Self-immolative lipids for RNA delivery

Abstract

The present disclosure describes compounds of Formula (I) and pharmaceutically acceptable salts thereof.

Description

Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO SELF-IMMOLATIVE LIPIDS FOR RNA DELIVERYTECHNICAL FIELD

[0001] Embodiments herein relate generally to lipids. In particular, embodiments herein relate to lipids and lipid compositions that facilitate the intracellular delivery of biologically active and therapeutic molecules.BACKGROUND

[0002] 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 (siNA), and microRNA (miRNA) for use in RNA interference (RNAi), antisense molecules, ribozymes, antagomirs, and aptamers.

[0003] For these inherently powerful therapeutic approaches to work effectively they need to be directed to appropriate tissues / cell type of disease relevance. In some of these modalities the RNA used is in its native, chemically unmodified form, and hence need to be protected from the chemical and enzymatic degradation while in circulation, and until it reaches cytoplasm where the endogenous machinery mostly operates.

[0004] One of the clinically validated and commercially successful methods [1-5] of protecting the mRNA drug from degradation is by encapsulating it in a ball of fatty molecules to form lipid nanoparticles (LNPs). Ionizable lipids, the key component of the LNPs designed for the delivery of nucleic acid-based therapeutics, has made a long way since their introduction by Bailey and Cullis in 1994 [6] in which they described the use of pH gradients to control the transbilayer asymmetry of the amino lipids thus controlling membrane fusion. Subsequently, a number of properties important to nucleic acid encapsulation and delivery have been discovered and studied, including pKa of the ionizable lipid. However, it was concluded that optimal pKa value is a relevant but not sufficient requirement for good in vivo activity, and other structural features, such as the nature of the linker between the head group and the lipid tails, can also contribute substantially to the in vivo activity. [7] Since the efficiency and tolerability of lipid nanoparticles are attributed to the ionizable lipid, focused efforts on optimization of the linker and the tails of the lipid nanoparticles enabled a dramatic improvement of efficacy. The evolution of the newer generations of the ionizable lipids to achieve higher efficacy often had a direct and inverse effect on the biodegradability of the ester-based lipids. The multi-tail ionizable lipids exhibit a higher efficiency in delivering theApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO RNA based cargo as they produce a more cone-shaped structure with increased membrane disrupting effect leading to an increased endosomal escape and functional delivery [8, 9], However, the branched lipids generate a steric hindrance around the ester bond hindering the access of the enzymatic machinery to hydrolyze the molecule.

[0010]

[0005] Accordingly, there is a need to develop new lipids suitable for use in delivery of nucleic acids.SUMMARY

[0006] The present disclosure provides lipids of Formula (I) as described herein useful for lipid-based delivery of nucleic acids and other therapeutic agents for treating diseases.Additional features and advantages of the subject technology will be set forth in the description below, and in part will be apparent from the description, or may be learned by practice of the subject technology. The advantages of the subject technology will be realized and attained by the structures particularly pointed out in the written description and embodiments hereof as well as the appended drawings.

[0007] For the successful therapeutic application of LNPs, the ionizable lipid component needs to be readily degraded into non-toxic-metabolites after completing the intracellular delivery of the cargo such as nucleic acid into the cytosol. To maintain the high efficiency of multi-tail architecture of the ionizable lipids and to achieve full biodegradability, herein is provided lipids of Formula (I). As shown herein, the lipids of Formula (I) are “self-immolative lipids” (SIL). For example, the lipids of Formula (I) can be activated by glutathione (GSH) which is the tripeptide y-Glu-Cys-Gly present in the cytosol at a concentration of about 1000-fold higher than its concentration in the extracellular environment. The chemical activation by GSH, as opposed to the esterase / lipase dependent hydrolytic activation, ensures a kinetically independent degradation pathway that might be accurately translated across different species due to the ubiquity and high intracellular content of GSH across species, which is not the case with the varying concentrations and species dependent isozyme preference of hydrolytic enzymes such as esterases and lipases.Activation of the SIL by GSH initiates a sequence of chemical reactions and self-elimination to release the lipid tails which are readily biodegradable.

[0008] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0009] In some embodiments, the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof:(I),or a pharmaceutically acceptable salt thereof, wherein:R1and R2are each independently H or Ci-6 alkyl, orR1and R2taken together with the N atom to which they are attached form a heterocycloalkyl having 4 to 10 ring members, wherein the heterocycloalkyl optionally further comprises a second ring heteroatom N, O, or S;X1is absent or is C1-2 alkylene;X2is C2-5 alkylene;OJVT'V', or '''I™, wherein each asterisk (*) indicates the atom attached to L1and L2, and RYis H or C1-6 alkyl;L1and L2are each independently C1-8 alkylene;Z1and Z2are each independently absent or C4-11 alkylene;R3and R4are each independently C3-12 alkyl or C3-8 cycloalkyl-Ci-6 alkylene; and R5and R6are each independently C3-12 alkyl or C3-8 cycloalkyl-Ci-6 alkylene.

[0010] In some embodiments, the present disclosure provides a lipid nanoparticle, comprising a plurality of ligands, wherein each ligand is independently a compound described herein, wherein the plurality of ligands self-assembles to form the lipid nanoparticle comprising an interior and exterior.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0011] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the compound described herein or the lipid nanoparticle described herein, and a pharmaceutically acceptable excipient.

[0012] In some embodiments, the present disclosure provides a method of treating a disease in a subject in need thereof, comprising administering a therapeutically effective amount to the subject the compound described herein, the lipid nanoparticle described herein, or the pharmaceutical composition described herein.

[0013] In some embodiments, the present disclosure provides a method of delivering a nucleic acid to a subject in needed thereof, comprising encapsulating a therapeutically effective amount of the nucleic acid in the lipid nanoparticle described herein, and administering the lipid nanoparticle to the subject.BRIEF SUMMARY OF THE DRAWINGS

[0014] FIG. 1A shows an illustration of a disulfide-containing lipid and its biodegradation.

[0015] FIG. IB shows a chemical scheme of a disulfide-containing lipid and its biodegradation.

[0016] FIG. 2A shows the results of a lipid degradation and mRNA release gel electrophoresis assay.

[0017] FIG. 2B shows the results of an ex vivo HepG2 transfection and hEPO protein expression assay at 3 different concentrations.

[0018] FIG. 3A shows a schematic of glutathione-mediated degradation of compound LI into compound LI -DI and compund L1-D2.

[0019] FIG. 3B shows a chromatogram of time-dependent degradation of LI upon reaction with glutathione (GSH) at an initial timepoint of 0 h.

[0020] FIG. 3C shows a chromatogram of time-dependent degradation of LI upon reaction with glutathione (GSH) at an intermediate timepoint of 4 h.

[0021] FIG. 3D shows a chromatogram of time-dependent degradation of LI upon reaction with glutathione (GSH) at a terminal timepoint of 24h.

[0022] FIG. 3E shows a mass spectrum of the time-dependent degradation of LI upon reaction with glutathione (GSH) at an intermediate timepoint, wherein LI, LI -DI, and L1-D2 are detected.

[0023] FIG. 4 shows hEPO protein expression of L1-L6 LNPs encapsulating hEPO mRNA at 0.03 and 0.1 mg / Kg doses, compared to control ssPalm-OPhe and MC3 lipid LNPs.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0024] FIG. 5A shows the concentration of lipids LI, L2 and MC3 (control) remaining in plasma (ng / mL) on day 1, 7, 14, 21 and 28 post i.v. administration of LNPs to 8-10 week old Balb / C mice (n = 3 / timepoint).

[0025] FIG. 5B shows the concentration of lipids LI, L2 and MC3 (control) remaining in liver (ng / g) on day 1, 7, 14, 21 and 28 post i.v. administration of LNPs to 8-10 week old Balb / C mice (n = 3 / timepoint).

[0026] FIG. 5C shows the concentration of lipids LI, L2 and MC3 (control) remaining in spleen (ng / g) on day 1, 7, 14, 21 and 28 post i.v. administration of LNPs to 8-10 week old Balb / C mice (n = 3 / timepoint).DETAILED DESCRIPTIONI. GENERAL

[0027] It is understood that various configurations of the subject technology will become readily apparent to those skilled in the art from the disclosure, wherein various configurations of the subject technology are shown and described by way of illustration. As will be realized, the subject technology is capable of other and different configurations and its several details are capable of modification in various other respects, all without departing from the scope of the subject technology. Accordingly, the summary and detailed description are to be regarded as illustrative in nature and not as restrictive.

[0028] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The detailed description includes specific details for the purpose of providing a thorough understanding of the subject technology. However, it will be apparent to those skilled in the art that the subject technology may be practiced without these specific details.II. DEFINITIONS

[0029] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “Ci-6 alkyl” is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and Ce alkyl.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0030] The term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0031] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0032] The phrase “at least one of’ preceding a series of items, with the term “and” or “or” to separate any of the items, modifies the list as a whole, rather than each member of the list (i.e., each item). The phrase “at least one of’ does not require selection of at least one of each item listed; rather, the phrase allows a meaning that includes at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the phrases “at least one of A, B, and C” or “at least one of A, B, or C” each refer to only A, only B, or only C; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0033] The terms “include,” “have,” or the like is used in the description or the claims, such term is intended to be inclusive in a manner similar to the term “comprise” as “comprise” is interpreted when employed as a transitional word in a claim.

[0034] The term “cationic lipid” means amphiphilic lipids and salts thereof having a positive, hydrophilic head group; one, two, three, or more hydrophobic fatty acid or fatty alkyl chains; and a connector between these two domains. An ionizable or protonatable cationic lipid is typically protonated (i.e., positively charged) at a pH below its pKa and is substantially neutral at a pH above the pKa. Preferred ionizable cationic lipids are those having a pKa that is less than physiological pH, which is typically about 7.4. The cationic lipids of the disclosure may also be termed titratable cationic lipids. The cationic lipids canApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO be an “amino lipid” having a protonatable tertiary amine (e.g., pH-titratable) head group. Some exemplary amino lipids can include C18 alkyl chains; and ether, ester, or ketal linkages between the head group and alkyl chains. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, y-DLenDMA, DLin-K-DMA, DLin-KC2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-KC3 -DMA, DLin-KC4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-MC2-DMA (also known as MC2), DLin-MC3 -DMA (also known as MC3) and (DLin-MP-DMA)(also known as 1 -Bl 1).

[0035] The term “comprising” or “comprises” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the terms “consisting of’ and “consisting essentially of’ are thus also encompassed and disclosed.

[0036] The term “commercially available chemicals” and the chemicals used in the Examples set forth herein may be obtained from standard commercial sources, where such sources include, for example, Acros Organics (Pittsburgh, Pa.), Sigma-Adrich Chemical (Milwaukee, Wis.), Avocado Research (Lancashire, U. K.), Bionet (Cornwall, U. K.), Boron Molecular (Research Triangle Park, N. C.), Combi-Blocks (San Diego, Calif.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, N. Y.), Fisher Scientific Co.(Pittsburgh, Pa.), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Lancaster Synthesis (Windham, N. H.), Maybridge Chemical Co. (Cornwall, U. K.), Pierce Chemical Co. (Rockford, Ill.), Riedel de Haen (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, N. J.), TCI America (Portland, Or.), and Wako Chemicals USA, Inc. (Richmond, Va.).

[0037] The phrase “compounds described in the chemical literature” may be identified through reference books and databases directed to chemical compounds and chemical reactions, as known to one of ordinary skill in the art. Suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds disclosed herein, or provide references to articles that describe the preparation of compounds disclosed herein, include for example, “Synthetic Organic Chemistry”, John Wiley and Sons, Inc. New York; S. R. Sandler et al, “Organic Functional Group Preparations,” 2nd Ed., Academic Press, New York, 1983; H. O. House, “Modern Synthetic Reactions,” 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif., 1972; T. L. Glichrist, “Heterocyclic Chemistry,” 2nd Ed. John Wiley and Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure,” 5th Ed., Wiley Interscience, New York, 2001; Specific and analogous reactants may also be identified through the indices of known chemicals prepared by theApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through online databases (the American Chemical Society, Washington, D. C. may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (such as those listed above) provide custom synthesis services.

[0038] The term “effective amount” of an agent, as used herein, is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats a disease, an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of the disease, as compared to the response obtained without administration of the agent.

[0039] The term “fully encapsulated” means that the nucleic acid (e.g., mRNA) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free RNA. When fully encapsulated, preferably less than 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10%, and most preferably less than 5% of the nucleic acid in the particle is degraded. “Fully encapsulated” also means that the nucleic acid-lipid particles do not rapidly decompose into their component parts upon in vivo administration.

[0040] The term “compound,” is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.

[0041] The term “cycloalkyl,” or, alternatively, “carbocycle,” as used herein, alone or in combination, refers to a saturated or partially saturated monocyclic, or bicyclic alkyl radical wherein each cyclic moiety contains from 3 to 12 carbon atom ring members and which may optionally be a benzo fused ring system which is optionally substituted as defined herein. In some embodiments, a cycloalkyl may comprise from 3 to 8 carbon atoms, or from 7 to 12 carbon atoms. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, octahydronaphthyl, 2,3-dihydro-lH-indenyl, adamantyl and the like. “Bicyclic” and “tricyclic” as used herein are intended to include both fused ring systems, such as decahydronaphthalene, octahydronaphthalene as well as the multicyclic (multi centered) saturated or partially unsaturated type. The latter type of isomer is exemplified in general by, bicyclo[l.l.l]pentane, camphor, adamantane, and bicyclo[3.2.1]octane. In embodiments, the cycloalkyl ring is a monocyclic ring from 3- to 8-Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO carbons. In embodiments, the monocyclic ring has 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, or 8 carbons. In embodiments, the cycloalkyl ring is a bicyclic ring from 7- to 12-carbons. In embodiments, the bicyclic ring has 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, or 12 carbons.

[0042] The term “delivery” refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.

[0043] The term “fragment,” as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.

[0044] The terms “heteroatom” or “ring heteroatom,” as used herein, are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), selenium (Se), and silicon (Si). In embodiments, the terms “heteroatom” or “ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0045] The term “heteroaryl,” “heteroraomtic ring,” or “heteroaromatic group” refers to an aromatic group that contains at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heteroaromatic ring of the multiple rings). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.Non-limiting examples of heteroaryl groups include pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl, benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-pyrrolyl, 2-pyrrolyl, 3 -pyrrolyl, 3 -pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted heteroaryl ring systems are selected from the group of acceptable substituents described below. A “heteroarylene,” alone or as part of another substituent, means a divalent radical derived from a heteroaryl. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.

