Ionizable lipids

Abstract

The present disclosure provides, among other things, a compound of formula I, or a pharmaceutically acceptable salt thereof, and uses thereof.

Description

IONIZABLE LIPIDSCROSS REFERNCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 613,653, filed December 21, 2023, which is incorporated in its entirety by reference herein.BACKGROUND

[0002] Particles for delivery of nucleic acids have been the subject of much recent work. Certain particles, such as lipid nanoparticles (LNPs) are particularly useful for the transport of therapies such as nucleic acid therapies to cells. See Tenchov, et al., ACS Nano, 2021, 15, 11 16982-17015. LNPs comprise, among other things, cationic or ionizable lipids that, through electrostatic interaction, form stable complexes that encapsulate nucleic acids and thereby facilitate delivery into the cell.SUMMARY

[0003] There remains a need for cationic or ionizable lipids that are capable of forming complexes with nucleic acids, but have improved properties (e.g., improved transfection of nucleic acids) relative to previous lipid formulations. The present disclosure provides cationic or ionizable lipid compounds that comprise symmetric aliphatic tails which, surprisingly, improve particular properties of nucleic acid particles comprising said cationic or ionizable lipids. The present disclosure further provides, among other things, methods of preparing said cationic or ionizable lipids.

[0004] In some embodiments, the present disclosure provides a compound of formula I:or a pharmaceutically acceptable salt thereof, whereinM1and M2are each -(CH2)p1-;L1and L2are each selected from a bond, -OC(O)-, or -C(O)O-;T1and T2are each:L3is optionally substituted C1-C6aliphatic;G1is -OH, or -N(Ra)2, andRais H, optionally substituted C1-C6aliphatic, or optionally substituted C3-C6cycloaliphatic; p1 is an integer selected from 2-20; p2 and p3 each an integer selected from 1-20, inclusive, and p2 and p3 are the same.

[0005] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described herein (e.g., a cationic or ionizable lipid such as a compound of formula I) and a nucleic acid.

[0006] In some embodiments, the present disclosure provides a suspension comprising a dispersed phase and an aqueous phase, and wherein the dispersed phase comprises a pharmaceutical composition as described herein in the form of particles.

[0007] In some embodiments, the present disclosure provides a method of treating a disease, disorder, or condition comprising administering to a subject a suspension described herein.

[0008] In some embodiments, the present disclosure provides a method of increasing or causing increased expression of RNA in a target in a subject comprising administering to the subject a suspension described herein.

[0009] In some embodiments, the present disclosure provides a method of preparing a compound of formula III:or a pharmaceutically acceptable salt thereof, the method comprising contacting a compound of formula IV- 1 with a compound of formula IV-2 in the presence of heat:to provide a first intermediate product, and wherein the first intermediate product is contacted with a platinum catalyst and a first acid in the presence of hydrogen gas to provide a second intermediate product, and wherein the second intermediate product is contacted with a second acid to provide the compound of formula III, whereinM1and M2are independently selected from a bond or optionally substituted C2-C10aliphatic;L1and L2are each independently selected from a bond, -OC(O)- and -C(O)O-;T1and T2are each independently selected from optionally substituted C2-C20aliphatic;L3is optionally substituted C1-C6aliphatic;LG1is a suitable leaving group; andPG is a suitable alcohol protecting group.

[0010] In some embodiments, a compound of formula IV-2 is prepared by contacting a compound of formula V-1a and formula V-1b with a compound of formula V-2in the presence of a palladium coupling catalyst, a copper(I) cocatalyst, and an amine base to provide the compound of formula IV-2, wherein LG2is a suitable leaving group.

[0011] In some embodiments, the present disclosure provides a method of preparing a compound of formula III- 1 :or a pharmaceutically acceptable salt thereof, the method comprising contacting a compound of formula IV- 1 with a compound of formula IV-3 in the presence of heat:to provide a first intermediate product, and wherein the first intermediate product is contacted with an acid to provide the compound of formula III- 1 , whereinM1and M2are each independently selected from a bond or optionally substituted C2-C10aliphatic;T1and T2are each independently selected from optionally substituted C2-C20aliphatic;L3is optionally substituted C1-C6aliphatic;LG1is a suitable leaving group; andPG is a suitable alcohol protecting group.BRIEF DESCRIPTION OF THE DRAWING

[0012] FIG. 1 illustrates in vitro expression of LNPs prepared using cationic or ionizable lipid compounds described herein in HepG2 cells.

