Secondary alcohol lipids

Abstract

Compounds of Formula (I) or a pharmaceutically acceptable salt thereof are provided, (I) wherein X1, X2, R1, R2, R3, L1, L2, L3, L4, L5, and L6 are as defined herein. The present disclosure also describes compositions, preparations, lipid nanoparticles comprising compounds of Formula (I) and methods of their use.

Description

SECONDARY ALCOHOL LIPIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of and priority to U. S. Provisional Application No. 63 / 712,808, filed October 28, 2024, which is hereby incorporated in its entirety.BACKGROUND

[0002] Lipids such as ionizable lipids have been used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer conjugated lipids, to form lipid nanoparticles to encapsulate and transport therapeutic agents such as nucleic acids, small molecules compounds, and proteins both in vitro and in vivo. There remains an ongoing need to develop new lipids to encapsulate therapeutic agents and improve the safety, efficacy, and specificity of such lipid nanoparticle-based transport vehicles.SUMMARY

[0003] The present invention recognizes a need for compositions, preparations, and / or lipid nanoparticles and methods of their use.

[0004] The present disclosure recognizes that chemical structures of lipid compounds may confer improved properties compared to reference lipid structures.

[0005] In brief, embodiments of the present invention provide lipid compounds, including stereoisomers, pharmaceutically acceptable salts, or tautomers thereof, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including for example, all sterols) and / or their analogs, and / or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents.

[0006] In one aspect, the invention features a compound having a structure according to Formula (I),or a pharmaceutically acceptable salt thereof, wherein:X1is -NRARBor a 5- to 6-membered nitrogen-containing heterocycle;each of RAand RBis independently H or Ci-Ce alkyl;L1is Ci-C6alkylene;X2is a covalent bond, -O-, or -NH-;R1is C1-C10 alkyl;L2is Ci-C6alkylene;each of L3and L4is independently Ce-Cio alkylene;each of L5and L6is independently -C(O)O- or -OC(O)-; andeach of R2and R3is independently C4-C20 alkyl, C4-C20 alkenyl, or C4-C20 alkynyl; and wherein each heterocycle, alkyl, alkenyl, alkynyl, and alkylene group is optionally substituted.

[0007] Pharmaceutical compositions comprising one or more of the foregoing compounds of Formula (I) and a therapeutic agent are also provided. In some embodiments, the pharmaceutical compositions and / or LNPs further comprise one or more components selected from neutral lipids, charged lipids, steroids, and polymer conjugated lipids. The disclosed compositions are useful for formation of lipid nanoparticles for the delivery of the therapeutic agent.

[0008] In other embodiments, the present invention provides a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing a composition of lipid nanoparticles comprising the compound of Formula (I) or a pharmaceutically acceptable salt thereof and a therapeutic agent and delivering the composition to the patient.

[0009] Among other things, as described herein, the present disclosure may demonstrate surprising attributes of ionizable lipids (e.g., unexpected tropism, stabilization,bioavailability, degradation property(ies) and delivery efficacy of cargos such as therapeutic or prophylactic agents), and compositions, preparations, and / or lipid nanoparticles (e.g., LNPs and / or LNP-containing compositions, preparations, and / or lipid nanoparticles) thereof, and methods of their use.

[0010] Among other things, the present disclosure recognizes that lipid nanoparticle (LNP) compositions comprise one or more ionizable lipids.

[0011] In some embodiments, provided compositions, preparations, and / or lipid nanoparticles are for use in methods of treatment, delivery, producing polypeptides, or delaying / arresting progression of a disease or disorder.

[0012] In some embodiments, provided compositions, preparations, and / or lipid nanoparticles are for use in methods of manufacturing.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Definitions

[0013] About: The term “about” or “approximately,” when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” in that context. For example, in some embodiments, the term “about” may encompass 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 in either direction (greater than or less than) of the reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0014] Administration: The term “administration” refers to the administration of a composition to a subject or system. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal,intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, a pharmaceutical composition comprising lipid nanoparticles can be formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0015] Alkenyl. The term “alkenyl” refers to a substituted straight or branched hydrocarbon chain having at least one double bond and having (unless otherwise specified) 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-20, C2-I8, C2-I6, C2-14, C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted.

[0016] Alkyl: The term “alkyl” refers to a straight or branched hydrocarbon chain which is saturated, having, for example, from one to twenty-four carbon atoms (C1-C24 alkyl), four to twenty carbon atoms (C4-C20 alkyl), six to sixteen carbon atoms (Ce-Cie alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C15 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (Ci-Cs alkyl) or one to six carbon atoms (Ci-Ce alkyl), e.g., methyl, ethyl, n-propyl, 1 -methylethyl, (isopropyl), / / -butyl, w-pentyl, 1,1-dimethylethyl ( / -butyl), 3 -methylhexyl, 2-methylhexyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted.

[0017] Alkylene: The term “alkylene” refers to a bivalent alkyl group (i.e., a bivalent saturated hydrocarbon chain) that is a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted. Any of the above-mentioned monovalent alkyl groups may be an alkylene by abstraction of a second hydrogen atom from the alkyl. In some embodiments, an “alkylene” is a polymethylene group, i.e., -(CH₂)n-, wherein n is a positive integer, preferably from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1to 4, from 1 to 3, from 1 to 2, from 2 to 5, or from 4 to 8. Unless stated otherwise specifically in the specification, an alkylene group is optionally substituted.

[0018] Alkynyl. The term “alkynyl” refers to a hydrocarbon group having one or more triple bonds. In some embodiments, the term “alkynyl”, refers to a straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-20, C2-I8, C2-I6, C2-14, C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted.

[0019] Amino acid: The term “amino acid” refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(RAA)-COOH, wherein RAAis an amino acid side chain. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid.

[0020] Aryl: The term “aryl” refers to monocyclic and bicyclic carbocyclic ring systems having a total of six to fourteen ring members (e.g., C6-14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In some embodiments, “aryl” refers to an aromatic ring system which includes, but is not limited to, phenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless stated otherwise specifically in the specification, an aryl group may be optionally substituted.

[0021] Biocompatible: The term “biocompatible” refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. In certain embodiments, materials are “biocompatible” if they are not toxic to cells. In certain embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and / or their administration in vivo does not induce significant inflammation or other such adverse effects.

[0022] Biodegradable. The term “biodegradable” refers to materials that, when introduced into cells, are broken down (e.g., by cellular machinery, such as by enzymatic degradation, by hydrolysis, and / or by combinations thereof) into components that cells can either reuse ordispose of without significant toxic effects on the cells. In certain embodiments, components generated by breakdown of a biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, biodegradable polymer materials break down into their component monomers. In some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymer materials) involves hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymer materials) involves cleavage of urethane linkages. Exemplary biodegradable polymers include, for example, polymers of hydroxy acids such as lactic acid and glycolic acid, including but not limited to poly(hydroxyl acids), poly(lactic acid)(PLA), poly(glycolic acid)(PGA), poly(lactic-co-glycolic acid)(PLGA), and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates, poly(lactide-co-caprolactone), blends and copolymers thereof. Many naturally occurring polymers are also biodegradable, including, for example, proteins such as albumin, collagen, gelatin and prolamines, for example, zein, and polysaccharides such as alginate, cellulose derivatives and polyhydroxyalkanoates, for example, polyhydroxybutyrate blends and copolymers thereof. Those of ordinary skill in the art will appreciate or be able to determine when such polymers are biocompatible and / or biodegradable derivatives thereof (e.g., related to a parent polymer by substantially identical structure that differs only in substitution or addition of particular chemical groups as is known in the art).

[0023] Biologically active: The term “biologically active” refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.

[0024] Carrier: The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil,sesame oil and the like. In some embodiments, carriers are or include one or more solid components.

[0025] Cycloalkyl: The term “cycloalkyl” refers to an optionally substituted saturated hydrocarbon ring system of about 3 to about 10 ring carbon atoms. In some embodiments, cycloalkyl groups have 3-6 carbons. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Unless stated otherwise specifically in the specification, a cycloalkyl group may be optionally substituted.

[0026] Composition: Those skilled in the art will appreciate that the term “composition” may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc.

[0027] Comprising: A composition or method described herein as “comprising” one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method.

[0028] “Improve,'’'’ “increase", “inhibit” or “reduce”: The terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0029] Encapsulated: The term “encapsulated”, when used in the context of a lipid nanoparticle, refers to substances such as cargo or a therapeutic agent that is fully encapsulated, partially encapsulated, or both. In an embodiment, the therapeutic agent or cargo is fully encapsulated in the lipid nanoparticle.

[0030] Excipient: The term “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example,starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0031] Expression: The term “expression” of a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g, by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0032] Halogen. The term “halogen” or “halo” means fluorine, chlorine, bromine, or iodine.

[0033] Heteroaryl: The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Exemplary heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridonyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[l,2-a]pyrimidinyl, imidazo[l,2-a]pyridinyl, thienopyrimidinyl, triazolopyridinyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4 / 7 quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-l,4-oxazin-3(4H)-one, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or“heteroaromatic”. Unless stated otherwise specifically in the specification, a heteroaryl group may be optionally substituted.

[0034] Heteroatom: The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quatemized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+(as in N-substituted pyrrolidinyl)).

[0035] Heterocycle: The terms “heterocycle”, “heterocyclyl”, and “heterocyclic ring” are used interchangeably herein, and refer to a stable 3- to 8-membered monocyclic, a 7- to 12-membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+(as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic rings include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, thiamorpholinyl, and 1,4-diazepinyl. Unless stated otherwise specifically in the specification, a heterocyclic ring may be optionally substituted.

[0036] In vitro'. 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, etc., rather than within a multi-cellular organism.

[0037] In vivo: The term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0038] Linker: The term “linker” refers to that portion of a multi-element agent that connects different elements to one another.

[0039] Lipid Nanoparticle: The term “lipid nanoparticle” refers to a particle comprising one or more lipids having a diameter or at least one dimension of less than 1000 nanometers (nm). In some embodiments, the lipid nanoparticles of the invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g., an adverse immune response. In some embodiments, lipid nanoparticles described herein can have an average hydrodynamic diameter from about 30 to about 170 nm. In some embodiments, lipid nanoparticles described herein can have an average hydrodynamic diameter that is about 30 nm, 35 nm,40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from between 50 nm to 100 nm. The term “lipid nanoparticle preparation” can be used interchangeably with “lipid nanoparticle.”

[0040] Lipid nanoparticle composition: The term “lipid nanoparticle composition” refers to a composition that contains at least one lipid nanoparticle and at least one additional agent or ingredient. In some embodiments, a lipid nanoparticle composition contains a substantially uniform collection of lipid nanoparticles as described herein.

[0041] N: P Ratio. The term “N: P ratio” or “N / P ratio” refers to the ionizable lipid nitrogen: nucleic acid phosphate (N: P) molar ratio which represents the charge balance between the cationic tertiary amine of the ionizable cationic lipid and the anionic phosphate group in the nucleotide polymer backbone. Based on this property, ionizable lipids form a complex with nucleotides. In some embodiments, provided herein the lipid nanoparticle comprises an N: P ratio of between about 10:1 to about 3:1.

[0042] Nucleic acid. In its broadest sense, the term “nucleic acid” refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can beincorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, “ nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, “nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid" is or comprises RNA; in some embodiments, a “nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more “peptide nucleic acids’", which are known in the art and have peptide bonds instead of phosphodi ester bonds in the backbone, are considered within the scope of the present invention.Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5’-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaad enosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2 ’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0043] Patient: The term “patient” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disorder or condition. In some embodiments, a patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or presence of one or more tumors. In some embodiments, the patient is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition.

[0044] Pharmaceutical composition: The term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0045] Pharmaceutically acceptable: The term “pharmaceutically acceptable” refers 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 / or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0046] Pharmaceutically acceptable carrier: The term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; Ph buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0047] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt” refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).

[0048] Prevent or prevention: The terms “prevent” or “prevention” when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition, and / or to delaying onset of, and / orto reducing frequency and / or severity of, one or more characteristics or symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0049] Polypeptide. The term “polypeptide” generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity.

[0050] Protein. The term “protein” refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” may be used to refer to the multiple polypeptides that are physically associated and function together as the discrete unit. In some embodiments, proteins may include moi eties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that in some embodiments the term “protein” may refer to a complete polypeptide chain as produced by a cell (e.g., with or without a signal sequence), and / or to a form that is active within a cell (e.g., a truncated or complexed form). In some embodiments where a protein is comprised of multiple polypeptide chains, such chains may be covalently associated with one another, for example by one or more disulfide bonds, or may be associated by other means.

[0051] Pure An agent or entity is “pure” if it is substantially free of other components. For example, a preparation that contains more than about 90% of a particular agent or entityis typically considered to be a pure preparation. In some embodiments, an agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0052] Subject: The term “subject” refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0053] Substantially: 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.

[0054] Substituted or optionally substituted. Molecular groups herein may be substituted or unsubstituted (e.g., as described herein). For example, compounds of this disclosure may contain optionally substituted and / or substituted moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one ormore hydrogens that are either explicit or implicit from the structure (e.g., Q"R1refers to). The term “unsubstituted” means that the specified group bears no substituents. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituentat each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Groups described as being “substituted” can have between 1 and 4 substituents, such as 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.

[0055] A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known. Exemplary substituents may be selected from halogen, R’, -COR’, -CO2H, -CO2R’, -CN, -OH, -OR’, -OCOR’, -OCO2R’, -NH2, -NHR’, -N(R’)2, -SR’, and -SO2R’, wherein each instance of R’ independently is R” or Ci-C2o alkyl (e.g., Ci-C2o alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl, which may be unsubstituted or substituted), and wherein each instance of R” is selected from cycloalkyl (e.g., C3-C6 cycloalkyl), heterocyclyl (e.g., 3- to 6-membered heterocyclyl comprising 1, 2, or 3 heteroatoms), aryl (e.g., Ce-Cio aryl), and heteroaryl (e.g., 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms) according to any embodiments described herein, and wherein R” itself is optionally substituted with one or more groups selected from R’”, -COR’”, -CO2H, -CO2R’”, -CN, -OH, -OR’”, -OCOR’”, -OCO2R’”, -NH2, -NHR’”, -N(R’)2, -SR’”, and -SO2R’” as described herein, where R’” is selected from Ci-C2o alkyl, cycloalkyl (e.g., C3-C6 cycloalkyl), heterocyclyl (e.g., 3- to 6-membered heterocyclyl comprising 1, 2, or 3 heteroatoms), aryl (e.g., Ce-Cio aryl), and heteroaryl (e.g., 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms). In certain embodiments thereof, R’ independently is an unsubstituted alkyl (e.g., unsubstituted Ci-C2o alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’ independently is unsubstituted Ci-Ce alkyl.

[0056] Systemic: The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” have their art-understood meaning referring to administration of a compound or composition such that it enters the recipient’s system.

[0057] Therapeutically effective amount: The term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, inhibit, alleviate, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.

[0058] “Tissue” and / or “organ”: The terms “tissue” and / or “organ” refer to viable cellular materials in an aggregate form, e.g., small portions of an organ, as well as dispersed cells, e.g., cells dispersed, isolated and / or grown from muscle, heart muscle, liver or kidney, including bone marrow cells and progeny cells, blood bom stem cells and progeny, and the various other blood elements, unless otherwise specified. In some embodiments, the tissue and / or organ refers to kidney, heart liver, stomach, spleen, pancreas, lung, brain, eye, intestines, bladder, skin or dermal tissue, blood vessel, veins, arteries, heart valves, sperm, and oocyte(s). As used herein, the term “organ” encompasses both solid organs, e.g., kidney, heart, liver, lung, as well as functional parts of organs, e.g., segments of skin, sections of artery, veins, transplantable lobes of a liver, kidney, lung, and the like.

[0059] Treatment. The term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of thedisease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

[0060] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0061] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a13C- or14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.

[0062] Among other things, the present invention provides compositions, preparations, and / or lipid nanoparticles for delivery of therapeutic and / or prophylactic agents. For example, the present disclosure describes lipid compounds for use in compositions, preparations, and / or lipid nanoparticles. In some embodiments, compositions, preparations, and / or nanomaterials comprise LNPs carrying cargo to designated target cells, tissue, and / or organs.Lipid nanoparticles (LNPs)

[0063] The present invention provides for compositions, preparations, and / or nanomaterials that comprise lipid nanoparticles. In some embodiments, lipid nanoparticles comprise one or more components. In some embodiments, lipid nanoparticles comprise oneor more components such as compounds, ionizable lipids, sterols, conjugate-linker lipids, and phospholipids.

[0064] In some embodiments, lipid nanoparticles comprise one or more compounds as described herein. In some embodiments, lipid nanoparticles comprise one or more ionizable lipids as described herein. In some embodiments, lipid nanoparticles comprise one or more sterols as described herein. In some embodiments, lipid nanoparticles comprise one or more conjugate-linker lipids as described herein. In some embodiments, lipid nanoparticles comprise one or more phospholipids as described herein.Compounds

[0065] Among other things, the present disclosure describes compositions, preparations, and / or lipid nanoparticles that comprise one or more compounds of Formula (I) as described herein.

[0066] In one aspect, the invention features a compound having a structure according to Formula (I),or a pharmaceutically acceptable salt thereof, wherein each of X1, X2, L1, L2, L3, L4, L5, L6, R1, R2, and R3is independently according to any embodiment and combinations thereof as described herein.

[0067] In embodiments of Formula (I),X1is -NRARBor a 5- to 6-membered nitrogen-containing heterocycle;each of RAand RBis independently H or Ci-Ce alkyl;L1is Ci-C6alkylene;X2is a covalent bond, -O-, or -NH-;R1is Ci-Cio alkyl;L2is Ci-C6alkylene;each of L3and L4is independently Ce-Cio alkylene;each of L5and L6is independently -C(O)O- or -OC(O)-;each of R2and R3is independently C4-C20 alkyl, C4-C20 alkenyl, or C4-C20 alkynyl; wherein each heterocycle, alkyl, alkenyl, alkynyl, and alkylene group is optionally substituted.

[0068] In embodiments of Formula (I),each 5- to 6-membered nitrogen-containing heterocycle is optionally substituted with one or more substituents selected from Ci-Ce alkyl, NH2, NH(Ci-Ce alkyl), N(Ci-Ce alkyl)2, C3-C6 cycloalkyl, and 5- to 6-membered heterocycle optionally substituted with one or more Ci-C6alkyls;each Ci-Ce alkylene or Ce-Cio alkylene is optionally substituted with one or more halogens;each Ci-Ce alkyl or C1-C10 alkyl is optionally substituted with one or more substituents selected from halogen, OH, O(C1-C6alkyl), NH2, NH(Ci-Ce alkyl), N(Ci-Ce alkyl)2, C3-C6 cycloalkyl, and 5- to 6-membered heterocycle optionally substituted with one or more Ci-Ce alkyls, and / oreach C4-C20 alkyl, C4-C20 alkenyl, or C4-C20 alkynyl is optionally substituted with one or more substituents selected from halogen, OH, O(C1-C6alkyl), C(O)O(C4-C20 alkyl), OC(O)(C4-C20 alkyl), C(0)0(C4-C2o alkenyl), and OC(0)(C4-C2o alkenyl).

[0069] In embodiments, X1is -NRARB.

[0070] In embodiments, X1is a 5- to 6-membered nitrogen-containing heterocycle.

[0071] In embodiments, each of RAand RBis independently H or Ci-Ce alkyl that is optionally substituted with a 5- to 6-membered nitrogen-containing heteroaryl (e.g., pyridyl or imidazolyl).

[0072] In embodiments, RAis H.

[0073] In embodiments, RAis CH3.

[0074] In embodiments, RAis CH2CH3.

[0075] In embodiments, RBis H.

[0076] In embodiments, RBis CH3.

[0077] In embodiments, RBis CH2CH3.

[0078] In embodiments, RAand RBare the same.

[0079] In embodiments, RAand RBare different.

[0080] In embodiments, X1is -NH2, -N(CH3)2 or -N(CH2CH3)2.

[0081] In embodiments,

[0082] In embodiments,O

[0083] In embodiments, X1is

[0084] In embodiments, X1is a 5- to 6-membered nitrogen-containing heterocycle. In embodiments, X1is an unsubstituted 5- to 6-membered nitrogen-containing heterocycle. In embodiments, X1is a substituted 5- to 6-membered nitrogen-containing heterocycle.

[0085] In embodiments, X1is optionally substituted pyrrolidine, piperidine, piperazine, or morpholine.

[0086] In embodiments, X1is pyrrolidine. In embodiments, X1is unsubstituted pyrrolidine. In embodiments, X1is substituted pyrrolidine. In embodiments, X1is N-linked pyrrolidine. In embodiments, X1is C-linked pyrrolidine.

[0087] In embodiments,embodiments,

[0088] In embodiments, X1is piperidine. In embodiments, X1is unsubstituted piperidine. In embodiments, X1is substituted piperidine. In embodiments, X1is N-linked piperidine. Inembodiments, X1is C-linked piperidine. In embodiments, X1is

[0089] In embodiments, X1is piperazine. In embodiments, X1is unsubstituted piperazine. In embodiments, X1is substituted piperazine. In embodiments, X1is N-linked piperazine. Inembodiments, X1is C-linked piperazine. In embodiments, X1is

[0090] In embodiments, X1is morpholine. In embodiments, X1is unsubstituted morpholine. In embodiments, X1is substituted morpholine. In embodiments, X1is N-linked morpholine. In embodiments, X1is C-linked morpholine.

[0091] In embodiments, L1is Ci-Ce alkylene. In embodiments, L1is (CH2)1. In embodiments, L1is (CH2)2. In embodiments, L1is (CH2)2. In embodiments, L1is (CH2)4. In embodiments, L1is (CH2)5. In embodiments, L1is (CH2)6.

[0092] In embodiments, X2is a covalent bond, -O-, or -NH-.

[0093] In embodiments, X2is a covalent bond.

[0094] In embodiments, X2is -O-.

[0095] In embodiments, X2is -NH-.

[0096] In embodiments, R1is Ci-Cio alkyl. In embodiments, R1is CH3. In embodiments, R1is CH2CH3. In embodiments, R1is CH2CH2CH3. In embodiments, R1is CH2CH2CH2CH3. In embodiments, R1is CH2CH2CH2CH2CH3. In embodiments, R1is CH2CH2CH2CH2CH2CH3. In embodiments, R1is CH2CH2CH2CH2CH2CH2CH3. In embodiments, R1is CH2CH2CH2CH2CH2CH2CH2CH3. In embodiments, R1is CH2CH2CH2CH2CH2CH2CH2CH2CH3. In embodiments, R1is CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3.

[0097] In embodiments, L2is Ci-Ce alkylene. In embodiments, L2is -(CH2)-. In embodiments, L2is (CH2)2. In embodiments, L2is (CH2)2. In embodiments, L2is (CH2)4. In embodiments, L2is (CH2)5. In embodiments, L2is (CH2)6.

[0098] In embodiments, each of L3and L4is independently Ce-Cio alkylene. In embodiments, L3and L4are the same length. In other embodiments, L3and L4are different lengths.

[0099] In embodiments, L3is (CH2)6, (CH2)7, (CH2)8, (CH2)9, or (CH2)10.

[0100] In embodiments, L4is (CH2)6, (CH2)7, (CH2)8, (CH2)9, or (CH2)10.

[0101] In embodiments, each of L5and L6is independently -C(O)O- or -OC(O)-.

[0102] In embodiments, L5and L6are -C(O)O-.

[0103] In embodiments, L5and L6are -OC(O)-.

[0104] In embodiments, each of R2and R3is independently C4-C20 alkyl, C4-C20 alkenyl, or C4-C20 alkynyl.

[0105] In embodiments, R2is C4-C20 alkyl.

[0106] In embodiments, R2is C4-C20 alkenyl.