[0046] The term “lipid” means an organic compound that comprises an ester of fatty acid and is characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0047] The term “lipid delivery vehicle” means a lipid formulation that can be used to deliver a therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, and the like). The lipid delivery vehicle can be a nucleic acid-lipid particle, which can be formed from a cationic lipid, a non-cationic lipid (e.g., a phospholipid), a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), and optionally cholesterol.Typically, the therapeutic nucleic acid (e.g., mRNA) may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0048] The term “lipid encapsulated” means a lipid particle that provides a therapeutic nucleic acid such as an mRNA with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid particle.

[0049] The term “amphipathic lipid” or “amphiphilic lipid” means the material in which the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0050] The term “heteroalkyl,” as used herein, alone or in combination, refers to a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, fully saturated or containing from 1 to 3 degrees of unsaturation, consisting of the stated number ofApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized (i.e. bond to 4 groups). The heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group. Up to two heteroatoms may be consecutive, such as, for example, — CH2NHOCH3.

[0051] The term “linker” or “linking moiety” refers to a group of atoms, e.g., 5-100 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker may be of sufficient length as to not interfere with incorporation into an amino acid sequence. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkyl, heteroalkyl, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomeric units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers. Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond ( — S — S — ) or an azo bond ( — N=N — ), which can be cleaved using a reducing agent or photolysis. Non -limiting examples of a selectively cleavable bond include an amido bond, which can be cleaved for example by the use of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and / or photolysis, as well as an ester bond, which can be cleaved for example by acidic or basic hydrolysis.

[0052] The term “mammal” means a human or other mammal or means a human being.

[0053] The term “messenger RNA” (mRNA) refers to any polynucleotide which encodes a protein or polypeptide of interest and which is capable of being translated to produce the encoded protein or polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[0054] The term “modified” refers to a changed state or structure of a molecule of the disclosure. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, nucleic acid active ingredients are modified by the introduction of non-natural nucleosides and / or nucleotides, e.g., as it relates to the natural ribonucleotides A, U, G, and C. Noncanonical nucleotides such as the cap structures are not considered “modified” although they may differ from the chemical structure of the A, C, G, U ribonucleotides.

[0055] The term “naturally occurring” means existing in nature without artificial aid.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0056] The phrase “optionally substituted X” (e.g., optionally substituted alkyl) is intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein said alkyl is optionally substituted.”) It is not intended to mean that the feature “X” (e.g. alkyl) per se is optional.

[0057] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0058] The phrase “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a subject. Excipients may include, for example: anti adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0059] The phrase “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate,Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0060] The term “pharmacokinetic” refers to any one or more properties of a molecule or compound as it relates to the determination of the fate of substances administered to a living organism. Pharmacokinetics is divided into several areas including the extent and rate of absorption, distribution, metabolism and excretion. This is commonly referred to as ADME where: (A) Absorption is the process of a substance entering the blood circulation; (D) Distribution is the dispersion or dissemination of substances throughout the fluids and tissues of the body; (M) Metabolism (or Biotransformation) is the irreversible transformation of parent compounds into daughter metabolites; and (E) Excretion (or Elimination) refers to the elimination of the substances from the body. In rare cases, some drugs irreversibly accumulate in body tissue.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0061] The term “pharmaceutically acceptable solvate,” as used herein, means a compound of the disclosure wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N, N'-dimethylformamide (DMF), N, N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a “hydrate.”

[0062] The term “phosphate” is used in its ordinary sense as understood by those skilled in the art and includes its protonated forms, for exampleOHO = P — O 0=P — oO'and OH

[0063] As used herein, the terms “monophosphate,” “diphosphate,” and “triphosphate” are used in their ordinary sense as understood by those skilled in the art, and include protonated forms.

[0064] The term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying progression from an infection, a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and / or condition.

[0065] The term “RNA” refers to a ribonucleic acid and means a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2' position of a P-D-ribo-furanose moiety. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of oneApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of an interfering RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant disclosure can also comprise nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA. As used herein, the terms “ribonucleic acid” and “RNA” refer to a molecule containing at least one ribonucleotide residue, including siRNA, antisense RNA, single stranded RNA, microRNA, mRNA, noncoding RNA, and multivalent RNA.

[0066] The term “sample” or “biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.

[0067] The terms “significant” or “significantly” are used synonymously with the term “substantially.”

[0068] The phrase “single unit dose” is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event.

[0069] The term “siRNA” or small interfering RNA, sometimes known as short interfering RNA or silencing RNA, refers to a class of double-stranded RNA non-coding RNA molecules, typically 18-27 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation.

[0070] The term “solvate” means a physical association of a compound of this disclosure with one or more solvent molecules. This physical association involves varying degrees of ionic bonding, including hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolatableApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.

[0071] The term “stable” refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.

[0072] The terms “stabilize”, “stabilized,” “stabilized region” means to make or become stable.

[0073] The term “substituted” means substitution with specified groups other than hydrogen, or with one or more groups, moieties, or radicals which can be the same or different, with each, for example, being independently selected.

[0074] The term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0075] The phrase “substantially equal” relates to time differences between doses, the term means plus / minus 2%.

[0076] The phrase “substantially simultaneously” relates to plurality of doses, the term means within 2 seconds.

[0077] The phrase “suffering from” relates to an individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and / or condition.

[0078] The phrase “susceptible to” relates to an individual who is “susceptible to” a disease, disorder, and / or condition has not been diagnosed with and / or may not exhibit symptoms of the disease, disorder, and / or condition but harbors a propensity to develop a disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of a protein and / or nucleic acid associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with development of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection with a microbeApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO associated with development of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0079] The term “synthetic” means produced, prepared, and / or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present disclosure may be chemical or enzymatic.

[0080] The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect.

[0081] The term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0082] The term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, “treating” cancer may refer to inhibiting survival, growth, and / or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0083] The term “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.

[0084] Compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known inApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO the art, such as by resolution of racemic mixtures or by enantio-selective and / or stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[0085] Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3 / 7-imidazole, 1H-, 2H- and 4 / 7-1,2,4-triazole, 1H-and 2 / 7-isoindole, and 1H- and 2 / / -pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0086] Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium.

[0087] The compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.

[0088] The term “half-life” is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.

[0089] The term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0090] The term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0091] The term “monomer” refers to a single unit, e.g., a single nucleic acid, which may be joined with another molecule of the same or different type to form an oligomer. In some embodiments, a monomer may be an unlocked nucleic acid, i.e., a UNA monomer.

[0092] The term “neutral lipid” means a lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, forApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0093] The term “non-cationic lipid” means an amphipathic lipid or a neutral lipid or anionic lipid and is described herein.

[0094] The term “subject” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0095] The term “translatable” may be used interchangeably with the term “expressible” and refers to the ability of polynucleotide, or a portion thereof, to be converted to a polypeptide by a host cell. As is understood in the art, translation is the process in which ribosomes in a cell's cytoplasm create polypeptides. In translation, messenger RNA (mRNA) is decoded by tRNAs in a ribosome complex to produce a specific amino acid chain, or polypeptide. Furthermore, the term “translatable” when used in this specification in reference to an oligomer, means that at least a portion of the oligomer, e.g., the coding region of an oligomer sequence (also known as the coding sequence or CDS), is capable of being converted to a protein or a fragment thereof.

[0096] Abbreviations as used herein, are defined as follows: “1 x” for once, “2 x” for twice, “3 x” for thrice, “°C” for degrees Celsius, “eq” for equivalent or equivalents, “g” for gram or grams, “mg” for milligram or milligrams, “L” for liter or liters, “mL” for milliliter or milliliters, “pL” for microliter or microliters, “N” for normal, “M” for molar, “mmol” for millimole or millimoles, “min” for minute or minutes, “h” for hour or hours, “rt” for room temperature, “RT” for retention time, “RBF” for round bottom flask, “atm” for atmosphere, “psi” for pounds per square inch, “cone.” for concentrate, “RCM” for ring-closing metathesis, “sat” or “sat'd” for saturated, “SFC” for supercritical fluid chromatography “MW” for molecular weight, “mp” for melting point, “ee” for enantiomeric excess, “MS” or “Mass Spec” for mass spectrometry, “ESI” for electrospray ionization mass spectroscopy, “HR” for high resolution, “HRMS” for high resolution mass spectrometry, “LCMS” for liquid chromatography mass spectrometry, “HPLC” for high pressure liquid chromatography, “RP HPLC” for reverse phase HPLC, “TLC” or “tic” for thin layer chromatography, “NMR” for nuclear magnetic resonance spectroscopy, “nOe” for nuclear Overhauser effect spectroscopy, “1H” for proton, “8” for delta, “s” for singlet, “d” for doublet, “t” for triplet, “q” for quartet, “m” for multiplet, “br” for broad, “Hz” for hertz, and “a”, “ ”, “R”, “S”, “E”, and “Z” are stereochemical designations familiar to one skilled in the art.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WOBoc or BOC ter -buty 1 oxy carb ony 1DCM or CH2CI2 dichloromethaneCH3CN or ACN acetonitrileCDCI3 deutero-chloroformCDI carbonyldiimidazoleCRISPR Clustered Regularly Interspaced Short Palindromic Repeats DMAP 4-dimethylaminopyridineDMF N, N’-dimethyl formamideDMSO dimethyl sulfoxideEDC1 or EDC. HCl A-(3-dimethylaminopropyl)-A'-ethylcarbodiimide hydrochlorideEtiN or TEA triethylamineEA or EtO Ac ethyl acetateHC1 hydrochloric acidHEPES 4-(2-hydroxyethyl)piperazine- 1 -ethanesulfonic acidMeOH methanolMeOTf methyl tritiate or methyl trifluoromethanesulfonateMsCl mesyl chloride or methanesulfonyl chlorideMTBE methyl tert-butyl etherNaBEU Sodium borohydrideNaHCCE sodium bicarbonateNaOH sodium hydroxideNa2SO4 sodium sulfatePE petroleum etherTEA triethylamineTFA trifluoroacetic acidTHF tetrahydrofuran

[0097] While this disclosure has been described in relation to certain embodiments, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that this disclosure includes additional embodiments, and that some of the details described herein may be varied considerably without departing from this disclosure. This disclosure includes such additional embodiments, modifications, and equivalents. InApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO particular, this disclosure includes any combination of the features, terms, or elements of the various illustrative components and examples.III. COMPOUNDS

[0098] In some embodiments, the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof:or a pharmaceutically acceptable salt thereof, wherein:R1and R2are each independently H or Ci-6 alkyl, orR1and R2taken together with the N atom to which they are attached form a heterocycloalkyl having 4 to 10 ring members, wherein the heterocycloalkyl optionally further comprises a second ring heteroatom N, O, or S;X1is absent or is C1-2 alkylene;X2is C2-5 alkylene; / A N* A A / / 'A N* % A, or, wherein each asterisk (*) indicates the atom attached to L1and L2, and RYis H or C1-6 alkyl;L1and L2are each independently C1-8 alkylene;Z1and Z2are each independently absent or C4-11 alkylene;R3and R4are each independently C3-12 alkyl or C3-8 cycloalkyl-Ci-6 alkylene; and R5and R6are each independently C3-12 alkyl or C3-8 cycloalkyl-Ci-6 alkylene.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0099] In some embodiments, R1and R2are each independently H or Ci-6 alkyl. In some embodiments, R1and R2are each independently Ci-6 alkyl. In some embodiments, R1and R2are each independently C1-3 alkyl. In some embodiments, R1and R2are each methyl.

[0100] In some embodiments, R1and R2taken together with the N atom to which they are attached form a heterocycloalkyl having 4 to 10 ring members, wherein the heterocycloalkyl optionally further comprises a second ring heteroatom N, O, or S. In some embodiments, R1and R2taken together with the N atom to which they are attached form a heterocycloalkyl having 4 to 6 ring members, wherein the heterocycloalkyl optionally further comprises a second ring heteroatom N, O, or S. In some embodiments, R1and R2taken together with the N atom to which they are attached form a heterocycloalkyl having 5 to 6 ring members, wherein the heterocycloalkyl optionally further comprises a second ring heteroatom N, O, or S. In some embodiments, R1and R2taken together with the N atom to which they are attached form a pyrrolidine or piperidine.

[0101] In some embodiments, X1is absent.

[0102] In some embodiments, X1is C1-2 alkylene. In some embodiments, X1is ethylene.

[0103] In some embodiments, X2is C2-5 alkylene. In some embodiments, X2is C2-3 alkylene. In some embodiments, X2is ethylene. In some embodiments, X2is propylene.

[0104] In some embodiments, Y is CH. In some embodiments, Y is CH and X1is absent.

[0105] In some embodiments, X1is C1-2 alkylene and Y is CH.O O

[0106] In some embodiments, YisO1 1'n? / u', or, wherein each asterisk (*) indicates the atom attached to L1and L2, and RYis H or C1-6 alkyl. In some embodiments, Y isApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WOI > > AAnA ' Y embodiments, Y is, or R. In some embodiments, YO O / N* A S- jf- ■ <* N* A 0- i|-

[0107] In some embodiments, X1is ethylene, and Y is 'ZYV',indicates the atom attached to L1and L2, and RYis H or Ci-6 alkyl. In some embodiments, X1O 0 0 N* A ^N* A S-| >- N* A 0- >|-, or. In some embodiments, X1is ethylene, and Y isJ^nn, '^AnOIn some embodiments, X1is ethylene, and Y isI > MW ‘ Y '" T"", or R. In some embodiments, X1is ethylene, and Y is

[0108] In some embodiments, L1and L2are each independently Ci-8 alkylene. In some embodiments, L1and L2are each independently Ci-6 alkylene. In some embodiments, L1and L2are each independently Ci-6 alkylene. In some embodiments, L1and L2are each independently C2-4 alkylene. In some embodiments, L1and L2are each propylene.

[0109] In some embodiments, Z1and Z2are each independently absent.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0110] In some embodiments, Z1and Z2are each independently C4-11 alkylene. In some embodiments, Z1and Z2are each independently C4-8 alkylene. In some embodiments, Z1and Z2are each independently C4-6 alkylene. In some embodiments, Z1and Z2are each unbranched.

