049386-553001WO

[0249] A mixture of sn-glycero-3-Phosphocholine (3.12 g, 1.0 eq) and DBTO (3.03 g, 1. 0eq)was dissolved in 2-propanol (120 ml, 40 V) in a 300 mL round-bottom flask at 25 °C with N2 protection. The reaction mixture was warmed to 82 °C reflux and stirred for 4 h. The mixture was cooled to 25 °C and TEA (3 g, 2 eq) was added followed by myristoyl chloride (3 g, 1 eq) into the reactor. The reaction mixture was stirred 15 h at 25 °C. Water was (10 V) charged into the reaction and filtered. The crude mixture was dissolved in CH₂Cl₂ (250 mL) and silica gel (50g, type: ZCX-100-200 mesh) was added. The solvent was removed in vacuo and the silica gel containing adsorbed 6-2 was loaded atop a silica gel column (300g, type: ZCX-100-200 mesh) which was eluted with a gradient of CH₂Cl₂: MeOH from 100:0 to 90:10 followed by DCM: MeOH:H₂O ((v / v / v) (65:25:4) ) to elute the product. Qualified fractions were pooled and concentrated in vacuo to afford 6-2 (3.55g, 62%) as a white solid. LC-MS (+ mode): RT 0.783 min, Calcd. for Chemical Formula: C22H46NO7P, 467.50; Found 468.50; 1H-NMR (400MHz, CD3OD): d 4.31 (m, 2H), 4.20 (m, 2H), 4.14 (3H), 4.07 (m, 2H), 3.31 (s, 9H), 2.34 (m, 2H), 1.61 (m, 2H), 1.22-1.32 (21H), 0.90 (t, J = 6.0Hz, 3H).(2R)-2-(2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,ll,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl)oxy)acetoxy)-3-(tetradecanoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (6)

[0250] To a solution of 6-2 (2.0g) and 2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methy lhep tan-2 -y l)-2,3,4,7,8,9,10,11,12,13,14, 15,16,17 -tetradecahydro- 1 H-cyclopenta[a]phenanthren-3-yl)oxy)acetic acid (2.28g) in DCM (60 mL), cooled in an ice-water bath under nitrogen, was added in order DCC (2.65g) and DMAP (0.234g). The mixture was stirred for 30 minutes at 0°C after the additions were complete, then was warmed to room temperature and was allowed to stir for 48 hours. The mixture was concentrated in vacuo and the residue was dissolved in CH2CI2 (100 mL) and 30g silica gel was added. The solvent was removedAttorney Docket: 049386-553001WOin vacuo and the silica gel containing adsorbed 6 was loaded atop a silica gel column (250g, type: ZCX-100-200 mesh) which was eluted with a gradient of CH₂Cl₂:MeOH from 100:0 to 80:20 followed by DCM: MeOH: H2O ( v / v / v = 65:25:4). Eluent was concentrated and freeze dried to afford 6 (1.72 g, HPLC = 95.6%) as off white solid. Yield = 43.6%. LC-MS (+ mode): RT 2.46 min, Calcd. for C51H92NO9P 894.27, Found 894.8 (M+);1H NMR: (400 MHz, Chloroform-tZ) 8 5.37 - 5.24 (m, 2H), 4.31 (s, 3H), 4.22 - 4.07 (m, 3H), 3.98 (t, J= 6.4 Hz, 2H), 3.86 (s, 2H), 3.80-3.35 (s, 10H), 2.26 (q, J= 7.8 Hz, 4H), 2.06 - 1.89 (m, 2H), 1.89 - 1.78 (m, 3H), 1.51 (tdt, J = 20.0, 9.9, 5.6 Hz, 5H), 1.26 (d, 5.7 Hz, 24H), 1.14 (dd, J= 10.4, 5.8 Hz, 3H), 1.13 - 0.99 (m, 3H), 1.02 - 0.84 (m, 21H), 0.86 (d, J= 1.8 Hz, 3H), 0.68 (s, 3H).Example 7 Synthesis of Compound 1-7OH7-2b(a), i. sn-glycero-3-Phosphocholine, dibutyltin oxide, i-PrOH, 25°C to reflux ii. cool to 25°C, EtsN (b). DCC, DMAP, CHCI3, r.t. 12h, 45%.Scheme 1-7(R)-2-hydroxy-3-(oleoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (7-2)OH

[0251] A mixture of sn-glycero-3-Phosphocholine (2.57 g, 1.0 eq) and DBTO (2.49 g, 1. Oeq) was dissolved in 2-propanol (100 ml, 40 V) in a 300 mL round-bottom flask at 25 °C with N2 protection. The reaction mixture was warmed to 82 °C reflux and stirred for 4 h. The mixture was cooled to 25 °C and TEA (3 g, 2 eq) was added followed by oleoyl chloride (3 g, 1 eq) into theAttorney Docket: 049386-553001WOreactor. The reaction mixture was stirred 15 h at 25 °C. Water was (10 V) charged into the reaction and filtered. The crude mixture was dissolved in CH₂Cl₂ (250 mL) and silica gel (50g, type: ZCX-100-200 mesh) was added. The solvent was removed in vacuo and the material was loaded onto a silica gel column (300g, type: ZCX- 100-200 mesh) and eluted with a gradient of CH₂Cl₂: MeOH from 100:0 to 90:10 followed by DCM: MeOH: H2O ((v / v / v) (65:25:4) ) to elute the product. Qualified fractions were pooled and concentrated in vacuo to afford 7-2 (3.56g, 68%) as a white solid.