[0013] FIG. 2 illustrates in vitro expression of LNPs prepared using cationic or ionizable lipid compounds described wherein said LNPs comprise an anionic amphiphile selected from DMGS, DPGS, and SMGS.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0014] The present disclosure provides, among other things, the surprising discovery of particular cationic or ionizable lipids comprising heterocyclic head group and symmetric aliphatic tails. Such lipids exhibit improved properties relative to lipids having asymmetric aliphatic tails. For example, as illustrated in the Examples provided herein, lipids having a heterocyclic head group and symmetric tails exhibit improved transfection relative to other lipid formulations.Compounds and Definitions

[0015] Compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75thEd. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5thEd., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0016] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, Table 1 shows one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and / or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.

[0017] Unless otherwise indicated, structures depicted herein are 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 replacement of hydrogen by deuterium or tritium, or replacement of a carbon by13C- or14C-enriched carbon are within the scope of this disclosure.

[0018] About or approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In general, those skilled in the art, familiar within the context, will appreciate the relevant degree of variance encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" may encompass a range of values that are within (i.e., ±) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0019] Administering: As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. 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 be parenteral. In some embodiments, administration may be oral. In some particular embodiments, administration may be intravenous. In some particular embodiments, administration may be subcutaneous. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. 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) forat least a selected period of time. In some embodiments, administration may comprise a prime- and-boost protocol. A prime-and-boost protocol can include administration of a first dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) followed by, after an interval of time, administration of a second or subsequent dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine). In the case of an immunogenic composition, a prime-and-boost protocol can result in an increased immune response in a patient.

[0020] Aliphatic: The term “aliphatic” refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “cycloaliphatic”), that has a single point or more than one points of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., C1-6). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6alkyl.

[0021] Alkyl: The term “alkyl”, used alone or as part of a larger moiety, refers to a saturated, optionally substituted straight or branched chain hydrocarbon group having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C1-12, C1-10, C1-8, C1-6, C1-4, C1-3, or C1-2). Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0022] Alkylene: The term “alkylene” is refers to a bivalent alkyl group. In some embodiments, “alkylene” is a bivalent straight or branched alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, e.g., from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is optionally replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group and also include those described in the specification herein. It will be appreciatedthat two substituents of the alkylene group may be taken together to form a ring system. In certain embodiments, two substituents can be taken together to form a 3- to 7-membered ring. The substituents can be on the same or different atoms. The suffix “-ene” or “-enyl” when appended to certain groups herein are intended to refer to a bifunctional moiety of said group. For example, “-ene” or “-enyl”, when appended to “cyclopropyl” becomes “cyclopropylene” or“cyclopropylenyl” and is intended to refer to a bifunctional cyclopropyl group, e.g.,

[0023] Alkenyl: The term “alkenyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain or cyclic hydrocarbon group having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms(e.g., C2-12, C2-10, C2-8, C2-6, C2-4, orC2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term “cycloalkenyl” refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0024] Alkynyl: The term “alkynyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0025] Aryl: The term “aryl” refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, an “aryl” group contains between six and twelve total ring members (e.g., C6-C12). The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons. In some embodiments, an “aryl” ring system is an aromatic ring (e.g., phenyl) that is fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aryl rings include that are fused include

[0026] Biological sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0027] Carbocyclyl: The terms “carbocyclyl,” “carbocycle,” and “carbocyclic ring” as used herein, refer to saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having from 3 to 14 members, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, “carbocyclyl” (or “cycloaliphatic”) refers to an optionally substituted monocyclic C3-C8hydrocarbon, or an optionally substituted C7-C10bicyclic hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. The term “cycloalkyl” refers to an optionally substituted saturated 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. The term “cycloalkenyl” refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0028] Carrier: As used herein, 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.