[0111] In some embodiments, R3and R4are each independently C3-12 alkyl or C3-8 cycloalkyl-Ci-6 alkylene. In some embodiments, R3and R4are each independently C3-12 alkyl. In some embodiments, R3and R4are each independently C4-10 alkyl. In some embodiments, R3and R4are each independently C5-8 alkyl. In some embodiments, R3and R4are each independently Ce-7 alkyl. In some embodiments, R3and R4are each independently C3-8 cycloalkyl-Ci-6 alkylene. In some embodiments, R3and R4are each independently C3-8 cycloalkyl-C2-3 alkylene. In some embodiments, R3and R4are each independently C5-6 cycloalkyl-Ci-6 alkylene. In some embodiments, R3and R4are each independently C5-6 cycloalkyl-C2-3 alkylene. In some embodiments, R3and R4are the same. In some embodiments, R3and R4are each unbranched Ce-7 alkyl. In some embodiments, R3and R4areeach

[0112] In some embodiments, R5and R6are each independently C3-12 alkyl or C3-8 cycloalkyl-Ci-6 alkylene. In some embodiments, R5and R6are each independently C3-12 alkyl. In some embodiments, R5and R6are each independently C4-10 alkyl. In some embodiments, R5and R6are each independently C5-8 alkyl. In some embodiments, R5and R6are each independently Ce-7 alkyl. In some embodiments, R5and R6are each independently C3-8 cycloalkyl-Ci-6 alkylene. In some embodiments, R5and R6are each independently C3-8 cycloalkyl-C2-3 alkylene. In some embodiments, R5and R6are each independently C5-6 cycloalkyl-Ci-6 alkylene. In some embodiments, R5and R6are each independently C5-6 cycloalkyl-C2-3 alkylene. In some embodiments, R5and R6are the same. In some embodiments, R5and R6are each unbranched Ce-7 alkyl. In some embodiments, R5and R6areeach—

[0113] In some embodiments, R3, R4, R5and R6are the same. In some embodiments, R3, R4, R5and R6are each C3 -12 alkyl or C3-8 cycloalkyl-Ci-6 alkylene. In some embodiments, R3, R4, R5and R6are each C3 -12 alkyl. In some embodiments, R3, R4, R5and R6are each C4-10 alkyl. In some embodiments, R3, R4, R5and R6are each C5-8 alkyl. In some embodiments, R3, R4, R5and R6are each Ce-7 alkyl. In some embodiments, R3, R4, R5and R6are each C3-8Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO cycloalkyl-Ci-6 alkylene. In some embodiments, R3, R4, R5and R6are each C3-8 cycloalkyl-C2-3 alkylene. In some embodiments, R3, R4, R5and R6are each C5-6 cycloalkyl-Ci-6 alkylene. In some embodiments, R3, R4, R5and R6are each C5-6 cycloalkyl-C2-3 alkylene. In some embodiments, R3, R4, R5and R6are each unbranched Ce-7 alkyl. In some embodiments, R3,R4, R5and R6are each

[0114] In some embodiments, X2is C2-3 alkylene, and R3, R4, R5and R6are each Ce-7 alkyl. In some embodiments, X2is C2-3 alkylene, and R3, R4, R5and R6are each unbranched Ce-7 alkyl. In some embodiments, X2is C2-3 alkylene, and R3, R4, R5and R6are each

[0115] In some embodiments, the compound is of formula la:or a pharmaceutically acceptable salt thereof, wherein W is O or S.O

[0116] In some embodiments, R1and R2are each methyl, X1is ethylene,Y isor, / vr~', L1and L2are each propylene, Z1and Z2are each absent, and the compound is of formula la:Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WOor a pharmaceutically acceptable salt thereof, wherein W is O or S, and wherein X2, R3, R4, R5, and R6are as defined for formula I. In some embodiments, X2is C2-3 alkylene. In some embodiments, R3and R4are each Ce-7 alkyl. In some embodiments, R3and R4are each. In some embodiments, R5and R6are each Ce-7 alkyl. In some embodiments,R5and R6are each. In some embodiments, R3, R4, R5and R6are eachunbranched Ce-7 alkyl. In some embodiments, R3, R4, R5and R6are each

[0117] In some embodiments, the compound has the formula lb:

[0118] In some embodiments, R1and R2are each methyl, X1is absent, Y is CH, L1and L2are each propylene, Z1and Z2are each absent, and the compound is of formula lb:Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WOor a pharmaceutically acceptable salt thereof, and X2, R3, R4, R5, and R6are as defined for formula I. In some embodiments, X2is C2-3 alkylene. In some embodiments, R3and R4areeach Ce-7 alkyl. In some embodiments, R3and R4are eachIn some embodiments, R5and R6are each Ce-7 alkyl. In some embodiments, R5and R6are each. In some embodiments, R3, R4, R5and R6are each unbranched Ce-7 alkyl. Insome embodiments, R3, R4, R5and R6are each

[0119] In some embodiments, the compound is selected from the group consisting of the compounds in Table 1 A and Table IB, or pharmaceutically acceptable salts thereof. In some embodiments, the compound is selected from the group consisting of the compounds in Table 1 A, or pharmaceutically acceptable salts thereof. In some embodiments, the compound is selected from the group consisting of the compounds in Table IB, or pharmaceutically acceptable salts thereof.Applicant Docket No.: LUNAR0011 WOMintz Docket No.: 049386-550F01 WOApplicant Docket No.: LUNAR0011 WOMintz Docket No.: 049386-550F01 WOTable IB:Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WOL8 / — / 0 / X' / ' 0 ' \,0 N— ''ZO i — ’ '■ o—1s J—1-*j j / A0'L9 / — ( 0x\ p OO ' ' 0, — ' ~i uQ—' s—1'^-0LIOCj p O N—1r^\, / J o / — 'NAQ—'V"0

[0120] In some embodiments, the present disclosure provides a lipid composition comprising a nucleic acid and a compound of the present disclosure. In some embodiments, the nucleic acid is RNA or DNA. In some embodiments, the nucleic acid is selected from anApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 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 self-replicating RNA. In some embodiments, the nucleic acid is a DNA plasmid. In some embodiments, the nucleic acid is an antisense oligonucleotide.

[0121] In some embodiments, the nucleic acid is an mRNA or a self-replicating RNA comprising a coding region that encodes a therapeutic protein of interest. In some embodiments, the nucleic acid is an mRNA comprising a coding region that encodes a therapeutic protein of interest. In some embodiments, the nucleic acid is a self-replicating RNA comprising a coding region that encodes a therapeutic protein of interest. In some embodiments, the therapeutic protein of interest is an enzyme, an antibody, an antigen, a receptor, or a transporter. In some embodiments, the therapeutic protein of interest is an enzyme. In some embodiments, the therapeutic protein of interest is an antibody. In some embodiments, the therapeutic protein of interest is an antigen. In some embodiments, the therapeutic protein of interest is a receptor. In some embodiments, the therapeutic protein of interest is a transporter. In some embodiments, the therapeutic protein of interest is a geneediting enzyme. In some embodiments, the gene-editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease. In some embodiments, the gene-editing enzyme is a TALEN. In some embodiments, the gene-editing enzyme is a CRISPR. In some embodiments, the gene-editing enzyme is a meganuclease. In some embodiments, the geneediting enzyme is a zinc finger nuclease.

[0122] In some embodiments, the lipid composition comprises liposomes, lipoplexes, or lipid nanoparticles. In some embodiments, the lipid composition comprises liposomes. In some embodiments, the lipid composition comprises lipoplexes. In some embodiments, the lipid composition comprises lipid nanoparticles.IV. LIPID FORMULATIONS AND NANOPARTICLESLipid-Based Formulations

[0123] 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 exogenousApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 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 acid-based 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 acid-based 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.

[0124] 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.

[0125] 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 CO VID-19 pandemic with several vaccines delivering mRNA encoding the spike protein of CO VID-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.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0126] 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

[0127] 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 colloidal dispersion 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.

[0128] 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.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0129] 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.Cationic Liposomes

[0130] 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 for in vivo clinical applications. Cationic lipids suitable for use in cationic liposomes are listed herein below.Lipid Nanoparticles

[0131] 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. LipidApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 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.

[0132] 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 i.v. 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.

[0133] 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 theLIPOFECT AMINE® 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 toxi cities, as well as immune system activation.

[0134] 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. In some embodiments, the plurality of ligands self-assembles to form the lipid nanoparticle comprising an interior and exterior.

[0135] In some embodiments, the average size of the lipid nanoparticle is about 100 nm. In some embodiments, the average 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 aboutApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 100 nm. In some embodiments, the average particle size of the lipid nanoparticle is about 50 nm to about 100 nm. In some embodiments, the average particle size of the lipid nanoparticle is about 65 nm to about 100 nm. In some embodiments, the average particle size of the lipid nanoparticle is about 50 nm to about 90 nm. In some embodiments, the average particle size of the lipid nanoparticle is about 55 nm to about 85 nm. In some embodiments, the average particle size of the lipid nanoparticle is about 65 nm to about 75 nm.

[0136] In some embodiments, the lipid nanoparticle further comprises a nucleic acid encapsulated in the interior. 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-repli eating RNA, a DNA plasmid, and an antisense oligonucleotide. In some embodiments, the nucleic acid is an mRNA or a self-replicating RNA comprising a coding region that encodes a therapeutic protein of interest. 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 embodiments, the nucleic acid is an mRNA comprising a coding region that encodes a therapeutic protein of interest. In some embodiments, the nucleic acid is a self-replicating RNA comprising a coding region that encodes a therapeutic protein of interest. In some embodiments, the therapeutic protein of interest is an enzyme, an antibody, an antigen, a receptor, or a transporter. In some embodiments, the therapeutic protein of interest is an enzyme. In some embodiments, the therapeutic protein of interest is an antibody. In some embodiments, the therapeutic protein of interest is an antigen. In some embodiments, the therapeutic protein of interest is a receptor. In some embodiments, the therapeutic protein of interest is a transporter. In some embodiments, the therapeutic protein of interest is a gene-editing enzyme. In some embodiments, the gene-editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease. In some embodiments, the gene-editing enzyme is a TALEN. In some embodiments, the gene-editing enzyme is a CRISPR. In some embodiments, the gene-editing enzyme is a meganuclease. In some embodiments, the geneediting enzyme is a zinc finger nuclease.

[0137] In some embodiments, the lipid nanoparticle further comprises a helper lipid selected from: dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), di stearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is DOPE. In some embodiments, the helper lipid is DMPC. InApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO some embodiments, the helper lipid is DSPC. In some embodiments, the helper lipid is DMPG. In some embodiments, the helper lipid is DPPC. In some embodiments, the helper lipid is PC.

[0138] In some embodiments, the lipid nanoparticle further comprises cholesterol.

[0139] In some embodiments, the lipid nanoparticle further comprises a polyethylene glycol(PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG.

[0140] In some embodiments, the lipid nanoparticle comprises about 45 mol% to 65 mol% of a compound of the present disclosure, about 2 mol% to about 15 mol% of a helper lipid, about 20 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate.

[0141] In some embodiments, the lipid nanoparticle comprises about 50 mol% to about 61 mol% of a compound of the present disclosure, about 5 mol% to about 9 mol% of the helper lipid, about 29 mol% to about 38 mol% of cholesterol, and about 1 mol% to about 2 mol% of the PEG-lipid conjugate.

[0142] In some embodiments, the lipid nanoparticle comprises about 56 mol% to about 58 mol% of a compound of the present disclosure, about 6 mol% to about 8 mol% of DSPC, about 31 mol% to about 34 mol% of cholesterol, and about 1.25 mol% to about 1.75 mol% of the PEG-lipid conjugate.

[0143] In some embodiments, the 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.

[0144] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure or a lipid nanoparticle of the present disclosure, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is a lyophilized composition. 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. In some embodiments, the pharmaceutical composition further comprises about 2.0 mg / mL to about 4.0 mg / mL of NaCl. In some embodiments, the pharmaceutical composition furtherApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 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 one or more cryoprotectants is sucrose. In some embodiments, the one or more cryoprotectants is glycerol. In some embodiments, the one or more cryoprotectants is a combination of sucrose and glycerol. 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 / mLLipid-Nucleic Acid Formulations

[0145] A nucleic acid or a pharmaceutically acceptable salt thereof can be incorporated into a lipid formulation (i.e., a lipid-based delivery vehicle). The terms “lipid formulation” and “lipid composition” are used interchangeably in the present disclosure.

[0146] In the context of the present disclosure, a lipid-based delivery vehicle typically serves to transport a desired nucleic acid (RNA or DNA, e.g., siRNA, plasmid DNA, mRNA, self-replicating RNA, etc.) to a target cell or tissue. The lipid-based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid-based delivery vehicle is a liposome, a cationic liposome, or a lipid nanoparticle containing a nucleic acid as described herein. In some embodiments, the lipid-based delivery vehicle comprises a nanoparticle or a bilayer of lipid molecules and a nucleic acid. In some embodiments, the lipid bilayer further comprises a neutral lipid or a polymer. In some embodiments, the lipid formulation (e.g. the lipid nanoparticle) comprises a liquid medium. In some embodiments, the lipid formulation encapsulates the nucleic acid. For example, the lipid nanoparticle encapsulates a nucleic acid. In some embodiments, the lipid formulation further comprises a nucleic acid and a neutral lipid or a polymer.

[0147] The description provides lipid formulations comprising one or more therapeutic nucleic acid molecules encapsulated within the lipid formulation. In some embodiments, the lipid formulation comprises liposomes. In some embodiments, the lipid formulation comprises cationic liposomes. In some embodiments, the lipid formulation comprises lipid nanoparticles. In some embodiments, the lipid nanoparticle encapsulates the nucleic acid.

[0148] In some embodiments, the nucleic acid is fully encapsulated within the lipid portion of the lipid formulation. In this case, the nucleic acid in the lipid formulation is resistant inApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO aqueous solution to nuclease degradation. In other embodiments, the lipid formulations described herein are substantially non-toxic to mammals such as humans.

[0149] The lipid formulations of the disclosure also typically have a total lipid: nucleic acid ratio (mass / mass ratio) of from about 1: 1 to about 100: 1, from about 1: 1 to about 50:1, from about 2:1 to about 45:1, from about 3:1 to about 40:1, from about 5:1 to about 38:1, or from about 6: 1 to about 40: 1, or from about 7: 1 to about 35: 1, or from about 8: 1 to about 30:1; or from about 10:1 to about 25:1; or from about 8:1 to about 12:1; or from about 13:1 to about 17:1; or from about 18:1 to about 24:1; or from about 20:1 to about 30:1. In some preferred embodiments, the total lipid: nucleic acid ratio (mass / mass ratio) is from about 10:1 to about 25:1. The ratio may be any value or subvalue within the recited ranges, including endpoints.

[0150] The lipid formulations of the present disclosure typically have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm, and are substantially non-toxic. The diameter may be any value or subvalue within the recited ranges, including endpoints. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease.

[0151] In some embodiments, the lipid formulations (e.g. lipid nanoparticle) comprise a nucleic acid, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugate and / or other lipid conjugate of the disclosure). The lipid formulations can also include cholesterol.