[0252] LC-MS (+ mode): RT 0.880 min, Calcd. for Chemical Formula: C26H52NO7P, 521.68; Found 522.60; 1H-NMR (400MHz, CD₃OD): 5.34 (dt, 2H), 4.32 (m, 2H), 4.30 (m, 2H), 4.14 (2H), 3.98 (m, 2H), 3.22 (s, 9H), 2.35 (m, 2H), 2.05 (m, 4H) 1.62 (m, 2H), 1.22-1.32 (m, 22H), 0.90 (t, J = 6.0Hz, 3H).(2R)-2-(2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,ll,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl)oxy)acetoxy)-3-(oleoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (7)7

[0253] To a solution of 7-2 (2.0g) and 2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methy lhep tan-2 -y l)-2,3,4,7,8,9,10,11,12,13,14, 15,16,17 -tetradecahydro- 1 H-cyclopenta[a]phenanthren-3-yl)oxy)acetic acid (2.28g) in DCM (60 mL), cooled in an ice-water bath under nitrogen, was added in order DCC (2.65g) and DMAP (0.261g). The mixture was stirred for 30 minutes at 0°C after the additions were complete, then was warmed to room temperature and was allowed to stir for 48 hours. The mixture was concentrated in vacuo and the residue was dissolved in CH2CI2 (100 mL) and 30g silica gel was added. The solvent was removed in vacuo, loaded atop a silica gel column (250g, type: ZCX-100-200 mesh) and was eluted with a gradient of CH2Ch: MeOH from 100:0 to 80:20 followed by DCM: MeOH: H2O ( v / v / v = 65:25:4). Eluent was concentrated and freeze dried to afford 7 (1.65 g, HPLC = 95.6%) as off white solid.IllAttorney Docket: 049386-553001WOYield = 44.6%. LC-MS (+ mode): RT 2.23 min, Calcd. for C55H98NO9P 948.36, Found 948.90 (M+); ‘H NMR: (300 MHz, Chloroform-d) δ 5.39 - 5.25 (m, 4H), 4.30 (s, 3H), 4.14 (q, J= 7.1, 6.2 Hz, 3H), 3.98 (t, J= 6.6 Hz, 2H), 3.80 (s, 2H), 3.64 (s, 1H), 3.36 (s, 9H), 2.42 - 2.22 (m, 4H), 2.01 (q, 6.1, 5.1 Hz, 7H), 1.91 - 1.80 (m, 10H), 1.62 - 1.43 (m, 3H), 1.29 (t, J= 8.8 Hz, 26H), 1.19 - 0.95 (m, 6H), 0.99 (s, 3H), 0.89 (dd, J= 14.6, 6.7 Hz, 13H), 0.67 (s, 3H).Example 8 Synthesis of Compound 1-8OH8-1 8-2b(a), i. sn-glycero-3-Phosphocholine, dibutyltin oxide, i-PrOH, 25°C to reflux ii. cool to 25°C, EtsN (b). DCC, DMAP, CHC13, r.t. 12h, 46.6%.Scheme 1-8(R)-2-hydroxy-3-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)propyl (2-(trimethylammonio)ethyl) phosphate (8-2)OH8-2

[0254] A mixture of sn-glycero-3 -phosphocholine (2.59 g, 1.0 eq) and DBTO (2.51 g, 1.0 eq) was dissolved in 2-propanol (104 ml, 40 V) in a 300 mL round-bottom flask at 25 °C with N2 protection. The reaction mixture was warmed to 82 °C reflux and stirred for 4 h. The mixture was cooled to 25 °C and TEA (3 g, 2 eq) was added followed by linoleoyl chloride (3 g, 1 eq) into the reactor. The reaction mixture was stirred 15 h at 25 °C. Water was (10 V) charged into the reaction and filtered. The crude mixture was dissolved in CH₂Cl₂ (250 mL) and silica gel (30g, type: ZCX-Attorney Docket: 049386-553001WO100-200 mesh) was added. The solvent was removed in vacuo and the silica gel containing adsorbed 8-2 was loaded atop a silica gel column (300g, type: ZCX-100-200 mesh) which was eluted with a gradient of CH₂Cl₂:MeOH from 100:0 to 90:10 followed by DCM: MeOH: H2O ((v / v / v) (65:25:4) ) to elute the product. Qualified fractions were pooled and concentrated in vacuo to afford 8-2 (3.6 g, 69%) as a white solid. LC-MS (+ mode): RT 0.82 min, Calcd. for Chemical Formula: C26H50NO7P, 519.66; Found 520.60; 1H-NMR (400MHz, CD3OD): 5.34 (m, 4H), 4.30 (m, 2H), 4.28 (m, 2H), 4.15 (3H), 3.95 (m, 2H), 3.29 (s, 9H), 2.77 (m, 2H), 2.35 (m, 2H), 2.07 (m, 4H), 1.62 (m, 2H), 1.22-1.32 (m, 15H), 0.90 (t, J= 6.0Hz, 3H).(2R)-2-(2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,ll,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl)oxy)acetoxy)-3-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)propyl (2- (trimethylammonio)ethyl) phosphate (8)