[0029] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents or modality(ies)). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality (ies) to a subject receiving the other agent(s) or modality (ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0030] Comparable. As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In someembodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0031] 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.

[0032] Cycloaliphatic. As used herein, the term “cycloaliphatic” refers to a monocyclic C3-8hydrocarbon or a bicyclic C6-10hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point or more than one points of attachment to the rest of the molecule.

[0033] Cycloalkyl. As used herein, the term “cycloalkyl” refers to an optionally substituted saturated ring monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0034] Deoxyribonucleic Acid (DNA). As used herein, the term “DNA” refers to a polymeric molecule of nucleotides that are typically double-stranded and comprise adenine, cytosine, guanine and thymine, and a deoxyribose sugar backbone structure as specified in the definition “Nucleic Acid / Polynucleotide.” In some embodiments, DNA is linear DNA, plasmid DNA, minicircle DNA, nanoplasmid DNA, doggybone DNA, or a transposon.

[0035] Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide” refers to unmodified and modified deoxyribonucleotides. For example, unmodified deoxyribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and thymine (T). Modified deoxyribonucleotides may include one or more modifications including,but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g, at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.

[0036] Dosing regimen or therapeutic regimen: Those skilled in the art will appreciate that the terms “dosing regimen” and “therapeutic regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0037] Excipient: As used herein, 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.

[0038] Heteroaliphatic. The term “heteroaliphatic” or “heteroaliphatic group”, as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, fromone to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. The term “nitrogen” also includes a substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2- CH2-O-CH2-CH2-O-CH3, and the like.

[0039] 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. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, 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, cycloaliphatic, 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, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H- -quinolizinyl, carbazolyl,acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, 4H-thieno[3,2-b]pyrrole, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.

[0040] Heteroatom: The term “heteroatom” as used herein refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.

[0041] Heterocycle: As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, a 6- to 10-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 radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms.

[0042] Nanoparticle: As used herein, the term “nanoparticle” refers to a discrete entity of small size, e.g., typically having a longest dimension that is shorter than about 1000 nanometers (nm) and often is shorter than 500 nm, or even 100 nm or less. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 μm and about 500 nm, or between about 1 nm and 1000 nm. In many embodiments, a population of microparticles is characterized by an average size (e.g., longest dimension) that is below about 1000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm. In many embodiments, a microparticle may be substantially spherical (e.g., so that its longest dimension may be its diameter). In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and / or biodegradable polymer.

[0043] Nucleic acid / Polynucleotide". As used herein, the term “nucleic acid” refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises a mixture of DNA and RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double- stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double- stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate, phosphorodithioate, phosphoramide, phosphite-borane complexes, or 5'-N- phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues.In some embodiments, a non-natural residue comprises a nucleoside analog (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-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or 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, 110, 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, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000,13.500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000,19.500, or 20,000 or more residues or nucleotides long.

[0044] Nucleic acid particle: A “nucleic acid particle” can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable lipid or lipid-like material and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0045] Nucleotide: As used herein, the term “nucleotide” refers to its art-recognized meaning. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0046] Parenteral: The phrases “parenteral administration” and “administered parenterally” as used herein have their art-understood meaning referring to modes of administration other thanenteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0047] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond between ring atoms. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (e.g., aryl or heteroaryl) moieties, as herein defined.

[0048] Patient or subject: As used herein, the term “patient” or “subject” 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 or subjects 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 or a subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient or subject displays one or more symptoms of a disorder or condition. In some embodiments, a patient or subject has been diagnosed with one or more disorders or conditions. In some embodiments, a patient or a subject is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition.

[0049] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic or dosing 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 apessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0050] Pharmaceutically acceptable: As used herein, the phrase “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 animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0051] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, 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).