[0107] In embodiments, R2is C4-C20 alkynyl.,

[0112] In embodiments,

[0113] In embodiments,

[0114] In embodiments,

[0115] In embodiments,

[0116] In embodiments,

[0117] In embodiments,

[0118] In embodiments,

[0119] In embodiments,

[0120] In embodiments,

[0121] In embodiments,

[0122] In embodiments,

[0123] In embodiments,

[0124] In embodiments, R3is C4-C20 alkyl.

[0125] In embodiments, R3is C4-C20 alkenyl.

[0126] In embodiments, R3is C4-C20 alkynyl.

[0130] In embodiments,

[0131] In embodiments,

[0132] In embodiments,

[0133] In embodiments,

[0134] In embodiments,

[0135] In embodiments,

[0136] In embodiments,

[0137] In embodiments,

[0138] In embodiments,

[0139] In embodiments,

[0140] In embodiments,

[0141] In embodiments,

[0142] In embodiments,

[0143] In embodiments, R2and R3are the same.

[0144] In embodiments, R2and R3are different.

[0145] In embodiments of any Formulas described herein, a 5- to 6-membered nitrogencontaining heterocycle is unsubstituted. In embodiments of any Formulas described herein, a 5- to 6-membered nitrogen-containing heterocycle is substituted. In embodiments of any Formulas described herein, a 5- to 6-membered nitrogen-containing heterocycle is substituted with one or more substituents selected from Ci-Ce alkyl, NH2, NH(Ci-Ce alkyl), N(Ci-Ce alkyl)2, and C3-C6 cycloalkyl. In embodiments of any Formulas described herein, a 5- to 6-membered heterocycle optionally substituted with one or more Ci-Ce alkyls.

[0146] In embodiments of any Formulas described herein, a 5- to 6-membered nitrogencontaining heterocycle is unsubstituted.

[0147] In embodiments of any Formulas described herein, a 5- to 6-membered nitrogencontaining heterocycle is substituted with one or more Ci-Ce alkyl.

[0148] In embodiments of any Formulas described herein, a 5- to 6-membered nitrogencontaining heterocycle is substituted with one or more NH2.

[0149] In embodiments of any Formulas described herein, a 5 - to 6-membered nitrogencontaining heterocycle is substituted with one or more NH(Ci-Ce alkyl).

[0150] In embodiments of any Formulas described herein, a 5- to 6-membered nitrogencontaining heterocycle is substituted with one or more N(Ci-Ce alkyl)2.

[0151] In embodiments of any Formulas described herein, a 5- to 6-membered nitrogencontaining heterocycle is substituted with one or more C3-C6 cycloalkyl.

[0152] In embodiments of any Formulas described herein, a 5- to 6-membered heterocycle substituted with one or more Ci-Ce alkyls.

[0153] In embodiments of any Formulas described herein, Ci-Ce alkylene or Ce-Cio alkylene is unsubstituted. In embodiments of any Formulas described herein, Ci-Ce alkylene or Ce-Cio alkylene is substituted. In embodiments of any Formulas described herein, Ci-Cealkylene or Ce-Cio alkylene is substituted with one or more halogens. In embodiments of any Formulas described herein, Ci-Ce alkylene or Ce-Cio alkylene is substituted with one or more fluorines. In embodiments of any Formulas described herein, Ci-Ce alkylene or Ce-Cio alkylene is substituted with one or more chlorines. In embodiments of any Formulas described herein, Ci-Ce alkylene or Ce-Cio alkylene is substituted with one or more bromines. In embodiments of any Formulas described herein, Ci-Ce alkylene or Ce-Cio alkylene is substituted with one or more iodines.

[0154] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is unsubstituted. In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted. In embodiments of any Formulas described herein, a Ci-Ce alkyl or Ci-Cio alkyl is substituted with one or more halogen, OH, O(C1-C6alkyl), NH2, NH(Ci-Ce alkyl), N(Ci-Cealkyl)2, C3-C6 cycloalkyl, and 5- to 6-membered heterocycle. In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more unsubstituted 5- to 6-membered heterocycle. In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more 5- to 6-membered heterocycle substituted with one or more Ci-Ce alkyls.

[0155] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is unsubstituted.

[0156] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more halogen. In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more fluorine. In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more chlorine. In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more bromine. In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more iodine.

[0157] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more OH.

[0158] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more O(C1-C6alkyl).

[0159] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more NH2.

[0160] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more NH(Ci-Ce alkyl).

[0161] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more N(Ci-Cealkyl)2.

[0162] In embodiments of any Formulas described herein, a Ci-Ce alkyl or Ci-Cio alkyl is substituted with one or more C3-C6 cycloalkyl.

[0163] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more unsubstituted 5- to 6-membered heterocycle.

[0164] In embodiments of any Formulas described herein, a Ci-Ce alkyl or C1-C10 alkyl is substituted with one or more 5- to 6-membered heterocycle substituted with one or more Ci-C6alkyls.

[0165] In embodiments of any Formulas described herein, a C4-C20 alkyl, C4-C20 alkenyl, or C4-C20 alkynyl is optionally substituted with one or more substituents selected from halogen, OH, O(Ci-C6alkyl), C(0)0(C4-C2o alkyl), OC(0)(C4-C2o alkyl), C(0)0(C4-C2o alkenyl), and OC(O) (C4-C20 alkenyl).

[0166] In embodiments of any Formulas described herein, a C4-C20 alkyl, C4-C20 alkenyl, or C4-C20 alkynyl is optionally substituted with one or more substituents selected from halogen, OH, and O(C1-C6alkyl).

[0167] In embodiments of any Formulas described herein, a C4-C20 alkyl, C4-C20 alkenyl, or C4-C20 alkynyl is optionally substituted with one or more substituents selected from C(0)0(C4-C2o alkyl), OC(0)(C4-C2o alkyl), C(0)0(C4-C2o alkenyl), and OC(O) (C4-C20 alkenyl).

[0168] In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more halogen. In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more fluorine. In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more chlorine. In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more bromine. In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more iodine.

[0169] In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more OH.

[0170] In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more O(C1-C6alkyl).

[0171] In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more C(O)O(C4-C20 alkyl).

[0172] In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more OC(O)(C4-C20 alkyl).

[0173] In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more C(O)O(C4-C20 alkenyl).

[0174] In embodiments of any Formulas described herein, a C4-C20 alkyl is optionally substituted with one or more OC(O)(C4-C20 alkenyl).

[0175] In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more halogen. In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more fluorine. In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more chlorine. In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more bromine. In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more iodine.

[0176] In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more OH.

[0177] In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more O(C1-C6alkyl).

[0178] In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more C(O)O(C4-C20 alkyl).

[0179] In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more OC(O)(C4-C20 alkyl).

[0180] In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more C(O)O(C4-C20 alkenyl).

[0181] In embodiments of any Formulas described herein, a C4-C20 alkenyl is optionally substituted with one or more OC(O)(C4-C20 alkenyl).

[0182] In embodiments of any Formulas described herein, each of m and n is independently an integer of 6-10.

[0183] In embodiments of any Formulas described herein, m is 6. In embodiments of any Formulas described herein, m is 7. In embodiments of any Formulas described herein, m is 8 In embodiments of any Formulas described herein, m is 9. In embodiments of any Formulas described herein, m is 10. In embodiments of any Formulas described herein, m is 7.

[0184] In embodiments of any Formulas described herein, n is 6. In embodiments of any Formulas described herein, n is 7. In embodiments of any Formulas described herein, n is 8. In embodiments of any Formulas described herein, n is 9. In embodiments of any Formulas described herein, n is 10. In embodiments of any Formulas described herein, n is 7.

[0185] In embodiments, a compound of Formula (I) has a structure according to Formula (II),or a pharmaceutically acceptable salt thereof, wherein each of X1, X2, L1, R1, R2, R3, m, and n is independently according to any embodiment and combinations thereof as described herein.

[0186] In embodiments, a compound of Formula (I) has a structure according to Formula (III),or a pharmaceutically acceptable salt thereof, wherein each of X1, L1, R1, R2, R3, m, and n is independently according to any embodiment and combinations thereof as described herein.

[0187] In embodiments, a compound of Formula (I) has a structure according to Formula (IV),(IV),or a pharmaceutically acceptable salt thereof, wherein each of X1, L1, R1, R2, R3, m, and n is independently according to any embodiment and combinations thereof as described herein.

[0188] In embodiments, a compound of Formula (I) has a structure according to Formula (V),or a pharmaceutically acceptable salt thereof, wherein each of X1, L1, R1, R2, R3, m, and n is independently according to any embodiment and combinations thereof as described herein.

[0189] In various embodiments, a compound of Formula (I) has a structure according to any one of Formula (II), (III), (IV), or (V), wherein:L1is (CH2)2or (CH2)3;R1is CH2CH2CH2CH2CH3, CH2CH2CH2CH2CH2CH3, or CH2CH2CH2CH2CH2CH2CH3;R2is C4-C20alkyl or C4-C2o alkenyl;R3is C4-C2o alkyl or C4-C2o alkenyl;m is 6, 7, or 8; andn is 6, 7, or 8.

[0190] In various embodiments, a compound of Formula (I) has a structure according to any one of Formula (II), (III), (IV), or (V), wherein:m is 6, 7, or 8; andn is 6, 7, or 8.

[0191] In various embodiments, a compound of Formula (I) has a structure according to any one of Formula (II), (III), (IV), or (V), wherein:m is 7; andn is 7.

[0192] In various embodiments, a compound of Formula (I) has a structure according to any one of Formula (II), (III), (IV), or (V), wherein:R2is C4-C20 alkyl or C4-C20 alkenyl;R3is C4-C20 alkyl or C4-C20 alkenyl;m is 6, 7, or 8; andn is 6, 7, or 8.

[0193] In various embodiments, a compound of Formula (I) has a structure according to any one of Formula (II), (III), (IV), or (V), wherein:m is 6, 7, or 8; andn is 6, 7, or 8.

[0194] In various embodiments, a compound of Formula (I) has a structure according to any one of Formula (II), (III), (IV), or (V), wherein:m is 7; andn is 7.

[0195] In various embodiments, a compound is selected from the group consisting of Compounds 1-1 through 1-27 as depicted in the table below.

[0196] In various embodiments, a compound is selected from the group consisting of Compounds 1-1 through 1-11 as depicted in the table below.

[0197] In embodiments, a compound is Compound 1-1, or a pharmaceutically acceptable salt thereof.

[0198] In embodiments, a compound is Compound 1-2, or a pharmaceutically acceptable salt thereof.

[0199] In embodiments, a compound is Compound 1-3, or a pharmaceutically acceptable salt thereof.

[0200] In embodiments, a compound is Compound 1-4, or a pharmaceutically acceptable salt thereof.

[0201] In embodiments, a compound is Compound 1-5, or a pharmaceutically acceptable salt thereof.

[0202] In embodiments, a compound is Compound 1-6, or a pharmaceutically acceptable salt thereof.

[0203] In embodiments, a compound is Compound 1-7, or a pharmaceutically acceptable salt thereof.

[0204] In embodiments, a compound is Compound 1-8, or a pharmaceutically acceptable salt thereof.

[0205] In embodiments, a compound is Compound 1-9, or a pharmaceutically acceptable salt thereof.

[0206] In embodiments, a compound is Compound 1-10, or a pharmaceutically acceptable salt thereof.

[0207] In embodiments, a compound is Compound 1-11, or a pharmaceutically acceptable salt thereof.

[0208] In embodiments, a compound is Compound 1-12, or a pharmaceutically acceptable salt thereof.

[0209] In embodiments, a compound is Compound 1-13, or a pharmaceutically acceptable salt thereof.

[0210] In embodiments, a compound is Compound 1-14, or a pharmaceutically acceptable salt thereof.

[0211] In embodiments, a compound is Compound 1-15, or a pharmaceutically acceptable salt thereof.

[0212] In embodiments, a compound is Compound 1-16, or a pharmaceutically acceptable salt thereof.

[0213] In embodiments, a compound is Compound 1-17, or a pharmaceutically acceptable salt thereof.

[0214] In embodiments, a compound is Compound 1-18, or a pharmaceutically acceptable salt thereof.

[0215] In embodiments, a compound is Compound 1-19, or a pharmaceutically acceptable salt thereof.

[0216] In embodiments, a compound is Compound 1-20, or a pharmaceutically acceptable salt thereof.

[0217] In embodiments, a compound is Compound 1-21, or a pharmaceutically acceptable salt thereof.

[0218] In embodiments, a compound is Compound 1-22, or a pharmaceutically acceptable salt thereof.

[0219] In embodiments, a compound is Compound 1-23, or a pharmaceutically acceptable salt thereof.

[0220] In embodiments, a compound is Compound 1-24, or a pharmaceutically acceptable salt thereof.

[0221] In embodiments, a compound is Compound 1-25, or a pharmaceutically acceptable salt thereof.

[0222] In embodiments, a compound is Compound 1-26, or a pharmaceutically acceptable salt thereof.

[0223] In embodiments, a compound is Compound 1-27, or a pharmaceutically acceptable salt thereof.

[0224] In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form).

[0225] In some embodiments, the present disclosure encompasses the recognition that provided compounds display certain desirable characteristics, e.g., as compared to reference compounds or other known compounds. For example, in some embodiments, provided compounds may exhibit more potent delivery to various cell types in one or more experiments described herein, and / or have one or more other characteristics that make them more suitable for delivery of cargos such as therapeutic or prophylactic agents than other known compounds. Without wishing to be bound by any particular theory, the present disclosure encompasses the recognition that provided compounds with secondary alcohols incombination with the head group when formulated as part of a lipid nanoparticle may display certain more desirable characteristics (e.g., more potent delivery to various cell types in one or more experiments described herein) than corresponding compounds lacking the same features.Preparing Provided Compounds

[0226] The compounds of Formula (I) may generally be made by the processes described in the ensuing schemes and examples.Ionizable lipids

[0227] Compounds described herein (e.g., a compound of any one of Formulas (I)-(V) such as any one of compounds 1-1 through 1-27, or any pharmaceutically acceptable salt thereof) can be used as ionizable lipids.

[0228] Among other things, the present disclosure describes compositions, preparations, and / or lipid nanoparticles that comprise one or more ionizable lipids as described herein.

[0229] For example, in some embodiments, compositions, preparations, and / or lipid nanoparticles having an ionizable lipid that is at about 50 mol percent or less, based on total moles of components of the lipid nanoparticle, may be useful and / or critical to functional activity of lipid nanoparticles such as desired tropisms, stabilization, and drug delivery efficacy as described herein.

[0230] In some embodiments, an ionizable lipid may include an amine-containing group on the head group. In some embodiments, an ionizable lipid is or comprises a compound described herein. In some embodiments, an ionizable lipid is present in a lipid nanoparticle (LNP) preparation from about 30 mole percent to about 70 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present from about 33 mol percent to about 60 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present from about 34 mol percent to about 55 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present from about 33 mol percent to about 51 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present at about 34.7 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid ispresent at about 50 mole percent, based on total moles of components of the lipid nanoparticle.

[0231] Among other things, in some embodiments, a lipid nanoparticle composition comprises an ionizable lipid. In some embodiments, a lipid nanoparticle preparation comprises an ionizable lipid; a phospholipid; a conjugate-linker lipid; and a cholesterol. In some embodiments, an ionizable lipid is or comprises a structure according to a compound described herein. In some embodiments, an ionizable lipid is present in an LNP preparation from about 30 mole percent to about 70 mole percent, based on total moles of components of the lipid nanoparticle.Sterols

[0232] Among other things, the present disclosure describes compositions, preparations, and / or lipid nanoparticles that comprise one or more sterols as described herein.

[0233] In some embodiments, a sterol is a cholesterol, or a variant or derivative thereof. In some embodiments, a cholesterol is modified. In some embodiments, a cholesterol is an oxidized cholesterol. In some embodiments, a cholesterol is esterified cholesterol.Unmodified cholesterol can be acted upon by enzymes to form variants that are side-chain or ring oxidized. In some embodiments, a cholesterol can be oxidized on the beta-ring structure or on the hydrocarbon tail structure. In some embodiments, a sterol is a phytosterol.Exemplary sterols that are considered for use in the disclosed lipid nanoparticles include but are not limited to 25-hydroxycholesterol (25-OH), 20a-hydroxy cholesterol (20a-OH), 27-hydroxycholesterol, 6-keto-5a-hydroxycholesterol, 7-ketocholesterol, 7P-hydroxycholesterol, 7a-hydroxycholesterol, 7P-25-dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof. In some embodiments, a side-chain oxidized cholesterol can enhance cargo delivery relative to other cholesterol variants. In some embodiments, a cholesterol is an unmodified cholesterol.

[0234] In some embodiments, an LNP preparation comprises from about 20 mol percent to about 50 mol percent sterol. In some embodiments, an LNP preparation comprises about 38 mol percent sterol. In some embodiments, an LNP preparation comprises about 38.5 mol percent sterol. In some embodiments, an LNP preparation comprises about 33.8 mol percent cholesterol.Conjugate-linker lipids

[0235] Among other things, the present disclosure describes compositions, preparations, and / or lipid nanoparticles that comprise one or more conjugate-linker lipids as described herein.

[0236] In some embodiments, a conjugate-linker lipid is or comprises a polyethylene glycol (PEG)-lipid or PEG-modified lipid. In some embodiments, PEG or PEG-modified lipids may be alternately referred to as PEGylated lipids or PEG-lipids. Inclusion of a PEGylating lipid can be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. In some embodiments, the PEGylation is reversible in that the PEG moiety is gradually released in blood circulation. Exemplary PEG-lipids include but are not limited to PEG conjugated to saturated or unsaturated alkyl chains having a length of C6-C20. PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEG-DAG), PEG-modified dialkylglycerols, and mixtures thereof. For example, in some embodiments, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or a PEG-DSPE lipid.

[0237] In some embodiments, a conjugate-linker lipid comprises a polyethylene glycol lipid. In some embodiments, the conjugate-linker lipid comprises DiMystyrlGlycerol (DMG), 1,2-Dipalmitoyl-rac-glycerol, methoxypolyethylene Glycol (DPG-PEG), or 1,2-Distearoyl-rac-glycero-3 -methylpolyoxyethylene (DSG - PEG). In some embodiments, a conjugate-linker lipid has an average molecular mass from about 500 Da to about 5000 Da. In some embodiments, a conjugate-linker lipid has an average molecular mass of about 2000 Da. In some embodiments, a LNP preparation comprises from about 0 mol percent to about 5 mol percent conjugate-linker lipid. In some embodiments, an LNP preparation comprises about 1.5 mol percent conjugate-linker lipid. In some embodiments, an LNP preparation comprises about 3 mol percent conjugate-linker lipid.Phospholipids

[0238] Among other things, the present disclosure describes compositions, preparations, and / or lipid nanoparticles that comprise one or more phospholipids as described herein. In some embodiments, the present disclosure describes compositions, preparations, and / or lipid nanoparticles that comprise one or more (poly)unsaturated lipids.

[0239] In some embodiments, one or more phospholipids may assemble into one or more lipid bilayers. In some embodiments, one or more phospholipids may include a phospholipid moiety. In some embodiments, one or more phospholipids may include one or more fatty acid moieties. In some embodiments, one or more phospholipids may include a phospholipid moiety and one or more fatty acid moieties. In some embodiments, a phospholipid moiety includes but is not limited to phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. In some embodiments, a fatty acid moiety includes but is not limited to lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alphalinolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful in functionalizing a lipid bilayer of a lipid nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a lipid nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0240] Exemplary phospholipids include but are not limited to l,2-distearoyl- wglycero-3-phosphocholine (DSPC), l,2-dioleoyl- w-glycero-3 -phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sw-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sw-glycerophosphocholine (DMPC), l,2-dioleoyl- w-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sw-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sw-glycerophosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sw-glycero-3 -phosphocholine (POPC), l,2-di-O-octadecenyl-sw-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoy l- w-glycero-3-phosphocholine (OchemsPC), 1 -hexadecyl snglycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sw-glycero-3 -phosphocholine, l,2-diarachidonoyl-sw-glycero-3 -phosphocholine, l,2-didocosahexaenoyl-sw-glycero-3 -phosphocholine, l,2-diphytanoyl- w-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sw-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sw-glycero-3 -phosphoethanolamine, l,2-dilinolenoyl-sw-glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sw-glycero-3 -phosphoethanolamine, 1,2-di docosahexaenoyl -s / 7-glycero-3 -phosphoethanol amine, l,2-dioleoyl-sw-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), l-stearoyl-2 oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or combinations thereof. In some embodiments, a phospholipid is DSPC. In some embodiments, a phospholipid is DMPC.

[0241] In some embodiments, the phospholipid comprises l,2-dioleoyl- w-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl PE), l,2-distearoyl- w-glycero-3-phosphocholine (DSPC), cholesterol, l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl-DPPE), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), or a combination thereof.Diameter

[0242] Among other things, the present disclosure describes compositions, preparations, and / or lipid nanoparticles that have an average hydrodynamic diameter from about 30 to about 220 nm. In some embodiments, compositions, preparations, and / or lipid nanoparticles described herein have an average hydrodynamic diameter that is about 30 nm, 35 nm,40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, compositions, preparations, and / or lipid nanoparticles described herein have an average hydrodynamic diameter from between 50 nm to 200 nm.

[0243] In some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from about 30 to about 220 nm. In some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter that is about 30 nm,35 nm,40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from between 50 nm to 200 nm.Polydispersity

[0244] Among other things, the present disclosure describes compositions, preparations, and / or lipid nanoparticles that have a polydispersity index (PDI) of about 0.01 to about 0.3. In some embodiments, compositions, preparations, and / or lipid nanoparticles described herein have a PDI that is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, compositions, preparations, and / or lipid nanoparticles described herein have a PDI from about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

[0245] In some embodiments, lipid nanoparticles described herein have a PDI from about 0.01 to about 0.3. In some embodiments, lipid nanoparticles described herein have a PDI that is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, lipid nanoparticles described herein have a PDI from about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.Encapsulation efficiency

[0246] Among other things, the present disclosure describes compositions, preparations, and / or lipid nanoparticles, wherein encapsulation efficiency of provided compositions, preparations, and / or lipid nanoparticles is from about 80% to about 100%. In some embodiments, encapsulation efficiency of compositions, preparations, and / or lipid nanoparticles described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, encapsulation efficiency of compositions, preparations, and / or lipid nanoparticles described herein is from about 90% to about 100%, about 95% to about 100%, about 95% to about 98%, or about 95.5% to about 97.5%. In some embodiments, encapsulation efficiency ofcompositions, preparations, and / or lipid nanoparticles described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0247] In some embodiments, encapsulation efficiency of lipid nanoparticles described herein is from about 80% to about 100%. In some embodiments, encapsulation efficiency of lipid nanoparticles described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, encapsulation efficiency of lipid nanoparticles described herein is from about 90% to about 100%, about 95% to about 100%, about 95% to about 98%, or about 95.5% to about 97.5%. In some embodiments, encapsulation efficiency of lipid nanoparticles described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.pKa

[0248] Among other things, the present disclosure describes compositions, preparations, and / or lipid nanoparticles that have a pKa from about 5 to about 9. In some embodiments, compositions, preparations, and / or lipid nanoparticles described herein have a pKa that is about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints defined by any two of the aforementioned values. In some embodiments, compositions, preparations, and / or lipid nanoparticles described herein have a pKa that is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range having endpoints defined by any two of the aforementioned values.

[0249] In some embodiments, lipid nanoparticles described herein have a pKa from about 5 to about 9. In some embodiments, lipid nanoparticles described herein have a pKa that is about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints defined by any two of the aforementioned values. In some embodiments, lipid nanoparticles described herein have a pKa that is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range having endpoints defined by any two of the aforementioned values.Exemplary LNP Preparations

[0250] The present invention provides for compositions, preparations, and / or lipid nanoparticles that comprise lipid nanoparticles. In some embodiments, a lipid nanoparticle preparation comprises about 30 mole percent to about 70 mole percent ionizable lipid, about5 mole percent to about 25 mole percent phospholipid, about 25 mole percent to about 45 mole percent cholesterol, and about 0 mole percent to about 5 mole percent conjugate-linker lipid.