[0255] To a solution of 8-2 (2.0g) and 2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methy lhep tan-2 -y l)-2,3,4,7,8,9,10,11,12,13,14, 15,16,17 -tetradecahydro- 1 H-cyclopenta[a]phenanthren-3-yl)oxy)acetic acid (2.05g) in DCM (60 mL), cooled in an ice-water bath under nitrogen, was added in order DCC (2.4g) and DMAP (0.235g). The mixture was stirred for 30 minutes at 0°C after the additions were complete, then was warmed to room temperature and was allowed to stir for 48 hours. The mixture was concentrated in vacuo and the residue was dissolved in CH₂Cl₂ (100 mL) and 30g silica gel was added. The solvent was removed in vacuo and the silica gel was loaded atop a silica gel column (250g, type: ZCX-100-200 mesh) which was eluted with a gradient of CH₂Cl₂: MeOH from 100:0 to 80:20 followed by DCM: MeOH: H2O ( v / v / v = 65:25:4). Eluent was concentrated and freeze dried to afford 8 (1.75 g, HPLC = 97%) as off white solid. Yield = 46.5%. LC-MS (+ mode): RT 2.37 min, Calcd. for C55H96NO9P 946.34, Found 946.85 (M+); ‘H NMR::(400 MHz, Chloroform- ) 8 5.44 - 5.25 (m, 6H), 4.33 (s, 3H), 4.14 (q, J= 7.1, 6.6 Hz, 3H), 4.03 - 3.93 (m, 2H), 3.86 - 3.79 (m, 2H), 3.37 (s, 10H), 3.05 (s, 2H),Attorney Docket: 049386-553001WO2.77 (t, J = 6.5 Hz, 2H), 2.32 - 2.18 (m, 3H), 2.10 - 1.97 (m, 5H), 1.97 - 1.78 (m, 3H), 1.57 (s, 4H), 1.58 - 1.41 (m, 4H), 1.41 - 1.22 (m, 19H), 1.21 - 0.83 (m, 25H), 0.67 (s, 3H).Example 9 Synthesis of Compound 1-9a. DCC, DMAP, DCM, 25 oC, 48h, 46.8%Scheme 1-9(2R)-2-((4-(((3 S,1 OR, 13R,17R)- 10, 13-dimethyl- 17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,ll,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoyl)oxy)-3-(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate (9)

[0256] To 45 mL anhydrous DCM kept in 250 mL round-bottom flask kept under argon was charged with (R)-2-hydroxy-3-(stearoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (4-4, 1.5 g, l. Oeq) and cholesteryl hemisuccinate (1.67 g, 1.2 eq) in dry DCM (45 mL, 30 V). The reaction mixture was cooled to 0 °C and charged with DCC (1.79 g, 3 eq) and DMAP (175.5 mg, 0.5eq) and stirred for 48 h at 20 - 25 °C. The reaction was mixed with 50 g silica gel and solvent was removed under reduced pressure at 35 °C to adsorb the material. The dry silica gel powder was applied onto atmospheric silica gel column (300 g, type: ZCX-2, 100-200 mesh) eluted with DCM: MeOH, gradient from 1 / 0 to 5:1, then eluted with DCM: MeOH: H2O ( v / v / v = 65:25:4 )Attorney Docket: 049386-553001WOand collected product. The eluent was concentrated and freeze dried to afford 9 (1.35 g, HPLC = 98.2%) as light-yellow solid. Yield = 46.80%. LC-MS (+ mode): RT 2.39 min, Calcd. for C57H102NO10P 992.41, Found 992.90 (M+); ‘H NMR: (400 MHz, Chloroform- ) 85.39 - 5.33 (m, 1H), 5.21 (qd, J= 5.4, 3.1 Hz, 1H), 4.58 (tt, J= 12.2, 5.6 Hz, 1H), 4.36 (dd, J= 12.2, 3.2 Hz, 1H), 4.32 (d, J= 6.8 Hz, 2H), 4.15 (dd, J= 12.1, 7.0 Hz, 1H), 3.97 (t, J= 6.3 Hz, 2H), 3.82 (d, J = 4.8 Hz, 2H), 3.49 (s, 2H), 3.37 (s, 9H), 2.61 (dtt, J= 12.7, 10.1, 6.4 Hz, 4H), 2.29 (dt, J= 11.9, 6.1 Hz, 4H), 1.99 (ddt, J= 21.4, 17.0, 4.2 Hz, 2H), 1.85 (ddd, J= 13.3, 8.8, 4.5 Hz, 3H), 1.65 -1.40 (m, 4H), 1.37 - 1.25 (m, 37H), 1.12 (qd, J= 11.0, 9.5, 5.0 Hz, 5H), 1.02 (s, 3H), 0.99 (d, J = 8.5 Hz, 1H), 0.97 - 0.83 (m, 15H), 0.68 (s, 3H).Example 10 General Synthesis of Compounds 1-2-1 to 1-2-9.1-5-1 to 1-5-9.1-6-1 to 1-6-9.1-7-1 to 1-7-9, 1-8-1 to 1-8-9, and 1-12 -1 to 1-12-9Step 1: General synthesis of 2-(cholesteryl-3-yl)oxyacetic acid analogues of sterolsRD-OH RD-OCH2C(O)-OHa. 2-Bromoacetic acid (2.5 equivalent), t-butyl lithium (IM in THF, 4.5-4.7 equivalent), N, N-dimethylacetamide (10V) / toluene (10V), 20-25 °C, 16-20 hours.