[0152] In some embodiments, the lipid nanoparticle further comprises a PEG-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG. In embodiments, PEG2000-DMG is the addition of polyethylene glycol to myristoyl diglyceride.

[0153] In the lipid-nucleic acid formulations, the nucleic acid may be encapsulated or complexed within the lipid portion of the formulation, thereby protecting the nucleic acidApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 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.

[0154] 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.

[0155] In other embodiments, the present disclosure provides a nucleic acid-lipid composition comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of nucleic acid-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of nucleic acid-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of nucleic acid-lipid nanoparticles.

[0156] In some embodiments, the lipid formulations (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%Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 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 or range therein) of the particles have the nucleic acid encapsulated therein. The amount may be any value or subvalue within the recited ranges, including endpoints.

[0157] 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.

[0158] 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.

[0159] According to some embodiments, expressible polynucleotides, nucleic acid active agents, and mRNA constructs can be lipid formulated. The lipid formulation is preferably selected from, but not limited to, liposomes, cationic liposomes, and lipid nanoparticles. In one preferred embodiment, a lipid formulation is a cationic liposome or a lipid nanoparticle (LNP) comprising:(a) a nucleic acid (mRNA, siRNA, etc.),(b) a lipid of the present disclosure, which may be cationic(c) optionally a non-cationic lipid (such as a neutral lipid), and (d) optionally, a sterol.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO Cationic Lipids

[0160] The lipid formulation preferably 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- trimethylammonium-propane) 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.

[0161] 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 l,2-Dioleyloxy-3-trimethyl aminopropane chloride salt), N-(l-(2,3-dioleyloxy)propyl)-N, N, N-trimethyl ammonium chloride (DOTMA), N, N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), l,2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N, N-dimethylaminopropane (y-DLenDMA), 1,2-Dilinoleylcarbamoyl oxy-3 -dimethylaminopropane (DLin-C-DAP), 1,2-Dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1, 2-Dilinoley oxy-3 -morpholinopropane (DLin-MA), l,2-Dilinoleoyl-3 -dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3 -dimethylaminopropane (DLin-S-DMA), l-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA. Cl), l,2-Dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP. C1), l,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-Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), l,l'-(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]-di oxolane (DLin-KC2-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-l-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 carboxy spermine (DOGS), l,2-dioleoyl-sn-3 -phosphoethanolamine (DOPE), l,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).

[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 Ci4 to C22 may 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.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[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] In some embodiments, the lipid formulation comprises a lipid of Formula I, Formula la, or Formula lb as described herein.Helper Lipids and Sterols

[0166] The lipid formulations (e.g. lipid nanoparticle) of the present disclosure can comprise a helper lipid, which can be referred to as a neutral lipid, a neutral helper lipid, noncationic lipid, non-cationic helper lipid, anionic lipid, anionic helper lipid, or a zwitterionic lipid. It has been found that 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-Ethanoalamine (DOPE) and l,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.

[0167] Non-limiting examples of non-cationic 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,Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), di oleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, 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.

[0168] 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).

[0169] Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof. One study concluded that as a helper lipid, 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, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof. In preferred embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.

[0170] 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 ofApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO cholesterol or a derivative thereof, e.g., a phospholipid-free lipid formulation. In some embodiments, the lipid nanoparticle further comprises cholesterol.

[0171] 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, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.

[0172] In some embodiments, the helper lipid comprises from about 1 mol% to about 50 mol%, from about 5 mol% to about 48 mol%, from about 5 mol% to about 46 mol%, about 25 mol% to about 44 mol%, from about 26 mol% to about 42 mol%, from about 27 mol% to about 41 mol%, from about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation. In some embodiments, the helper lipid comprises from about 1 mol% to about 20 mol%, about 2 mol% to about 12mol%, about 5 mol% to about 9 mol% or about 6 mol% to about 8 mol%.

[0173] In some embodiments, the total of helper lipid in the formulation comprises two or more helper lipids and the total amount of helper lipid comprises from about 20 mol% to about 50 mol%, from about 22 mol% to about 48 mol%, from about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, from about 26 mol% to about 42 mol%, from about 27 mol% to about 41 mol%, from about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation. In some embodiments, the helper lipids are a combination of DSPC and DOTAP. In some embodiments, the helper lipids are a combination of DSPC and DOTMA.

[0174] The cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 30 mol% to about 40 mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, or about 40 mol% of the total lipid present in the lipid formulation.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0175] 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, for example, by ± 5 mol%.Mechanism of Action for Cellular Uptake of Lipid Formulations

[0176] 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.

[0177] 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, temperature, 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.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO Lipid Formulation Manufacture

[0178] There are many different methods for the preparation of lipid formulations comprising a nucleic acid. (Curr. DrugMetabol. 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

[0179] 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 (ML Vs) 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

[0180] 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).Reverse Phase Evaporation

[0181] 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.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO Microfluidic Preparation

[0182] 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

[0183] 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

[0184] 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.Detergent Dialysis

[0185] 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. UnencapsulatedApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 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

[0186] 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.V. PHARMACEUTICAL COMPOSITIONS AND DELIVERY METHODS

[0187] To facilitate nucleic acid activity (e.g., mRNA expression, or knockdown by an ASO or siRNA) in vivo, the lipid formulation delivery vehicles described herein can be combined with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

[0188] The lipid formulations and pharmaceutical compositions of the present disclosure may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein (e.g., enzyme) production.

[0189] The pharmaceutical compositions disclosed herein can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit a sustainedApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO or delayed release (e.g., from a depot formulation of the nucleic acid); (4) alter the biodistribution (e.g., target the nucleic acid to specific tissues or cell types); (5) increase the activity of the nucleic acid or a protein expressed therefrom in vivo; and / or (6) alter the release profile of the nucleic acid or an encoded protein in vivo.

[0190] Preferably, 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).

[0191] Pharmaceutical compositions 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 not limited to the lung, trachea, and / or nasal passages, muscle, liver, eye, or the central nervous system.

[0192] 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 with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.

[0193] Pharmaceutical compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, 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, and the like, as suited to the particular dosage form desired.

[0194] 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, coreshell 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.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0195] Accordingly, 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.

[0196] Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the embodiments of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

[0197] 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. In some embodiments, the pharmaceutical composition comprises a nucleic acid lipid formulation that has been lyophilized.

[0198] 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 someApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO embodiments, a lyophilized nucleic acid-lipid nanoparticle formulation can be resuspended in a buffer as described herein.

[0199] 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.

[0200] 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), adsorption inhibitors (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.

[0201] 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 poly glycerin 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 aApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 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.

[0202] 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.

[0203] According to the present disclosure, a therapeutically effective dose of the provided composition, when administered regularly, results 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.

[0204] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the compounds described herein, or the lipid nanoparticle described herein, and a pharmaceutically acceptable excipient.

[0205] In some embodiments, the present disclosure provides a method of delivering a nucleic acid to a subject in needed thereof, comprising encapsulating a therapeutically effective amount of the a nucleic acid in the lipid nanoparticle described herein, and administering the lipid nanoparticle to the subject.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0206] In some embodiments, the present disclosure provides a method of delivering mRNA to a subject in needed thereof, comprising encapsulating a therapeutically effective amount of the mRNA in the lipid nanoparticle described herein, and administering the lipid nanoparticle to the subject.VI. METHOD OF TREATMENT

[0207] In some embodiments, the present disclosure provides a method of treating a disease in a subject in need thereof, comprising administering a therapeutically effective amount to the subject of the compound described herein, the lipid nanoparticle described herein, or the lipid composition described herein. In some embodiments, the compound, composition, or lipid nanoparticle is administered intravenously or intramuscularly. In some embodiments, the compound, composition, or lipid nanoparticle is administered intravenously. In some embodiments, the compound, composition, or lipid nanoparticle is administered intramuscularly.

[0208] In some embodiments, a method of treating a disease in a subject in need thereof is provided comprising administering to the subject a lipid nanoparticle described herein. In some embodiments, the lipid nanoparticle is administered intravenously or intramuscularly. In some embodiments, the lipid nanoparticle is administered intravenously. In some embodiments, the lipid nanoparticle is administered intramuscularly.

[0209] In some embodiments, a method of treating a disease in a subject in need thereof is provided comprising administering to the subject a lipid composition described herein. In some embodiments, the lipid composition is administered intravenously or intramuscularly. In some embodiments, the lipid composition is administered intravenously. In some embodiments, the lipid composition is administered intramuscularly.

[0210] In some embodiments, there are provided a methods of treating a disease or disorder in a mammalian subject. A therapeutically effective amount of a composition comprising a lipid, as disclosed herein, specifically a cationic lipid, a nucleic, an amphiphile, a phospholipid, cholesterol, and a PEG-linked cholesterol 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 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,Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO muscle dystrophies, immune disorder, cancer, renal disease, fibrotic disease, genetic abnormality, inflammation, and cardiovascular disorder.

[0211] In some embodiments, the present disclosure provides a method of delivering a nucleic acid to a subject in needed thereof, comprising encapsulating a therapeutically effective amount of a nucleic acid in a lipid nanoparticle as described herein, and administering the lipid nanoparticle to the subject.

[0212] In some embodiments, 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 dislcosure, or the pharmaceutical composition of the present disclosure. In some embodiments, the protein or polypeptide is an antigen, and expression of the antigen elicits an in vivo immunogenic response.VII. EXAMPLES

[0213] Starting materials and other reagents were purchased from commercial suppliers and were used without further purification unless otherwise indicated. The reactions were assayed by high-performance liquid chromatography (HPLC) and terminated as judged by ithe consumption of starting material. H NMR spectra were recorded on Varian or Bruker iinstruments operating at the field strength indicated. H NMR spectra are obtained as DMSO-d6 or CDC13 solutions as indicated (reported in ppm), using TMS as the reference. Mass spectra were obtained using liquid chromatography mass spectrometry (LC-MS) on a Schimadzu instrument using atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). Mass spectra were also measured by direct injection on a Perkin Elmer PE-SCIEX API- 150 instrument or Agilent-TRAP XCT instrument using electrospray (ESI) ionization.Synthesis of IntermediatesExample A. Synthesis of 2-((3-(dimethylamino)propyl)disulfaneyl)ethan-l-ol (1)

[0214] Step 1: N,N-dimethyl-3-(pyridin-2-yldisulfaneyl)propan-1-amine (la). To a 3 L three-neck round-bottle flask under N2 was added l,2-di(pyridin-2-yl)disulfane (230.0 g, 1.0 equiv) in MeOH (920 mL, 4 V) at room temperature. The reaction mixture was cooled to 0 °C in an ice / water bath. A solution of 3-(dimethylamino)propane-l-thiol (125.0 g, 1.0 equiv) in MeOH (115 mL, 0.5 V) was added dropwise at 0 °C. The resulting solution was warmed to room temperature and stirred overnight. The mixture was diluted with DCM (2 L) andApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO washed with 10% aqueous NaOH (1 L) followed by water (4 x 500 mL). The organic layer was dried and concentrated at room temperature. The residue was adsorbed on 350 g silica gel and purified on a 1.2 Kg silica gel column by eluting first with PE / EA (gradient from 100:0 to 70:30) followed by MeOH / DCM (gradient from 100:0 to 90:10). Fractions containing pure compound was pooled, evaporated and dried under vacuum over P2O5 to get 137 g (57%) of A, A-dimethyl-3-(pyridin-2-yldisulfaneyl)propan-l -amine (la). LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 1.20 min., hold 0.6 min): RT 0.63 min, m / e = 229.05 (M+H+). 'H NMR (400 MHz, CDCI3) d 8.4s (s, 1H), 7.73 (s, 1H), 7.65 (s, 1H), 7.08 (s, 1H), 2.83 (t, J = 7.2 Hz, 2H), 2.34 (t, J= 7.2 Hz, 2H), 2.21 (s, 6H), 1.86 (m, 2H).

[0215] Step 2: 3-((2-((tert-butyldimethylsilyl)oxy)ethyl)disulfaneyl)-N, N-dimethylpropan-l-amine. To a 5 L three-neck round-bottle flask was added N,N-dimethyl-3-(pyridin-2-yldisulfaneyl)propan-1-amine (la) (137.0 g, 1.0 equiv) in pyridine (68.5 mL, 0.5 V) and MeOH (2.74 L, 20 V) at room temperature followed by 2-((tert-butyldimethylsilyl)oxy)ethane-l -thiol (288.4 g, 2.5 equiv). The resulting solution was stirred for 15 h at room temperature. The mixture was diluted with DCM (5 L, 36.5 V) and washed with 10% aqueous NaOH (2 L, 14.6 V), 10% aqueous citric acid (2 L, 14.6 V) and H2O (1 L, 7.3 V). The residue was purified on a 1.2 Kg silica gel column by eluting with DCM / MeOH (gradient from 100:0 to 90:10). Fractions containing pure compound were pooled, evaporated and dried under vacuum over P2O5 to get 67 g (36% yield) 3-((2-((tert-butyldimethylsilyl)oxy)ethyl)disulfaneyl)-N, N-dimethylpropan-l-amine as a white solid. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 1.20 min., hold 0.6 min): RT 0.80 min, m / e = 310.10 (M+H+). 'HNMR (400 MHz, CDC13) d 3.80 (t, J = 6.4 Hz, 2H), 3.09 (m, 2H), 2.79 (s, 6H), 2.77 (m, 4H), 2.26 (m, 2H), 0.85 (s, 9H), 0.03 (t, 6H).

[0216] Step 3. 2-((3-(dimethylamino)propyl)disulfaneyl)ethan-l-ol (1). To a 5 L threenecked round-bottle flask was added 3-((2-((tert-butyldimethylsilyl)oxy)ethyl)disulfaneyl)-N, N-dimethylpropan-l -amine (67.0 g, 1.0 equiv) in ACN (800 mL, 12 V) and H2O (1.2 L, 18 V) at room temperature. The pH of the mixture was adjusted to 2.5-3.5 with TFA. Most of the ACN and part of water was removed by evaporation over 3 h to about 13 V. The TBS was removed in this evaporation. The water phase was extracted with MTBE (2 x 500 mL, 15 V) to remove the impurities. The pH value of water phase was adjusted to 8-9 with NaHCO3solid. The water phase was extracted with DCM (15 x 1 L, 224 V). The combined the organicApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO phase was washed with water (200 mL, 3 V). The organic layer was dried and concentrated at room temperature to result in 34 g (80% yield) 2-((3-(dimethylamino)propyl)disulfaneyl)ethan-l-ol (1) as an oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 1.20 min., hold 0.6 min): RT 0.39 min, m / e = 196.10 (M+H+). 'H NMR (400 MHz, CDC13) d 3.87 (t, J = 6.4 Hz, 2H), 2.88 (t, J = 7.6 Hz, 2H), 2.78 (t, J = 9.6 Hz, 2H), 2.43 (t, J = 9.2 Hz, 2H), 2.24 (s, 6H), 1.9 (m, 2H).Example B. Synthesis of bis(l,5-dicyclohexylpentan-3-yl) 4„4,-((tert-butoxycarbonvDazanedivDdibutyrate (SM-C1).