[0052] Physiological conditions: as used herein, has its art-understood meaning referencing conditions under which cells or organisms live and / or reproduce. In some embodiments, the term refers to conditions of the external or internal mileu that may occur in nature for an organism or cell system. In some embodiments, physiological conditions are those conditions present within the body of a human or non-human animal, especially those conditions present at and / or within a surgical site. Physiological conditions typically include, e.g., a temperature range of 20 - 40°C, atmospheric pressure of 1, pH of 6-8, glucose concentration of 1-20 mM, oxygen concentration at atmospheric levels, and gravity as it is encountered on earth. In some embodiments, conditions in a laboratory are manipulated and / or maintained at physiologic conditions. In some embodiments, physiological conditions are encountered in an organism.

[0053] Polycyclic'. As used herein, the term “polycyclic” refers to a saturated or unsaturated ring system having two or more rings (for example, heterocyclyl rings, heteroaryl rings, cycloalkyl rings, or aryl rings), having between 7 and 20 atoms, in which one or more carbon atoms are common to two adjacent rings. For example, in some embodiments, a polycyclic ring system refers to a saturated or unsaturated ring system having three or more rings (for example, heterocyclyl rings, heteroaryl rings, cycloalkyl rings, or aryl rings), having between 14 and 20 atoms, in which one or more carbon atoms are common to two adjacent rings. The rings in apolycyclic ring system may be fused (i.e., bicyclic or tricyclic), spirocyclic, or a combination thereof. An example polycyclic ring is a steroid.

[0054] Polypeptide: The term “polypeptide” or “peptide”, as used herein, typically has its art- recognized meaning of a polymer of at least three amino acids or more. 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, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining at least one activity) of such complete polypeptides. In some embodiments, polypeptides may contain L-amino acids, D-amino acids, or both and / or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics).

[0055] Prevent or prevention: As used herein, the terms “prevent” or “prevention”, when used in connection with the occurrence of a disease, disorder, and / or condition, refer to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of 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.

[0056] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0057] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0058] Ribonucleic acid (RNA): As used herein, the term “RNA” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc ), or a messenger RNA (mRNA). In some embodiments where an RNA is a mRNA. In some embodiments where an RNA is a mRNA, a RNA typically comprises at its 3 ’ end a poly(A) region. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0059] Substituted or optionally substituted: As described herein, compounds of the invention may contain “optionally 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 or more hydrogens that are either explicit or implicit from the structure (e.g., refers to at least). Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at 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 invention 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 provided herein. Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.

[0060] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)0-4R°; -(CH2)0-4OR°; -O(CH2)0-4R°, -O- (CH2)0-4C(O)OR°; -(CH2)0-4CH(OR°)2; -(CH2)0-4SR°; -(CH2)0-4Ph, which may be substituted with R°; -(CH2)0-4O(CH2)0-1Ph which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)0-4O(CH2)0-1-pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)0-4N(R°)2; -(CH2)0-4N(R°)C(O)R°; -N(R°)C(S)R°; -(CH2)O-4N(R°)C(O)NR°2; -N(R°)C(S)NR°2; -(CH2)0-4N(R°)C(O)OR°;N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2)0-4C(O)R°; C(S)R°; -(CH2)0-4C(O)OR°; -(CH2)0-4C(O)SR°; -(CH2)0-4C(O)OSIR°3; -(CH2)0-4OC(O)R°; -OC(O)(CH2)0-4SR°; -(CH2)0-4SC(O)R°; -(CH2)0-4C(O)NR°2; -C(S)NR°2; -C(S)SR°; - SC(S)SR°, -(CH2)0-4OC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; - C(NOR°)R°; -(CH2)0-4SSR°; -(CH2)0-4S(O)2R°; -(CH2)0-4S(O)2OR°; -(CH2)0-4OS(O)2R°; - S(O)2NR°2; -(CH2)0-4S(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; - P(O)2R°; -P(O)R°2; -OP(O)R°2; -OP(O)(OR°)2; SiR°3; -(C1-4straight or branched alkylene)O- N(R°)2; or — (C1-4straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C1-6aliphatic, -CH2Ph, -O(CH2)0-1Ph, -CH2-(5- to 6-membered heteroaryl ring), or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0061] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)0-2R●, -(haloR●), -(CH2)0-2OH, -(CH2)0-2OR●, -(CH2)0-2CH(OR●)2, -O(haloR●), -CN, -N3, -(CH2)0-2C(O)R●, -(CH2)0-2C(O)OH, -(CH2)0-2C(O)OR●, -(CH2)0-2SR●, -(CH2)0-2SH, -(CH2)0-2NH2, - (CH2)o-2NHR●, -(CH2)0-2NR●2, -NO2, -SIR●3, -OSIR●3, -C(O)SR●— (C1-4straight or branched alkylene)C(O)OR●, or -SSR●wherein each R●is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4aliphatic, - CH2Ph, -O(CH2)0-1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =O and =S.