[0257] Cholesterol derivative RDis conjugated to 2-bromoacetic acid through nucleophilic attack.Step 2: General synthesis of compound of Formula Iba. i. sn-glycero-3 -phosphocholine, dibutyltin oxide, i-PrOH, 25 °C to reflux ii. cool to 25°C, EtsN; b.2-(cholesteryl-3-yl)oxyacetic acid, DCC, DMAP, CHCI3.Attorney Docket: 049386-553001WO

[0258] Fatty acid acyl chloride is attached to sn-glycero-3-phosphocholine. Then the 2-(cholesteryl-3-yl)oxyacetic acid obtained from step 1 is conjugated to the resulting compound to obtain Compound of Formula I.Example 11 General Synthesis of Compounds 1-9-1 to 1-9-9.1-13-1 to 1-13-9, 1-14-1 to 1-14-9, 1-15-1 to 1-15-9, 1-16-1 to 1-16-9, 1-17-1 to 1-17-9

[0259] Cholesterol derivative RDis reacted with succinic anhydride to form the corresponding succinates that are combined with various fatty acids to obtain further exemplary compounds of Formula I.Step 1: General synthesis of Sterol SuccinateRD-OH RD-OC(O)CH2CH2C(O)-OHa. N, N -Dimethylaminopyridine (catalytic), pyridine, room temperature, 7 days or until reaction is complete.

[0260] Cholesterol derivative RDis reacted with succinic anhydride to obtain sterol succinate.Step 2: General synthesis of compound of Formula Ia RD-OC(O)CH2CH2C(O)-OH OHa. DCC, DMAP, DCM, 25 °C, 48h

[0261] The fatty acid ester of sn-glycero-3 -phosphocholine obtained from step 2 of Example 10 is reacted with the sterol succinate with a coupling agent to obtain exemplary compounds of Formula I.III. Biological ExamplesExample A: Preparation of lipid nanoparticle

[0262] Exemplary compounds of Formula I of the present disclosure were used to form lipid nanoparticles using the general method described below. The resulting lipid nanoparticles wereAttorney Docket: 049386-553001WOcharacterized in terms of particle size (PS), polydispersity index (PDI), and encapsulation efficiency (ee%). PS, PDI, ee% were assessed again once the lipid particles have undergone a cycle of freeze-thaw. Results are shown in Table 2.

[0263] The LNPs were prepared by mixing lipids in ethanol with mRNA in an acidic aqueous buffer as previously described. In general, lipid excipients including ionizable lipid, phospholipid of the present disclosure, cholesterol, and PEG-DMG were dissolved in ethanol. The lipids were mixed, at a mRNA / total lipid weight / weight ratio of about 0.037, with mRNA solution prepared in pH 4.0 citrate buffer using a commercially available mixer (MIVM-1L, Holland Applied Technologies, Burr Ridge, IL). The freshly formed LNPs were stabilized by sequential dilution with phosphate buffer at pH 6.5, followed by HEPES buffer at pH 8.0. To concentrate the formulation, the diluted formulations were processed with tangential flow filtration (TFF) using PES hollow fiber membrane (100 kDa MWCO, Repligen, Waltham, MA), and further diafiltered with lOx volume of HEPES buffer and concentrated by centrifuging at 2000 RPM for 15-30 minutes in an Amicon® Ultra filtration tube with a100 kD NMWL cellulose membrane (Millipore Sigma, USA). After filtering the formulation with 0.2 pm PES filter, an in-process RNA concentration determination was performed using a RiboGreen® assay following vendor (Thermofisher Scientific, USA) protocol, and the formulation concentration was adjusted to the desired concentration by cryoprotectant addition. After sterile filtration, bulk formulation is aseptically filled into glass vials and stored frozen at - 70°C

[0264] The particle size and poly dispersity index (PDI) were characterized using a Zen3600 (Malvern Instruments, with Zetasizer 7.1 software, Malvern, U. K). A volume of 50 pL of formulations were diluted into 950 pL of Tris buffer at pH = 7.4 and equilibrated to 25 °C prior to analysis in a 1 mL cuvette using the following settings for measurement: material refractive index of 1.5, dispersant viscosity of 1.1 cP, and refractive index of 1.3. Each sample was analyzed for up to 30 runs. Encapsulation efficiency was calculated by determining the unencapsulated mRNA content by measuring the fluorescence intensity (Fi) upon the addition of RiboGreen (Molecular Probes, Eugene, OR, USA) to the LNP and comparing this value to the total fluorescence intensity (Ft) of the RNA content that is obtained upon lysis of the LNPs by 1% Triton X-100, where % encapsulation = (Ft - Fi) / Ft x 100).Attorney Docket: 049386-553001WOTable 2. Lipid particle characteristicsBulk formulation After 1 freeze-thaw cyclePS Encapsulation PS Encapsulation PCA* (nm) PDI efficiency (ee %) (nm) PDI efficiency (ee %) 1-2 86.6 0.139 99.3 76.3 0.204 99.31-3 83.9 0.040 99.7 75.9 0.194 99.41-4 83.5 0.057 98.2 78.9 0.160 99.3DSPC 75.5 0.076 99.6 84.4 0.231 99.8* PCA: Phosphatidylcholine analogue