[0251] In some embodiments, a lipid nanoparticle preparation comprises about 45 mole percent ionizable lipid, about 9 mole percent phospholipid, about 44 mole percent cholesterol, and about 2 mole percent conjugate-linker lipid. In some embodiments, a lipid nanoparticle preparation comprises about 50 mole percent ionizable lipid, about 9 mole percent phospholipid, about 38 mole percent cholesterol, and about 3 mole percent conjugatelinker lipid.

[0252] In some embodiments, the lipid nanoparticle preparation comprises about 47.5 mole percent ionizable lipid, about 10 mole percent phospholipid, about 40 mole percent cholesterol, and about 2.5 mole percent conjugate-linker lipid.

[0253] In some embodiments, the lipid nanoparticle preparation comprises about 50 mole percent ionizable lipid, about 10 mole percent phospholipid, about 38.5 mole percent cholesterol, and about 1.5 mole percent conjugate-linker lipid.

[0254] In some embodiments, a lipid nanoparticle preparation comprises about 40 mole percent to about 60 mole percent ionizable lipid of any provided compound, about 5 mole percent to about 15 mole percent l-2-distearoyl-sn-glycero-3-phosphocholine, about 1 mole percent to about 5 mole percent C14PEG2000, and about 30 mole percent to about 47 mole percent cholesterol, based on the total moles of these four ingredients.

[0255] In some embodiments, a lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):mRNA from about 2:1 and 50:1. In some embodiments, an LNP preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):mRNA of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1. In some embodiments, a lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):mRNA of about 11.7:1 and 19:1.

[0256] In some embodiments, a lipid nanoparticle preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): siRNA from about 2:1 and 50:1. In some embodiments, an LNP preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): siRNA of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about24:l, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1. In some embodiments, a lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): siRNA of about 11.7:1 and 19:1.

[0257] In some embodiments, a lipid nanoparticle preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): NA from about 2:1 and 50:1. In some embodiments, an LNP preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): NA of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about24:l, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1. In some embodiments, a lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): NA of about 11.7:1 and 40:1.

[0258] In some embodiments, NA comprises a base editor and gNRA as described herein. In some embodiments, a mass ratio of base editorgNRA is 1: 1. In some embodiments, a mass ratio of base editorgNRA is 2:1. In some embodiments, a mass ratio of base editorgNRA is 3:1. In some embodiments, a mass ratio of base editorgNRA is 4:1. In some embodiments, a mass ratio of base editorgRNA is 5:1. In some embodiments, a mass ratio of base editorgNRA is 6:1. In some embodiments, A mass ratio of base editorgRNA is 7:1. In some embodiments, A mass ratio of base editorgNRA is 8:1. In some embodiments, a mass ratio of base editorgRNA is 9:1. In some embodiments, A mass ratio of base editorgRNA is 10:1. In some embodiments, a mass ratio of base editorgRNA is 1:2. In some embodiments,a mass ratio of base editorgRNA is 1:3. In some embodiments, a mass ratio of base editorgRNA is 1:4. In some embodiments, a mass ratio of base editorgRNA is 1:5. In some embodiments, a mass ratio of base editorgRNA is 1:6. In some embodiments, a mass ratio of base editorgRNA is 1:7. In some embodiments, a mass ratio of base editorgRNA is 1:8. In some embodiments, a mass ratio of base editorgRNA is 1:9. In some embodiments, a mass ratio of base editorgRNA is 1:10.

[0259] In some embodiments, provided the lipid nanoparticle particle product comprises anN: P ratio of about 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1. In some embodiments, provided the lipid nanoparticle particle product comprises an N: P ratio of about between 5: 1 to 7: 1. In some embodiments, provided the lipid nanoparticle particle product comprises an N: P ratio of about 6:1.Pharmaceutical compositions

[0260] The present invention provides for compositions, preparations, and / or lipid nanoparticles that comprise pharmaceutical compositions. Among other things, in some embodiments, pharmaceutical compositions comprise lipid nanoparticles and lipid nanoparticle preparations described herein. For example, in some embodiments, lipid nanoparticles and lipid nanoparticle preparations described herein can be formulated in whole or in part as pharmaceutical compositions.

[0261] In some embodiments, pharmaceutical compositions may include one or more lipid nanoparticle compositions described herein. For example, a pharmaceutical composition may comprise one or more lipid nanoparticle compositions including one or more different therapeutic and / or prophylactics including but not limited to one or more nucleic acids of different types or encode different agents. In some embodiments, a pharmaceutical composition comprises one or more pharmaceutically acceptable excipients or accessory ingredients including but not limited to a pharmaceutically acceptable carrier.

[0262] A pharmaceutical composition may be administered to a subject. In some embodiments, a pharmaceutical composition is administered as described herein. In some in vivo approaches, the lipid nanoparticle compositions disclosed herein are administered to a subject in a therapeutically effective amount as described herein.

[0263] In some embodiments, the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to devise an appropriate dosage level and dosing regimen using the pharmaceutical compositions described herein fortreatment of various conditions in various patients. For example, in some embodiments, a selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. In some embodiments, generally dosage levels of about 0.001 mg to about 5 mg of nucleic acid per kg of body weight are administered each dosage to mammals. More specifically, in some embodiments, a preferential dose for nucleic acids within the disclosed lipid nanoparticles is about 0.1 mg / kg to about 1.0 mg / kg. For the disclosed lipid nanoparticles, generally dosage levels of about 0.2 mg to about 100 mg of four components (ionizable lipid, cholesterol, conjugate-linker conjugate, and phospholipid) / kg of body weight are administered to mammals. More specifically, in some embodiments, a preferential dose of the disclosed lipid nanoparticles is about 0.5 mg / kg to about 5 mg / kg of the four components / kg of body weight.

[0264] In some embodiments, a pharmaceutical composition described herein is administered locally, for example by injection directly into a site to be treated. Typically, the injection causes an increased localized concentration of the composition which is greater than that which can be achieved by systemic administration. In some embodiments, a pharmaceutical composition described herein can be combined with a matrix as described herein to assist in creating an increased localized concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides out of the site to be treated.Preparations for parenteral administration

[0265] In some embodiments, the compositions, preparations, and / or lipid nanoparticles disclosed herein, including those containing lipid nanoparticles, are administered in an aqueous solution, by parenteral injection. In some embodiments, a preparation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of a lipid nanoparticle, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), Ph and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol,polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.Controlled delivery polymeric matrices

[0266] In some embodiments, the compositions, preparations, and / or lipid nanoparticles disclosed herein can also be administered in controlled release formulations. In some embodiments, controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (such as a rod, cylinder, film, disk) or injection (such as microparticles). In some embodiments, a matrix can be in the form of microparticles such as microspheres. In some embodiments, an agent is dispersed within a solid polymeric matrix or microcapsules. In some embodiments, a core is of a different material than a polymeric shell of any of the described compositions, preparations, and / or lipid nanoparticles. In some embodiments, a peptide is dispersed or suspended in a core, which may be liquid or solid in nature, of any of the described compositions, preparations, and / or lipid nanoparticles. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. In some embodiments, a polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

[0267] In some embodiments, non-biodegradable matrices are used for delivery of the described compositions, preparations, and / or lipid nanoparticles. In some embodiments, biodegradable matrices are used for delivery of the described compositions, preparations, and / or lipid nanoparticles. In some embodiments, biodegradable matrices are preferred. In some embodiments, biodegradable matrices comprise natural or synthetic polymers. In some embodiments, synthetic polymers are preferred due to the better characterization of degradation and release profiles. In some embodiments, a polymer is selected based on the period over which release is desired. In some embodiments, linear release may be most useful, although in others a pulse release or “bulk release” may provide more effective results. In some embodiments, a polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water) and can optionally be crosslinked with multivalent ions or polymers.Cargo

[0268] Among other things, the present invention provides for compositions, preparations, and / or lipid nanoparticles that comprise cargo as described herein. In some embodiments, the compositions, preparations, and / or lipid nanoparticles include a therapeutic or prophylactic agent for delivery to a subject. In some embodiments, a therapeutic or prophylactic agent is encapsulated by a lipid nanoparticle. In some embodiments, a lipid nanoparticle is loaded with one or more nucleic acids.Therapeutic and / or prophylactic agents

[0269] Cargo delivered via an LNP preparation may be a biologically active agent. In some embodiments, the cargo is or comprises one or more biologically active agents, such as mRNA, circular RNA (circRNA), guide RNA (gRNA), nucleic acid, RNA-guided DNA-binding agent, expression vector, template nucleic acid, antibody (e.g., monoclonal, chimeric, humanized, nanobody, and fragments thereof etc.), cholesterol, hormone, peptide, protein, chemotherapeutic and other types of antineoplastic agent, low molecular weight drug, vitamin, co-factor, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex forming oligonucleotide, antisense DNA or RNA composition, chimeric DNA: RNA composition, allozyme, aptamer, ribozyme, decoys and analogs thereof, plasmid and other types of vectors, and small nucleic acid molecule, RNAi agent, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA) and “self-replicating RNA” (encoding a replicase enzyme activity and capable of directing its own replication or amplification in vivo) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), and iRNA (asymmetrical interfering RNA). The above list of biologically active agents is exemplary only and is not intended to be limiting. Such compounds may be purified or partially purified, and may be naturally occurring or synthetic, and may be chemically modified.

[0270] Cargo delivered via an LNP preparation may be an RNA, such as an mRNA molecule or circular RNA (circRNA) encoding a protein of interest.

[0271] For example, in some embodiments, an mRNA for expressing a protein such as green fluorescent protein (GFP), an RNA-guided DNA-binding agent, or a Cas nuclease is described herein. LNP preparations that include a Cas nuclease mRNA, for example a Class2 Cas nuclease mRNA that allows for expression in a cell of a Class 2 Cas nuclease such as a Cas9 or Cpfl protein are provided. Further, cargo may contain one or more guide RNAs or nucleic acids encoding guide RNAs. A template nucleic acid, e.g., for repair or recombination, may also be included in the composition or a template nucleic acid may be used in the methods described herein. In some embodiments, cargo comprises an mRNA that encodes a Streptococcus pyogenes Cas9, optionally and an S. pyogenes gRNA. In some embodiments, cargo comprises an mRNA that encodes Neisseria meningitidis Cas9, optionally and an nme gRNA.

[0272] “mRNA” refers to a polynucleotide and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acyl ated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2’-methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2’-methoxy ribose residues, or a combination thereof. In general, mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). An mRNA can contain modified uridines at some or all of its uridine positions.

[0273] Circular RNA (circRNA) is a type of single-stranded RNA that forms a circular structure through covalent bonds, and which lack free 3' and 5' ends. In embodiments, a circRNA encodes a protein (e.g., a therapeutically effective protein). In embodiments, a circRNA encodes a protein (e.g., an antigen) that induces an adaptive immune response when administered to a patient (e.g., in methods described herein).CRISPR / Cas Cargo

[0274] In some embodiments, the disclosed compositions, preparations, and / or lipid nanoparticles comprise an mRNA encoding an RNA-guided DNA-binding agent, such as a Cas nuclease. In particular embodiments, the disclosed compositions, preparations, and / or lipid nanoparticles comprise an mRNA encoding a Class 2 Cas nuclease, such as S. pyogenes Cas9.

[0275] Cas nuclease”, as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents. Cas cleavases / nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the CaslO, Csml, or Cmr2 subunitthereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. As used herein, a “Class 2 Cas nuclease” is a single chain polypeptide with RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases / nickases (e.g., H840A, D10A, or N863 A variants), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase / nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, C2c3, HF Cas9 (e.g., N497A, R661 A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(l.l) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpfl protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpfl sequences of Zetsche are incorporated by reference in their entirety herein. See, e.g., Zetsche, Tables SI and S3. See, e.g, Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015), the contents of which are hereby incorporated in its entirety herein.

[0276] In some embodiments, cargo for an LNP preparation includes at least one guide RNA comprising guide sequences that direct an RNA-guided DNA binding agent, which can be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA. gRNA may guide the Cas nuclease or Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, a gRNA binds with and provides specificity of cleavage by a Class 2 Cas nuclease. In some embodiments, a gRNA and the Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex such as a CRISPR / Cas9 complex. In some embodiments, a CRISPR / Cas complex may be a Type-II CRISPR / Cas9 complex. In some embodiments, a CRISPR / Cas complex may be a Type-V CRISPR / Cas complex, such as a Cpfl / guide RNA complex. Cas nucleases and cognate gRNAs may be paired. gRNA scaffold structures that pair with each Class 2 Cas nuclease vary with the specific CRISPR / Cas system.

[0277] Guide RNA”, “gRNA”, and simply “guide” are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). Guide RNAs can include modified RNAs as described herein. The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. trRNA may be a naturally occurring sequence, or a trRNA sequence with modifications or variations compared to naturally occurring sequences.

[0278] Certain embodiments of the present disclosure also provide delivery of base editors (e.g., adenine base editors (“ABEs”) or cytosine base editors (“CBEs”)) using the LNPs compositions, preparations, nanoparticles, and / or nanomaterials described herein. Certain embodiments of the present disclosure provide delivery of ABEs using the LNPs compositions, preparations, nanoparticles, and / or nanomaterials described herein. Certain embodiments of the present disclosure provide delivery of CBEs using the LNPs compositions, preparations, nanoparticles, and / or nanomaterials described herein. ABEs and methods of their use are described, e.g., in U. S. Patent No. 10,113,163 and U. S. Patent Publication No. 2021 / 0130805, and CBEs and methods of their use are described, e.g., in U. S. Patent Nos. 10,167,457 and 9,840,699, the contents of each of which are hereby incorporated by reference in their entireties.Modified RNAs

[0279] In certain embodiments, the disclosed compositions, preparations, and / or lipid nanoparticles comprise modified nucleic acids, including modified RNAs.

[0280] Modified nucleosides or nucleotides can be present in an RNA, for example a gRNA or mRNA. A gRNA or mRNA comprising one or more modified nucleosides or nucleotides, for example, is called a “modified” RNA to describe the presence of one or more non-naturally and / or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified RNA is synthesized with a non-canonical nucleoside or nucleotide, here called “modified.”

[0281] Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2’ hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3’ end or 5’ end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3’ or 5’ cap modifications may comprise a sugar and / or backbone modification);and (vii) modification or replacement of the sugar (an exemplary sugar modification). Certain embodiments comprise a 5’ end modification to an mRNA, gRNA, or nucleic acid. Certain embodiments comprise a 3’ end modification to an mRNA, gRNA, or nucleic acid. A modified RNA can contain 5’ end and 3’ end modifications. A modified RNA can contain one or more modified residues at non-terminal locations. In certain embodiments, a gRNA includes at least one modified residue. In certain embodiments, an mRNA includes at least one modified residue.

[0282] Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the RNAs (e.g., mRNAs, gRNAs) described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.

[0283] Accordingly, in some embodiments, RNA or nucleic acids in the disclosed the disclosed compositions, preparations, and / or lipid nanoparticles comprise at least one modification which confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms “modification” and “modified” as such terms relate to the nucleic acids provided herein, include at least one alteration which preferably enhances stability and renders the RNA or nucleic acid more stable (e.g., resistant to nuclease digestion) than the wild-type or naturally occurring version of the RNA or nucleic acid. As used herein, the terms “stable” and “stability” as such terms relate to the nucleic acids of the present invention, and particularly with respect to the RNA, refer to increased or enhanced resistance to degradation by, for example nucleases (i.e., endonucleases or exonucleases) which are normally capable of degrading such RNA. Increased stability can include, for example, less sensitivity to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing the residence of such RNA in the target cell, tissue, subject and / or cytoplasm. The stabilized RNA molecules provided herein demonstrate longer half-lives relative to their naturally occurring,unmodified counterparts (e.g., the wild-type version of the mRNA). Also contemplated by the terms “modification” and “modified” as such terms related to the mRNA of the LNP preparations disclosed herein are alterations which improve or enhance translation of mRNA nucleic acids, including for example, the inclusion of sequences which function in the initiation of protein translation (e.g., the Kozac consensus sequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987), the contents of which are hereby incorporated by reference herein in its entirety).

[0284] In some embodiments, an RNA or nucleic acid of the disclosed compositions, preparations, and / or lipid nanoparticles disclosed herein have undergone a chemical or biological modification to render it more stable. Exemplary modifications to an RNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. The phrase “chemical modifications” as used herein, includes modifications which introduce chemistries which differ from those seen in naturally occurring RNA, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such RNA molecules).

[0285] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both ofthe linking oxygens. The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.mRNA

[0286] In some embodiments, the disclosed compositions, preparations, and / or lipid nanoparticles comprise an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease, or Class 2 Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease or Class 2 Cas nuclease, is provided, used, or administered. An mRNA may comprise one or more of a 5’ cap, a 5’ untranslated region (UTR), a 3’ UTRs, and a polyadenine tail. The mRNA may comprise a modified open reading frame, for example to encode a nuclear localization sequence or to use alternate codons to encode the protein.

[0287] mRNA in the disclosed compositions, preparations, and / or lipid nanoparticles may encode, for example, a secreted hormone, enzyme, receptor, polypeptide, peptide, or other protein of interest that is normally secreted. In one embodiment of the invention, the mRNA may optionally have chemical or biological modifications which, for example, improve the stability and / or half-life of such mRNA or which improve or otherwise facilitate protein production.

[0288] In addition, suitable modifications include alterations in one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable than the codon found in the wild-type version of the mRNA. For example, an inverse relationship between the stability of RNA and a higher-number cytidines (C’s) and / or uridines (U’s) residues has been demonstrated, and RNA devoid of C and U residues have been found to be stable to most RNAses (Heidenreich, et al. J Biol Chem 269, 2131-8 (1994), the disclosure of which is hereby incorporated by reference herein in its entirety). In some embodiments, the number of C and / or U residues in an mRNA sequence is reduced. In another embodiment, the number ofC and / or U residues is reduced by substitution of one codon encoding a particular amino acid for another codon encoding the same or a related amino acid. Contemplated modifications to the mRNA nucleic acids of the present invention also include the incorporation of pseudouridines. The incorporation of pseudouridines into the mRNA nucleic acids of the present invention may enhance stability and translational capacity, as well as diminishing immunogenicity in vivo. See, e.g., Kariko, K., et al., Molecular Therapy 16 (11): 1833-1840 (2008), the contents of which is hereby incorporated by reference herein in its entirety.Substitutions and modifications to the mRNA of the present invention may be performed by methods readily known to one or ordinary skill in the art.

[0289] The term modification also includes, for example, the incorporation of nonnucleotide linkages or modified nucleotides into the mRNA sequences of the present invention (e.g., modifications to one or both the 3’ and 5’ ends of an mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail), the alteration of the 3’ UTR or the 5’ UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an mRNA molecule (e.g., which form secondary structures).Template nucleic acid

[0290] The compositions, preparations, and / or lipid nanoparticles and methods disclosed herein may include a template nucleic acid. A template may be used to alter or insert a nucleic acid sequence at or near a target site for an RNA-guided DNA binding protein such as a Cas nuclease, e.g., a Class 2 Cas nuclease. In some embodiments, the methods comprise introducing a template to the cell. In some embodiments, a single template may be provided. In some embodiments, two or more templates may be provided such that editing may occur at two or more target sites. For example, different templates may be provided to edit a single gene in a cell, or two different genes in a cell.Methods of manufacturing LNPs

[0291] Methods of manufacturing lipid nanoparticles are known in the art. In some embodiments, the described compositions, preparations, and / or lipid nanoparticles are manufactured using microfluidics. For instance, exemplary methods of using microfluidics to form lipid nanoparticles are described by Leung, A. K. K, et al., J Phys Chem, 116: 18440-18450 (2012), Chen, D., et al., J Am Chem Soc, 134:6947-6951 (2012), and Belliveau, N. M.,et al., Molecular Therapy- Nucleic Acids, 1: e37 (2012), the disclosures of which are hereby incorporated by reference in their entireties.

[0292] Briefly, a cargo, such as a cargo described herein, is prepared in a first buffer solution. The other lipid nanoparticle components (such as ionizable lipid, conjugate-linker lipids, cholesterol, and phospholipid) are prepared in a second buffer solution. In some embodiments, a syringe pump introduces the two solutions into a microfluidic device. The two solutions come into contact within the microfluidic device to form lipid nanoparticles encapsulating the cargo.Methods of use

[0293] Among other things, the present disclosure describes methods of using compositions, preparations, and / or lipid nanoparticles described herein. For example, in some embodiments, the present disclosure describes methods of using compositions, preparations, and / or lipid nanoparticles to deliver cargo to specific cells, tissues, or organs, as described herein. As another example, in some embodiments, the present disclosure describes methods of treatment and / or delaying and / or arresting progression of a disease or disorder using compositions, preparations, and / or lipid nanoparticles as described herein. In some embodiments, compositions, preparations, and / or lipid nanoparticles described herein are for use in medicine.

[0294] In some embodiments, compositions, preparations, and / or lipid nanoparticles described herein deliver therapeutic or prophylactic agents to specific cells or organs in a subject in need thereof. In some embodiments, the compositions, preparations, and / or lipid nanoparticles deliver therapeutic or prophylactic agents to specific cells or organs in a subject in need thereof in the absence of a targeting ligand. In some embodiments, the compositions, preparations, and / or lipid nanoparticles are useful to treat or prevent diseases in a subject in need thereof.Methods of delivering cargo to cells, tissue, or organs

[0295] Among other things, in some embodiments, compositions, preparations, and / or lipid nanoparticles disclosed herein target a particular type or class of cells (e.g., cells of a particular organ or system thereof), tissues, and / organs. In some embodiments, the present disclosure provides methods of delivering one or more cargos described herein to a subject in need thereof. In some embodiments, such methods comprise in vivo and / or in vitro delivery.In some embodiments, such methods comprise in vivo delivery. In some embodiments, such methods comprise in vitro delivery. In some embodiments, the present disclosure provides for methods of delivering one or more therapeutic and / or prophylactic nucleic acids to a subject in need thereof are described herein.

[0296] In some embodiments, a composition, preparation, and / or lipid nanoparticle comprises a therapeutic and / or prophylactic of interest that may be specifically delivered to liver cells in the subject. Exemplary liver cells include but are not limited to hepatocytes.

[0297] In some embodiments, a composition, preparation, and / or lipid nanoparticle comprises a therapeutic and / or prophylactic of interest that may be specifically delivered to spleen cells in the subject. Exemplary spleen cells include but are not limited to splenic monocytes, splenic T cells, splenic memory B cells, or splenic B cells.

[0298] In some embodiments, a composition, preparation, and / or lipid nanoparticle comprises a therapeutic and / or prophylactic of interest that may be specifically delivered to bone marrow cells in the subject. Exemplary bone marrow cells include but are not limited to bone marrow monocytes, bone marrow B cells, bone marrow memory B cells, or bone marrow T cells.

[0299] In some embodiments, a composition, preparation, and / or lipid nanoparticle comprises a therapeutic and / or prophylactic of interest that may be specifically delivered to immune cells in the subject. Exemplary immune cells include but are not limited to CD8+, CD4+, or CD8+CD4+ cells.

[0300] In some embodiments, a composition, preparation, nanoparticle, and / or nanomaterial comprises a therapeutic and / or prophylactic of interest that may be specifically delivered to hematopoietic stem cells in the subject. Unless otherwise specified, it is understood that the terms “hematopoietic stem cells (HSCs)” and “hematopoietic stem and progenitor cells (HSPCs)” are used interchangeably in the present disclosure.