[0062] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =O (“oxo”), =S, =NNR●2, =NNHC(O)R●, =NNHC(O)OR●, =NNHS(O)2R●, =NR●, =NOR●, -O(C(R●2))2-3O-, or -S(C(R●2))2-3S-, wherein each independent occurrence of R●is selected from hydrogen, C1-6aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR●2)2-3O-, wherein each independent occurrence of R●is selected from hydrogen, C1-6aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0063] Suitable substituents on the aliphatic group of R●include halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2, wherein each R●is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 3- to 6-membered saturated,partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0064] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group includewherein each is independently hydrogen, C1-6aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of taken together with theirintervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0065] Suitable substituents on the aliphatic group ofare independently halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2, wherein each R●is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 3- to 6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0066] Small molecule: As used herein, the term “small molecule” means a low molecular weight organic and / or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer.

[0067] In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and / or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and / or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and / or does not comprise a polysaccharide; for example, in some embodiments, asmall molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid.

[0068] In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting agent or an activating agent). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent.

[0069] Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and / or utilized in any of a variety of forms such as, for example, crystal forms (e.g., polymorphs, solvates, etc), salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, etc.

[0070] Those of ordinary skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form.

[0071] Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms.

[0072] Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g.,2H or3H for H;11C,13C or14C for12C;13N or15N for14N;17O or18O for16O;36Cl for35Cl or37Cl;18F for19F;131I for127I; etc.). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof.

[0073] In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form.

[0074] In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and / or utilized in accordance in the present disclosure in a form different from that in which it exists or is found in nature. Those of ordinary skill in the art will appreciate that, in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of the compound, or of a particular form thereof, that is different from the absolute or relative (with respect to another component of the preparation including, for example, another form of the compound) amount of the compound or form that is present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source) is distinct from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compound may be considered to be a different form from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form of the compound from one that contains the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc.

[0075] Those skilled in the art will further appreciate that, in small molecule structures, the symbol , as used herein, refers to a point of attachment between two atoms. Additionally or alternatively, the symbol refers to a point of attachment ring in a spirocyclic manner.

[0076] Treat: As used herein, the terms “treat,” “treatment,” or “treating” refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.Cationic or Ionizable Lipids

[0077] The present disclosure provides, among other things, cationic or ionizable lipids Compounds described herein are also referred to as “ionizable” or “cationic” lipids. Such lipids are intended to mean compounds that, in some embodiments, are capable of becoming cationic (i.e., becoming positively charged) less than physiological pH. In some embodiments, example lipids described herein become positively charged at a pH less than 7, less than 6.5, less than 6, less than 5.5, less than 5, less than 4.5, or less than 4. In some embodiments, a composition comprising a plurality of cationic or ionizable lipids described herein is characterized in that 10% or more of the cationic or ionizable lipids in the composition comprise a positive charge.

[0078] In some embodiments, the present disclosure provides compound of formula I:or a pharmaceutically acceptable salt thereof, whereinM1and M2are each independently selected from -(CH2)p1-;L1and L2are each selected from a bond, -OC(O)-, or -C(O)O-;T1and T2are each:L3is optionally substituted C1-C6aliphatic;G1is -OH, or -N(Ra)2, andRais H, optionally substituted C1-C6aliphatic, or optionally substituted C3-C6cycloaliphatic; p1 is an integer selected from 2-20, inclusive; p2 and p3 each an integer selected from 1-20, inclusive, and p2 and p3 are the same.