[0265] Further exemplary compounds of Formula I were used to form LNPs with an ionizable cationic lipid LI. DSPC was used as a positive control. Results are shown in Table 3.Table 3. Lipid particle characteristicsBulk formulation After 1 freeze-thaw cycle LNP PS (nm) PDI EE (%) PS (nm) PDI EE(%) Ll+ I-5 70.93 0.106 96.3 71.36 0.15 93.6 Ll+ I-6 67.5 0.084 96.3 77.6 0.28 87.9 Ll+ I-7 71.45 0.047 92.9 74.61 0.12 92.4 Ll+ I-9 66.93 0.12 96.2 67.18 0.11 92.5 Ll+ I-8 66.86 0.09 96.9 69.39 0.12 94.4 Ll+ I-2 65.21 0.104 95.9 66.83 0.12 94.8Ll+DSPC 65.81 0.113 94.6 67.47 0.17 94.2

[0266] As shown in the tables above, the compounds of Formula I were able to form uniform LNPs of particle size less than 100 nm, while maintaining a consistently high encapsulation efficiency.Example B: siRNA knock down assay in mice

[0267] Lipid particle formulations using compounds 1-2, 1-3, or 1-4 were then tested with FVII siRNA as exemplary nucleic acid to be encapsulated to assess capability of the compounds of Formula I to form functional lipid particles. The formed particles were then used in FVII mRNA knock down experiments. DSPC lipid nanoparticles were used as positive control. PBS was used as negative control. FVII mRNA knock down as represented by the reduction in FVII plasma protein (as a percentage of base value) at 48 hours after mice are administered intravenously with FVII siRNA encapsulated LNPs at dose levels of 0.01 and 0.03 mg / Kg of FVII siRNA. ResultsAttorney Docket: 049386-553001WOare shown in FIG. 1 LNPs formed with the compounds of Formula I of the present disclosure were able to knock down expression of the FVII target gene.Example C: Serum hEPO expression in mice and NHPs with phosphatidyl choline LNPs

[0268] LNPs formulated with various phosphatidylcholine derivatives (DSPC, DMPC, DPPC and DAPC) using hEPO mRNA as the nucleic acid. The formulated LNPs were administered to mice and non-human primates (NHPs). hEPO protein expression was measured. Results are shown in FIG. 2. FIG. 2A shows Mouse serum hEPO protein expressions (ng / mL) at 6 hours post intravenous administration of hEPO mRNA in LNPs formulated with DSPC, DMPC, DPPC and DAPC each as helper lipids and LI (di(pentadecan-8-yl) 4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)dibutyrate ) as the principal ionizable cationic lipid (iCL) at 0.3 mg / Kg doses. FIG. 2B shows hEPO expressions in NHP serum at 0.3 mg / kg dose of hEPO mRNA encapsulated with DSPC, DMPC, DPPC and DAPC each as helper lipids and LI as the principal iCL.

[0269] In each case, relative to the negative control, hEPO protein expression was observed.Example D: Serum hEPO expression in mice and NHP serum with LNPs of the present disclosure

[0270] The LNPs encapsulating hEPO mRNA were formed with compounds 1-2, 1-5, 1-6, 1-7, 1-8, or 1-9 and the ionizable cationic lipid LI using methods as described in Example A. The resulting LNPs were administered to mice. DSPC was used as a positive control. PBS was used as negative control. Mouse serum hEPO protein levels 6 h post administration of 0.3 mg / Kg of hEPO mRNA formulated in LNPs were assessed. Results are shown in FIG. 3. As shown, the LNPs of the present disclosure made with compounds of Formula I were able to deliver hEPO mRNA to the animal, leading to hEPO expression.

[0271] The LNPs encapsulating hEPO mRNA were formed with compound 1-2 or 1-3 and the ionizable cationic lipid LI using methods as described in Example A. These LNPs were compared with hEPO mRNA containing LNPs made with DSPC + ionizable cationic lipid LI, DAPC+L1, DAPC, MC3+DSPC, MC3+DAPC. The LNPs were administered to mice at 0.1 or 0.3 mg hEPO / kg. hEPO expression was measured after 6 hours post intravenous administration. Results are shown in FIG. 4. The LNPs were also tested on NHP serum at 0.3 mg / kg. Results are shown in FIG. 5Attorney Docket: 049386-553001WO

[0272] As shown, the LNPs of the present disclosure, prepared using the compounds of Formula I were able to produce hEPO expression in a dose dependent fashion.Example E; Serum hEPO expression in NHP serum with LNPs of the present disclosure with different ionizable cationic lipids

[0273] Compound 1-2 was formulated into LNPs with different ionizable cationic lipids (LI, L8, L9, LIO, Lil, LI 2, L13, LI 4, L15, LI 6, LI 7) to encapsulate exemplary nucleic acid hEPO mRNA. DSPC was used as a positive control. PBS was used as negative control. The LNPs were administered to NHP ((n = 3), 0.3 mg / Kg dose). Expression level of hEPO protein in NHP serum was measured. Results are shown in FIG. 6A-6F. As shown, compound 1-2 containing LNPs were able to achieve higher expression level than DSPC containing LNPs.

[0274] The present disclosure provides reference to various embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the present disclosure. The description is made with the understanding that it is to be considered an exemplification of the claimed subject matter, and is not intended to limit the appended claims to the specific embodiments illustrated.