[0301] In some embodiments, the lipid nanoparticles can be formulated to be delivered in the absence of a targeting ligand to a mammalian liver hepatocyte, liver immune cells, spleen T cells, or lung endothelial cells. Specific delivery to a particular class or type of cells indicates that a higher proportion of lipid nanoparticles are delivered to target type or class of cells. In some embodiments, specific delivery may result in a greater than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold compared to delivery using a conventional nanoparticle system (e.g., MC3 -containing LNPs).Methods of producing a polypeptide

[0302] Among other things, in some embodiments, methods of using compositions, preparations, and / or lipid nanoparticles disclosed herein are used for methods of producing a polypeptide. Among other things, in some embodiments, lipid nanoparticles described herein can be used for producing a polypeptide in a target cell in a subject in need thereof. For example, in some embodiments, lipid nanoparticles described herein can be used for producing a polypeptide in a target cell in a subject in need thereof. In some embodiments, compositions, preparations, and / or lipid nanoparticles disclosed herein comprise one or more nucleic sequences to be delivered to a cell.

[0303] In some embodiments, one or more nucleic acids are expressed in a cell. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.Methods of gene regulation

[0304] Among other things, in some embodiments, methods of using compositions, preparations, and / or lipid nanoparticles disclosed herein are used for gene regulation. Among other things, in some embodiments, lipid nanoparticles described herein can be used for reducing and / or increasing gene expression in a target cell in a subject in need thereof. For example, in some embodiments, lipid nanoparticles described herein can deliver one or more nucleic acids to a target cell in the subject without a targeting ligand. In some embodiments, a nucleic acid is an inhibitor nucleic acid. In some embodiments, an inhibitory nucleic acid is an siRNA. In some embodiments, a nucleic acid is a nucleic acid described herein. As another example, in some embodiments, lipid nanoparticles described herein can deliver cargo to a target cell in the subject without a targeting ligand. In some embodiments, cargo is any cargo described herein.

[0305] Among other things, in some embodiments, methods of using compositions, preparations, and / or lipid nanoparticles disclosed herein for editing of a gene in a cell in a subject in need thereof.

[0306] In some embodiments, a cell that is targeted for gene regulation is an immune cell. The immune cell can be a T cell, such as CD8+ T cell, CD4+ T cell, or T regulatory cell. Other exemplary immune cells for gene editing include but are not limited to macrophages, dendritic cells, B cells or natural killer cells. In some embodiments, the cell that is targeted for gene regulation in a hepatocyte.

[0307] Exemplary genes that can be targeted include but are not limited to T cell receptors, B cell receptors, CTLA4, PD1, FOXO1, FOXO3, AKTs, CCR5, CXCR4, LAG3, TIM3, Killer immunoglobulin-like receptors, GITR, BTLA, LFA-4, T4, LFA-1, Bp35, CD27L receptor, TNFRSF8, TNFRSF5, CD47, CD52, ICAM-1, LFA-3, L-selectin, Ki-24, MB1, B7, B70, M-CSFR, TNFR-II, IL-7R, OX-40, CD137, CD137L, CD30L, CD40L, FasL, TRAIL, CD257, LIGHT, TRAIL-R1, TRAILR2, TRAIL-R4, TWEAK -R, TNFR, BCMA, B7DC, BTLA, B7-H1, B7-H2, B7-H3, ICOS, VEGFR2, NKG2D, JAG1, GITR, CD4, CCR2, GATA-3, MTORC1, MTORC2, RAPTOR, GATOR, FOXP3, NF AT, IL2R, and IL7. Other exemplary genes that can be targeted include but are not limited to OCT, G6Pase, Mut, PCCA, PCCB, PCSK9, ALAS1, and PAH. Exemplary tumor-associated antigens that can be recognized by T cells and are contemplated for targeting, include but are not limited to MAGE1, MAGE3, MAGE6, BAGE, GAGE, NYESO-1, MARTl / Melan A, MC1R, GP100, tyrosinase, TRP-1, TRP-2, PSA, CEA, Cyp-B, Her2 / Neu, Htert, MUC1, PRAME, WT1, RAS, CDK-4, MUM-1, KRAS, MSLN and P-catenin.Methods of inducing an adaptive immune response

[0308] Among other things, in some embodiments, methods of using compositions, preparations, and / or lipid nanoparticles disclosed herein are used for methods of inducing an adaptive immune response. Among other things, in some embodiments, lipid nanoparticles described herein can be used for inducing an immune response in vivo by administering to the subject a lipid nanoparticle comprising a nucleic acid therapeutic.

[0309] Accordingly, in some embodiments, lipid nanoparticles disclosed herein comprise one or more nucleic sequences to be delivered to a cell. In embodiments, a nucleic acid is an RNA. In embodiments, a nucleic acid is an mRNA or a circular RNA. In embodiments, a nucleic acid is an mRNA (e.g., an mRNA encoding an antigen). In embodiments, a nucleicacid is circular RNA (circRNA) such as a circular RNA encoding an antigen. In embodiments, a nucleic acid (e.g., mRNA or circRNA) encodes a protein that is an antigen (e.g., an antigen from an infectious agent or an antigen associated with cancer). In embodiments, an antigen is from an infectious agent (e.g., a virus). In embodiments, an antigen is associated with cancer (e.g., associated with a cancer of a subject or identified from a cancer cell of a subject).

[0310] In embodiments, compositions, preparations, and / or lipid nanoparticles (e.g., a lipid nanoparticle comprising a cargo or therapeutic agent that is circular RNA) disclosed herein are suitable for use in vaccine compositions. In further embodiments, a vaccine comprises compositions, preparations, and / or lipid nanoparticles disclosed herein.

[0311] In another aspect, the present invention provides a method of delivering a vaccine in vivo comprising administering to a subject a vaccine or vaccine composition comprising a lipid nanoparticle described herein (e.g., a lipid nanoparticle comprising a cargo or therapeutic agent that is circular RNA). In some embodiments, a vaccine or vaccine composition is administered subcutaneously, intradermally, intramuscularly, or intravenously.Subjects to be treated

[0312] In some embodiments, subjects who are treated are mammals experiencing cancer, autoimmune disease, infections disease, organ transplant, organ failure, protein deficiency, or a combination thereof. In some embodiments, a subject is a human. In some embodiments, methods described herein may cause hepatocytes to translate certain proteins. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a hepatocyte. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a splenic T cell. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a splenic B cell. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a splenic monocyte. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a bone marrow cell.

[0313] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0314] While the invention has been particularly shown and described with reference to specific preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.Exemplification

[0315] The present disclosure exemplifies compositions, preparations, formulations, and / or lipid nanoparticles described herein. The present disclosure also exemplifies methods of preparing, characterizing, and validating compositions, preparations, formulations, and / or lipid nanoparticles described herein.

[0316] The Exemplification provides exemplary materials and methods of preparing, characterizing, and validating certain compositions, preparations, and / or lipid nanoparticles described herein. Detailed description of the present Example can be found at Example 1 of WO 2022 / 140252, herein incorporated by reference.

[0317] For example, provided herein are exemplary materials and methods of preparing, characterizing, and validating ionizable lipids as described herein. As described in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It will be appreciated that, although the general methods depict the synthesis of certain compounds of the present disclosure, the following general methods and other methods known to one of ordinary skill in the art can be applied to all compounds and subclasses and species of each of these compounds, as described herein.List of AbbreviationsACN acetonitrileCDCl₃ deuterated chloroformDCC N, N'-dicyclohexylcarbodiimideDCM dichloromethaneDIPEA N, N-diisopropylethylamineDMAP 4-dimethylaminopyridineDMF N, N-dimethylformamideDMSO dimethyl sulfoxideDMSO-d₆ deuterated dimethyl sulfoxideDSPC distearoylphosphatidylcholineEDC HCl N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride eqv equivalent(s)EtI ethyl iodideEtOAc ethyl acetateEtOH ethanolh hour(s)LAH lithium aluminum hydridem multipletMeOH methanolmin minute(s)MS mass spectrometerNMR nuclear magnetic resonanceo.d. outside diameterRt retention times singletSM starting materialt tripletTBAF tetrabutylammonium fluorideTBDMSC1 tert-butyldimethylsilyl chlorideTEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxylTHF tetrahydrofuranTLC thin-layer chromatographyUPLC ultra performance liquid chromatographyw.r.t. with respect tow / w weight per weight

[0318] For example, provided herein are representative methods of synthesizing exemplary compounds 1-1 to 1-27.General:

[0319] 1H NMR: NMR spectra were recorded in CDCh and DMSO-de solution in 5 mm o.d. tubes [Wilma NMR tubes (Sigma- Aldrich), 5mm Thin Wall, 7” Length] at 27 °C and were collected on Bruker Advance NMRS-400 at 400 MHz forXH. The chemical shifts (8) in DMSO-de are relative to dimethyl sulfoxide (DMSO = 2.50 ppm) and expressed in ppm. Thechemical shifts in CDCI3 are relative to tetramethylsilane (TMS = 0.00 ppm) and expressed in ppm.General UPLC Methods:Method-A (5 min run)

[0320] Column: XTERRA RP 18 (4.6 x 50 mm), 5 pm, (mobile phase: initially 50%[0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN: THF]; then to 2% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN: THF] in 2.65 min, held this mobile phase composition up to 3.75 min, and finally back to initial condition, i.e., 50%[0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN: THF] in 4.90 min, held this mobile phase composition up to 5.10 min. Flow =1.2 mL / min.Method-B (12 min run)

[0321] Column: XTERRA RP 18 (4.6 x 50 mm), 5 pm, (mobile phase: initially 80%[0.1% HCOOH in water] and 20% [0.1% HCOOH in (70:30) ACN: THF]; held this initial condition for 0.75 min; then to 65% [0.1% HCOOH in water] and 35% [0.1% HCOOH in (70:30) ACN: THF] in 3.0 min, then to 2% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN: THF] in 6.0 min, held this mobile phase composition up to 9.0 min, and finally back to initial condition, i.e., 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH in (70:30) ACN: THF] in 11.00 min, held this mobile phase composition up to 12.10 min. Flow =1.2 mL / min.HPLC Method-A: (30 min run)

[0322] Reversed phase HPLC was carried out on Agilent 1200 series instrument with Charged Aerosol Detector (CAD) as well as Diode Array Detector (DAD). Column:ACQUITY BEH C18 (1.7 pm, 150 x 2.1 mm) with a flow rate of 0.2 mL / min. Two mobile phases were used. Mobile phase A: 0.1% NH3 in MeOH; mobile phase B: 0.1% NH3 in ACN, and they were employed to run a gradient conditions: Mobile phase from A: 98% [0.1% NH3 in MeOH] and mobile phase B: 0.2% [0.1% NH3 in ACN] for 0.01 min, Mobile phase from 98% [0.1% NH3in MeOH] and 0.2% [0.1% NH3 in ACN] for 20.0 min, 0.0% [0.1% NH3 in MeOH] and 100% [0.1% NH3 in ACN] for 21.0 min held in this composition up to 24.0 min, then returned to initial composition in 26.0 min and held this condition up to 30.0 min (total run time 30.0 min). Diluent: Acetonitrile. An injection volume of 1.0 pl was used.Synthesis of Starting MaterialsSynthesis of 2-(Aminoethyl)(ethyl)methylamine (5-(II)):

[0323] To a stirred solution of tert-butyl N-{2-[(methyl)amino]ethyl}carbamate (la) (10 g, 57.39 mmol) in DMF (40 mL) was added CS2CO3 (22.4 g, 68.87 mmol) and was stirred for 5 min at 25 °C. Then was added EtI (8.9 g, 57.39 mmol) diluted in DMF (5 mL) dropwise to the reaction mixture and stirred at 25 °C for 2 h. Upon completion, cold water was added to the reaction mixture, extracted with ethyl acetate (2 x 100 mL). The organic layer was washed with water, followed by brine, and concentrated under reduced pressure. The obtained crude material was purified by combiflash chromatography, eluted with 5% MeOH-DCM to afford tert-butyl N-{2-[ethyl(methyl)amino]ethyl}carbamate (2a) (9 g, 77% yield) as colorless liquid.

[0324] 'H NMR (400 MHz, CDCI3) 6 1.1 (t, J = 7.6 Hz, 3H), 1.43 (s, 9H), 2.3 (s, 3H), 2.50 - 2.56 (m, 4H), 3.26 (t, 2H).

[0325] To a stirred solution of (2a) (9.0 g, 51.65 mmol) in dioxane (20.0 mL) was added HC1 in dioxane (2 M in dioxane, 25 mL, 103.3 mmol) at 0 °C and stirred the reaction mixture at 25 °C for 1 h. After completion, the excess solvent was decanted out and the sticky compound was washed with diethyl ether and dried under rotary evaporator. The HC1 salt of crude material was dissolved in MeOH (20 mL) and neutralized with dropwise addition of ammonia in MeOH (7 M NH3 in MeOH). White precipitation formed and was filtered, and the filtrate was evaporated under reduced pressure to afford (5-(II)) (5.0 g, 94% yield), which may be directly used without purification.

[0326] 1H NMR (400 MHz, DMSO-d6) 8 1.23-1.26 (t, J= 7.6 Hz, 3H), 2.75 (s, 3H), 3.8 -3.98 (m, 4H).Syntheses of 4-({[2-(Pyrrolidin-l-yl)ethyl]carbamoyl}oxy)decanoic acid (8-1):

[0327] To a stirred solution of 5-hexyldihydrofuran-2(3H)-one (1) (8 g, 47.00 mmol) in THF (80 mL) at 0 °C was added 2.4 M LAH in THF (39.1 mL, 94.00 mmol) portion-wise ina period of 10 min. Next, the reaction mixture was allowed to stir at 25 °C for 12 h. After completion, the reaction was quenched with Na2SC>4 IOH2O under a cooling condition and filtered through celite to remove solid precipitate and washed with 10% MeOH in DCM (50 mL). The organic layer was evaporated under reduced pressure to afford decane- 1,4-diol (2) (8 g, crude) as colorless liquid.

[0328] 1H NMR (400 MHz, CDCI3) 60.87 (t, J= 5.9 Hz, 3H), 1.23-1.27 (m, 8H), 1.39-1.49 (m, 4H), 1.61-1.7(m, 6H), 3.57-3.71 (m, 3H).

[0329] To a stirred solution of (2) (8 g, 45.90 mmol) In DCM (80 mL) was added imidazole (4.7 g, 68.55 mmol) and TBDMSC1 (10.4 g, 68.55 mmol) portion wise at 0 °C. The reaction mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (2 x 60 mL). The organic layer was dried over anhydrous Na2SC>4 and evaporated under reduced pressure to afford l-((tert-butyldimethylsilyl)oxy)decan-4-ol (3) (8.6 g, 63%, two steps yield) as colorless liquid.

[0330] 1H NMR (400 MHz, CDCI3) 60.05 (s, 6H), 0.88 (m, 12H), 1.27 (m, 7H), 1.42 (m, 4H), 1.62 (m, 3H), 3.59-3.65 (m, 3H).

[0331] To a stirred solution of (3) (1.0 eqv) in DCM (20 mL) at 0 °C was added 4-nitrophenyl chloroformate (4) (2.0 eqv) followed by pyridine (2.0 eqv) and DMAP (0.2 eqv) and stirred at 25 °C for 2 h. Then, l-(2-aminoethyl)pyrrolidine (5-1) (2 eqv) was added to this reaction mixture, followed by DIPEA (3 eqv) and further was stirred for 12 h. The reaction mixture was diluted with dichloromethane (40 mL) and washed with 1 M aqueous sodium carbonate (2 x 50 mL), water (50 mL), and brine. The organic layer was dried over anhydrous Na2SC>4. The resulting dichloromethane layer was concentrated and purified by flash column chromatography and eluted with 5% MeOH-DCM to afford l-((tert-butyldimethylsilyl)oxy)decan-4-yl (2-(pyrrolidin-l-yl)ethyl)carbamate (6-1) as faint yellow liquid.

[0332] Yield: 4.3 g, 72%.

[0333] UPLC: Purity is 100%; Rt 0.34 min MS calculated: 429.34 [M+H], MS found: 429.71 [M+H],

[0334] To a stirred solution of (6-1) (1.0 eqv) in THF (50 mL) was added 1 M solution of TBAF in THF (2.0 eqv) at 0 °C and stirred at 25 °C for 3 h. The reaction progress was checked with TLC, which showed consumption of (6-(I)). The reaction was quenched bywater (100 mL) and extracted with 10% MeOH-DCM (2 x 50 mL). The organic layers were washed with brine, dried over anhydrous Na2SC>4 and concentrated under reduced pressure. The crude mass was purified by combiflash chromatography and eluted with 10% MeOH-DCM to afford 1 -hydroxy decan-4-yl (2-(pyrrolidin-l-yl)ethyl)carbamate (7-1).

[0335] Yield: 2.5 g, 79%.

[0336] UPLC: Purity is 100%; Rt 0.34 min MS calculated: 315.26 [M+H], MS found: 315.61 [M+H],

[0337] To a solution of (7-1) (1 eqv), dissolved in Acetone (30 mL), cooled in an ice-water bath, was added Jone’s reagent (1.2 eqv, 2 M solution in H2O), dropwise. The cooling bath was removed, and stirring was continued for 16 h at 25 °C. Upon completion, isopropyl alcohol was added, and the mixture was filtered. The filtrate was concentrated under reduced pressure. The crude mass was diluted with water and the pH of the aqueous layer was adjusted to 6, and the mixture was extracted with 5% MeOH-DCM (3 x 100 mL). The combined organic layers were dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford 4-({[2-(pyrrolidin-l-yl)ethyl]carbamoyl}oxy)decanoic acid (8-(I)), which may be used without further purification.

[0338] Yield: 2.8 g, 92%.

[0339] UPLC-MS: Method-A was performed for UPLC. Purity is 100%; Rt 0.34 min., MS calculated: 329.24 [M+H], MS found 329.41 [M+H],Synthesis of 4-[({2-[Ethyl(methyl)amino]ethyl}carbamoyl)oxy]decanoic acid (8-II):

[0340] With the substitution of the compound (2-aminoethyl)(ethyl)methylamine (5-II) for the compound (5-1), the compound (8-II) was prepared using the same procedures used to prepare (8-1) above.

[0341] UPLC: Purity is 100%; Rt 0.34 min MS calculated: 317.23 [M+H], MS found: 317.49 [M+H],Syntheses of 4-([{(l-Ethylpiperidin-3-yl)methoxy}carbonyl]oxy)decanoic acid (12):

[0342] To a stirred solution of l-((tert-butyldimethylsilyl)oxy)decan-4-ol (3) (2 g, 6.9 mmol) in DCM (20.0 ml) at 0 °C was added 4-nitrophenyl chloroformate (4) (2.7 g, 13.8 mmol), followed by pyridine (1.09 ml, 13.8 mmol) and DMAP (0.23 g, 1.38 mmol) and stirred at 25 °C for 2 h. (1-ethylpiperi din-3 -yl)m ethanol (9) (1.19 g, 8.32 mmol) was added to this reaction mixture, followed by DIPEA (2.68 g, 20.79 mmol), and again stirred for 12 h. After completion, the reaction mixture was diluted with DCM and quenched with saturated sodium bicarbonate solution, then extracted with DCM and dried over anhydrous sodium sulfate and evaporated under reduced pressure, to get crude product which was purified using flash chromatography eluting 40% EtOAc-hexane to afford l-((tert-butyldimethylsilyl)oxy)decan-4-yl((l-ethylpiperidin-3-yl)methyl)carbonate (10) (1.2 g, 37% yield) as faint yellow liquid.

[0343] 'H NMR (400 MHz, CDC13) 80.86 (t, J= 5.9 Hz, 6H), 0.98-1.02 (t, 3H), 1.15 (m, 6H), 1.25 (m, 19H) 1.62-1.65 (m, 25H). 1.75 (m, 8H), 2.03 (m, 3H), 2.17 (m, 2H), 2.50-2.56 (t, 4H), 3.34 - 3.44 (m, 2H), 3.64 (t, 4H), 3.93-3.96 (m, 2H), 4.03-4.13 (m, 2H), 4.71-4.75 (t, 2H).

[0344] To a stirred solution of (10) (1.5 g, 3.28 mmol) in THF (5.0 mL) at 0 °C was added 1 M solution of TBAF in THF (4.26 mL, 4.26 mmol) dropwise and stirred at 25 °C for 3 h. After completion reaction mixture was concentrated under reduced pressure and purified with flash column chromatography using 10% MeOH-DCM to afford 4-({[(l-ethylpiperidin-3-yl)methoxy]carbonyl}oxy)decan-l-ol (11) (800 mg, 71% yield) as faint yellow liquid.

[0345] UPLC: Purity is 100%; Rt 0.34 min MS calculated: 317.23 [M+H], MS found: 317.49 [M+H],

[0346] To a stirred solution of (11) (500 mg, 1.457 mmol) in acetone (5.0 mL) added CrOs H2SO4 (1.45 mL, 2.91 mmol) dropwise at 0 °C and stirred at 25 °C for 12 h. After completion reaction mixture was quenched with 1-propanol (1.0 mL), filtered through celite and filtrate was evaporated under reduced pressure. The crude mass was then diluted withwater and pH was adjusted to ~8 using sodium bicarbonate. Further, this reaction mixture was acidified to pH ~4 using 1.0 N HC1 solution, then water layer was extracted with 10% MeOH: DCM (3 x 30 mL). Combined organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to afford 4-([{(l-ethylpiperidin-3-yl)methoxy}carbonyl]oxy)decanoic acid (12) (400 mg, crude), which was used for next step without further purification.

[0347] UPLC: Purity is 91%; Rt 0.35 min MS calculated: 358.25 [M+H], MS found: 358.48 [M+H],Synthesis of 4-[{4-(Dimethylamino)butanoyl}oxy]decanoic acid (16):

[0348] To a stirred solution of 4-(dimethylamino)butyric acid (13) ( 2.27 g, 17.33 mmol) in DCM (40 mL) at 0 °C, was added EDC HC1 (4.98 g, 25.99 mmol) followed by DMAP (0.63 g, 5.19 mmol) and stirred for 15 min then added DIPEA (9.33 mL, 51.99 mmol) and (3) (5 g, 17.33 mmol) in DCM (5 mL) and further was stirred at 25 °C for 8 h. After completion, the reaction mixture was diluted with DCM and quenched with saturated sodium bicarbonate then extracted with DCM and dried over anhydrous, sodium sulfate and evaporated under reduced pressure. The crude material was purified with flash column chromatography, eluted with 30% EtOAc: hexane to afford l-{(tert-butyldimethylsilyl)oxy}decan-4-yl 4-(dimethylamino)butanoate (14) (3.9 g, 55% yield) as colorless liquid.

[0349] UPLC: Method-A was performed for UPLC; purity is 99.21%; Rt 0.52 min MS calculated: 402.33 [M+H], MS found: 402.62 [M+H],

[0350] To a stirred solution of (14) (3.9 g, 9.70 mmol) in THF (5.0 mL) at 0 °C was added 1 M solution of TBAF in THF (12.6 mL, 12.6 mmol) dropwise and stirred at 60 °C for 4 h. After completion, reaction mixture was concentrated under reduced pressure and purified with Flash column chromatography eluting 10% MeOH: DCM to afford 1 -hydroxy decan-4-yl 4-(dimethylamino)butanoate (15) (2.2 g, 78% yield) as faint yellow liquid.