[0079] In some embodiments the present disclosure recognizes a surprising benefit when moieties T1and T2are the same, and where each of p2 and p3 are the same in compounds of formula I.

[0080] As described herein, M1and M2are each independently selected from -(CH2)p1-. In some embodiments, M1and M2are the same. In some embodiments, M1and M2are different. In some embodiments, M1is -(CH2)2-10-. In some embodiments, M1is -(CH2)4-6-. In some embodiments, M1is -(CH2)4-. In some embodiments, M1is -(CH2)5-. In some embodiments, M1is -(CH2)6-.

[0081] In some embodiments, M2is -(CH2)2-10-. In some embodiments, M2is -(CH2)4-6-. In some embodiments, M2is -(CH2)4-. In some embodiments, M2is -(CH2)5-. In some embodiments, M2is -(CH2)6-.

[0082] . In some embodiments, M1and M2are each -(CH2)2-10-. In some embodiments,M1and M2are each -(CH2)4-6-. In some embodiments, M1and M2are each -(CH2)4-. In some embodiments, M1and M2are each -(CH2)5-. In some embodiments, M1and M2are each -(CH2)6-

[0083] As described herein, p1 is an integer selected from 2-20, inclusive. In some embodiments, p1 is an integer selected from 2-10, inclusive. In some embodiments, p1 is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, p1 is an integer selected from 4, 5, or 6.

[0084] As described herein, L1and L2are each selected from a bond, -OC(O)-, and -C(O)O-. In some embodiments, L1and L2are the same. In some embodiments, L1and L2are different. In some embodiments, L1and L2are selected from -OC(O)- and -C(O)O-. In some embodiments, L1is a bond. In some embodiments, L1is -OC(O)-. In some embodiments, L1is -C(O)O-. In some embodiments, L2is a bond. In some embodiments, L2is -OC(O)-. In some embodiments, L2is - C(O)O-. In some embodiments, L1and L2are each a bond. In some embodiments, L1and L2are each -OC(O)-. In some embodiments, L1and L2are each -C(O)O-.

[0085] As described herein, T1and T2are each:

[0086] In some embodiments, T1and T2are the same.

[0087] As described herein, p2 and p3 each an integer selected from 1-20, inclusive, and p2 and p3 are the same. In some embodiments, p2 and p3 are each an integer selected from 3-9, inclusive. In some embodiments, p2 and p3 are each 3, 4, 5, 6, 7, 8, or 9. In some embodiments, p2 and p3 are each 3. In some embodiments, p2 and p3 are each 4. In some embodiments, p2 andp3 are each 5. In some embodiments, p2 and p3 are each 6. In some embodiments, p2 and p3 are each 7. In some embodiments, p2 and p3 are each 8. In some embodiments, p2 and p3 are each 9. In some embodiments, p2 and p3 are each 4, 5, or 6.

[0088] In some embodiments, T1and T2are each selected from:

[0089] In some embodiments, a total number of carbon atoms in -M1-L1-T1is between 20-30, inclusive. In some embodiments, -M1-L1-T1is represented by:

[0090] wherein p2 and p3 are each an integer selected from 2-10, inclusive. In some embodiments, -M1-L1-T1is represented by:wherein p2 and p3 are each an integer selected from 4, 5, or 6.

[0091] In some embodiments, a total number of carbon atoms in -M2-L2-T2is between 20-30, inclusive. In some embodiments, -M2-L2-T2is represented by:

[0092] wherein p2 and p3 are each an integer selected from 2-10, inclusive. In some embodiments, -M2-L2-T2is represented by:wherein p2 and p3 are each 4, 5, or 6.

[0093] In some embodiments, M1-L1-T1and M2-L2-T2are the same.

[0094] In some embodiments, M1-L1-T1and M2-L2-T2are each selected from

[0095] As described herein, L3is optionally substituted C1-C6aliphatic. In some embodiments, L3is optionally substituted C1-C6alkylene. In some embodiments, L3is -(CH2)1-6- . In some embodiments, L3is -(CH2)3- or -(CH2)4-. In some embodiments, L3is -(CH2)3-. In some embodiments, L3is -(CH2)4-.