Claims

Attorney Docket: 049386-553001WOCLAIMS1. A compound of Formula I:IwhereinX1, X2, and X3are each independently O or S,Ra, Rb, and Rcare each independently H, or C1-6 alkyl, orRais H or C1-6 alkyl, and Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, the heterocyclyl being optionally substituted with one or more C1-3 alkyl,n is 2 to 6,eitherwhereinY1is absent, O, or S,L1is C3-40 alkyl, C3-40 alkenyl, or C3-40 alkynyl,either(a) Y2is absent or C1-4 alkylene, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl, optionally the alkyl, alkenyl, and alkynyl are branched, or(b) Y2is C1-4 alkylene, optionally substituted with oxo, W is absent, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative; or(ii) R1is and R2isAttorney Docket: 049386-553001WOwhereinY1is absent, O, or S,L1is C3-40 alkyl, C3-40 alkenyl, or C3-40 alkynyl,Y2is C1-4 alkylene, W is absent or carbonyl, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative.

2. The compound of claim 1, wherein the compound is of a compound of Formula I-I:(I-I)or a pharmaceutically acceptable salt thereof,whereinX1, X2, and X3are each independently O or S,Ra, Rb, and Rcare each independently H, or C1-6 alkyl, orRais H or C1-6 alkyl, and Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, the heterocyclyl being optionally substituted with one or more C1-3 alkyl,n is 2 to 6,Y1is absent, O, or S,L1is C3-40 alkyl, C3-40 alkenyl, or C3-40 alkynyl,Y2is absent or C1-4 alkylene, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl, optionally the alkyl, alkenyl, and alkynyl are branched, orAttorney Docket: 049386-553001WOY2is Ci-4 alkylene, optionally substituted with oxo, W is absent, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative.

3. The compound of claim 1 or 2, wherein X1, X2, and X3are each independently O.

4. The compound of any one of claims 1 to 3, wherein Rband Rcare each independently Cn 6 alkyl.

5. The compound of any one of claims 1 to 4, wherein Rais H.

6. The compound of any one of claims 1 to 4, wherein Rais Ci-6 alkyl.

7. The compound of any one of claims 1 to 3, wherein Ra, Rband Rcare each independently Ci -6 alkyl.

8. The compound of claim 7, wherein Ra, Rb, and Rcare each independently C1-3 alkyl.

9. The compound of claim 7 or 8, wherein Ra, Rb, and Rcare each independently methyl.

10. The compound of any one of claims 1 to 3, wherein Rais C1-6 alkyl, and Rband Rctaken together with the N they are attached to form a 3- to 8-membered heterocyclyl, optionally substituted with one or more C1-3 alkyl.

11. The compound of claim 10, wherein Rais C1-3 alkyl, optionally Rais methyl.

12. The compound of claim 10 or 11, wherein Rband Rctaken together with the N they are attached to form a 5- to 6-membered heterocyclyl, optionally substituted with one or more C1-3 alkyl.

13. The compound of any one of claims 10 to 12, wherein Rband Rctaken together with the N they are attached to form a piperazinyl, optionally substituted with one or more C1-3 alkyl.

14. The compound of any one of claims 1 to 13, wherein n is 2 to 4.

15. The compound of any one of claims 1 to 14, wherein n is 2.

16. The compound of any one of claims 1 to 15, wherein Y1is absent.

17. The compound of any one of claims 1 to 15, wherein Y1is O or S.Attorney Docket: 049386-553001WO18. The compound of any one of claims 1 to 17, wherein L1is Ce-25 alkyl, Ce-25 alkenyl, or Ce-25 alkynyl.

19. The compound of claim 18, wherein L1is Ce-25 alkyl.

20. The compound of claim 18 or 19, wherein L1is C10-20 alkyl.

21. The compound of any one of claims 1 to 20, where L1is branched.

22. The compound of any one of claims 1 to 21, wherein Y2is absent or C1-4 alkylene, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl.

23. The compound of claim 22, wherein Y2is C1-4 alkylene, W is O or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl.

24. The compound of claim 23, wherein Y2is absent, W is absent, O, or S, and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl, optionally W is absent.

25. The compound of claim 23 or 24, wherein W is O.

26. The compound of any one of claims 23 to 25, wherein L2is selected from Ce-25 alkyl, Ce- 25 alkenyl, and Ce-25 alkynyl.

27. The compound of any one of claims 23 to 26, wherein L2is selected from C10-20 alkyl, Cio-20 alkenyl, and C10-20 alkynyl, optionally L2is C10-20 alkyl.

28. The compound of any one of claims 22 to 26, wherein L2 is C6-25 alkyl.

29. The compound of claim 28, wherein L2is C10-20 alkyl.

30. The compound of any one of claims 22 to 29, wherein L2is branched.

31. The compound of any one of claims 1 to 22, wherein Y2is C1-4 alkylene, W is absent, and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or cholesterol derivative.

32. The compound of claim 31, wherein the cholesterol derivative is selected from Betasitosterol, stigmasterol, lanosterol, thiocholesterol, stigmastanol, campesterol, fucosterol, brassicasterol and ergosterol33. The compound of claim 31 or 32, wherein Y2is C1-2 alkylene.Attorney Docket: 049386-553001WO34. The compound of any one of claims 31 to 33, where L2is cholesterol attached to Y2via an O atom of the cholesterol.

35. The compound of claim 1 or 2, wherein the compound is of Formula IAIA,or a pharmaceutically acceptable salt thereof.

36. The compound of claim 35, wherein n is 2 to 4, optionally n is 2.

37. The compound of claim 35 or 36, wherein Y2is absent and L2is selected from C3-40 alkyl, C3-40 alkenyl, and C3-40 alkynyl.