[0217] Into a 500 mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was charged a solution of 4,4'-((tert-butoxycarbonyl)azanediyl)dibutyric acid (7.8 g, 27.0 mmol) in DCM (117 mL). To the resulting mixture cooled to 0 °C was added l,5-dicyclohexylpentan-3-ol (13.7 g, 54.3 mmol) followed by EDC1 (20.7 g, 0.108 mol) and DMAP (3.3 g, 27.1 mmol) and stirred at 20 °C for 16 h. The crude mixture was adsorbed ontol5.6 g of silica gel and purified on 156 g of silica gel using heptane / acetone (gradient from 100: 0 to 90: 10). After TLC analysis (heptane / acetone =6: 1) qualified fractions were pooled, combined and evaporated under reduced pressure to obtain 17 g (22.4 mmol, 82.9 %) SM-C1 as an oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 2.0 min., hold 0.5 min): RT 0.85 min, m / e = 658.60 (M-Boc). This material was used without further characterization.Example C. Synthesis of 2-((2-(dimethylamino)ethyl)disulfaneyl)ethan-l-ol (2).

[0218] Step 1: N,N-dimethyl-2-(pyridin-2-yldisulfaneyl)ethan-1-amine (2a). To a 3 L three-neck round-bottle flask under N2 was added l,2-di(pyridin-2-yl)disulfane (40.0 g, 0.18 mmo, 1.0 equiv) in MeOH (160 mL, 4 V) at room temperature. The reaction mixture was cooled to 0 °C in an ice / water bath. A solution of 2-(dimethylamino)ethane-l -thiol (19.1 g, 0.18 mmol, 1.0 equiv) in MeOH (20 mL, 0.5 V) was added dropwise at 0 °C. Resulting solution was warmed to room temperature and stirred overnight. The mixture was diluted with DCM (400 mL) and washed with 10% aqueous NaOH (200 mL) followed by water (4 x 200 mL). The organic layer was dried and concentrated at room temperature. The residue was adsorbed on 80 g silica gel and purified on a 800 g silica gel column by eluting first with PE / EA (gradient from 100:0 to 70:30) followed by MeOH / DCM (gradient from 100:0 toApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 90: 10). Fractions containing pure compound was pooled, evaporated and dried under vacuum over P2O5 to get 20 g (51%) of A, A-dimethyl-2-(pyridin-2-yldisulfaneyl)ethan-l -amine as an oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 2.00 min., hold 1.2 min): RT 0.64 min, m / e = 215.10 (M+H+).

[0219] Step 2: 2-((2-(dimethylamino)ethyl)disulfaneyl)ethan-l-ol (2). To a 5 L three-neck round-bottle flask was added A, A-dimethyl-2-(pyri din-2 -yldisulfaneyl)ethan-l -amine (2a) (40.0 g, 187 mmol, 1.5 equiv) in methanol (800 mL, 20V) was added pyridine (20 mL, 0.5 V) at room temperature followed by 2-((tert-butyldimethylsilyl)oxy)ethane-l -thiol (23.9 g, 124 mmol, 1.0 equiv). The resulting solution was stirred for 15 h at room temperature. The reaction was then quenched by the addition of 10% aqueous NaOH solution (400 ml, 10 V). The aqueous layer was extracted with MTBE (2 x 400 ml, 10 V). The organic layer was washed with 10% aqueous citric acid (2 x 400 ml, 10 V) and collected the organic layer. The pH value of the aqueous layer was adjusted to 8 with NaOH (1 mol / L, 800 ml). The aqueous layer was extracted with MTBE (2 x 400 ml, 10 V). All the combined organic layers were dried over anhydrous sodium sulfate and concentrated at room temperature. The residue was adsorbed on 80 g silica gel and purified on 800 g silica gel column by eluting with PE / EA (gradient from 100: 0 to 50: 50). Fractions containing pure compound were pooled, evaporated, and dried under vacuum over P2O5 to get 6 g (33.1 mmol, 30% yield) 2-((2-(dimethylamino)ethyl)disulfaneyl)ethan-l-ol (2) as light-yellow oil. 'H NMR (400 MHz, Chloroform-d,82.900- 2.769 (m, 2H), 2.622 (dd, J= 8.4, 6.1 Hz, 2H), 2.281 (s, 4H).Example D. Synthesis of 2-((3-(dimethylamino)propyl)disulfaneyl)ethane-l-thiol (3).

[0220] To a 500 mL three-necked round-bottom flask was added ethane- 1,2-dithiol (2.44 g, 0.026 mol, 1.0 equiv) in THF (90 mL, 15 V). A solution of 7V, A-dimethyl-3-(pyridin-2-yldisulfaneyl)propan-l -amine (la) (6 g, 0.026 mol, 1.0 equiv) in tetrahydrofuran (90 mL, 15V) was added to the reactor and stirred for 0.5 hour at room temperature. Solvent was removed under reduced pressure and the crude residue was purified reverse phase silica gel column on a combi-flash system (type: C18, ACN: H2O with 0.5% TFA, 5%-35%, 30 min). The fraction was concentrated below 20 °C to remove most of the solvent and freeze-dried. 2-((3-(dimethylamino)propyl)disulfaneyl)ethane-l-thiol (3) was obtained as its trifluoroacetate (2.5 g, 31%) as a light-yellow oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 2.00 min., hold 0.7 min): RT 0.8 min, m / e = 212.05 (M+H+). This material was immediately used as such in subsequent reaction without further purification.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WOGeneral Scheme 1: Synthesis of LI, L2, L3, L4, and L5:SM-A1: R-, = C7H15SM-A2: R.| = C7H15SM-B1: R-, = C6H13SM-B2: R-, = C6H13SM-C1: R-i = -ethylcyclohexyl SM-C3: R-i = -ethylcyclohexyl(L1): R1= C7H15; X = O; n = 2 (L2) R-i = C6H13; X = O; n = 2Int-C: Ri = -ethylcyclohexyl (L3) RT = C7H15; X = O; n = 1(L4) R-, = ethylcyclohexyl; X = O; n = 2(L5) R-, = C7H15; X = S; n = 2Example 1. Synthesis of LI (as shown in General Scheme 1)

[0221] Step a. Preparation of SM-A2. Into a 3 -necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of SM-A1 (prepared as in Rajappan et al J. Med. Chem. 2020, 63 (21), 12992-13012), in EA (10 V) and cooled to 15 °C. To this solution was added 4 M HC1 in EA (5 V) and the mixture was stirred overnightApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO at room temperature and was concentrated under vacuum. The residue was adsorbed on silica gel (1.2-1.5 X by weight) and purified on a silica gel (5X by weight) column by eluting first with PE / EA (gradient from 100:0 to 70:30) followed by MeOH / DCM (gradient from 100:0 to 90:10). Fractions containing pure compound were pooled, evaporated, and dried under vacuum over P2O5 to generate SM-A2 in quantitative yield.

[0222] Di(pentadecan-8-yl) 4,4'-azanediyldibutyrate hydrochloride (SM-A2). Yield 99%, LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 1.30 min., hold 0.3 min): RT 0.83 min, m / e = 610.50 (M+H+). 'H NMR (400 MHz, CDCI3) d 4.85 (m, 2H), 3.10 (s, 4H), 2.48 (t, J= 6.4 Hz, 4H), 2.20 (m, 4H), 1.51 (m, 8H), 1.31 (m, 40H), 0.89 (t, 12H).

[0223] Step b. Preparation of Int-A. Under nitrogen atmosphere, the amine hydrochloride salt SM-A2 (1.0 equiv.) in DCM (20 V) was taken in a 3-necked round-bottom bottle and CDI (2.0 equiv.) was added followed by Et3N (4.0 equiv.) and the mixture was stirred for 4 h at room temperature. The resulting solution was washed with 3% aqueous citric acid (15 V), H2O (3 x 6V) and brine (6V). The organic phase was dried with anhydrous ISfeSCU and concentrated at 35 °C under vacuum. The residue was adsorbed on silica gel (2X by weight) and purified on a silica gel (8X by weight) column by eluting with PE / EA (volume gradient from 100:0 to 50:50). Fractions were analyzed on TLC, pooled, evaporated, and dried under vacuum over P2O5 to get pure Int-A.

[0224] Di(pentadecan-8-yl) 4,4'-((4H-imidazole-5-carbonyl)azanediyl)dibutyrate (Int-A).Yield 75%. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 3.0 min., hold 1.00 min): RT 1.15 min, m / e = 704.6 (M+H+). *HNMR (400 mHz, CDC13): 8 = 7.91 (m, 1 H), 7.25 (m, 1 H), 7.12 (m, 1 H), 4.80 (m, 2 H), 3.47 (m, 4 H), 2.33 (m, 4 H), 1.97 (m, 4 H), 1.42-1.51 (40 H), 0.85 (m, 12 H).

[0225] Step c. Preparation of LI. Under nitrogen atmosphere, 2-((3-(dimethylamino)propyl)disulfaneyl)ethan-l-ol (1) (1.5 equiv) was dissolved in THF (lOx V), cooled in an ice / water bath, and charged with NaH (60%, 1.35 equiv.). The reaction was stirred for 1 hour at room temperature. In a separate flask Int-A (1.0 equiv) was taken in THF (lOx V), cooled to ice-bath temperature and a solution of TMA in THF (2 mol / L, 0.15 equiv) was added followed by 3 equivalents of tri ethyl amine. This solution was slowly added to the above solution of 2-((3-(dimethylamino)propyl)disulfaneyl)ethan-l-ol and NaH. The ice / water bath was removed, and the reaction was stirred overnight at room temperature. TheApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO mixture was filtered, and the filter cake was washed with THF (lOx V). The filtrate was concentrated under reduced pressure and purified by combi-flash (type: C-18, IPA: H2O with 0.5% TFA, 50%-80%, 30 min). Fractions were pooled, combined, and concentrated below 20 °C. The pH value of residue was adjusted to 8 with aqueous NaHCCf and extracted with DCM (lOOx V). The combined organic layers were washed with H2O (2 x 50x V), dried with anhydrous ISfeSC and concentrated below 20 °C. This material was dissolved in heptane (300 ml, 25 V) and washed with 5% Na2CO3(10 time, 15x V each), H2O (3 times, 15x V each). The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to get LI as yellow oil.

[0226] Pentadecan-8-yl 2-m ethyl- 11 -oxo- 12-(4-oxo-4-(pentadecan-8-yloxy )butyl)-10-oxa-6,7-dithia-2,12-diazahexadecan-16-oate (LI). Yield 31%. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 3.0 min., hold 1.00 min): RT 1.91 min, m / e = 831.62 [M+H]+; 'H-NMR (400 MHz, CDC, ppm) 84.86 (p, J= 6.3 Hz, 2H), 4.32 (t, J= 6.6 Hz, 2H), 3.27 (bs, 4H), 2.91 (t, J= 6.6 Hz, 2H), 2.74 (t, J= 7.2 Hz, 2H), 2.38 (t, = 7.1 Hz, 2H), 2.27 (d, J= 18.6 Hz, 10H), 1.90-1.82 (m, 6H), 1.51 (bs, 8H), 1.27 (bs, 40H), 0.88 (t, J= 6.7 Hz, 12H). HPLC purity 97.7%.Example 2. Synthesis of L2 (as shown in General Scheme 1)NS\s

[0227] L2 was prepared using the method of Example 1, replacing SM-A1 with SM-B1 as a starting material in step a. SM-B1 was prepared as in Rajappan et al J. Med. Chem. 2020, 63 (21), 12992-13012).

[0228] Di(tridecan-7-yl) 4,4'-azanediyldibutyrate hydrochloride (SM-B2). Yield 99%, LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 2.0 min., hold 0.5 min): RT 0.84 min, m / e = 554.50 (M+H+). *HNMR (400 MHz, CDC13) d 4.8 (m, 2H), 3.09 (s, 4H), 2.49 (t, J= 6.4 Hz, 4H), 2.20 (m, 4H), 1.51 (m, 8H), 1.31 (m, 32H), 0.89 (t, 12H).Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0229] Di(tridecan-7-yl) 4,4'-((lH-imidazole-l-carbonyl)azanediyl)dibutyrate (Int-B). Yield 66%. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 3.0 min., hold 1.00 min): RT 0.89 min, m / e = 648.5 (M+H+). *HNMR (400 mHz, CDC13): 8 = 7.92 (m, 1 H), 7.25 (m, 1 H), 7.12 (m, 1 H), 4.88 (m, 2 H), 3.47 (m, 4 H), 2.33 (m, 4 H), 2.00 (m, 4 H), 1.42-1.51 (34 H), 0.90 (m, 12 H).

[0230] Tridecan-7-yl 2-methyl-l l-oxo-12-(4-oxo-4-(tridecan-7-yloxy)butyl)-10-oxa-6,7-dithia-2,12-diazah exadecan- 16-oate (L2). Yield 32%. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 2.0 min., hold 1.00 min): RT 0.93 min, m / e = 775.55 [M+H]+; ‘H-NMR (400 MHz, CDC13, ppm) 64.882 (p, J= 6.3 Hz, 2H), 4.346 (t, J= 6.6 Hz, 2H), 3.288 (d, J= 8.0 Hz, 4H), 2.936 (t, J= 6.6 Hz, 2H), 2.773 (t, J = 7.2 Hz, 2H), 2.490 (bs, 2H), 2.331-2.291 (m, 10H), 1.960-1.841 (m, 6H), 1.522 (bs, 8H), 1.287 (bs, 32H), 0.991-0.803 (m, 12H). HPLC purity 97.9%.Example 3. Synthesis of L3 (as shown in General Scheme 1)

[0231] L3 was prepared using the method of Example 1, replacing 2-((3- (dimethylamino)propyl)disulfaneyl)ethan-l-ol (1) with 2-((2- (dimethylamino)ethyl)disulfaneyl)ethan-l-ol (2) in step c.