[0096] As described herein, G1is -OH, or -N(Ra)2. In some embodiments, G1is -OH. In some embodiments, G1is -N(Ra)2. In some embodiments, G1is -N(H)(Ra). In some embodiments, G1is -N(C1-C6aliphatic)(H). In some embodiments, G1is -N(C1-C6aliphatic)2. In some embodiments, G1is -N(CH3)2.

[0097] In some embodiments, -L3-G1is -(CH2)3-OH or -(CH2)4-OH. In some embodiments, -L3-G1IS -(CH2)3-N(CH3)2or -(CH2)4- N(CH3)2.

[0098] In some embodiments, a compound of formula I is represented by formula II- 1 :or a pharmaceutically acceptable salt thereof, wherein G1, L3, L1, L2, p2, and p3 are as defined in classes and subclasses herein, both singly and in combination, and wherein * represents a stereopure carbon atom. In some embodiments of formula II- 1, one of the carbon atoms indicated with * is (R) and the other is (S). In some embodiments, both carbon atoms indicated with * are (R). In some embodiments, both carbon atoms indicated with * are (S).

[0099] In some embodiments, a compound of formula I is represented by formula II-2:

[0100] or a pharmaceutically acceptable salt thereof, wherein G1, L3, L1, and L2are as defined in classes and subclasses herein, both singly and in combination, and wherein * represents astereopure carbon atom. In some embodiments of formula II-2, one of the carbon atoms indicated with * is (R) and the other is (S). In some embodiments, both carbon atoms indicated with * are (R). In some embodiments, both carbon atoms indicated with * are (S).

[0101] In some embodiments, a compound of formula I is represented by formula II-3 :or a pharmaceutically acceptable salt thereof, wherein L3, L1, L2, p2, and p3 are as defined in classes and subclasses herein, both singly and in combination, and wherein * represents a stereopure carbon atom. In some embodiments of formula II-3, one of the carbon atoms indicated with * is (R) and the other is (S). In some embodiments, both carbon atoms indicated with * are (R). In some embodiments, both carbon atoms indicated with * are (S).

[0102] In some embodiments, a compound of formula I is represented by formula II-4:or a pharmaceutically acceptable salt thereof, wherein L3is as defined in classes and subclasses herein, and wherein * represents a stereopure carbon atom. In some embodiments of formula II- 4, one of the carbon atoms indicated with * is (R) and the other is (S). In some embodiments, both carbon atoms indicated with * are (R). In some embodiments, both carbon atoms indicated with * are (S).

[0103] In some embodiments, a compound of formula I is selected from Table 1 :Table 1In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to salts thereof, unless otherwise indicated.

[0104] As described herein, in some embodiments, a compound of formula I can be characterized by having a Wiener path of between about 15,000 and 30,000 and an MMFF94 energy between -15 and 0. In some embodiments a compound of formula I has a Wiener path of between about 15,000 and 30,000. A Wiener path (i.e., a Wiener index) is a topological index of a molecule, defined as the sum of the lengths of the shortest paths between all pairs of vertices in the chemical graph representing the non-hydrogen atoms in the molecule. In some embodiments, a compound of formula I has a MMFF94 energy between about -15 and 0. A MMFF94 energy refers to a Merck molecular force field and provides conformational energy for certain hydrogen- bonded complexes. See T. Halgren, J. Comput. Chem., 1999 May; 20(7):730-748.