38. The compound of claim 37, wherein L2is C3-40 alkyl.

39. The compound of claim 37 or 38, wherein L2 is C6-25 alkyl.

40. The compound of any one of claims 37 to 39, wherein L2is C10-20 alkyl.

41. The compound of any one of claims 35 to 40, wherein W is absent or O.

42. The compound of claim 35 or 36, wherein Y2is C1-4 alkylene and L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or via an O or S atom of the cholesterol derivative.

43. The compound of claim 42, wherein Y2is C1-3 alkylene and L2is cholesterol.

44. The compound of claim 42 or 43, wherein Y2is substituted with one oxo.

45. The compound of claim 44, wherein L2is attached to Y2via an ester linkage.

46. The compound of claim 1 or 2, wherein the compound is of Formula IBAttorney Docket: 049386-553001WOIBor a pharmaceutically acceptable salt thereof.

47. The compound of claim 46, wherein n is 2 to 4, optionally n is 2.

48. The compound of claim 1 or 2, wherein the compound is for Formula ICICor a pharmaceutically acceptable salt thereof.

49. The compound of claim 48, wherein n is 2 to 4, optionally n is 2.

50. The compound of any one of claims 46 to 49, wherein L2is selected from C3-40 alkyl, C3- 40 alkenyl, and C3-40 alkynyl.

51. The compound of claim 50, wherein L2is C3-40 alkyl.

52. The compound of claim 50 or 51, wherein L2 is C6-25 alkyl.

53. The compound of any one of claims 50 to 52, wherein L2is C10-20 alkyl.

54. The compound of any one of claims 46 to 53, wherein L2is branched.

55. The compound of claim 1 or 2, wherein the compound is for Formula IDAttorney Docket: 049386-553001WOIDor a pharmaceutically acceptable salt thereof,wherein m is 1 to 3.

56. The compound of claim 55, wherein L2is cholesterol or cholesterol derivative attached to Y2via an O atom of the cholesterol or cholesterol derivative.

57. The compound of claim 55 or 56, wherein L2is cholesterol.

58. The compound of any one of claims 55 to 57, wherein m is 1 or 2, optionally 1.

59. The compound of any one of claims 35 to 58, wherein Ra, Rb, and Rcare each independently C1-3 alkyl.

60. The compound of any one of claims 35 to 59, wherein L1is Ce-25 alkyl, Ce-25 alkenyl, or Ce-25 alkynyl.

61. The compound of claim 60, wherein L1is Ce-25 alkyl.

62. The compound of claim 61, wherein L1is Cioto20 alkyl.

63. The compound of claim 60, wherein L1is Ce-25 alkenyl.

64. The compound of claim 63, wherein L1is C10-20 alkenyl.

65. The compound of any one of claims 35 to 64, wherein L1is branched.

66. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the compounds of Table A, the compounds of Table B, and the compounds of Table C.Attorney Docket: 049386-553001WO67. A pharmaceutical composition comprising a compound of any one of claims 1 to 66, and a pharmaceutically acceptable excipient.

68. The pharmaceutical composition of claim 67, further comprising one or more additional therapeutic agents.

69. A lipid composition comprising a nucleic acid and a compound of any one of claims 1 to 66.

70. The lipid composition of claim 69, wherein the nucleic acid is RNA or DNA.

71. The lipid composition of claim 69 or 70, wherein the nucleic acid is selected from an siRNA, an mRNA, a self-replicating RNA, a DNA plasmid, and an antisense oligonucleotide.

72. The lipid composition of any one of claims 69 to 71, wherein the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a protein.

73. The lipid composition of claim 72, wherein the protein is an enzyme, and antibody, an antigen, a receptor, or a transporter.

74. The lipid composition of claim 72 or 73, wherein the protein is a gene-editing enzyme.

75. The lipid composition of claim 74, wherein the gene-editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease.

76. The lipid composition of any one of claims 69 to 75, wherein the lipid composition comprises liposomes, lipoplexes, or lipid nanoparticles.

77. A lipid nanoparticle comprising a compound of Formula I of any one of claims 1 to 66 or a salt thereof.

78. The lipid nanoparticle of claim 77, wherein the compound of Formula I or the salt thereof self-assembles to form the lipid nanoparticle.

79. The lipid nanoparticle of claim 77 or 78, wherein the average particle size of the lipid nanoparticle is less than about 100 nm.

80. The lipid nanoparticle of any one of claims 77 to 79, wherein the average particle size of the lipid nanoparticle is about 40 nm to about 100 nm, about 50 nm to about 100 nm, aboutAttorney Docket: 049386-553001WO60 nm to about 100 nm, about 50 nm to about 95 nm, about 50 nm to about 90 nm, or about 60 nm to about 90 nm.

81. The lipid nanoparticle of any one of claims 77 to 80, wherein the lipid nanoparticle further comprises a nucleic acid.

82. The lipid nanoparticle of claim 81, wherein the nucleic acid is encapsulated in the lipid nanoparticle.

83. The lipid nanoparticle of claim 82, wherein the nucleic acid is RNA or DNA.

84. The lipid nanoparticle of claim 82 or 83, wherein the nucleic acid is selected from an siRNA, an mRNA, a self-replicating RNA, a DNA plasmid, and an antisense oligonucleotide.

85. The lipid nanoparticle of any one of claims 81 to 84, wherein the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a protein.

86. The lipid nanoparticle of claim 85, wherein the protein is an enzyme, an antibody, an antigen, a receptor, or a transporter.