[0232] Pentadecan-8-yl 2-methyl-10-oxo-l l-(4-oxo-4-(pentadecan-8-yloxy)butyl)-9-oxa-5,6-dithia-2, 11 -di azapentadecan- 15-oate (L3). Yield 34%. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 2.0 min., hold 1.00 min): RT 1.83 min, m / e = 817.7 [M+H]+; 'H-NMR (400 MHz, CDC13, ppm) 54.809 (p, J= 6.2 Hz, 2H), 4.327 (t, J= 6.6 Hz, 2H), 3.331 - 3.194 (m, 4H), 2.913 (t, J= 6.6 Hz, 2H), 2.741 (t, J= 7.2 Hz, 2H), 2.377 (t,.7= 7.2 Hz, 2H), 2.242 (s, 10H), 1.858 (m, 4H), 1.513 (m, 8H), 1.293 - 1.051 (m, 42H), 1.014 (m, 12H). HPLC purity 98.2%.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WOExample 4. Synthesis of L4 (as shown in General Scheme 1)

[0233] L4 was prepared using the method of Example 1, replacing SM-A1 with SM-C1 as a starting material in step a to generate crude SM-C2, which was used directly in step b without purification.

[0234] Bis(l,5-dicyclohexylpentan-3-yl) 4,4'-azanediyldibutyrate (SM-C2). LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 3.0 min., hold 1.80 min): RT 1.66 min, m / e = 658.55 (M+H+).

[0235] Bis(l,5-dicyclohexylpentan-3-yl) 4,4'-((lH-imidazole-l- carbonyl)azanediyl)dibutyrate (Int-C). Yield 89%. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 3.0 min., hold 1.00 min): RT 1.89 min, m / e = 752.6 (M+H+). 'H NMR (400 mHz, CDC13): 8 = 7.918 (s, 1H), 7.259 (d, J= 8.5 Hz, 2H), 4.808 (p, J= 6.2 Hz, 2H), 3.524 - 3.322 (m, 4H), 2.314 (t, J= 7.1 Hz, 5H), 2.028 - 1.917 (m, 4H), 1.782 - 1.604 (m, 22H), 1.582 - 1.438 (m, 8H), 1.303 - 1.024 (m, 25H), 0.852 (tdd, J= 13.1, 8.2, 3.0 Hz, 9H).

[0236] l,5-Dicyclohexylpentan-3-yl 12-(4-((l,5-dicyclohexylpentan-3-yl)oxy)-4-oxobutyl)-2-methyl-l l-oxo-10-oxa-6,7-dithia-2,12-diazahexadecan-16-oate (L4). Yield 34%. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 2.0 min., hold 1.00 min): RT 1.66 min, m / e = 879.39 [M+H]+; ‘H-NMR (400 MHz, CDC13, ppm) 64.809 (t, J= 6.2 Hz, 2H), 4.327 (t, J= 6.6 Hz, 2H), 3.326 - 3.195 (m, 4H), 2.913 (t, J = 6.6 Hz, 2H), 2.741 (t, J= 7.2 Hz, 2H), 2.377 (t, J= 7.2 Hz, 2H), 2.242 (t, 3H), 1.902 (s, 6H),Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 1.861 (pd, J= 7.4, 4.2 Hz, 6H), 1.767 - 1.592 (m, 20H), 1.516 (q, J= 7.3 Hz, 8H), 1.301 -1.044 (m, 25H), 0.927 - 0.788 (m, 8H). HPLC purity 96.2%.Example 5. Synthesis of L5 (as shown in General Scheme 1)S\s

[0237] A solution of Int-A (2 g, 2.84 mmol) in THF (20 mL, 10 V) was inlOO ml round bottom flask was cooled to 0 - 5 °C, MeOTf (650 mg, 3.98 mmol) was added, and the mixture was stirred for 1 h at 0 - 5 °C. To this mixture was added TEA (2.03 g, 19.89 mmol) and stirred for another 0.5 h at 0 - 5 °C. A THF (20 mL, 10 V) solution of 2-((3-(dimethylamino)propyl)disulfaneyl)ethane-l-thiol (3) (70% pure, 2.0 g, 4.3 mmol) was charged into the reactor and the mixture was stirred for 0.5 h at room temperature. The crude mixture was filtered through a pad of Celite™ and the filtrate was purified by combi-flash (type: C-18, IPA: H2O with 0.5% TFA, 50%~80%, 30 min). Fractions containing product were combined and concentrated under reduced pressure to remove most of the solvent. The pH of the residue was adjusted to 8 with aqueous NaHCCh and extracted with heptane (2x60 mL, 60 V). The combined organic layer was washed with H2O (30 mL, 15 V), dried with anhydrous Na2SO4 and concentrated below 20 °C. The material was adsorbed on silica gel and purified on a silica gel column (Combiflash, Teledyne ISCO) by eluting with DCM / MeOH (gradient from 100: 0 to 97.5: 2.5). Fractions were analyzed, pooled, and concentrated below 20 °C and dried under vacuum to get 750 mg of L5 as light-yellow oil.

[0238] Pentadecan-8-yl 2-methyl-l l-oxo-12-(4-oxo-4-(pentadecan-8-yloxy)butyl)-6,7, 10-trithia-2,12-diazahexadecan- 16-oate (L5). Yield: 31%. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B at 2.0 min., hold 1.00 min): RT 1.06 min, m / e = 847.6 [M+H]+; 'HNMR (400 MHz, Chloroform-d, ppm)'. 84.868 (p, J= 6.2 Hz, 2H), 3.375 (s, 4H), 3.281 - 3.161 (m, 2H), 2.923 -2.835 (m, 2H), 2.754 (t, J= 7.2Hz,Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO 2H), 2.470 (d, J= 8.3 Hz, 2H), 2.303 (s, 10H), 1.921 (dt, J= 14.4, 6.8 Hz, 6H), 1.511 (d, J = 6.2 Hz, 8H), 1.422 - 1.158 (m, 40H), 0.951 - 0.762 (m, 12H).Example 6. Synthesis of L6

[0239] Step a. Di(pentadecan-8-yl) 5-oxononanedioate (5). Into a 250 mL round-bottom flask purged and maintained under inert atmosphere of nitrogen was added 5-oxoazelaic acid (10.0 g, 49.5 mmol) in DCM (150 mL, 15 V). This was followed by the addition of pentadecan-8-01 (24.9 g, 0.11 mol) and DMAP (6.0 g, 49.5 mmol). The temperature was reduced to 0 °C in an ice / water batch and EDC. HC1 (20.8 g, 0.11 mol) was added. The resulting solution was stirred for 16 h at room temperature. The reaction was then quenched by the addition of aqueous HC1 (1 mol / L, 150 mL, 15 V) and was extracted with DCM (2 x 150 mL, 30 V). Organic layers were combined and washed with brine (2 x 150 mL, 30 V). The crude product was adsorbedApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO on 60g silica gel and purified on a 240 g silica gel column with PEZEA gradient (from 100 / 0 to 40 / 60). Fractions were pooled and evaporated under reduced pressure to obtain 15.4 g (50% yield) of 5 as yellow oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA 80:20 to 20:80 A / B at 3.0 min., hold 1.75 min): RT 2.42 min, m / e = 623.6 [M+H]+; 'H NMR (400 MHz, Chloroform-d, ppni 54.860 (m, 2H), 2.467 (m, 4H), 2.306 (m, 4H), 1.888 (m, 4H), 1.514 (m, 8H), 1.485 (m, 40H), 0.951 - 0.762 (m, 12H).

[0240] Step b. Di(pentadecan-8-yl) 5-hydroxynonanedioate (6). Into a 250 mL roundbottom flask under inert atmosphere of nitrogen was taken 5 (8.0 g, 12.8 mmol) in THF / H2O (10:1, 80 mL, 10 V). The temperature was reduced to 0 °C NaBH4 (0.97 g, 25.6 mmol) was added, and the resulting solution was stirred for 4 h at room temperature. The reaction was then quenched by the addition of water / ice (160 mL, 20 V) and the resulting solution was extracted with ethyl acetate (3 x 160 mL, 60 V) and the combined organic solution was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to get 7.4 g (92%) of crude 6 as light-yellow oil which was used as such in the next reaction. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B CH3CN / 0.05% TFA 80:20 to 20:80 A / B at 3.0 min., hold 1.75 min): RT 2.18 min, m / e = 647.6 [M+Na]+. *HNMR (400 MHz, Chloroform-d, ppni) 84.870 (m, 2H), 3.595 (m, 1H), 2.321 (m, 4H), 1.766 (m, 6H), 1.727 (m, 12H), 1.703 (m, 39H), 1.403 (m, 12H).

[0241] Step c. Di(pentadecan-8-yl) 5-((methylsulfonyl)oxy)nonanedioate (7). To a 250 mL round-bottom flask with mechanical agitation under N2, Compound 6 (7.4 g, 11.8 mmol) was taken in DCM (74 mL, 10 V) cooled to 0 °C in an ice / water bath. This was followed by the addition of TEA (2.4 g, 23.8 mmol) and MsCl (2.7 g, 23.6 mmol) dropwise. The resulting solution was stirred for 5 h at room temperature. The reaction was then quenched by the addition of H2O (150 mL, 20 V), the phases were separated, and the aqueous layer was extracted with DCM (150 mL, 20 V). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to get 6.7 g (81% yield) of 7 as yellow oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B CH3CN / 0.05% TFA 80:20 to 20:80 A / B at 3.0 min., hold 1.75 min): RT 2.06 min, m / e = 725.6 [M+Na]+. This material was used as such in the next reaction.

[0242] Step d. Di(pentadecan-8-yl) 5-mercaptononanedioate (8). Into a 100 mL roundbottom flask containing DMF (60 mL, 10V) and kept at ice-bath temperature under inert atmosphere was added 7 (6.0 g, 9.36 mmol) followed NaHS (2.4 g, 46.8 mmol). The resulting solution was stirred for 5 h at room temperature and then quenched by the addition of water / ice (250 mL, 41.7 V). The resulting mixture was extracted with ethyl acetate (3 x 100 mL, 50 V)Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO and the organic layers combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to get 3.3 g (60%) of 8 as light-yellow oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B CH3CN / 0.05% TFA 80:20 to 0:100 A / B at 3.0 min., hold 3 min): RT 2.31 min, m / e = 663.6 [M+Na]+

[0243] Step e. Di(pentadecan-8-yl) 5-((3-(dimethylamino) propyl)disulfaneyl) nonanedioate (L6). To a 500 mL round-bottom flask was added 8 (3.0 g, 46.8 mmol) in pyridine (1.5 mL, 0.5 V) and MeOH (60 mL, 20 V) at room temperature. To the mixture was added A, A-dimethyl-3-(pyridin-2-yldisulfaneyl)propan-l-amine (la) (2.7 g, 11.7 mmol) stirred for 15 h at room temperature. The mixture was diluted with DCM (109 mL, 36.5 V) and washed with 10% aqueous NaOH (43.8 mL, 14.6 V), 10% aqueous citric acid (43.8 mL, 14.6 V) and H2O (21.9 mL, 7.3 V). The mixture was purified by pre-HPLC (Column: C18 Column; Mobile Phase A: Water (10 mmol / L TFA), Mobile Phase B: IP A; Flow rate: 100 mL / min; Gradient: 60% B to 90% B in 30 min, 73% B). Fractions were pooled and concentrated under reduced pressure to obtain 1.1 g (32%) L6 as a yellow oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B CH3CN / 0.05% TFA 100:0 to 0:100 A / B at 2.0 min., hold 1.20 min): RT 1.68 min, m / e = 758.6 [M+H]+'HNMR (400 MHz, Chloroform-d, ppm) 84.86 (t, J= 4.9 Hz, 2H), 2.67 (dt, J = 2.6, 2.8 Hz, 3H), 2.39 (t, J= 2.4 Hz, 2H), 2.27 (d, J= 2.3 Hz, 10H), 1.86-1.76 (m, 12H), 1.56 (s, 7H), 1.26 (s, 40H), 1.00-0.80 (m, 12H).Example 7. Nanoparticle characterization and mRNA encapsulation

[0244] Measurement of pKa in Formulated Lipid Nanoparticles Using the TNS Assay: The apparent pKa of ionizable lipid in the lipid nanoparticle was determined using 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS reagent, Sigma-Aldrich, St. Louis, MO). Lipid nanoparticles were diluted in PBS to a concentration of 1 mM total lipid. TNS was prepared as a 1 mg / mL stock solution in DMSO and then further diluted using distilled water to a working solution of 60 pg / mL (179 mM). Lipid nanoparticle samples were diluted to 90 pM lipid in 165 pL of buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, and final TNS working solution of 1.33 pg / mL (4 pM) where the pH ranged from 3.8 to 12. Following pipette mixing and incubation at room temperature in the dark for 15 min, fluorescence intensity was measured at room temperature in a BioTek Cytation3 imaging reader using excitation and emission wavelengths of 321 and 445 nm, respectively. The fluorescence signal was plotted as a function of the pH and analyzed using a nonlinear (Boltzmann) regression analysis with the apparent pKaApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO determined as the pH giving rise to half maximal fluorescence intensity. Amino lipid apparent pKa values were calculated using the Henderson-Hasselbalch equation.

[0245] mRNA Formulation into Lipid Nanoparticles: Lipid nanoparticles encapsulating mRNA were prepared by mixing an ethanolic solution of lipids with an aqueous solution of mRNA as previously described (nanomedicine 2022). Briefly, lipid excipients ATX (proprietary ionizable amino lipids), DSPC (l,2-distearoyl-sn-glycero-3 -phosphocholine) (Avanti Polar Lipids), cholesterol (Avanti Polar Lipids), and DMG-PEG (1,2-Dimyristoylsn-glycerol, methoxypolyethylene glycol, PEG chain molecular weight: 2000) (NOF America Corporation) are dissolved in ethanol. An aqueous solution of the mRNA is prepared in citrate buffer pH 3.5. The lipid mixture is then combined with the RNA solution at a flow rate ratio of 1:3 (V / V) using the commercially available mixer device. The mixed material was then diluted three times with HEPES buffer pH 7.4 after leaving the micromixer outlet which further reduced the ethanol content. The diluted LNP slurry was purified by tangential flow filtration with hollow fiber membranes (mPES Kros membranes, Repligen), and A total of 10 diavolumes were exchanged, effectively removing the ethanol. Concentration of the formulation is adjusted to the final target RNA concentration using 100,000 MWCO Amicon Ultra centrifuge tubes (Millipore Sigma) followed by filtration through a 0.2 pm PES sterilizing-grade filter. Particle size was determined by dynamic light scattering (ZEN3600, Malvern Instruments). Encapsulation efficiency was calculated by determining unencapsulated RNA content by measuring the fluorescence upon the addition of RiboGreen (Molecular Probes) to the LNP slurry (Fi) and comparing this value to the total RNA content that is obtained upon lysis of the LNPs by 1% Triton X-100 (Ft), where % encapsulation = (Ft - Fi) / Ft x 100.