[0351] UPLC: Method-A was performed for UPLC, Purity is 100.0 %; Rt 0.34 min MS calculated: 288.25 [M+H], MS found: 288.57 [M+H],

[0352] To a stirred solution of (15) (1.3 g, 4.52 mmol) in acetone (5 mL) added CrCh H2SO4 (11.3 mL, 11.3 mmol) dropwise at 0 °C and stirred at 25 °C for 12 h. The reaction was quenched by isopropanol (2 mL) and extracted with 10% MeOH-DCM (2 x 50 mL). The organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude mass was purified by combi-flash chromatography, eluted with 10% MeOH-DCM to afford 4-[{4-(dimethylamino)butanoyl}oxy]decanoic acid (16) (600 mg, 45% yield), which was used in next step without further purification. Due to loss of compound during the work-up process, the yield may be low.Synthesis of 3-Pentyloctan-l-ol (21-III):

[0353] To a stirred solution of tri ethyl phosphonoacetate (17) (3 g, 13.38 mmol) in THF (30 mL) was added NaH (60% dispersion in mineral oil) (535 mg, 13.38 mmol) at 0 °C portion wise for a period of 20 min and stirred at 25 °C for 30 min. Reaction mixture was cooled to 0 °C and was added dipentyl ketone (18) (2.96 g, 17.39 mmol) at 0 °C. The reaction mixture was gradually warmed to 25 °C and heated at 70 °C for 16 h. Upon completion, the reaction mixture was cooled and quenched with cold water (60 mL) and extracted with ethyl acetate (3 x 100 mL). Combined organic layer was washed with brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by combiflash column chromatography using 10% EtOAc-hexane to afford ethyl-3-pentyloct-2-enoate (19) (2.2 g, 68% yield) as colorless liquid.

[0354] 1H NMR (400 MHz, CDCI3) 60.87 - 0.89 (m, 6H), 1.19 - 1.32 (m, 15H), 1.39 -1.58 (m, 6H), 2.11 (t, J = 7.72 Hz, 2H), 2.36 (t, J = 7.56 Hz, 2H), 2.56 (t, J = 8.04 Hz, 2H), 4.07 -4.14 (m, 2H), 5.59 (s, 1H).

[0355] To a stirred solution of (19) (2.2 g, 9.15 mmol) in EtOH (30 mL) was added Pd(OH)2 (50% (w / w), 257 mg, 1.83 mmol) portion wise at 25 °C and stirred for 2 h under H2 atmosphere. Upon completion the reaction mixture was filtered through celite bed, washed with EtOH (60 mL) and concentrated under reduced pressure to afford ethyl 3-pentyloctanoate (20) (2 g, 90% yield) as colorless liquid.

[0356] 1H NMR (400 MHz, CDCh) 80.84 - 0.88 (m, 9H), 1.22 - 1.30 (m, 23H), 1.82 (m, 1H), 2.19 - 2.21 (d, J = 6.84 Hz, 1H), 2.36 (t, J = 7.56 Hz, 1H), 4.08 - 4.13 (m, 2H).

[0357] To a stirred solution of (20) (2 g, 8.25 mmol) in THF (30 mL) was added LAH in THF (2.4 M, 4.1 mL, 9.90 mmol) dropwise at 0 °C and stirred for 1 h. Upon completion, the reaction mixture was quenched with Na2SC>4 IOH2O and filtered through celite bed, washed with THF (80 mL), and concentrated under reduced pressure to afford 3-pentyloctan-l-ol (21-III) (12 g, 72% yield) as colorless liquid.

[0358] 1H NMR (400 MHz, CDCh) 60.85 - 0.89 (m, 6H), 1.24 - 1.33 (m, 15H), 1.40 (m, 2H), 1.48 - 1.54 (m, 2H), 3.65 (t, J = 7.08 Hz, 2H).Syntheses of 9-((tert-butyldimethylsilyl)oxy)heptadecane-l,17-diol (27-1):

[0359] To a stirred solution of NaH / THF (2.0 eqv) was added in DMF (0.5 mL / mmol) under a nitrogen atmosphere. The resulting mixture was cooled to -10 °C and a solution of 8-bromooctan-l-ol (22-1) (1.0 eqv) in DMF (0.5 mL / mmol) was added dropwise. The resulting mixture was warmed up to 25 °C and stirred for 2 h. After 2 h, the resulting mixture was cooled to 0 °C and benzyl bromide (23) (1.5 eqv) was added dropwise. The resultant reaction mixture was stirred at 25 °C for 14 h. After completion, the reaction mixture was quenched with ice-cold water and extracted with ethyl acetate. The combined organic layer was washed with water, then brine, and dried over anhydrous Na2SC>4, filtered, and evaporated. The crude material was purified by column chromatography eluting with 20-30% EtOAc / hexane to afford (((8-bromooctyl)oxy)methyl)benzene (24-1) (60-62% yields) as a colorless liquid.

[0360] Yield: 880 mg, 60%

[0361] 1H NMR (400 MHz, CDCh) 6 1.26 - 1.42 (m, 8H), 1.55 - 1.66 (m, 2H), 1.78 - 1.90 (m, 2H), 3.39 (t, J= 6.8 Hz, 2H), 3.46 (t, J= 6.6 Hz, 2H), 4.49 (s, 2H), 7.20 - 7.33 (m, 5H).

[0362] To freshly activated Mg turnings (5.0 eqv) was added 70 mL dry THF, followed by an addition of 1,2-dibromoethane (cat. amount) into the reaction mixture under a nitrogen atmosphere. A solution of (24-1) (1.0 eqv) in 30 mL of dry THF was added slowly at 0 °C.Then, the reaction mixture was heated to reflux for 1 h. After 1 h, the reaction mixture was cooled to 25 °C, methyl formate (1.0 eqv) was added slowly, and the reaction mixture stirredat 25 °C for 16 h. After completion the reaction mixture was quenched with 1 N HC1 solution and extracted with ethyl acetate, and the combined organic layer was washed with water, brine, and dried over sodium sulfate and concentrated under vacuum. The crude material was diluted with ethanol:water mixture (4: 1) and KOH (2.0 eqv) was added to the reaction mixture and stirred at 25 °C for 16 h. Then, ethanol was concentrated and diluted with ethyl acetate and washed with 1 N HC1 solution (100 mL). The separated organic layer was washed with water, brine, and dried over anhydrous sodium sulfate and concentrated under vacuum. The crude material was purified through combiflash chromatography using 0-40% ethyl acetate in hexane to obtain l,17-bis(benzyloxy)heptadecan-9-ol (25-1) as a sticky gum.

[0363] Yield: 680 mg, 20%

[0364] 1H NMR (400 MHz, CDC13) 6 1.27 - 1.41 (m, 23H), 1.48 - 1.54 (m, 6H), 1.56 -1.64 (m, 4H), 3.45 (t, J= 6.6 Hz, 4H), 3.49 - 3.63 (m, 1H), 4.49 (s, 4H), 7.22 - 7.33 (m, 10H).

[0365] To a stirred solution of (25-1) (1.0 eqv) in THF (1 mL / mmol) and cooled to 0 °C.Imidazole (4.0 eqv) and TBDMSC1 (2.0 eqv) were added in the reaction mixture. The resultant reaction mixture was stirred at 25 °C for 16 h. After 16 h, the reaction mixture was quenched with ice-cold water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layer was separated and washed with water (50 mL) and brine (50 mL) and dried over anhydrous sodium sulfate and concentrated under vacuum. This crude obtained was purified through combiflash chromatography using 3-4% ethyl acetate in hexane to afford (l,17bis(benzyloxy)heptadecan-9-yl)oxy)(tert-butyl)dimethylsilane (26-1) as alight-yellow liquid.

[0366] Yield: 710 mg, 84%

[0367] 1H NMR (400 MHz, CDCI3) 60.01 (s, 6H), 0.86 (s, 9H), 1.23 - 1.28 (m, 13H), 1.55 - 1.61 (m, 4H), 1.78 - 1.85 (m, 11H), 3.44 (t, J= 6.6 Hz, 4H), 3.57 - 3.69 (m, 1H), 4.48 (s, 4H), 7.24 - 7.32 (m, 10H).

[0368] To a stirred solution of (26-1) (1 eqv) in ethyl acetate (100 mL), was added 10% Pd / C (2 g). The resulting reaction mixture was stirred under a hydrogen balloon pressure at 25 °C for 2 h. After 2 h, reaction mixture was filtered through celite pad and filtrate was concentrated under reduced pressure to afford 9-((tert-butyldimethylsilyl)oxy)heptadecane-1, 17-diol (27-1) as colorless oil.

[0369] Yield: 1.0 g, 90%

[0370] 1H NMR (400 MHz, CDC13) 60.02 (s, 6H), 0.87 (s, 9H), 1.03 - 1.45 (m, 25H), 1.52 - 1.60 (m, 4H), 3.61 - 3.65 (m, 4H).General Scheme for Syntheses of Examples 1 to 5Example 1: l,17-Bis(heptadecan-9-yl)-9-[[4-([[2-(pyrrolidin-l-yl)ethyl]carbamovnoxy) decanoyl] oxy] heptadecanedioate (Compound 1-1)Compound 1-1

[0371] Step 1: To a stirred solution of 9-((tert-butyldimethylsilyl)oxy)heptadecane-l, 17-diol (27-1) (7.6 g, 18.89 mmol) in ACN: H2O (60 mL, 1:1) and TEMPO (1.47 g, 9.44 mmol) was added in the reaction mixture. Diacetoxyiodo benzene (24.33 g, 75.55 mmol) was added in one portion, and the reaction was allowed to continue to stir at 25 °C for 16 h. After completion the reaction mixture was quenched with 15% aqueous sodium thiosulfate solution (60 mL). The reaction mixture was diluted with water and extracted with EtOAc (3 x 60 mL). The combined organic layer was washed with brine (60 mL) and dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude material was purified through combiflash chromatography using 15-20% ethyl acetate in hexane to afford 9-((tert-butyldimethylsilyl)oxy)heptadecanedioic acid (28) (4.8 g, 60% yield) as a light-yellow sticky gum.

[0372] 1H NMR (400 MHz, DMSO-d6) 80.02 (s, 6H), 0.85 (s, 9H), 1.11 - 1.28 (m, 16H), 1.31 - 1.39 (m, 4H), 1.43 - 1.51 (m, 4H), 2.17 (t, J= 7.4Hz, 4H), 3.59 - 3.66 (m, 1H), 11.66 - 12.39 (m, 2H).

[0373] Step 2: To a stirred solution of (28) (1.0 eqv) in DCM (10 mL) were added DIPEA (6.0 eqv), EDC HQ (3 eqv), DMAP (1 eqv), and heptadeca-9-ol (21-1) (2.2 eqv). The reaction mixture was stirred at 25 °C for 12 h. After completion, quenched with water (50 mL) and extracted with ethyl acetate (3 x 50 mL). Then, the combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduce pressure. The crude material was purified by combiflash column chromatography eluting 1-2% ethyl acetate-hexane to afford l,17-bis(heptadecan-9-yl)-9-[(tert-butyldimethylsilyl)oxy]heptadecanedioate (29-1) (51-63% yields) as a light-yellow oil.

[0374] Yield: 330 mg, 52%

[0375] 'HNMR (400 MHz, CDC13) 60.02 (s, 6H), 0.77 - 0.99 (m, 19H), 1.20 - 1.32 (m, 71H), 1.35 - 1.40 (m, 3H), 1.43 - 1.52 (m, 8H), 2.26 (t, J= 7.2 Hz, 4H), 3.49 - 3.59 (m, 1H), 4.73 -4.91 (m, 2H).

[0376] Step 3: To a stirred solution of (29-1) (1.0 eqv) in THF (5.0 mL) at 0 °C added TBAF (1.5 eqv) dropwise and stirred at 60 °C for 2 h. After completion reaction mixture was concentrated under reduced pressure and diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL) and washed with brine (20 mL). Combined organic layer was dried overanhydrous Na2SC>4 and concentrated under reduced pressure to afford l,17-bis(heptadecan-9-yl)-9-hydroxyheptadecanedioate (30-1) (47-97% yields) as a light-yellow liquid.

[0377] Yield: 260 mg, 90%

[0378] 'HNMR (400 MHz, CDC13) 60.86 (t, J= 6.4 Hz, 12H), 1.24 - 1.43 (m, 65H), 1.48 - 1.52 (m, 10H), 1.58 - 1.63 (m, 5H), 2.26 (t, J= 7.4 Hz, 4H), 3.50 - 3.60 (m, 2H), 4.80 -4.86 (m, 2H).

[0379] Step 4: To a stirred solution of 4-({[2-(pyrrolidin-l-yl)ethyl]carbamoyl}oxy)decanoic acid (8-1) (1.0 eqv) in DCM (10 mL) were added DMAP (0.5 eqv) and DCC (1.5 eqv). The reaction mixture was stirred for 15 min and was added 30-(I) (1.0 eqv), stirring at 25 °C for 6 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with cold water (2 x 50 mL), then brine (50 mL). Organic layer was dried over anhydrous Na2SO4 and concentrated under reduce pressure. The crude material was purified by combiflash chromatography, eluted with 50-60% EtOAc-hexane to afford 1,17-bis(heptadecan-9-yl)-9-{[4-({[2-(pyrrolidin-l-yl)ethyl]carbamoyl}oxy)decanoyl]oxy}heptadecanedioate (Compound 1-1).

[0380] Yield: 110 mg, 27%, a colorless Gum.

[0381] UPLC-MS: Method-A was performed for UPLC. Purity is 99.37%; Rt 2.59 min., MS calculated: 1103.98 [M+H], MS found 1104.52 [M+H],

[0382] 1H NMR (400 MHz, CDCI3) 80.86 (t, J= 6.8 Hz, 12H), 1.23 - 1.30 (m, 72H), 1.44 - 1.71 (m, 23H), 1.77 - 1.93 (m, 5H), 2.17 - 2.40 (m, 6H), 2.56 - 2.95 (m, 5H), 3.30 -3.42 (m, 2H), 4.61 - 5.03 (m, 4H), 5.30 - 5.56 (m, 1H).Example 2: Difheptadecan-9-yl)9-ff4-fff2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)decanoyl)oxy)heDtadecanedioate (Compound 1-2)Compound 1-2

[0383] With the substitution of the compound (8-II) for the compound (8-1) in Step 4, the compound of Example 2 was prepared using the same procedures used to prepare the compound of Example 1 above.

[0384] UPLC-MS: Method-B was performed for UPLC. Purity is 100%; Rt 7.82 min., MS calculated: 1091.98 [M+H], MS found 1092.64 [M+H],

[0385] HPLC: CAD (96.83%), DAD (100 %)Example 3: l,17-Bis[(2Z)-non-2-en-l-yl]9-[[4-([[2-(pyrrolidin-l-yl)ethyl]carbamoynoxy)decanoyl]oxy}heDtadecanedioate (Compound 1-3)Compound 1-3

[0386] With the substitution of the compound (Z)-non-2-en-l-ol (21-11) for the compound 21-1 in Step 2, the compound of Example 3 was prepared using the same procedures used to prepare the compound of Example 1 above.

[0387] UPLC-MS: Method-A was performed for UPLC. Purity is 92.34%; Rt 2.15 min., MS calculated: 875.70 [M+H], MS found 876.13 [M+H],Example 4: Di((Z)-non-2-en-l-yl)9-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl) oxy)decanoyl)oxy)heptadecanedioate (Compound 1-4)Compound 1-4

[0388] With the substitution of the compound (Z)-non-2-en-l-ol (21-11) for the compound 21-1 in Step 2 and the substitution of the compound (8-II) for the compound (8-1) in Step 4, the compound of Example 4 was prepared using the same procedures used to prepare the compound of Example 1 above.

[0389] UPLC-MS: Method-A was performed for UPLC. Purity is 100.0%; Rt 6.83 min., MS calculated: 863.70 [M+H], MS found 864.20 [M+H],Example 5: Bis(3-pentyloctyl)9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy) decanoyl)oxy)heptadecanedioate (Compound 1-5)Compound 1-5

[0390] With the substitution of the compound 3-pentyloctan-l-ol (21-III) for the compound 21-1 in Step 2, the compound of Example 5 was prepared using the same procedures used to prepare the compound of Example 1 above.

[0391] UPLC-MS: Method-A was performed for UPLC. Purity is 98.69%; Rt 2.72 min., MS calculated: 991.86 [M+H], MS found 992.34 [M+H],General Scheme for the Synthesis of Examples 6 to 9Example 6: l,17-Bis(heptadecan-9-yl)9-[[4-([[(l-ethylpiperidin-3-yl)methoxy]carbonynoxy) decanoylloxy] heptadecanedioate (Compound 1-6)Compound 1-6

[0392] To a stirred solution of 4-([{(l-ethylpiperidin-3-yl)methoxy}carbonyl]oxy)decanoic acid (12) (1.0 eqv) in DCM (10 mL) were added DMAP (0.5 eqv) and DCC (1.5 eqv). The reaction mixture was stirred for 15 min and then was added l,17-bis(heptadecan-9-yl)9-hydroxyheptadecanedioate (30-1) (1.0 eqv) and further was stirred at 25 °C for 2 h. Reaction mixture was diluted with ethyl acetate (50 mL) and washed with cold water (2 x 50 mL), then brine (50 mL). The organic layer was dried over anhydrous Na2SC>4 and concentrated under reduced pressure. The crude material was purified by combiflash chromatography, eluting with 50-60% EtOAc-hexane to afford the compound of Example 6.

[0393] Yield: 100 mg, 35%, a light-yellow sticky gum

[0394] UPLC-MS: Method-B was performed for UPLC. Purity is 96.76 %; Rt 5.82 min., MS calculated: 1133.00 [M+H], MS found 1133.47 [M+H],Example 7: l,17-Bis[(2Z)-non-2-en-l-yl]9-{[4-({[(l-ethylpiperidin-3-yl)methoxy] carbonvnoxy)decanoyl]oxy} heptadecanedioate (Compound 1-7)Compound 1-7

[0395] With the substitution of the compound 30-11 for the compound 30-1, the compound of Example 7 was prepared using the same procedures used to prepare the compound of Example 6 above.

[0396] Yield: 50 mg, 31%, a yellow gel.

[0397] UPLC-MS: Method-A was performed for UPLC. Purity is 97.15%; Rt 2.29 min., MS calculated: 904.72 [M+H], MS found 905.10 [M+H],Example 8: l,17-Bis(3-pentyloctyl)9-([4-(([(l-ethylpiperidin-3-yl)methoxy]carbonyl) oxy)decanoyl]oxy}heptadecanedioate (Compound 1-8)Compound 1-8

[0398] With the substitution of the compound 30-III for the compound 30-1, the compound of Example 8 was prepared using the same procedures used to prepare the compound of Example 6 above.

[0399] Yield: 135 mg, 47%, a colorless gum

[0400] UPLC-MS: Method-B was performed for UPLC. Purity is 94.80 %; Rt 5.58 min., MS calculated: 1020.87 [M+H], MS found 1021.36 [M+H],

[0401] 1H NMR (400 MHz, CDCl3) δ0.87 (t, J= 6.9 Hz, 12H), 1.22 - 1.37 (m, 70H), 1.54 - 1.61 (m, 14H), 1.73 - 2.00 (m, 4H), 2.22 - 2.39 (m, 6H), 2.44 - 2.72 (m, 2H), 2.76 -3.22 (m, 2H), 3.96 -4.10 (m, 5H), 4.61 - 4.87 (m, 2H).General Scheme for Synthesis of Examples 9 to 11Example 9: l,17-Bis(heptadecan-9-yl)9-[(4-l[4-(dimethylamino)butanoyl] oxy)decanoyl)oxy]heptadecanedioate (Compound 1-9)Compound 1-9

[0402] To a stirred solution of 4-[{4-(dimethylamino)butanoyl}oxy]decanoic acid (16) (1.0 eqv) in DCM (10 mL) were added DMAP (0.5 eqv) and DCC (1.5 eqv). The reaction mixture was stirred for 15 min and was added l,17-bis(heptadecan-9-yl)9-hydroxyheptadecanedioate (30-1) (1.0 eqv). The reaction was stirred at 25 °C for 2 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with cold water (2 x 50 mL), then brine (50 mL). The organic layer was dried over anhydrous Na2SO4 andconcentrated under reduced pressure. The crude material was purified by combiflash chromatography, eluted with 50-60% EtOAc-hexane to afford the compound of Example 9.

[0403] Yield: 100 mg, 35%, a light-yellow sticky gum

[0404] UPLC-MS: Method-A was performed for UPLC. Purity is 98.86%; Rt 2.61 min, MS calculated: 1076.97 [M+H], MS found 1077.46 [M+H],Example 10: 1.17-Bisl(2Z)-non-2-en-l-yl|9-|(4-!l4- (dimethylamino)bntanoyl]oxy}decanoyl) oxylheptadecanedioate (Compound 1-10)Compound 1-10

[0405] With the substitution of the compound (30-11) for the compound (30-1), the compound of Example 10 was prepared using the same procedure used to prepare the compound of Example 9 above.

[0406] Yield: 70 mg, 31%, a light-yellow gum.

[0407] UPLC-MS: Method-A was performed for UPLC. Purity is 100%; Rt 2.16 min, MS calculated: 848.69 [M+H], MS found 849.12 [M+H],Example 11: l,17-Bis(3-pentyloctyl)9-[(4-[[4-(dimethylamino)butanoyl]oxy}decanoyl) oxylheptadecanedioate (Compound 1-11)Compound 1-11

[0408] With the substitution of the compound (30-III) for the compound (30-1), the compound of Example 11 was prepared using the same procedure used to prepare the compound of Example 9 above.

[0409] Yield: 62 mg, 29%, a light brown gum

[0410] UPLC-MS: Method-A was performed for UPLC. Purity is 96.98 %; Rt 2.39 min., MS calculated: 964.85 [M+H], MS found 965.28 [M+H],Example 12: Bis(3,7,ll-trimethyldodeca-l.,6,10-trien-3-yl) 9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)heDtadecanedioate (Compound 1-12)Molecular Weight: 1035.59Compound 1-12

[0411] To a stirred solution of 8-bromooctanoic acid (2 g, 8.96 mmol) in DCM (20 mL) were added EDC (2.08 g, 13.45 mmol), DMAP (0.55 g, 4.48 mmol) and DIPEA (4.67 mL, 26.89 mmol) at 25 °C and stirred for 30 min. Then 3,7,11 -trimethyldodeca- 1, 6, 10-trien-3-ol (2.39 g, 10.76 mmol) was added portion wise and stirred at 25 °C for 6 h in an inert atmosphere. Upon completion, reaction mixture was diluted with water (20 mL) and extracted with DCM (3 X 20 mL). Then combined organic layers were washed with saturated NaHCCh solution (40 mL) and brine (30 mL). Organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to provide crude, which was purified through ELSD combi flash column chromatography, eluted with 30% EtOAc-heptane to afford 3,7,ll-trimethyldodeca-l,6,10-trien-3-yl 8-bromooctanoate (680 mg, 48% yield; w.r.t recovery SM) as a yellow liquid.

[0412] 1H NMR (400 MHz, CDCl3) δ6.02 - 5.89 (m, 1H), 5.19 - 5.02 (m, 4H), 3.51 (t, J = 6.7 Hz, 1H), 3.39 (t, J= 6.8 Hz, 2H), 2.29 - 2.20 (m, 2H), 2.10 - 1.91 (m, 6H), 1.91 - 1.69 (m, 4H), 1.62 - 1.50 (m, 13H), 1.48 - 1.38 (m, 2H), 1.36 - 1.28 (m, 4H).

[0413] To a stirred solution of 3,7,1 l-trimethyldodeca-l,6,10-trien-3-yl 8-bromooctanoate (210 mg, 0.49 mmol) and TosMIC (47.96 mg, 0.25 mmol) in DMSO: Ether (1:1) (3 mL) was added NaH (21.62 mg, 0.54 mmol) at 0 °C in an inert atmosphere. Then TBAI (18.15 mg, 0.05 mmol) was added to the reaction mixture at 0 °C in an inert atmosphere. Then reaction mixture was stirred at 25 °C for 4h in an inert atmosphere. Upon completion, reaction mixture was quenched with water (10 mL) and extracted with ethyl acetate (3 x 15 mL). The combined organic layers were washed with brine (30 mL). Organic part was dried over Na2SO4, filtered and concentrated under reduced pressure to provide crude, which was purified through ELSD combi-flash column chromatography with 20% ethyl acetate-hex to afford 1, 17-bis(3,7, 1 l-trimethyldodeca-l,6,10-trien-3-yl) 9-isocyano-9-[(4-methylbenzene)sulfonyl]heptadecanedioate (210 mg, 42% yield) as a colorless liquid.