87. The lipid nanoparticle of claim 85 or 86, wherein the protein is a gene-editing enzyme.

88. The lipid nanoparticle of claim 87, wherein the gene-editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease.

89. The lipid nanoparticle of any one of claims 77 to 88, wherein the lipid nanoparticle further comprises a helper lipid selected from: dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC).

90. The lipid nanoparticle of claim 89, wherein the helper lipid is distearoylphosphatidylcholine (DSPC).

91. The lipid nanoparticle of any one of claims 77 to 90, further comprising cholesterol.

92. The lipid nanoparticle of any one of claims 77 to 91, further comprising a polyethylene glycol(PEG)-lipid conjugate.Attorney Docket: 049386-553001WO93. The lipid nanoparticle of claim 92, wherein the PEG-lipid conjugate is PEG-DMG.

94. The lipid nanoparticle of claim 93, wherein the PEG-DMG is PEG2000-DMG.

95. The lipid nanoparticle of any one of claims 77 to 94, further comprising an ionizable cationic lipid.

96. The lipid nanoparticle of claim 95, wherein, based on the total amount of lipids in the lipid nanoparticle, the lipid nanoparticle comprises about 5 mol% to 15 mol% of a combined amount of the compound of any one of claims 1 to 66 and the helper lipid, about 30 mol% to about 70 mol% of the ionizable cationic lipid, about 20 mol% to about 60 mol% of cholesterol, and about 0.5 mol% to about 5 mol% of a PEG-lipid conjugate.

97. The lipid nanoparticle of claim 96, wherein, based on the total amount of lipids in the lipid nanoparticle, the lipid nanoparticle comprises about about 5 mol% to 10 mol% of a combined amount of the compound of any one of claims 1 to 66 and the helper lipid, about 40 mol% to about 60 mol% of the ionizable cationic lipid, about 30 mol% to about 50 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate.

98. The lipid nanoparticle of claim 96 or 97, wherein, based on the total amount of lipids in the lipid nanoparticle, the lipid nanoparticle comprises about 5 mol% to 12 mol% of a combined amount of the compound of any one of claims 1 to 66 and the helper lipid, about 45 mol% to about 55 mol% of the ionizable cationic lipid, about 35 mol% to about 45 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate.

99. The lipid nanoparticle of any one of claims 77 to 98, wherein the lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 50:1 to about 10:1.

100. The lipid nanoparticle of claim 99, wherein the lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 40: 1 to about 20: 1.

101. The lipid nanoparticle of claim 99, wherein the lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 35:1 to about 25:1.

102. The lipid nanoparticle of claim 99, wherein the lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 32: 1 to about 28: 1.

103. The lipid nanoparticle of claim 99, wherein the lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 31:1 to about 29: 1.Attorney Docket: 049386-553001WO104. A pharmaceutical composition comprising the compound of any one of claims 1 to 66, or the lipid nanoparticle of any one of claims 77 to 103, and a pharmaceutically acceptable excipient.

105. The pharmaceutical composition of claim 104, wherein the pharmaceutical composition is a lyophilized composition.

106. The pharmaceutical composition of claim 103 or 104, wherein the pharmaceutical composition comprises a HEPES buffer at a pH of about 7.4.

107. The pharmaceutical composition of claim 104, wherein the HEPES buffer is at a concentration of about 7 mg / mL to about 15 mg / mL.

108. The pharmaceutical composition of any one of claims 104 to 107, wherein the pharmaceutical composition further comprises about 2.0 mg / mL to about 4.0 mg / mL of NaCl.

109. The pharmaceutical composition of any one of claims 104 to 108, wherein the pharmaceutical composition further comprises one or more cryoprotectants.

110. The pharmaceutical composition of claim 109, wherein the one or more cryoprotectants are selected from sucrose, glycerol, or a combination of sucrose and glycerol.

111. The pharmaceutical composition of claim 110, wherein the pharmaceutical composition comprises a combination of sucrose at a concentration of about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of about 50 mg / mL to about 70 mg / mL.

112. A method of treating and / or preventing a disease in a subject in need thereof, comprising administering a therapeutically effective amount to the subject, the lipid nanoparticle of any one of claims 77 to 103, or the pharmaceutical composition of any one of claims 104 to 111.

113. The method of claim 112, wherein the pharmaceutical composition or lipid nanoparticle is administered intravenously or intramuscularly.

114. The method of claim 112 or 113, wherein the disease or disorder is associated with expression or overexpression of a gene, and / or wherein the disease or disorder is cancer or inflammatory disease, and / or wherein the disease is selected from the group consisting ofAttorney Docket: 049386-553001WOcentral nervous system disorders, peripheral nervous system disorders, muscle atrophies, muscle dystrophies, immune disorder, cancer, renal disease, fibrotic disease, genetic abnormality, inflammation, and cardiovascular disorder.

115. A method of expressing a protein or polypeptide in a target cell, comprising contacting the target cell with a lipid nanoparticle of any one of claims 77 to 103, or the pharmaceutical composition of any one of claims 104 to 111.

116. The method of claim 115, wherein the protein or polypeptide is an antigen, and expression of the antigen elicits an in vivo immunogenic response.

117. A method of delivering a nucleic acid to a subject in need thereof, comprising encapsulating a therapeutically effective amount of the nucleic acid in the lipid nanoparticle of any one of claims 77 to 103, and administering the lipid nanoparticle to the subject.

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