[0246] Fragment analysis: The integrity of mRNA in LNPs was measured with the aid of 5400 Fragment Analyzer Instrument (Agilent Technologies, Santa Clara, CA, USA) equipped with a Charged Coupled Device and CCD detector (Agilent Technologies). The separation gel is initially introduced into the capillary of the fragment analyzer. Samples are then introduced into the capillary inlet electrokinetically at 5.0 kV for 4 seconds and run for 40 minutes at 8.0kV. The LED light source excites at 460nm through the sample window, and the emitted fluorescent from the intercalating dye into specific band pass filters and is spatially imaged onto the CCD detector. The RNA ladder marker of 15 nucleotides were added into every sample well. A designated sample well containing the RNA STD ladder will be used to determine the size of each sample. The remaining sample wells can contain RNA samples. Each RNA sample is compared to the RNA STD ladder to determine the size ofApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO RNA. Test articles were analyzed in this manner. Data were processed using Prosize data analysis (Agilent Technologies).

[0247] Characteristics ofLl-L6 and control LNPs: Lipids L1-L6 were formulated into lipid nanoparticles with optimal physico-chemical parameters and specifications as provided in Table 2. The hydrodynamic diameter of these particles is within the 70 ±5 nm range, while PDI is <0.3 with % encapsulation >90. In contrast, a disulfide containing commercially available lipid ssPalmO-Phe formulated into significantly larger particles with <90% encapsulation efficiency. Lipid MC3 was used as another comparator which showed similar particle characteristics as L1-L6, and were consistent with literature precedent. The integrity of mRNA in LNPs was measured with the aid of 5400 Fragment Analyzer Instrument equipped with a Charged Coupled Device and detector which performs parallel separations of degraded and intact mRNA simultaneously via multiplexed capillary electrophoresis. The percentage of intact mRNA was measured in all the LNPs and compared against the standard unencapsulated mRNA, and found to be mostly intact across all LNPs. This demonstrated that neither the Ll-L6 lipids nor the control lipids had any adverse effect on the integrity of the hEPO mRNA used in these formulations.Table 2.Lipid Diameter (nm) PDI % encap FAaLI 73.2 0.163 94.2 98L2 73.2 0.21 93.3 97L3 69.29 0.192 89.8 98L4 69.53 0.13 94.1 94L5 74.94 0.172 94.3 95L6 70.26 0.175 91.8 99MC3 74.03 0.142 92.4 95ssPalmO-Phe 97.91 0.101 89.4 96aFA = fragment analysis; a measure of the purity relative amount of full-length mRNA.Example 8. In vitro transfection and RNA release assay

[0248] Cell culture: HEPA1-6 (mouse, ATCC CRL-1830) and HEPG2 (human, ATCC HB-8065) were cultured in DMEM (Gibco: 11995-065) with 10% FBS (Fisher: SH3007103HI) at 37 °C with 5% CO2. Cells were seeded on Collagen Coated flask.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0249] in vitro Transfection and RNA release assay. Cells were seeded on a 96 well plate coated with collagen (Thermo: A1048301) at a cell density of lOk / well and incubated overnight. Lipid nanoparticles with different concentrations of RNA (lOOng, 50ng, 25ng / well) were added to the cells, LNP dilutions were prepared in DMEM (Gibco: 11995-065) with 10% FBS (Fisher: SH3007103HI) and lOug / mL of ApoE (Sigma: SRPG303-50ug). Incubate for 24 hrs at 37 °C with 5% CO2. Culture medium were collected to run on MSD U-Plex Human EPO Assay (MSD: K151VXK-2)

[0250] Lipid nanoparticles were incubated with 10 mM concentration of glutathione in 10 mM HBS (pH 7.3) at 37 °C for 20 h. Released RNA was visualized by 2 % agarose gel electrophoresis. 2% Agarose gel was prepared by adding 4g of UltraPure Agarose (Thermo: 16500500) into 200mL of IX TBE Buffer (Thermo: 15581-044) with SYBR Safe DNA Gel Stain (Thermo: S33102). Samples were loaded at 60uL per well (1,5ug total per well) and run with IX TBE for 15min at 100V. The image was taken by Azure Imager (Azure Biosystems: Azure 400 AZI400).

[0251] Lipid nanoparticles prepared with lipids L1-L6, along with control nanoparticles with two other lipids, were incubated with 10 mM concentration of glutathione (GSH) in 10 mM HBS (pH 7.3) at 37 °C for 20 h. Released RNA was visualized by 2 % agarose gel electrophoresis (FIG. 2A). Cells were seeded on a 96 well plate coated with collagen and incubated overnight. Lipid nanoparticles with different concentrations (lOOng, 50ng, 25ng / well) of RNA were added to HepG2 cells, followed by lOug / mL of ApoE and incubated for 24 hrs at 37 °C with 5% CO2. Culture medium were collected to run on MSD U-Plex human EPO Assay.

[0252] Results. As can be seen from FIG. 2A, MC3 (which does not have a glutathione-reducible disulfide bond) did not release mRNA from its repspective LNP. Each of lipids LI, L2, L3, L4, L5, L6, and ssPalmO-Phe released mRNA from the respective LNPs. Band intensities show that LI, L3, L6 and Coatsome® lipid ssPalmO-Phe all showed greater mRNA release compared to L2, L4 and L5. This quantitative divergence in mRNA release with physiologically relevant GSH concentration of 10 mM, albeit in an artificial system, might disrupt the LNP enough to leak the mRNA, but the residual principal lipid component may have its own residual sequestering ability.

[0253] Figure 2B shows the EPO expression profile of these lipids upon transfection into HepG2 cells. As can be seen LI has similar expression profile as MC3, while L2 has superior dose dependant EPO expression. L3 and L6 shows relatively lower EPO expression due to their low pKa and consequent lower release of mRNA into the cytoplasm. L4 has cyclohexylsApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO in the tail portion that may have influenced the packing and EPO protein expression. L5 has a thiocarbamate linker unlike the carbmate linker in close analoges LI that may have affected the rate of self immolation and release of mRNA.

[0254] To test the possibility that these disulfide lipids would self-immolate in the predictive fashion as shown pictorially in FIG. IB, one of the lipids (LI) was incubated with 10 mM concentration of glutathione in 10 mM HBS (pH 7.3) at 37 °C for 20 h to determine whether chemical intermediates and final products would be detected as shown in FIG. 3 A. Aliquots of the reaction mixture were collected, and contents were analyzed by LC-MS using an Agilent Zorbax Eclipse plus RRHD C18 column (FIG. 3B, FIG. 3C, and FIG. 3D). Both the chromatogram and mass spectrum showed a time dependent transition of LI to its disulfide reduced degradant (LI -DI) and the self-immolated product (L1-D2) resulting from sequential elimination of episulfide and CO2. As can be clearly seen at 4 hours (FIG. 3C), LI was partially converted to LI -DI with a retention time (RT) of 14.35 minutes and at 24 hours was fully converted to L1-D2 (RT = 7.5 minutes) (FIG. 3D). Further, the mass spectrum showed the presence of all 3 species present at an intermediate stage (FIG. 3E). These intermediates were not present at T = 0, nor were LI or Ll-Dl present at T = 24h (FIG. 3D).Example 9. In vivo release and EPO expression in mice

[0255] Having demonstrated the GSH dependent bio-degradability of an exemplary lipid LI, the in vivo potency of L1-L6 LNPs were tested, along with control LNPs using MC3 and ssPalmO-Phe. For this purpose, a human erythropoietin (hEPO) reporter mRNA was used as the encapsulant so that the secreted serum hEPO protein level would be easily measured using Elisa / MSD. Female Balb / C mice, 8 to 10 weeks old, were administered with LNPs intravenously at 0.1 and 0.03 mg / kg with a dosing volume of 10 mL / kg. Six hours post administration, mice were anesthetized via 2.5% isoflurane and blood was collected retro-orbitally into and processed to serum. Human EPO protein expression levels were quantified using ELISA. As opposed to the ex vivo EPO expression data, in vivo EPO expression was the highest for LI LNP while L2, L3 and L4 LNPs showed similar level protein expression (FIG. 4). LI, L2, L and L5 LNPs were clearly superior to disulfide containing control lipid ssPalm-OPhe. Also, Ll-LNP was almost 2x potent than one of the most widely used and commercially available ionizable lipid MC3, which has poor biodegradability with a mouse plasma half-life of more than 72h (see Maier, M. A.; Jayaraman, M. et al. BiodegradableApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21, 1570-1578).

[0256] The plasma and tissue (liver and spleen) clearance of LI and L2 was then tested. Lipid MC3 was used as a control. Female Balb / C mice (8-10 weeks old) were administered with LNPs containing hEPO mRNA at 0.5 mg / kg by intravenous injection via the lateral tail vein. Blood, liver, and spleen were collected at 0 hr, 24 hr, D7, D14, D21, and D28 post-dose (n = 3 / timepoint). All samples were stored at -80 °C before analyzing for lipid quantification by liquid chromatography with tandem mass spectrometry detection (LC / MS / MS).

[0257] As shown in FIG. 5 A, FIG. 5B, and FIG. 5C, LI and L2 concentration in plasma at day 1 was almost negligible compared to that of liver and spleen concentrations, respectively. This was readily supported by the fact that the LI and L2 LNPs were mostly cleared from the plasma in less than an hour and any free lipid that may have existed in plasma due to particle istability, if any, would be readily degraded by the plasma GSH which is present at ~20 pM concentration. The liver clearance of lipid LI and L2 showed a longer half-life (Table 3) which was surprising considering that the GSH concentration in the liver cells is -1000 fold higher than that in the plasma and in the extracellular environment vide supra (Wu, G.; Fang, Y. et al. Glutathione metabolism and its implications for health. J. Nutr. 2004, 134, 489-492), while overall tissue concentration of GSH is the highest in the liver of an 8-10-week-old mice (Stohs, S. J.; Hassing, J. M. et al. Glutathione levels in hepatic and extrahepatic tissue of mice as a function of age. Age. 1980, 3, 11-14) (Hazelton, G. A.; Lang, C. A. Glutathione Contents of Tissues in the Aging Mouse. Biochem. J. 1980, 188, 25-30.). This may be due to two main factors. Firstly, GSH is used for multiple essential processes in the liver and the available GSH for LI or L2 lipids which are present in substantial concentration immediately after administration due to rapid plasma clearance. Secondly, some concentration of the lipid as LNP is still sequestered in the endosomal compartment and not available for reduction by GSH. It is to be noted that only 33.4% administered LI is detected at To (day 1). This shows that substantial amount of lipid LI may have been reduced immediately, and this may have caused temporary change in GSH level.Table 3. Quantification of lipids LI, L2 and MC3 in plasma, liver and kidney and tissue halflives.AUC 0-t Half-life (T1 / 2)Lipid Organ / tissue(ng / mL*d) daysPlasma ND. ND.LI Liver 708297 4.2Spleen 69020 4.6Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO Plasma ND. ND.L2 Liver 715353 2.6Spleen 45095 5.1Plasma 3329 7.1MC3 Liver 912452 7Spleen 847719 37.8Example 10. LCMS analysis of degradants upon glutathione incubation

[0258] Lipid nanoparticles were incubated with 10 mM concentration of glutathione in 10 mM HBS (pH 7.3) at 37 °C for 20 h. 20 pL aliquots of the reaction mixture were collected and diluted with 80 pL of acetonitrile (MeCN) containing 1.25% formic acid. Contents of the final mixture (25% water, 75% MeCN, 1% formic acid) were analyzed by LC-MS. The separation was conducted using an Agilent Zorbax Eclipse plus RRHD C18 column 1.8 μm (2.1x100 mm) column (Cat.# 959758-902) at a flow rate of 0.4 mL / min. The column was heated to 40° C and used a gradient solvent system. The solvent system; 0-8 min with 20:80 of Mobile phase A (0.1% Formic acid in Water) and Mobile Phase B (0.1% Formic acid in Methanol), 8-12 min mobile phase B (0.1% Formic acid in Methanol), 18-20 min with 20:80 of Mobile phase A (0.1% Formic acid in Water) and Mobile Phase B (0.1% Formic acid in Methanol). All of the solutions contained 1% formic acid and used positive MS mode.Example 11. Mouse plasma stability and Pharmacokinetic analysis

[0259] 8-10 weeks old female Balb / C mice were obtained from Charles River Laboratories (Hollister, CA, USA). Mice were administered with LNP containing EPO mRNA at 0.5 mg / kg by intravenous injection via the lateral tail vein. Blood, liver, and spleen were collected at 0 hr, 24 hr, D7, D14, D21, and D28 post-dose (n=3 / timepoint). Mice were perfused with saline before tissue collection. Blood samples were processed to obtain plasma. All samples were stored at -80 °C before analyzing for lipid quantification by liquid chromatography with tandem mass spectrometry detection (LC / MS / MS). Tissue samples (~50 mg) were weighed frozen and homogenized in 45 / 50 / 5 (v / v) (10 mM ammonium formate with 0.2% formic acid) / (50 / 50 acetonitrile / methanol with 0.1% formic acid) / dimethylformamide (1:9 weight / volume). Standards and QC samples were freshly prepared in plasma acidified by 1 / 10 of the volume of 10% formic acid in 0.5M citric acid. The plasma samples or tissue homogenates (25 pL) were extracted by adding 400 pL of 0.1% FA in 1: 1 MeOH-IPA containing 100 ng / mL of IS (Internal Standard). The plate was vortex mixed for 5 minutes and centrifuged at 2800 rpm for 10 min. The supernatant was transferred to a clean 96-wellApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO plate and diluted by adding 400 pL of 0.1% FA in 70 / 25 / 5 solvent (v / v):(MPB) / (MPA) / (DMF). The plate was briefly vortexed and centrifuged for 10 minutes at 2800 rpm and placed in the LC-MS / MS for analysis. LC conditions were given in the table. The calibration curve ranged from 25 ng / mL to 25000 ng / mL with an LLOQ of 25 ng / mL for plasma samples and 250 ng / g tissue to 250000 ng / g tissue with an LLOQ of 250 ng / g tissue for tissue samples.Example 12. In vivo protein (hEPO) expression in mice

[0260] Female Balb / C mice, 8 to 10 weeks old, were purchased from Charles River Laboratories (Hollister, CA). The mice were housed in a pathogen-free environment in Innovive disposable IVC rodent caging system with a 12 h light / dark cycle and ad libitum access to rodent chow and water. LNPs were administered intravenously at 0.1 and 0.03 mg / kg with a dosing volume of 10 mL / kg. Six hours post administration, mice were anesthetized via 2.5% isoflurane and blood was collected retro-orbitally into microcollection tubes (BD Microtainer® Tube with BD Microgard™ Closure; BD Biosciences, San Diego, CA) and processed to serum by centrifuging coagulated blood at 10,000 rpm for 10 min. Serum was stored at -80°C for later analysis for hEPO protein expression levels.