[0414] 1H NMR (400 MHz, CDCl3) δ7.84 (d, J= 8.0 Hz, 2H), 7.40 (d, J= 8.0 Hz, 2H), 6.02 - 5.89 (m, 2H), 5.18 - 5.05 (m, 11H), 2.28 - 2.19 (m, 5H), 2.08 - 1.90 (m, 17H), 1.90 -1.69 (m, 5H), 1.69 - 1.50 (m, 22H), 1.31 - 1.27 (m, 18H).

[0415] To a stirred solution of l,17-bis(3,7,ll-trimethyldodeca-l,6,10-trien-3-yl) 9-isocyano-9-[(4-methylbenzene)sulfonyl]heptadecanedioate (130 mg, 0.15 mmol) in MeOH: H2O (1: 1 eqv) (2 mL) was added HC1 (23 pL, 0.73 mmol) at 0 °C. Then reaction mixture was refluxed for 3 h. Upon completion, reaction mixture was diluted with water (10 mL) and extracted DCM (3 X 10 mL). Then combined organic part was washed with brine (25 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to provide crude, which was purified through ELSD combi-flash column chromatography eluted with 2-5% EtOAc-heptane to afford l,17-bis(3,7,ll-trimethyldodeca-l,6,10-trien-3-yl) 9-oxoheptadecanedioate (60 mg, 46% yield) as a colorless liquid.

[0416] 1H NMR (400 MHz, CDCI3) 67.58 (d, J= 7.9 Hz, 1H), 7.33 (d, J= 7.9 Hz, 1H), 6.01 - 5.89 (m, 2H), 5.18 - 5.14 (m, 1H), 5.14 - 5.05 (m, 6H), 2.42 (s, 2H), 2.36 (t, J= 7.5 Hz, 4H), 2.28 - 2.18 (m, 4H), 2.10 - 1.91 (m, 12H), 1.90 - 1.68 (m, 4H), 1.67 (s, 12H), 1.62 - 1.50 (m, 33H), 1.34 - 1.20 (m, 15H).

[0417] To a stirred solution of 1, 17-bis(3,7, 1 l-trimethyldodeca-l,6,10-trien-3-yl) 9-oxoheptadecanedioate (80 mg, 0.11 mmol) in MeOH (2 mL) was added NaBH4 (6.28 mg, 0.17 mmol) at 0 °C in an inert atmosphere. Then reaction mixture was stirred at 25 °C for 2 h in an inert atmosphere. Upon completion, reaction mixture was quenched with aqueous ammonium chloride solution (10 mL), extracted with EtOAc (2 X 15 mL). The combinedorganics were washed with brine, dried over Na2SC>4, filtered and concentrated under reduced pressure to provide crude, which was purified through ELSD combi flash column chromatography (eluting with 20 EtOAc-heptane) to afford l,17-bis(3,7,l 1-trimethyldodeca- l,6,10-trien-3-yl) 9-hydroxyheptadecanedioate (40 mg, 48% yield) as a colorless liquid.

[0418] 1H NMR (400 MHz, CDCl3) δ6.06 - 5.85 (m, 1H), 5.18 - 5.07 (m, 4H), 2.28 -2.19 (m, 2H), 2.06 - 1.93 (m, 4H), 1.91 - 1.69 (m, 2H), 1.64 - 1.50 (m, 20H), 1.43 - 1.38 (m, 4H), 1.35 - 1.17 (m, 16H), 0.90 - 0.80 (m, 4H).

[0419] To a stirred solution of 1, 17-bis(3,7, 1 l-trimethyldodeca-l,6,10-trien-3-yl) 9-hydroxyheptadecanedioate (150 mg, 0.21 mmol) and 4-({[2-(pyrrolidin-l-yl)ethyl]carbamoyl}oxy)decanoic acid (8-1) (101.91 mg, 0.31 mmol) in toluene (2 mL) was added PPI13 (163.21 mg, 0.62 mmol) and DBAD (142.89 mg, 0.62 mmol) at 25 °C in an inert atmosphere. Then the reaction mixture was stirred at 25 °C for 21 h in an inert atmosphere. Upon completion, the reaction mixture was diluted with water (10 mL) and extracted with DCM (3 X 10 mL). Then combined organic layers were washed with brine (20 mL). Organic part was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to provide crude, which was purified through ELSD combi flash chromatography eluent with 10% MeOH-DCM to afford the compound of Example 12 as a light-yellow gum.

[0420] UPLC-MS: Method-A was performed for UPLC. Purity is 100%; Rt 7.33 min., MS calculated: 1036.60 [M+H], MS found: 1036.0 [M+H],

[0421] HPLC-MS: Method-A was performed for HPLC. Purity is 90.08% in CAD and no response in DAD.Example 13: Di((Z)-non-2-en-l-yl)9-((5-cyclopentyl-4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)pentanoyl)oxy)heptadecanedioate (Compound 1-13)Compound 1-13

[0422] To a stirred solution of 3,4-dihydro-2H-pyran (5 g, 59.44 mmol) in DCM (500 mL) was added PPTS (4.48 g, 17.83 mmol) and butane- 1,4-diol (10.71 g, 118.88 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 4 h. Upon completion the reaction mixture was diluted with DCM (2x150 mL) and washed with water and brine (100 mL). Organic layerwas dried over anhydrous Na2SC>4 and concentrated under reduced pressure. Crude compound thus obtained was purified by combi flash column chromatography, eluted with 20% ethyl acetate-hexane to afford 4-(oxan-2-yloxy)butan-l-ol (5 g, 48%) as colorless oil.

[0423] 1H NMR (400 MHz, CDCl3) δ 1.50 - 1.70 (m, 8H), 1.74 - 1.84 (m, 1H), 2.13 -2.17 (m, 1H), 3.37 - 3.52 (m, 2H), 3.64 (t, J = 5.9 Hz, 2H), 3.74 - 3.87 (m, 2H), 4.58 (t, J = 3.5 Hz, 1H).

[0424] To a stirred solution of 4-(oxan-2-yloxy)butan-l-ol (5 g, 28.7 mmol) in DCM (50 mL) were added NaOAc (3.53 g, 43.05 mmol) and PCC (9.28 g, 43.05 mmol) at ice cooled condition. Reaction mixture was stirred at 25 °C for 2 h. Upon completion, (as checked by TLC 40% EtOAc-hexanes (stained by DNP and KMnCh), the reaction mixture was filtered through celite bed and concentrated under reduced pressure to provide 5.0 g crude. The crude material was purified by column chromatography eluted by (0-15% EtOAc / heptanes) to afford 4-(oxan-2-yloxy)butanal (3.3 g, 67%) as a colorless liquid.

[0425] 1H NMR (400 MHz, CDCl3) δ 1.51 - 1.60 (m, 4H), 1.67 - 1.80 (m, 2H), 1.91 -1.97 (m, 2H), 2.50 - 2.55 (m, 2H), 3.36 - 3.45 (m, 1H), 3.45 - 3.52 (m, 1H), 3.76 - 3.83 (m, 2H), 4.55 (t, J = 3.4 Hz, 1H), 9.75 - 9.80 (m, 1H).

[0426] To a stirred solution of Mg (698 mg, 29.07 mmol) in THF (10 mL) was added a catalytic amount of b (as initiator). (Bromomethyl)cyclopentane (1.9 g, 11.628 mmol) was added to the reaction mass gradually drop by drop under nitrogen atmosphere. A sudden exotherm of the reaction was maintained by cold water bath outside. Reaction was stirred at 25 °C for 2 h. A grey color solution was generated 4. To a stirred solution of 4-(oxan-2-yloxy)butanal (3.0 g, 40.519 mmol) in THF (30 mL) was added 4 (crude) under nitrogen atmosphere at 0 °C. Reaction mass was stirred at 25 °C for 3 h. Saturated ammonium chloride solution (30 mL) was added in the reaction mass, extracted with EtOAc (2 x 30 mL). Organic layer was dried over anhydrous sodium sulfate, evaporated under reduced pressure. Crude compound thus obtained was purified by Combiflash chromatography, eluent 10% ethyl acetate-hexane to afford l-cyclopentyl-5-(oxan-2-yloxy)pentan-2-ol (800 mg, 54 %) as a colorless liquid.

[0427] 1H NMR (400 MHz, CDCl3) δ0.99 - 1.16 (m, 2H), 1.32 - 1.43 (m, 1H), 1.43 -1.65 (m, 10H), 1.65 - 1.99 (m, 8H), 3.33 - 3.56 (m, 2H), 3.60 - 3.71 (m, 1H), 3.73 - 3.90 (m, 2H), 4.56 - 4.62 (m, 1H).

[0428] To a solution of l-cyclopentyl-5-(oxan-2-yloxy)pentan-2-ol (1.5 g, 5.85 mmol) in DCM (10 mL) were added pyridine (1.18 mL, 14.63 mmol), DMAP (0.36 g, 2.92 mmol) and 4-nitrophenyl chloroformate (1.18 g, 5.85 mmol) at 0 °C, then pH of the reaction mixture was 12-13 (checked by pH paper), the resulting mixture was stirred at 25 °C for 2 h. TLC was checked SM was consumed (30% EtOAc / hexanes). The reaction mixture was diluted with H2O and the organic part was washed with Na2COs (3 times) and brine. The organic part was dried over Na2SO4, filtered and concentrated under reduced pressure to provide 3.0 g crude. The crude residue was purified by combiflash column chromatography (eluted by 0-3% EtOAc / heptanes) to afford l-cyclopentyl-5-((tetrahydro-2H-pyran-2-yl)oxy)pentan-2-yl (4-nitrophenyl) carbonate (2.3 g, 93%) as a colorless oil.

[0429] 1H NMR (400 MHz, CDCl3) δ0.99 - 1.16 (m, 2H), 1.32 - 1.43 (m, 1H), 1.43 -1.65 (m, 10H), 1.65 - 1.99 (m, 8H), 3.33 - 3.56 (m, 2H), 3.60 - 3.71 (m, 1H), 3.73 - 3.90 (m, 2H), 4.56 - 4.62 (m, 1H).

[0430] To a stirred solution of l-cyclopentyl-5-((tetrahydro-2H-pyran-2-yl)oxy)pentan-2-yl (4-nitrophenyl) carbonate (800,0 mg, 3.12 mmol) in DCM (5.0 mL) were added 4-nitrophenylchloroformate (391.931 mg, 3.432 mmol), pyridine (2.0 mL, 24.962 mmol) and DMAP (571.801 mg, 4.68 mmol) at 0 °C and stirred for 2 h. Then were added l-(2-aminoethyl)pyrrolidine (783.862 mg, 6.865 mmol) and DIPEA (4.0 mL, 24.962 mmol) and was further stirred at 25 °C for 16 h. After completion, the reaction mixture was diluted with DCM and washed with saturated solution of sodium carbonate (3 x 20 mL) and extracted with DCM (3 x 20 mL). Combined organic parts were dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Crude material was purified using flash chromatography, eluted with (0-4% MeOH / DCM) to afford l-cyclopentyl-5-(oxan-2-yloxy)pentan-2-yl N-[2-(pyrrolidin-l-yl)ethyl]carbamate (820 mg, 60%) as a colorless oil.

[0431] 1H NMR (400 MHz, CDCl3) δ 1.43 - 1.84 (m, 16H), 2.61 - 2.70 (m, 6H), 3.02 (s, 3H), 3.31 - 3.52 (m, 3H), 3.67 - 3.77 (m, 2H), 3.80 - 3.89 (m, 2H), 4.52 - 4.59 (m, 2H), 4.78 - 4.82 (m, 2H), 5.25 - 5.29 (m, 2H), 6.36 - 6.64 (m, 1H), 8.18 - 8.22 (m, 1H).

[0432] To a stirred solution of l-cyclopentyl-5-(oxan-2-yloxy)pentan-2-yl N-[2-(pyrrolidin-l-yl)ethyl]carbamate (500 mg, 1.26 mmol) in MeOH (5 mL) pTSA»H2O (239.84 mg, 1.26 mmol) was added. The reaction mixture was stirred at 60 °C for 2 h. TLC was checked SM was consumed. Then the mixture was concentrated under reduced pressure, diluted with EtOAc washed with NaHCCh (pH -9-10), dried over Na2SO4, filtered andconcentrated under reduced pressure to provide l-cyclopentyl-5-hydroxypentan-2-yl (2-(pyrrolidin-l-yl)ethyl)carbamate (340 mg, crude). The crude was used for the next step without any purification.

[0433] UPLC-MS: Column- XTERRA RP 18 (4.6 x 50 mm), 5um, (mobile phase: initially 50% [0.1% HCOOH in WATER] and 50% [0.1% HCOOH in (70:30) ACN: THF]; then to 2.0 % [0.1% HCOOH in WATER] and 98% [0.1% HCOOH in (70:30) ACN: THF] in 1.2 min, held this mobile phase composition up to 2.50 min; then back to initial composition i.e., 50% [0.1% HCOOH in WATER] and 50% [0.1% HCOOH in (70:30) ACN: THF] in 2.85 min, held this mobile phase composition up to 3.10 min. Flow =1.5 ml / min.

[0434] Rt 0.54 min., MS calculated: 313.20 [M+H], MS found: 313.3 [M+H],

[0435] To a stirred solution of the primary alcohol above (1 g, 3.2 mmol) in DCM (10 mL), DMP (2.04 g, 4.8 mmol) was added portion wise at 0 °C for 2 h. TLC and UPLC was checked SM was consumed. Then the reaction mixture was quenched with sat. NaHCCh (10 mL), extracted with EtOAc, dried over Na2SC>4, filtered and concentrated under reduced pressure to afford 1.0 g crude. The crude material was purified by Combiflash column chromatography to afford l-Cyclopentyl-5-oxopentan-2-yl (2-(pyrrolidin-l-yl)ethyl)carbamate (700 mg, crude).

[0436] UPLC-MS: Column- XTERRA RP 18 (4.6 x 50 mm), 5u, (mobile phase: initially 50% [0.1% HCOOH in WATER] and 50% [0.1% HCOOH in (70:30) ACN: THF]; then to 2.0 % [0.1% HCOOH in WATER] and 98% [0.1% HCOOH in (70:30) ACN: THF] in 1.2 min, held this mobile phase composition up to 2.50 min; then back to initial composition i.e., 50% [0.1% HCOOH in WATER] and 50% [0.1% HCOOH in (70:30) ACN: THF] in2.85 min, held this mobile phase composition up to 3.10 min. Flow =1.5 ml / min.

[0437] Rt 0.54 min., MS calculated: 311.20 [M+H], MS found: 311.3 [M+H],

[0438] To a stirred solution of the aldehyde above (600 mg, 1.93 mmol) in tert-BuOH (5.0 mL) and H2O (1.0 mL) were added NaH2PO4 (463.79 mg, 3.87 mmol) and NaClO2 (262.2 mg, 2.9 mmol) to the reaction mixture. The reaction mixture was stirred for 6 h at 25 °C. TLC was checked one polar spot observed in TLC and desired mass showed in UPLC. The reaction mixture was concentrated under reduced pressure to provide crude. The crude residue was washed with water, dried over Na2SC>4, filtered and concentrated under reduced pressure to provide 900 mg crude. Thus, the crude material was purified by Combiflashcolumn chromatography to afford 5-cyclopentyl-4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)pentanoic acid (INT-64) (300 mg, crude) as a sticky gel.

[0439] UPLC-MS: Column- XTERRA RP 18 (4.6 x 50 mm), 5u, (mobile phase: initially 50% [0.1% HCOOH in WATER] and 50% [0.1% HCOOH in (70:30) ACN: THF]; then to 2.0 % [0.1% HCOOH in WATER] and 98% [0.1% HCOOH in (70:30) ACN: THF] in 1.2 min, held this mobile phase composition up to 2.50 min; then back to initial composition i.e., 50% [0.1% HCOOH in WATER] and 50% [0.1% HCOOH in (70:30) ACN: THF] in 2.85 min, held this mobile phase composition up to 3.10 min. Flow =1.5 ml / min.

[0440] Rt 0.54 min., MS calculated: 327.2 [M+H], MS found: 327.3 [M+H],

[0441] To a stirred solution l,17-bis[(2Z)-non-2-en-l-yl]-9-hydroxyheptadecanedioate (30-11) (130 mg, 0.4 mmol) and INT-64 (269.95 mg, 0.48 mmol) in Toluene (5 mL), DBAD (275.1 mg, 1.2 mmol) and triphenylphosphine (313.26 mg, 1.2 mmol) were added. The reaction mixture was stirred for 2 h at 80 °C. UPLC and TLC was checked SM was consumed. Upon completion the mixture was diluted with EtOAc (20 mL) washed with water, dried over Na2SO4, filtered and concentrated under reduced pressure to provide 360 mg crude. Thus, the crude residue was purified by combiflash column chromatography to afford the compound of Example 13 (90 mg, 26%) as a yellow liquid.

[0442] 1H NMR (400 MHz, MeOD) δ5.69 - 5.59 (m, 2H), 5.57 - 5.47 (m, 2H), 4.83 -4.75 (m, 1H), 4.61 (d, J = 6.9 Hz, 4H), 3.29 - 3.24 (m, 1H), 2.74 - 2.67 (m, 6H), 2.39 - 2.26 (m, 6H), 2.12 (q, J = 7.1 Hz, 4H), 1.88 - 1.71 (m, 9H), 1.65 - 1.47 (m, 16H), 1.43 - 1.20 (m, 32H), 1.17 - 1.09 (m, 3H), 0.93 - 0.86 (m, 6H).

[0443] UPLC-MS: Method-B was performed for UPLC. Purity is 97.42%; Rt 6.91 min., MS calculated: 873.7 [M+H], MS found 873.9 [M+H],

[0444] HPLC-MS: Method-A was performed for HPLC. Purity is 94.08% (CAD) and no response in DAD.Synthesis of Intermediates for Examples 14 through Example 27:Synthesis of INT-2: 4-hydroxydecanoic acid

[0445] To a stirred solution of 5-hexyldihydrofuran-2(3H)-one (5 g, 29.369 mmol) in EtOH: Water (3:1, 100 mL), was added NaOH (2.3 g, 58.738 mmol). Then the reaction mixture was stirred at 25 °C for 16 h. After completion, reaction solvent was (EtOH) concentrated under vacuum and then diluted with water, acidified with 1(N) HC1 and then solid precepted was filtered through sintered funnel, washed with water (75 mL) and then dried over high vacuum pump for 2 h to obtain the product 4-hydroxydecanoic acid (INT-2) (4.9 g, crude) as off-white solid which was used for the next step without purification.

[0446] 1H NMR (400 MHz, DMSO) 60.86 (t, J= 6.7 Hz, 3H), 1.20 - 1.31 (m, 10H), 1.38 - 1.51 (m, 1H), 1.54 - 1.67 (m, 1H), 2.15 - 2.35 (m, 2H), 3.33 - 3.41 (m, 1H), 3.91 - 4.96 (m, 1H), 11.55 - 12.40 (m, 1H).Synthesis of INT-4: Benzyl 4-hydroxydecanoate

[0447] To a stirred solution of INT-2 (1.3 g, 6.905 mmol) in THF (15 mL), was added 3 (1.6 g,6.905 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 48 h. The reaction was monitored by TEC (30% EtOAc -heptane). The reaction mixture was filtered through celite pad and washed with ethyl acetate (150 mL). Then the eluted part was concentrated under reduced pressure to provide a crude product (2.2 g), which was purified by combi flash chromatography, eluted with (15-18% EtOAc-Heptane) to provide INT-4 (1.5 g, 78%, 2 step) as colorless liquid.

[0448] 1H NMR (400 MHz, CDC13) 60.83 - 0.91 (m, 3H), 1.23 - 1.31 (m, 8H), 1.37 -1.47 (m, 3H), 1.63 - 1.76 (m, 1H), 1.78 - 1.91 (m, 1H), 2.46 - 2.56 (m, 2H), 3.55 - 3.64 (m, 1H), 5.11 (s, 2H), 7.27 - 7.40 (m, 5H).Synthesis of INT-5A: Benzyl 4-(((4-nitrophenoxy)carbonyl)oxy)decanoate

[0449] To a solution of INT-4 (4 g, 14.368 mmol) in dichloromethane (50 mL), were added pyridine (2.9 mL, 35.921 mmol), DMAP (527 mg, 4.311 mmol) and 4-nitrophenyl carb onochlori date (5.8 g, 28.737 mmol) were added and stirred at 25 °C for 2 h. The consumption of starting material was judged by TLC (20% EtOAc-heptane). The reaction mixture was diluted with ethyl acetate (500 mL), washed with IM sodium carbonate (3 x 150 mL), water (150 mL), brine (150 mL) and finally dried over anhydrous Na2SC>4. The resulting organic layer was concentrated to obtain crude (6.5 g) which was purified by combi-flash column chromatography (eluted with 5-7% EtOAc in heptane) to obtain INT-5A (3.8 g, 60%) as light-yellow liquid.

[0450] 1H NMR (400 MHz, CDCh) 60.83 - 0.91 (m, 3H), 1.25 - 1.43 (m, 8H), 1.57 -1.67 (m, 1H), 1.67 - 1.79 (m, 1H), 1.91 -2.04 (m, 1H), 2.04 - 2.16 (m, 1H), 2.49 (t, J= 7.5 Hz, 2H), 4.81 - 4.91 (m, 1H), 5.12 (s, 2H), 7.28 - 7.45 (m, 7H), 8.20 - 8.30 (m, 2H).Synthesis of INT-7O: Benzyl 4-(((2-(pyrrolidin-l-yl)ethoxy)carbonyl)oxy)decanoate

[0451] To a solution of INT-5A (6 g, 13.529 mmol) in THF (75 mL), was added 2- (pyrrolidin-l-yl)ethan-l-ol (3.1 g, 27.058 mmol) followed by the addition of DIPEA (7.1 mL, 40.587 mmol) and DMAP (826 mg, 6.765 mmol) and then stirred at 70 °C for 16 h. The consumption of starting material was judged by TLC (10% MeOH DCM). The reaction mixture was diluted with ethyl acetate (350 mL), washed with IM sodium carbonate (2 x 175 mL), water (175 mL), brine and finally dried over anhydrous Na2SC>4. The resulting organic layer was concentrated to obtain crude (5.95 g) which was purified by flash column chromatography (eluted with 3-4% MeOH-DCM) to obtain INT-7O (4.1 g, 72%) as lightyellow sticky liquid.

[0452] UPLC-MS: Method-B was performed for UPLC. Purity is 100%; Rt 3.99 min., MS calculated: 420.27 [M+H], MS found: 420.4 [M+H],Synthesis of INT-8O: 4-(((2-(pyrrolidin-l-yl)ethoxy)carbonyl)oxy)decanoic acid

[0453] A solution of INT-7O (4.1 g, 9.772 mmol) in MeOH (50 mL) was degassed under argon atmosphere for 5 min then added Pd / C (50% moist) (900 mg) at 25 °C. Reaction mixture was stirred at 25 °C under hydrogen balloon pressure for Ih. Upon completion, TLC (10% MeOH-DCM) was checked, SM was fully consumed. Then it was filtered through celite bed and washed with MeOH and concentrated under reduced pressure to obtain INT-80 (2.9 g, 90%) as light-yellow sticky gum which was used directly for the next step.

[0454] UPLC-MS: Method-B was performed for UPLC. Purity is 100%; Rt 2.11 min., MS calculated: 330.22 [M+H], MS found: 330.4 [M+H],

[0455] 1H NMR (400 MHz, DMSO) 60.85 (t, = 6.4 Hz, 3H), 1.19 - 1.32 (m, 8H), 1.47 - 1.57 (m, 2H), 1.61 - 1.77 (m, 5H), 1.77 - 1.89 (m, 1H), 2.17 - 2.26 (m, 2H), 2.44 - 2.51 (m, 4H), 2.66 (t, J= 5.7 Hz, 2H), 4.15 (t, J= 5.7 Hz, 2H), 4.58 - 4.69 (m, 1H). Note: One -COOH proton is missing.

[0456] HPLC-MS: Method-A was performed for HPLC. Purity is 99.49% in CAD and No response in DAD.General Scheme for Syntheses of Compounds 37-1 through 37 -XIwhere R2is defined by the alkyl groups of inputs 9-1 through 9-X described below.

[0457] To a stirred solution of 28 (500 mg, 1.16 mmol) in DCM (10,0 mL), were sequentially added, EDC•HCl (556.86 mg, 2.90 mmol) DMAP (42.58 mg, 0.34 mmol), DIPEA (0.6 mL, 4.64 mmol) under N2 atmosphere at 25°C and was stirred for 10 min. Then octan-l-ol (9-1) (378.29 mg, 2.90 mmol) was added and stirred at 25°C for 16 h. Upon completion the reaction mixture was diluted with water and extracted with EtOAc (2 x 40 mL), organic layer was washed with brine, dried over Na2SO4h, filtered and concentrated under reduced pressure to provide crude. The crude was first purified by combi flash column chromatography (eluted by 0-0.5% EtOAc-Heptane) and then purified by combi-flash column chromatography (eluted by 0-20% EtOAc-Heptane) to afford 36-1 (520 mg, 68%) as a colorless liquid.

[0458] 'H NMR (400 MHz, CDC13) 64.04 (t, J = 6.7 Hz, 4H), 3.64 - 3.54 (m, 1H), 2.27 (t, J = 7.5 Hz, 4H), 1.64 - 1.56 (m, 8H), 1.40 - 1.20 (m, 40H), 0.92 - 0.82 (m, 15H), 0.02 (s, 6H).

[0459] To a stirred solution of 36-1 (500 mg, 0.76 mmol) in THF (5.0 mL), was added TBAF (1.5 mL, 1.52 mmol) at 0 °C. The reaction mixture was allowed to warm to 25 °C and stirred for 6h at the same temperature. TLC was checked SM was not consumed. After that the reaction mixture was warmed at 80 °C for 6 h. Upon completion the reaction mixture was cooled and quenched with water. The water part was extracted with EtOAc (2 times), driedover Na2SO4, filtered and concentrated under reduced pressure to provide crude. The crude was purified by combi flash column chromatography (eluted by 0-10% EtOAc-Heptane) to afford dioctyl 9-hydroxyheptadecanedioate (37-1) (320 mg, 77%) as a colorless liquid.

[0460] 'H NMR (400 MHz, CDC13) 64.04 (t, J = 6.7 Hz, 4H), 3.61 - 3.49 (m, 1H), 2.28 (t, J = 7.5 Hz, 4H), 1.64 - 1.56 (m, 8H), 1.43 - 1.39 (m, 4H), 1.36 - 1.19 (m, 33H), 0.87 (t, 6H).Example 14: Di((Z)-non-2-en-l-yl) 9-((4-(((2-(pyrrolidin-l-yl)ethoxy)carbonyl)oxy)decanoyl)oxy)heptadecanedioate (Compound 1-14)(Compound 1-14)

[0461] To a stirred solution of INT-8O (2.1 g, 6.373 mmol) in DCM (35 mL), were added EDC’HCl (1.6 g, 8.497 mmol), DIPEA (3.7 mL, 21.243 mmol) and DMAP (260 mg, 2.124 mmol) at 25 °C. The reaction mixture was stirred for 5 min and then was added 30-11 (2.4 g, 4.249 mmol) to the solution. Then the reaction mixture was stirred at 25 °C for 16h. The reaction was monitored by TLC (10% MeOH-DCM). Then it was diluted with water (100 mL) and extracted with EtOAc (100 mL X 3). The organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure to provide crude product (4.2 g), which was purified by combi flash chromatography, eluted with (1.2-2% MeOH-DCM) to provide the compound of Example 14 (2.1 g, 56%) as yellow liquid.

[0462] UPLC-MS: Method-B was performed for UPLC: Purity is 100 %; Rt 6.81 min, MS calculated: 876.69 [M+H], MS found 876.8 [M+H],

[0463] HPLC: Purity is 95.27% in CAD and 98.23% in DAD.

[0464] 1H NMR (400 MHz, MeOD) 60.83 - 0.94 (m, 9H), 1.28 - 1.44 (m, 40H), 1.49 - 1.65 (m, 10H), 1.75 - 1.88 (m, 5H), 1.91 -2.00 (m, 1H), 2.12 (q, J = 7.2 Hz, 4H), 2.30 (t, J = 7.4 Hz, 4H), 2.36 (t, J = 7.3 Hz, 2H), 2.56 - 2.65 (m, 4H), 2.78 (t, J = 5.7 Hz, 2H), 4.25 (t, J = 5.7 Hz, 2H), 4.61 (d, J = 6.8 Hz, 4H), 4.70 - 4.78 (m, 1H), 4.86 - 4.91 (m, 1H), 5.46 - 5.57 (m, 2H), 5.58 - 5.69 (m, 2H).Example 15: Dioctyl 9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy) heptadecanedioate (Compound 1-15)Compound 1-15

[0465] To a stirred solution of 8-1 (100 mg, 0.30 mmol) in DCM (5.0 mL), were sequentially added, EDC*HC1 (146.0 mg, 0.76 mmol) DMAP (11.16 mg, 0.09 mmol), DIPEA (0.1 mL, 1.21 mmol) under N2 atmosphere at 25°C and was stirred for 10 min. Then 37-1 (148.30 mg, 0.27 mmol) was added and stirred at 25°C for 16 h. Upon completion the reaction mixture was diluted with water and extracted with EtOAc (2 x 40 mL), organic layer was washed with brine, dried over NaiSC, filtered and concentrated under reduced pressure to provide crude. The crude was purified by combi flash column chromatography (eluted by 0-5% MeOH / DCM) to afford the compound of Example 15 (120 mg, 46%) as a yellow liquid.

[0466] 1H NMR (400 MHz, MeOD) 84.92 - 4.84 (m, 3H), 4.80 - 4.66 (m, 1H), 4.05 (t, J = 6.7 Hz, 4H), 2.69 (s, 6H), 2.39 - 2.26 (m, 6H), 1.86 - 1.81 (m, 7H), 1.66 - 1.50 (m, 14H), 1.31 (t, J = 6.6 Hz, 43H), 0.93 - 0.86 (m, 9H).

[0467] UPLC-MS: Method-B was performed for UPLC. Purity is 100.00%; Rt 6.78 min., MS calculated: 851.7 [M+H], MS found 851.8 [M+H],

[0468] HPLC-MS: Method- A was performed for HPLC. Purity is 97.77% (CAD) and 94.65% in DAD.Example 16: Dihexyl 9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)heptadecanedioate (Compound 1-16)Compound 1-16

[0469] To a stirred solution of 8-1 (100 mg, 0.30 mmol) in DCM (5.0 mL), were sequentially added, EDC*HC1 (145.91 mg, 0.76 mmol) DMAP (11.15 mg, 0.09 mmol), DIPEA (0.1 mL, 1.21 mmol) under N2 atmosphere at 25°C and was stirred for 10 min. Then 37-11 (132.83 mg, 0.27 mmol) was added and stirred at 25°C for 16 h. Upon completion the reaction mixture was diluted with water and extracted with EtOAc (2 x 40 mL), organic layer was washed with brine, dried over NaiSC, filtered and concentrated under reduced pressure to provide crude. The crude was purified by combi flash column chromatography (eluted by 0-5% MeOH / DCM) to afford the compound of Example 16 (120 mg, 50%) as a yellow liquid.

[0470] 'H NMR (400 MHz, MeOD) 64.97 - 4.85 (m, 1H), 4.80 - 4.66 (m, 1H), 4.06 (t, J = 6.6 Hz, 4H), 3.30 - 3.23 (m, 2H), 2.70 - 2.62 (m, 6H), 2.39 - 2.26 (m, 6H), 2.04 - 1.70 (m, 6H), 1.66 - 1.50 (m, 14H), 1.37 - 1.26 (m, 36H), 0.95 - 0.85 (m, 9H).

[0471] UPLC-MS: Method-B was performed for UPLC. Purity is 100.00%; Rt 6.37 min., MS calculated: 795.6 [M+H], MS found 795.8 [M+H],

[0472] HPLC-MS: Method-A was performed for HPLC. Purity is 97.77% (CAD) and 94.65% in DAD.Example 17: Dibutyl 9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy) heptadecanedioate (Compound 1-17)Compound 1-17

[0473] To a stirred solution of 8-1 (180 mg, 0.55 mmol) in DCM (10 mL), was added EDC’HCl (157.83 mg, 0.82 mmol), DMAP (33.48 mg, 0.27 mmol) and DIPEA (0.28 mL, 1.64 mmol). Reaction mass was stirred at an ambient temperature for 5 min, finally was added 37-III (187.93 mg, 0.44 mmol) and it was further stirred for 16 h. After completion thereaction mixture was diluted with EtOAc and washed with (2x50 mL) and brine (50 mL). Organic layer was dried over anhydrous Na2SO4, evaporated under reduced pressure. Crude compound (630 mg) was purified by repeat combi flash column chromatography, eluted with 2-5% MeOH-DCM to afford Example 17 (150 mg, 37%) as light-yellow gum.

[0474] UPLC-MS: Method-B was performed for UPLC. Purity is 100.00%; Rt 5.95 min., MS calculated: 739.58 [M+H], MS found 739.7 [M+H],

[0475] HPLC-MS: Method-A was performed for HPLC. Purity is 97.75% (CAD) and 82.45% in DAD.

[0476] 'H NMR (400 MHz, MeOD) 64.79 - 4.66 (m, 1H), 4.06 (t, J= 6.6 Hz, 4H), 3.28 -3.23 (m, 2H), 2.70 -2.62 (m, 6H), 2.39 -2.26 (m, 6H), 1.92 - 1.73 (m, 6H), 1.66 - 1.50 (m, 13H), 1.44 - 1.24 (m, 30H), 0.98 - 0.86 (m, 9H).Example 18: Di(undecan-5-yr)9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)heptadecanedioate (Compound 1-18)Compound 1-18

[0477] To a stirred solution of 8-1 (158 nig, 0.48 mmol) in DCM (12 mL), were added EDC’HCl (123 mg, 0.64 mmol), DIPEA (0.28 mL, 1.6 mmol) and DMAP (20 mg, 0.16 mmol) at RT. The reaction mixture was stirred for 20 min and was added 37-IV (200 mg, 0.32 mmol) to the solution. Then the reaction mixture was stirred at RT for 16 h. The reaction was monitored by TLC (10% MeOH-DCM). Then it was diluted with 50 ml water and extracted with EtOAc (50 mL x 3). The organic layer was washed with water, brine, dried over NazSOi and concentrated under reduced pressure to provide a crude product (325 mg), which was purified by combi flash chromatography, eluted with (2-3% MeOH-DCM) to provide Example 18 (140 mg, 47%) as light-yellow liquid.

[0478] 'H NMR (400 MHz, MeOD) 64.93 - 4.82 (m, 3H), 4.77 - 4.68 (m, 1H), 3.30 (p, J = 1.7 Hz, 2H), 2.77 - 2.73 (m, 6H), 2.39 - 2.25 (m, 6H), 1.95 - 1.72 (m, 6H), 1.64 - 1.48 (m, 18H), 1.38 - 1.23 (m, 48H), 0.94 - 0.85 (m, 15H).

[0479] UPLC-MS: Method-B was performed for UPLC. Purity is 100.00%; Rt 7.09 min., MS calculated: 935.79 [M+H], MS found 935.9 [M+H],

[0480] HPLC-MS: Method-A was performed for HPLC. Purity is 96.45% (CAD) and No response in DAD.Example 19: Bis(2-butylheptyl)9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)heptadecanedioate (Compound 1-19)Compound 1-19

[0481] To a stirred solution of 8-1 (100 mg, 0.30 mmol) in DCM (5.0 mL), were sequentially added, EDC•HCl (145.91 mg, 0.76 mmol), DMAP (11.15 mg, 0.09 mmol), DIPEA (0.2 mL, 1.21 mmol) under N2 atmosphere at 25°C and was stirred for 10 min. Then 37-V (190.29 mg, 0.30 mmol) was added and stirred at 25°C for 16 h. Upon completion the reaction mixture was diluted with water and extracted with EtOAc (2 x 40 ml.), organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to provide 310 mg crude. The crude was purified by combi flash column chromatography (eluted by 0-5% MeOH / DCM) to afford Example 19 (120 mg, 42%) as a yellow liquid.

[0482] 'H NMR (400 MHz, MeOD) 64.80 - 4.66 (m, 1H), 3.99 (d, J = 5.6 Hz, 4H), 3.29 - 3.19 (m, 2H), 2.67 - 2.63 (m, 6H), 2.38 - 2.27 (m, 6H), 1.86 - 1.79 (m, 4H), 1.63 - 1.58 (m, 6H), 1.56 - 1.50 (m, 6H), 1.36 - 1.27 (m, 55H), 0.95 - 0.86 (m, 15H).

[0483] UPLC-MS: Method-B was performed for UPLC. Purity is 99.35%; Rt 7.22 min., MS calculated: 879.7 [M+H], MS found 880.0 [M+H],

[0484] HPLC-MS: Method-A was performed for HPLC. Purity is 97.85% (CAD) and no response in DAD.Example 20: Di(pentadecan-8-yl) 9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)heptadecanedioate (Compound 1-20)Compound 1-20

[0485] To a stirred solution 8-1 (100 mg, 0.3 mmol) in DCM (10 mL), wasadded EDC’HCl (87.69 mg, 0.46 mmol), DMAP (18.6 mg, 0.15 mmol) and DIPEA (0.15 mL, 0.91 mmol). Reaction mass was stirred at an ambient temperature for 5 min, finally was added 37-VI (179.57 mg, 0.24 mmol) and it was further stirred for 16 h. After completion the reaction mixture was filtered and added water (25 mL) to the reaction mass and it was extracted with DCM (2 X 50 mL). Organic layer was dried over anhydrous Na2SO4, evaporated under reduced pressure. Crude compound (350 mg) was purified by repeat combi flash column chromatography, eluted with 2-5% MeOH-DCM to afford Example 20 (150 mg, 47%) as a light-yellow gum.

[0486] 1H NMR (400 MHz, MeOD) 64.79 - 4.69 (m, 1H), 2.72 - 2.68 (m, 6H), 2.37 - 2.25 (m, 6H), 1.87 - 1.82 (m, 6H), 1.65 - 1.50 (m, 19H), 1.36 - 1.26 (m, 68H), 0.90 (t, J= 6.8 Hz, 15H).

[0487] UPLC-MS: Method-B was performed for UPLC. Purity is 97.23%; Rt 7.52 min., MS calculated: 1047.92 [M+H], MS found 1048.0 [M+H],

[0488] HPLC-MS: Method-A was performed for HPLC. Purity is 98.99% (CAD) and No response in DAD.Example 21: Bis(5-methyloctyl)9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)heptadecanedioate (Compound 1-21)Compound 1-21

[0489] To a stirred solution of 8-1 (150 mg, 0.46 mmol) and 37-VII (259.82 mg, 0.46 mmol) in Toluene (5 mL), DBAD (316.04 mg, 1.37 mmol) and triphenylphosphine (359.24mg, 1.37 mmol) were added. The reaction mixture was stirred for 2h at 80 °C. Upon completion the reaction mixture was diluted with ethyl acetate and washed the organic part with water (2 times), dried over Na2SC>4, filtered and concentrated under reduced pressure to provide 450 mg. The crude was purified by combi flash column chromatography (two times) to afford Example 21 (230 mg, CAD purity 87%) as light-yellow liquid. Thus prep-HPLC was submitted. After prep-HPLC purification, Example 21 was collected (110 mg, 27%) with 97% CAD purity as a light-yellow liquid.

[0490] 'H NMR (400 MHz, MeOD) 84.79 - 4.65 (m, 1H), 4.06 (t, J = 6.5 Hz, 4H), 3.29 -3.23 (m, 2H), 2.68 -2.63 (m, 6H), 2.38 -2.26 (m, 6H), 2.01 - 1.68 (m, 6H), 1.66 - 1.46 (m, 14H), 1.45 - 1.21 (m, 39H), 1.18 - 1.07 (m, 4H), 0.93 - 0.84 (m, 15H).

[0491] UPLC-MS: Method-B was performed for UPLC. Purity is 99.35%; Rt 7.22 min., MS calculated: 879.7 [M+H], MS found 880.0 [M+H],

[0492] HPLC-MS: Method-A was performed for HPLC. Purity is 97.85% (CAD) and no response in DAD.Example 22: Bis(4,5-dimethyloctyl)9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)heDtadecanedioate (Compound 1-22)Compound 1-22

[0493] To a stirred solution of 8-1 (100 mg, 0.3 mmol) in DCM (5.0 mL), were sequentially added, EDC»HC1 (87.55 mg, 0.46 mmol), DMAP (11.16 mg, 0.09 mmol), DIPEA (0.17 mL, 1.22 mmol) under N2 atmosphere at 25°C and was stirred for 10 min. Then 37-VIII (181.75 mg, 0.3 mmol) was added and stirred at 25°C for 16 h. Upon completion the reaction mixture was diluted with water and extracted with EtOAc (2 x 20 mL), organic layer was washed with brine, dried over Na2SC>4, filtered and concentrated under reduced pressure to provide crude. The crude was purified by Combiflash column chromatography (eluted by 0-5% MeOH / DCM) to afford Example 22 (120 mg, 43%) as a yellow liquid.

[0494] 'H NMR (400 MHz, MeOD) 64.97 - 4.87 (m, 2H), 4.78 - 4.68 (m, 1H), 4.05 (t, J = 6.5 Hz, 4H), 3.31 - 3.27 (m, 2H), 2.76 - 2.72 (m, 6H), 2.30 (t, J = 7.3 Hz, 6H), 1.90 - 1.83(m, 5H), 1.78 - 1.48 (m, 15H), 1.47 - 1.18 (m, 39H), 1.18 - 1.01 (m, 3H), 0.97 - 0.74 (m, 18H).

[0495] UPLC-MS: Method-B was performed for UPLC. Purity is 100%; Rt 7.16 min., MS calculated: 907.7 [M+H], MS found 907.9 [M+H],

[0496] HPLC-MS: Method-A was performed for HPLC. Purity is 95.85% (CAD) and no response in DAD.Example 23: Bis(3,7-dimethyloctyl) 9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)heptadecanedioate (Compound 1-23)Compound 1-23

[0497] To a stirred solution of 37-IX (200 mg, 0.34 mmol) and 8-1 (132.05 mg, 0.4 mmol) in toluene (10 mL), was added triphenylphosphine (263.62 mg, 1 mmol) followed by the addition of di-tert-butyl azodi carb oxy late (DBAD) (231.43 mg, 1 mmol) at 0°C. The reaction mixture was stirred at RT for 16h. The reaction was monitored by TLC (10% MeOH-DCM). The reaction mixture was diluted with 50 ml water and extracted with EtOAc (75 mL X 2). The organic layer was washed with water, brine, dried over Na2SC>4 and concentrated under reduced pressure to provide crude (350 mg) which was purified through Combiflash column chromatography (eluted with 3-4% MeOH-DCM) to obtain Example 23 (170 mg, 56%) as light brown liquid.

[0498] 1H NMR (400 MHz, MeOD) 64.79 - 4.66 (m, 1H), 4.16 - 4.03 (m, 4H), 3.00 -2.71 (m, 6H), 2.72 -2.58 (m, 1H), 2.40 -2.32 (m, 2H), 2.29 (t, J= 7.3 Hz, 4H), 1.90 - 1.86 (m, 6H), 1.72 - 1.46 (m, 18H), 1.47 - 1.21 (m, 32H), 1.21 - 1.09 (m, 6H), 0.94 - 0.85 (m, 21H).

[0499] UPLC-MS: Method-B was performed for UPLC. Purity is 97.75%; Rt 6.84 min., MS calculated: 907.76 [M+H], MS found 907.9 [M+H],

[0500] HPLC-MS: Method-A was performed for HPLC. Purity is 97.66% (CAD) and 89.34% in DAD.Example 24: Di((Z)-oct-5-en-l-yl) 9-((4-(((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)heptadecanedioate (Compound 1-24)Compound 1-24

[0501] To a stirred solution of 8-1 (99.41 mg, 0.3 mmol) were added EDC*HC1 (96.42 mg, 0,5 mmol), DIPEA (0.22 mL, 1,26 mmol) and DMAP (15,36 mg, 0.13 mmol) at RT. The reaction mixture was stirred for 5 min and added 37-X (135 mg, 0.25 mmol) to the solution. Then the reaction mixture was stirred at RT for 16h. The reaction was monitored by TLC (10% MeOH-DCM) and checked by UPLC. Then the reaction mixture was diluted with 50 ml water and extracted with EtOAc (50 mL X 3). The organic layer was washed with water, brine, dried over Na₂SO₄ and concentrated under reduced pressure to provide crude (250 mg) which was purified through Combiflash column chromatography (eluted with 3-4% MeOH-DCM) to obtain Example 24 (75 mg) as a light-yellow liquid.

[0502] 1H NMR (400 MHz, MeOD) 65.43 - 5.25 (m, 4H), 4.80 - 4.64 (m, 1H), 4.10 - 4.02 (m, 4H), 2.74 -2.70 (m, 6H), 2.40 -2.25 (m, 6H), 2.12 - 1.99 (m, 8H), 1.87 - 1.83 (m, 6H), 1.69 - 1.50 (m, 9H), 1.47 - 1.37 (m, 6H), 1.38 - 1.20 (m, 30H), 0.99 - 0.92 (m, 6H), 0.92 - 0.86 (m, 3H).

[0503] UPLC-MS: Method-B was performed for UPLC. Purity is 95.61%; Rt 6.52 min., MS calculated: 847.67 [M+H], MS found 847.8 [M+H],

[0504] HPLC-MS: Method- A was performed for HPLC. Purity is 94.04% (CAD) and 70.52% in DAD.Example 25: Di((Z)-non-2-en-l-yl) 9-((4-(((3- (diethylamino)propoxy)carbonyl)oxy)decanoyl)oxy)heptadecanedioate (Compound I- 25)Compound 1-25

[0505] To a solution of INT-5A (1 g, 2.25 mmol) in THF (20 mL), was added 3-(diethylamino)propan-l-ol (38) (0.59 g, 4.51 mmol) followed by the addition of DIPEA (1.18 mL, 6.76 mmol) and DMAP (0.14 g, 1.13 mmol) and then stirred at 70 °C for 16 h. The consumption of starting material was judged by TLC (10% MeOH DCM). The reaction mixture was diluted with ethyl acetate (200 mL), washed with IM sodium carbonate (2 x 100 mL), water (100 mL), brine and finally dried over anhydrous Na₂SO₄. The resulting organic layer was concentrated to obtain crude (1.1 g) which was purified by flash column chromatography (eluted with 3-4% MeOH-DCM) to obtain benzyl 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoate (39) (610 mg, 62%) as a colorless sticky liquid.

[0506] 'H NMR (400 MHz, DMSO) 87.40 - 7.29 (m, 5H), 5.07 (s, 2H), 4.68 - 4.60 (m, 1H), 4.14 - 4.01 (m, 2H), 2.42 - 2.34 (m, 8H), 1.90 - 1.83 (m, 1H), 1.83 - 1.70 (m, 1H), 1.67 (t, J= 6.8 Hz, 2H), 1.54 - 1.48 (m, 2H), 1.23 (s, 8H), 0.91 (t, J= 7.1 Hz, 6H), 0.88 - 0.81 (m, 3H).