[0261] Measurement of human Erythropoietin (hEPO) protein in serum of mice was determined using the U-PLEX Human EPO Assay (Meso Scale Discovery, Catalog Number# K151VXK). This kit is specific to Human EPO, and there is no cross-reactivity with endogenous Mouse EPO. 96-well plates provided with the kit were coated for 1 hour in agitation at room temperature with 25ul of coating solution, composed by 200ul of biotinylated capture antibody diluted in 3.3 mL of DiluentlOO. Then, plates were washed 3 times with lx MSD Wash Buffer, and 25ul of Diluent43 were added to each well.Immediately after that, 25ul of either Calibrator Standards or serum samples diluted 1:2000 in DiluentlOO were added and plates were incubated for 1 hour in agitation at room temperature. Plates were washed 3 times with lx MSD Wash Buffer and incubated with 50ul of lx Detection Antibody Solution for 1 hour in agitation at room temperature. Finally, plates were washed 3 times with lx MSD Wash Buffer, and 150pL of MSD GOLD Read Buffer B was added to each well. Human EPO electrochemiluminescence (ECL) signal was generated and recorded on a QuickPlex 120 instrument (Meso Scale Discovery, Catalog Number# AH AA-0). ECL signal from the Calibrator Standards was used to generate a standard curve with the Discovery Workbench Analysis Software, and samples were interpolated and reported as ng hEPO / ml serum.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO

[0262] Conclusions. Redox-responsive delivery systems have been studied extensively in biomedicine. Redox-responsive drug delivery systems can respond to the high intracellular level of glutathione and release the loaded cargoes rapidly. With liver being the primary organ for deposition of LNPs and the concentration of GSH is naturally high in the hepatocytes the disulfide-containing lipids L1-L6 were tested for their potency for effective delivery of loaded mRNA. Using optimized formulation conditions, lipids L1-L6 indeed formed LNPs with good particle characteristics of <100 nm hydrodynamic size, >90% encapsulation of mRNA, <0.2 polydispersity index and robust protection of mRNA from hydrolysis. These LNPs were used in both in vitro and in vivo studies to deliver encapsulated mRNA effectively into the hepatocytes and express protein. These LNPs may be useful for tumor specific RNA therapeutic delivery because of much higher concentration of GSH in the tumor cells, and the possibility that LNPs can be readily altered for tumor targeting.

[0263] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO REFERENCES CITED IN THE PRESENT DISCLOSURE, THE CONTENT OF WHICH IS INCORPORATED BY REFERENCE1. Arcturus Therapeutics Announces Positive Interim ARCT-021 (LUNAR-COV19) Phase 1 / 2 Study Results for both Single Shot and Prime-Boost Regimens, and up to $220 Million in Additional Financial Commitments fromSingapore https: / / ir.arcturusrx.com / node / 10176 / pdf (2020).2. Ong, E. Z.; Yee, J. X. et al. Immune gene expression analysis indicates the potential of a self-amplifying Covid-19 mRNA vaccine. NPJ Vaccines 2022, 7, 154.3. Aldrich, C.; Leroux-Roels, I. et. al. Proof-of-concept of a low-dose unmodified mRNA-based rabies vaccine formulated with lipid nanoparticles in human volunteers: a phase 1 trial. Vaccine 2021, 39, 1310-1318.4. Mulligan, M. J.; Lyke, K. E. et al. Phase I / II study of COVID-19 RNA vaccine BNT162b1 in adults. Nature 2020, 586, 589-593.5. Kremsner, P.; Mann, P. et al. Safety and immunogenicity of an mRNA-lipid nanoparticle vaccine candidate against SARS-CoV-2: a phase 1 randomized clinical trial. Wien Klin. Wochenschr. 2021, 133, 931-941.6. Bailey, A. L.; Cullis, R. P. Modulation of membrane fusion by transbilayer distributions of amino lipids. Biochemistry, 1994, 33, 12573-12580.7. Jayaraman, M.; Ansell, S. M.; Mui, B. L.; Tam, Y. K.; Chen, J.; Du, X.; Butler, D.;Eltepu, L.; Matsuda, S.; Narayanannair, J. K.; Rajeev, K. G.; Hafez, I. M.; Akinc, A.; Maier, M. A.; Tracy, M. A.; Cullis, P. R.; Madden, T. D.; Manoharan, M.; Hope, M. J. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem., Int. Ed. 2012, 51, 8529-8533.8. Hashiba, K.; Sato, Y. et al. Branching ionizable lipids can enhance the stability, fusogenicity, and functional delivery of mRNA. Small Sci. 2023, 3, 2200071.9. Hajj, K. A.; Ball, R. L. et al. Branched-Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH. Small, 2019, 15, 1805097.10. Rajappan, K.; Tanis, S. P. et al. Property driven design and development of lipids for efficient delivery of siRNA. J. Med. Chem. 2020, 63, 12992-13012.

Claims

Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WOWHAT IS CLAIMED IS:

1. A compound of formula (I)(I),or a pharmaceutically acceptable salt thereof, wherein:R1and R2are each independently H or Ci-6 alkyl, orR1and R2taken together with the N atom to which they are attached form a heterocycloalkyl having 4 to 10 ring members, wherein the heterocycloalkyl optionally further comprises a second ring heteroatom N, O, or S;X1is absent or is C1-2 alkylene;X2is C2-5 alkylene;O■"7"", or ''“i'"', wherein each asterisk (*) indicates the atom attached to L1and L2, and RYis H or C1-6 alkyl;L1and L2are each independently C1-8 alkylene;Z1and Z2are each independently absent or C4-11 alkylene;R3and R4are each independently C3-12 alkyl or C3-8 cycloalkyl-Ci-6 alkylene; and R5and R6are each independently C3-12 alkyl or C3-8 cycloalkyl-Ci-6 alkylene.

2. The compound of claim 1, wherein R1and R2are each independently C1-6 alkyl.

3. The compound of claim 1 or 2, wherein R1and R2are each methyl.

4. The compound of any one of claims 1 to 3, wherein X1is absent.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO5. The compound of any one of claims 1 to 3, wherein X1is ethylene.O / •» N* x S-s|- 6. The compound of any one of claims 1 to 5, wherein Yis CH,, orO7. The compound of claim 4, wherein Y is CH.O O / A > A A•» N* S-|- * N* O-t|- 8. The compound of claim 5, whereinY is 'n^v' or9. The compound of any one of claims 1 to 8, wherein L1and L2are each propylene.

10. The compound of any one of claims 1 to 9, wherein Z1and Z2are each absent.

11. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein the compound has the formula:wherein W is O or S.

12. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has the formula:Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO13. The compound of any one of claims 1 to 12, wherein X2is C2-3 alkylene.

14. The compound of any one of claims 1 to 13, wherein R3and R4are each Ce-7 alkyl.

15. The compound of any one of claims 1 to 13, wherein R3and R4are each16. The compound of any one of claims 1 to 15, wherein R5and R6are each Ce-7 alkyl.

17. The compound of any one of claims 1 to 15, wherein R5and R6are each18. The compound of claim 1, 11, or 12, wherein:X2is C2-3 alkylene;R3and R4are each independently Ce-7 alkyl; andR5and R6are each independently Ce-7 alkyl.

19. The compound of claim 1, 11, or 12, wherein:X2is C2-3 alkylene;R5and R6are each20. The compound of claim 1, which is:Applicant Docket No.: LUNAR0011 WOMintz Docket No.: 049386-550F01 WOL2,Applicant Docket No.: LUNAR0011 WOMintz Docket No.: 049386-550F01 WOL5, oror a pharmaceutically acceptable salt thereof.

21. The compound of claim 1, which is:Applicant Docket No.: LUNAR0011 WOMintz Docket No.: 049386-550F01 WOApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO22. A lipid composition comprising a nucleic acid and a compound of any one of claims 1 to 21.

23. The lipid composition of claim 22, wherein the nucleic acid is RNA or DNA.

24. The lipid composition of claim 22 or 23, wherein the nucleic acid is selected from an siRNA, an mRNA, a self-replicating RNA, a DNA plasmid, and an antisense oligonucleotide.

25. The lipid composition of any one of claims 22 to 24, wherein the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a therapeutic protein of interest.

26. The lipid composition of claim 25, wherein the therapeutic protein of interest is an enzyme, and antibody, an antigen, a receptor, or a transporter.

27. The lipid composition of claim 25 or 26, wherein the therapeutic protein of interest is a gene-editing enzyme.

28. The lipid composition of claim 27, wherein the gene-editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease.

29. The lipid composition of any one of claims 22 to 28, wherein the lipid composition comprises liposomes, lipoplexes, or lipid nanoparticles.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO30. A lipid nanoparticle comprising a plurality of lipids, wherein each lipid is independently a compound of any one of claims 1 to 21, wherein the plurality of lipids self-assembles to form the lipid nanoparticle comprising an interior and exterior.

31. The lipid nanoparticle of claim 30, wherein the average particle size of the lipid nanoparticle is less than about 100 nm.

32. The lipid nanoparticle of claim 30 or 31, wherein the average particle size of the lipid nanoparticle is about 50 nm to about 100 nm, about 65 nm to about 100 nm, or about 65 nm to about 75 nm.

33. The lipid nanoparticle of any one of claims 30 to 32, wherein the lipid nanoparticle further comprises a nucleic acid encapsulated in the interior.

34. The lipid nanoparticle of claim 33, wherein the nucleic acid is RNA or DNA.

35. The lipid nanoparticle of claim 33 or 34, wherein the nucleic acid is selected from an siRNA, an mRNA, a self-replicating RNA, a DNA plasmid, and an antisense oligonucleotide.

36. The lipid nanoparticle of any one of claims 33 to 35, wherein the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a therapeutic protein of interest.

37. The lipid nanoparticle of claim 36, wherein the therapeutic protein of interest is an enzyme, and antibody, an antigen, a receptor, or a transporter.

38. The lipid nanoparticle of claim 36 or 37, wherein the therapeutic protein of interest is a gene-editing enzyme.

39. The lipid nanoparticle of claim 38, wherein the gene-editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease.

40. The lipid nanoparticle of any one of claims 30 to 39, wherein the lipid nanoparticle further comprises a helper lipid selected from: dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholineApplicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO(DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), andphosphatidylcholine (PC).

41. The lipid nanoparticle of claim 40, wherein the helper lipid isdi stearoylphosphatidylcholine (D SPC).

42. The lipid nanoparticle of any one of claims 30 to 40, further comprising cholesterol.

43. The lipid nanoparticle of any one of claims 30 to 42, further comprising a polyethylene glycol(PEG)-lipid conjugate.

44. The lipid nanoparticle of claim 43, wherein PEG-lipid conjugate is PEG-DMG.

45. The lipid nanoparticle of claim 44, wherein the PEG-DMG is PEG2000-DMG.

46. The lipid nanoparticle of any one of claims 30 to 45, wherein the lipid nanoparticle comprises about 45 mol% to 65 mol% of the compound of any one of claims 1 to 20, about 2 mol% to about 15 mol% of a helper lipid, about 20 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate.

47. The lipid nanoparticle of claim 46, wherein the lipid nanoparticle comprises about 50 mol% to about 61 mol% of the compound of any one of claims 1 to 21, about 5 mol% to about 9 mol% of the helper lipid, about 29 mol% to about 38 mol% of cholesterol, and about 1 mol% to about 2 mol% of the PEG-lipid conjugate.

48. The lipid nanoparticle of claim 47, wherein the lipid nanoparticle comprises about 56 mol% to about 58 mol% of the compound of any one of claims 1 to 21, about 6 mol% to about 8 mol% of DSPC, about 31 mol% to about 34 mol% of cholesterol, and about 1.25 mol% to about 1.75 mol% of the PEG-lipid conjugate.

49. The lipid nanoparticle of any one of claims 30 to 48, wherein the lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 50:1 to about 10:1.

50. The lipid nanoparticle of claim 49, wherein the lipid nanoparticle has a total lipid: nucleic acid weight ratio of about 40: 1 to about 20: 1.

51. The lipid nanoparticle of claim 49, wherein the lipid nanoparticle has a total lipid: nucleic acid weight ratio of about 35:1 to about 25:1.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO52. The lipid nanoparticle of claim 49, wherein the lipid nanoparticle has a total lipid: nucleic acid weight ratio of about 32: 1 to about 28: 1.

53. The lipid nanoparticle of claim 49, wherein the lipid nanoparticle has a total lipid: nucleic acid weight ratio of about 31:1 to about 29: 1.

54. A pharmaceutical composition comprising the compound of any one of claims 1 to 21, or the lipid nanoparticle of any one of claims 30 to 53, and a pharmaceutically acceptable excipient.

55. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition is a lyophilized composition.

56. The pharmaceutical composition of claim 54 or 55, wherein the pharmaceutical composition comprises a HEPES buffer at a pH of about 7.4.

57. The pharmaceutical composition of claim 56, wherein the HEPES buffer is at a concentration of about 7 mg / mL to about 15 mg / mL.

58. The pharmaceutical composition of any one of claims 54 to 57, wherein the pharmaceutical composition further comprises about 2.0 mg / mL to about 4.0 mg / mL ofNaCl.

59. The pharmaceutical composition of any one of claims 54 to 58, wherein the pharmaceutical composition further comprises one or more cryoprotectants.

60. The pharmaceutical composition of claim 59, wherein the one or more cryoprotectants are selected from sucrose, glycerol, or a combination of sucrose and glycerol.

61. The pharmaceutical composition of claim 60, 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.

62. A method of treating 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 30 to 53, or the pharmaceutical composition of any one of claims 54 to 61.Applicant Docket No.: LUNAR0011 WO Mintz Docket No.: 049386-550F01 WO63. The method of claim 62, wherein the pharmaceutical composition or lipid nanoparticle is administered intravenously or intramuscularly.

64. 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 30 to 53, or the pharmaceutical composition of any one of claims 54 to 61.

65. The method of claim 64, wherein the protein or polypeptide is an antigen, and expression of the antigen elicits an in vivo immunogenic response.

66. A method of delivering a nucleic acid to a subject in needed thereof, comprising encapsulating a therapeutically effective amount of the nucleic acid in the lipid nanoparticle of any one of claims 30 to 53, and administering the lipid nanoparticle to the subject.

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