[0507] UPLC-MS: Method-B was performed for UPLC. Purity is 100%; Rt 4.40 min., MS calculated: 436.3 [M+H], MS found 436.4 [M+H],Synthesis of INT-40: 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoic acid

[0508] A solution of 39 (610 mg, 1.4 mmol) in MeOH (20 mL) was degassed under argon atmosphere for 5 min the added Pd / C (50% moist) (100 mg) at RT. Reaction mixture was stirred at 25°C under hydrogen balloon pressure for 2h. Upon completion, TLC (10% MeOH-DCM) was checked, SM was fully consumed. Then it was filtered through celite bed and washed with MeOH and concentrated under reduced pressure to obtain INT-40 (450 mg, 93%) as a light-yellow sticky gum.

[0509] 'H NMR (400 MHz, DMSO) 64.67 - 4.59 (m, 1H), 4.09 (t, J= 6.4 Hz, 2H), 2.49 - 2.39 (m, 5H), 2.25 - 2.16 (m, 2H), 1.88 - 1.77 (m, 1H), 1.77 - 1.64 (m, 3H), 1.54 - 1.48 (m, 2H), 1.33 - 1.16 (m, 9H), 0.93 (t, J= 7.1 Hz, 6H), 0.85 (t, J= 6.5 Hz, 3H).

[0510] UPLC-MS: Method-B was performed for UPLC. Purity is 100%; Rt 2.64 min., MS calculated: 346.25 [M+H], MS found 346.3 [M+H],

[0511] To a stirred solution of INT-40 (146.78 mg, 0.42 mmol) in DCM (12 mL), were added DIPEA (0.31 mL, 1.77 mmol), DMAP (21.63 mg, 0.18 mmol) and EDC•HCl (135.75 mg, 0.71 mmol) at RT. The reaction mixture was stirred for 5 min and then was added 30-11 (200 mg, 0.35 mmol) to the solution. Then the reaction mixture was stirred at RT for 16 h. After completion, reaction mixture was diluted with ethyl acetate (150 mL) and washed with water (100 mL X 2), brine (100 mL). Then the organic part was dried over Na₂SO₄and concentrated under reduced pressure to provide crude (325 mg) which was purified with combi flash column chromatography (eluted with 2-3% MeOH-DCM) to obtain the product Example 25 (160 mg, 51%) as a colorless liquid.

[0512] 'H NMR (400 MHz, MeOD) 65.71 - 5.60 (m, 2H), 5.59 - 5.48 (m, 2H), 4.80 -4.70 (m, 1H), 4.63 (d, J = 6.9 Hz, 4H), 4.17 (t, J = 6.2 Hz, 2H), 2.67 - 2.57 (m, 6H), 2.40 -2.28 (m, 6H), 2.14 (q, J= 7.2 Hz, 4H), 2.04 - 1.77 (m, 3H), 1.69 - 1.50 (m, 10H), 1.35 - 1.30 (m, 42H), 1.08 (t, J = 7.2 Hz, 6H), 0.96 - 0.88 (m, 9H).

[0513] UPLC-MS: Method-B was performed for UPLC. Purity is 100%; Rt 6.92 min., MS calculated: 892.72 [M+H], MS found 893.0 [M+H],

[0514] HPLC-MS: Method-A was performed for HPLC. Purity is 92.98% (CAD) and 90.29% in DAD.Example 26: Di((Z)-non-2-en-l-yl) 9-((4-((4-(pyrrolidin-l-yl)butanoyl)oxy)decanoyl)oxy)heptadecanedioate [Compound 1-26)Compound 1-26

[0515] To a stirred solution of 4-(pyrrolidin-l-yl)butanoic acid (500 mg, 3.18 mmol) in DCM (15 mL), were added EDCHICl (915.97 mg, 4.77 mmol), DIPEA (1.66 mL, 9.54 mmol) and DMAP (116.57 mg, 0.95 mmol). The reaction mixture was stirred for 15 min and then 4 (1062.48 mg, 3.82 mmol) was added to the solution. The reaction mixture wasstirred for 16h at 25°C. The reaction was monitored by TLC (10% MeOH-DCM). The reaction mixture was diluted with 75 ml water and extracted with ethyl acetate (100 mL X 2). The organic layer was washed with brine and dried over Na2SO4 and concentrated under reduced pressure to provide crude product, which was purified by combi flash column chromatography using (4-5% MeOH-DCM) to provide benzyl 4-((4-(pyrrolidin-l-yl)butanoyl)oxy)decanoate (44) (870 mg, 65%) as a light-yellow thick liquid.

[0516] UPLC-MS: Method-B was performed for UPLC. Purity is 100%; Rt 4.27 min., MS calculated: 418.29 [M+H], MS found 418.4 [M+H],Synthesis of INT-45: 4-((4-(pyrrolidin-l-yl)butanoyl)oxy)decanoic acid

[0517] A solution of 44 (850 mg, 2.04 mmol) in MeOH (15 mL) was degassed under argon atmosphere for 5 min then added Pd / C (50% moist) (150 mg) at RT. Reaction mixture was stirred at 25°C under hydrogen balloon pressure for 2h. Upon completion, TLC (10% MeOH-DCM) was checked, SM was fully consumed. Then it was filtered through celite bed and washed with MeOH and concentrated under reduced pressure to obtain INT-45 (615 mg, 92%) as a light-yellow sticky gum.

[0518] 'H NMR (400 MHz, DMSO) 64.87 - 4.77 (m, 1H), 2.61 - 2.53 (m, 1H), 2.47 -2.35 (m, 6H), 2.30 (t, J= 7.2 Hz, 2H), 2.21 - 2.14 (m, 2H), 1.74 - 1.62 (m, 8H), 1.53 - 1.43 (m, 2H), 1.25 - 1.21 (m, 8H), 0.89 - 0.81 (m, 3H).

[0519] UPLC-MS: Method-B was performed for UPLC. Purity is 98.35%; Rt 4.98 min., MS calculated: 328.24 [M+H], MS found 328.3 [M+H],

[0520] To a stirred solution of INT-45 (139.13 mg, 0.42 mmol) and 30-11 in toluene (10 mL), was added triphenylphosphine (278.6 mg, 1.06 mmol), followed by the addition of di-tert-butyl azodi carb oxy late (DBAD) (244.58 mg, 1.06 mmol) at 0°C. The reaction mixture was stirred at RT for 16 h. The reaction was monitored by TLC (10% MeOH-DCM). The reaction mixture was diluted with 200 ml water and extracted with ethyl acetate (200 mL X 2). The organic layer was washed with water and brine and dried over Na₂SO₄ andconcentrated under reduced pressure to provide crude product (345 mg), which was purifiedtwo times by combi flash chromatography using (2-3% MeOH-DCM) to provide Example 26 (95 mg, 31%) as a light-yellow gum.

[0521] 'H NMR (400 MHz, MeOD) 65.71 - 5.61 (m, 2H), 5.59 - 5.49 (m, 2H), 4.63 (d, J = 6.9 Hz, 4H), 2.77 - 2.73 (m, 4H), 2.70 - 2.63 (m, 2H), 2.39 - 2.28 (m, 6H), 2.14 (q, J= 7.2 Hz, 4H), 1.95 - 1.86 (m, 8H), 1.64 - 1.52 (m, 10H), 1.46 - 1.21 (m, 46H), 0.96 - 0.88 (m, 9H).

[0522] UPLC-MS: Method-B was performed for UPLC. Purity is 93.62%; Rt 7.71 min., MS calculated: 874.71 [M+H], MS found 875.0 [M+H],

[0523] HPLC-MS: Method-A was performed for HPLC. Purity is 91.87% (CAD) and No response in DAD.Example 27: Di((Z)-non-2-en-l-yl) 9-((4-(((2- (diethylamino)ethyl)carbamoyl)oxy)decanoyl)oxy)heptadecanedioate (Compound 1-27)Compound 1-27

[0524] To a solution of INT-5A (500 mg, 1.127 mmol) in dichloromethane (10 mL), was added l-(2-aminoethyl)pyrrolidine (262 mg, 2.255 mmol) followed by the addition of DIPEA (0.6 mL, 3.382 mmol) and stirred at 25 °C for 16 h. The consumption of starting material was judged by TLC (10% MeOH DCM). The reaction mixture was diluted with di chloromethane (150 mL), washed with IM sodium carbonate (2 x 100 mL), water (100 mL), brine and finally dried over anhydrous Na2SC>4. The resulting dichloromethane layer was concentrated to obtain crude (575 mg) which was purified by flash column chromatography (eluted with 3-4% MeOH-DCM) to obtain benzyl 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)decanoate (47) (310 mg, 65%) as a light-yellow sticky liquid.

[0525] 'H NMR (400 MHz, DMSO) 67.40 - 7.29 (m, 5H), 6.87 - 6.82 (m, 1H), 5.07 (s, 2H), 4.65 - 4.60 (m, 1H), 3.00 - 2.96 (m, 2H), 2.46 - 2.31 (m, 8H), 1.83 - 1.78 (m, 1H), 1.73 - 1.63 (m, 1H), 1.47 - 1.42 (m, 2H), 1.23 (s, 8H), 0.91 (t, J= 7.1 Hz, 6H), 0.87 - 0.79 (m, 3H).Synthesis of INT-48: 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)decanoic acid

[0526] A solution of 47 (310 mg, 0.737 mmol) in MeOH (10 mL) was degassed under argon atmosphere for 5 min then added Pd / C (50% moist) (75 mg) at RT. Reaction mixture was stirred at 25°C under hydrogen balloon pressure for 2h. Upon completion, TLC (10% MeOH-DCM) was checked, SM was fully consumed. Then it was filtered through celite bed and washed with MeOH and concentrated under reduced pressure to obtain INT-48 (230 mg, 94%) as light-yellow sticky gum.

[0527] 'H NMR (400 MHz, DMSO) 66.87 - 6.79 (m, 1H), 4.63 - 4.58 (m, 1H), 3.04 -2.95 (m, 2H), 2.53 -2.37 (m, 6H), 2.22 -2.15 (m, 2H), 1.76 - 1.71 (m, 1H), 1.68 - 1.56 (m, 1H), 1.47 - 1.42 (m, 2H), 1.35 (s, OH), 1.35 - 1.14 (m, 9H), 0.93 (t, J= 7.1 Hz, 6H), 0.85 (t, J = 6.6 Hz, 3H).

[0528] To a stirred solution of INT-48 (130 mg, 0.39 mmol) and 30-11 in toluene (15 mL) were added PPhs (309.54 mg, 1.18 mmol), 42 (222.22 mg, 0.39 mmol) and DBAD (271.74 mg, 1.18 mmol). Reaction mixture was stirred at 25 °C for 16h. Upon completion, reaction mixture was diluted with EtOAc and washed with water (2 x 150 mL) and brine (50 mL). Organic layer was dried Na₂SO₄ and concentrated under reduced pressure. Crude mass (460 mg) was purified by repeat combi flash column chromatography, eluted with 2-3% MeOH-DCM to afford Example 27 (165 mg, 48%) as a colorless gum.

[0529] 'H NMR (400 MHz, MeOD) 65.69 - 5.58 (m, 2H), 5.57 - 5.46 (m, 2H), 4.78 -4.66 (m, 1H), 4.61 (d, J = 6.9 Hz, 4H), 3.21 (t, J = 7.1 Hz, 2H), 2.67 - 2.57 (m, 7H), 2.43 -2.24 (m, 7H), 2.12 (q, J= 7.2 Hz, 4H), 1.90 - 1.85 (m, 1H), 1.82 - 1.70 (m, 1H), 1.63 - 1.49 (m, 8H), 1.44 - 1.19 (m, 40H), 1.07 (t, J = 7.2 Hz, 6H), 0.93 - 0.85 (m, 9H).

[0530] UPLC-MS: Method-B was performed for UPLC. Purity is 99.58%; Rt 6.94 min., MS calculated: 877.72 [M+H], MS found 877.8 [M+H],

[0531] HPLC-MS: Method-A was performed for HPLC. Purity is 95.26% (CAD) and No response in DAD.Example 28: Utilization of LNP preparations for base editing in spleen, liver, and bone marrow

[0532] Among other subject matters, the present Example provides exemplary LNP compositions, preparations, and / or lipid nanoparticles as described herein.

[0533] The present Example provides exemplary LNP compositions, preparations, and / or lipid nanoparticles that confer gene editing (e.g., using base editors) in a variety of cell types. The screening platforms described herein can be used to identify LNP preparations to determine what type of LNP preparation would be most potent for base editing of a variety of cell types in vivo (e.g., can perform base editing of cells in mice). The present example can be used to demonstrate that provided lipids can be used to perform base editing in a variety of cell types, including bone marrow, spleen, and liver, and / or improve delivery of LNP preparations to particular cell types. The present example can also be used to demonstrate that mRNA contained with LNP preparations containing an exemplary compound of are more stabilized in the LNP during delivery.Methods

[0534] Exemplary LNP preparations are selected to determine each preparation’s ability to perform base editing in a Balb / C mouse model as described herein.

[0535] Lipid nanoparticle components were dissolved in 100% ethanol at specified lipid component molar ratios. Nucleic acid (NA) cargo was dissolved in 10 mM citrate, 100 mM NaCl, pH 4.0, resulting in a concentration of NA cargo of approximately 0.22 mg / mL.

[0536] LNP preparations were prepared with 47.5% ionizable lipid (e.g., a compound provided herein): 40% cholesterol: 2.5% PEG-2000 DMG: 10% DPSC and an N / P ratio range of 4.5-6. LNP preparations were formed by microfluidic mixing of the lipid and NA solutions using Precision Nanosystems NanoAssemblr Spark or Benchtop series Instruments, according to the manufacturer’s protocol.

[0537] A ratio of aqueous to organic solvent of approximately 2:1 or 3: 1 was maintained during mixing using differential flow rates. After mixing, LNP preparations were collected, diluted in phosphate buffer saline (PBS) or Tris-buffered saline (TBS) (approximately 1:1 v / v). Further buffer exchange was conducted using dialysis in PBS or TBS at 4 °C for 4 to 24 hours against a 20 kDa filter. After this initial dialysis, each individual LNP preparation may be characterized via dynamic light scattering (DLS) to measure the size (e.g., diameter) and polydispersity. LNP preparations falling within specific diameter and poly dispersity ranges were pooled, and further dialyzed against PBS or TBS at 4 °C for 1 to 4 hours against a 100kDa dialysis cassette. After the second dialysis, LNP preparations were sterile filtered using 0.22 pm filter and stored at 4 °C for further use.LNP Characterization

[0538] DLS - LNP preparation hydrodynamic diameter and poly dispersity index (PDI) is measured using high throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt). LNP preparations are diluted IX PBS to an appropriate concentration and analyzed. Concentration & Encapsulation Efficiency

[0539] Concentration of NA is determined by Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) per manufacturer’s instructions. Encapsulation efficiency is determined by measuring unlysed and lysed LNPs.ALAS and PCSK9 Base editing

[0540] Male Balb / C mice aged approximately 8-12 weeks are used in the experiments described herein. Each mouse is temporarily restrained, and LNP preparations are administered IV via tail vein injection. Age-matched mice are also used to administer vehicle (IX PBS) via tail vein injection as a control. Four to six days post-dose, tissues including liver, spleen, heart, and muscle are collected (only liver and muscle (quadricep) are reported). Genomic DNA is isolated and fragmented and adapter-ligated using the Nextera DNA Flex Library Prep Kit (Illumina) using the 96-well plate Nextera indexing primers (Illumina), according to the manufacturer’s instructions. Library size and concentration is confirmed by Fragment Analyzer (Agilent) and sent to Novogene for whole genome sequencing using an Illumina HiSeq.

[0541] For base editing, mRNA encoding an adenine base editor (ABE) and a guide RNA (sgRNA) targeting ALAS1 or PCSK9 at 1: 1 (mass ratio) is co-encapsulated in LNP preparations as described herein. LNP preparations are administrated into Balb / C mice through tail vein injections at concentrations ranging from 0.03 to 0.1 mg / kg total RNA. At 4 days after dosing, mice are euthanized, and liver, spleen and bone barrow may be harvested. Base editing is determined by performing targeted deep sequencing analysis at the ALAS1 or PCSK9 target site using extracted genomic DNA.

[0542] Characterization and ALAS1 or PCSK9 liver base editing data for LNP preparations prepared using the provided ionizable lipids is summarized in Table A. The LNPs of Table A have a formulation ratio of 47.5:40:2.5:10.Table A.Equivalents

[0543] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A compound having a structure according to compound of Formula (I)or a pharmaceutically acceptable salt thereof, wherein:X1is -NRARBor a 5- to 6-membered nitrogen-containing heterocycle;each of RAand RBis independently H or Ci-Ce alkyl;L1is Ci-C6alkylene;X2is a covalent bond, -O-, or -NH-;R1is C1-C10 alkyl;L2is Ci-C6alkylene;each of L3and L4is independently Ce-Cio alkylene;each of L5and L6is independently -C(O)O- or -OC(O)-; andeach of R2and R3is independently C4-C20 alkyl, C4-C20 alkenyl, or C4-C20 alkynyl; wherein each heterocycle, alkyl, alkenyl, alkynyl, and alkylene group is optionally substituted;each 5- to 6-membered nitrogen-containing heterocycle is optionally substituted with one or more substituents selected from Ci-Ce alkyl, NH2, NH(Ci-Ce alkyl), N(Ci-Ce alkyl)2, C3-C6 cycloalkyl, and 5- to 6-membered heterocycle optionally substituted with one or more Ci-C6alkyls;each Ci-Ce alkylene or Ce-Cio alkylene is optionally substituted with one or more halogens;each Ci-Ce alkyl or C1-C10 alkyl is optionally substituted with one or more substituents selected from halogen, OH, O(C1-C6alkyl), NH2, NH(Ci-Ce alkyl), N(Ci-Ce alkyl)2, C3-C6 cycloalkyl, and 5- to 6-membered heterocycle optionally substituted with one or more Ci-Ce alkyls; and / oreach C4-C20 alkyl, C4-C20 alkenyl, or C4-C20 alkynyl is optionally substituted with one or more substituents selected from halogen, OH, O(C1-C6alkyl), C(O)O(C4-C20 alkyl), OC(O)(C4-C20 alkyl), C(0)0(C4-C2o alkenyl), and OC(0)(C4-C2o alkenyl).

2. The compound of claim 1, wherein each of RAand RBis Ci-Ce alkyl.

3. The compound of claim 1 or 2, wherein each of RAand RBis independently H, methyl, or ethyl.

4. The compound of any one of claims 1-3, wherein each of L3and L4is Ce-Cio alkylene.

5. The compound of any one of claims 1-4, wherein each of L5and L6is -C(O)O-.

6. The compound of any one of claims 1-5, having a structure according to the following formula (II),or a pharmaceutically acceptable salt thereof, wherein each of m and n is independently an integer of 6-10.

7. The compound of any one of claims 1-6, having a structure according to the following formula (III),or a pharmaceutically acceptable salt thereof.

8. The compound of any one of claims 1-6, having a structure according to the following formula (IV),or a pharmaceutically acceptable salt thereof.

9. The compound of any one of claims 1-6, having a structure according to the following formula (V),or a pharmaceutically acceptable salt thereof.

10. The compound of any one of claims 1-9, wherein L1is -CH2-, -CH2CH2-, or -CH2CH2CH2-.

11. The compound of any one of claims 1-10, wherein R1is w-hexyl.

12. The compound of any one of claims 1-11, wherein each m and n is an integer of 7.

13. The compound of any one of claims 1-12, wherein each of R2and R3is C4-C20 alkyl or C4-C20 alkenyl.

14. The compound of claim 13, wherein each of R2and R3is independently selected froma group consisting of:

15. The compound of claim 14, wherein each of R2and R3is independently selected from16. The compound of any one of claims 1-15, wherein X1is selected from a groupconsisting of:

17. The compound of claim 1, selected from a group consisting of:(1-26); andor a pharmaceutically acceptable salt thereof.

18. A lipid nanoparticle (LNP) preparation comprising a compound according to any one of claims 1-17, or a pharmaceutically acceptable salt thereof.

19. A lipid nanoparticle (LNP) preparation comprising:a compound according to any one of claims 1-17, or a pharmaceutically acceptable salt thereof;a phospholipid;a cholesterol; anda conjugate-linker lipid (e.g., polyethylene glycol lipid).

20. The LNP preparation of claim 18 or 19, further comprising a therapeutic and / or prophylactic agent.

21. The LNP preparation of any one of claims 18-20, wherein the therapeutic and / or prophylactic agent is or comprises one or more nucleic acids.

22. The LNP preparation of claim 21, wherein the one or more nucleic acids is or comprises RNA.

23. The LNP preparation of claim 21 or 22, wherein the one or more nucleic acids is or comprises DNA.

24. The LNP preparation of any one of claims 18-23, wherein the LNP preparation is formulated to deliver the therapeutic and / or prophylactic agent to target cells.

25. The LNP preparation of claim 24, wherein the target cells are or comprise spleen cells (e.g., splenic B cells, splenic T cells, splenic monocytes), liver cells (e.g., hepatocytes), bone marrow cells (e.g., bone marrow monocytes), immune cells, muscle cells (e.g., myocytes), heart cells (e.g., cardiomyocytes), kidney cells, or cells in the central nervous system.

26. The LNP preparation of claim 24, wherein the target cells are or comprise hematopoietic stem cells.

27. The LNP preparation of claim 24, wherein the target cells are or comprise liver cells.

28. A pharmaceutical composition comprising an LNP preparation of any one of claims 18-27 and a pharmaceutically acceptable excipient.

29. A method for administering a therapeutic and / or prophylactic agent to a subject in need thereof, the method comprising administering the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28 to the subject.

30. A method for treating a disease or a disorder in a subject in need thereof, the method comprising administering the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28 to the subject, wherein the therapeutic and / or prophylactic agent is effective to treat the disease.

31. A method for delaying and / or arresting progression a disease or a disorder in a subject in need thereof, the method comprising administering the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28 to the subject, wherein the therapeutic and / or prophylactic agent is effective to treat the disease.

32. A method of delivering a therapeutic and / or prophylactic agent to a mammalian cell derived from a subject, the method comprising contacting the cell of the subject having been administered the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28.

33. A method of producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the LNP preparation of any one of claims 18-27 or thepharmaceutical composition of claim 28, wherein the therapeutic and / or prophylactic agent is or comprises an mRNA, and wherein the mRNA encodes the polypeptide of interest, whereby the mRNA is capable of being translated in the cell to produce the polypeptide of interest.

34. A method of inhibiting production of a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28, wherein the therapeutic and / or prophylactic agent is or comprises an RNA, whereby the RNA is capable of inhibiting production of the polypeptide of interest.

35. A method of specifically delivering a therapeutic and / or prophylactic agent to a mammalian organ, the method comprising contacting a mammalian organ with the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28, whereby the therapeutic and / or prophylactic agent is delivered to the organ.

36. The method of claim 35, comprising administering to a subject the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28 to the subject.

37. A method of vaccinating by administering the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28.

38. A method of inducing an adaptive immune response in a subject, comprising administering to the subject an effective amount of a composition comprising at least one RNA; wherein the composition comprises an LNP preparation comprising a compound of any one of claims 1-17, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof.

39. The method of claim 38, wherein the LNP preparation is the LNP preparation of anyone of claims 18-27 and / or the pharmaceutical composition is the pharmaceutical composition of claim 28.

40. A method of base editing a genome of a cell, the method comprising contacting the cell of the subject having been administered the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28.

41. A method of base editing a genome of a cell in a subject, the method comprising administering to the subject an effective amount of a composition comprising the LNP preparation of any one of claims 18-27 or the pharmaceutical composition of claim 28.

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