[0105] In some embodiments, a pharmaceutical composition described herein (e.g., a composition that is or comprises lipid nanoparticles) comprises about 20 to about 70 mol% of a cationic lipid (e.g., a compound of formula I or any of II- 1 to II-4) relative to the total amount of lipids in the composition. In some embodiments, a pharmaceutical composition comprises about 40 to about 50 mol% of a cationic lipid relative to the total amount of lipids in the composition. In some embodiments, a pharmaceutical composition comprises about 30 to about 60 mol% of a cationic lipid relative to the total amount of lipids in the composition.Lipid Nanoparticles

[0106] In some embodiments, a compound of formula I described herein is complexed with a nucleic acid to form a nucleic acid particle. In some embodiments, nucleic acid particles are lipid nanoparticles. In some embodiments, lipid nanoparticles are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., ones described herein), a nucleic acid (e.g., RNA and / or DNA), and one or more additional lipids (e.g., a polymer-conjugated lipid, a steroid, an anionic amphiphile, and / or a neutral lipid). Lipid nanoparticles (LNPs) have proven useful for the delivery of nucleic acid cargo to tissue of interest. LNPs are used, for example, in certain commercial vaccines for treatment of COVID-19.

[0107] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a cationic or ionizable lipid compound (e.g., a compound of formula I, II- 1, II-2, II-3, or II-4) described herein and a nucleic acid. In some embodiments, a cationic or ionizable lipid compound (e.g., a compound of formula I, II-l, II-2, II-3, or II-4) and a nucleic acid are in the form of a lipid nanoparticle (LNP).

[0108] Nucleic acid particles (e.g., ribonucleic acid particles or deoxyribonucleic acid particles) described herein can be characterized by an “N / P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N / P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N / P) in RNA. It is understood that a cationic group is one that is either in cationic form (e.g., N+), or one that is ionizable to become cationic. Use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number over 1, e.g., an N / P ratio of about 4 is intended to mean about 4: 1. In some embodiments, an N / P ratio of a pharmaceutical composition described herein (e.g., in a nucleic acid particle such as a lipid nanoparticle) is from about 3 to about 12. In some embodiments, an N / P ratio is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Insome embodiments, an N / P ratio is about 4 to about 8. In some embodiments, an N / P ratio is 4. In some embodiments, an N / P ratio is 5. In some embodiments, an N / P ratio is 6. In some embodiments, an N / P ratio is 7. In some embodiments, an N / P ratio is 8.

[0109] In some embodiments, a pharmaceutical composition comprising a cationic or ionizable lipid compound (e.g., a compound of formula I, II- 1, II-2, II-3, or II-4) described herein and a nucleic acid further comprises an additional lipid. In some embodiments, a pharmaceutical composition comprising a cationic or ionizable lipid compound (e.g., a compound of formula I, II- 1, II-2, II-3, or II-4) described herein and a nucleic acid further comprises one or more of a neutral lipid, a polymer-conjugated lipid, a steroid, or an anionic amphiphile.

[0110] In some embodiments, a pharmaceutical composition comprising a cationic or ionizable lipid compound (e.g., a compound of formula I, II- 1, II-2, II-3, or II-4) described herein and a nucleic acid further comprises a neutral lipid, a polymer-conjugated lipid, and a steroid. In some embodiments, a pharmaceutical composition comprising a cationic or ionizable lipid compound (e.g., a compound of formula I, II- 1, II-2, II-3, or II-4) described herein and a nucleic acid further comprises a neutral lipid, an anionic amphiphile, and a steroid. In some embodiments, a pharmaceutical composition comprising a cationic or ionizable lipid compound (e.g., a compound of formula I, II- 1, II-2, II-3, or II-4) described herein and a nucleic acid further comprises a neutral lipid, an anionic amphiphile, and a steroid, and wherein said pharmaceutical composition does not comprise a polymer-conjugated lipid.(i) Neutral lipids

[0111] As described herein, in some embodiments, a pharmaceutical composition described herein further comprises neutral lipid. In some embodiments, a neutral lipid is a phospholipid. In some embodiments, a neutral lipid is or comprises 1 ,2-distearoyI-sn -glycero-3 -phosphocholine (DSPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phosphatidyl ethanol amines such as 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), sphingomyelins (SM), 1 ,2-diacylglyceryl- 3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), ceramides, cholesterol, steroids, such as sterols and their derivatives.

[0112] In some embodiments, a neutral lipid is or comprises phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. In some embodiments, a neutral lipid is or comprises diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, including, for example diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3- phospho-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM). In some embodiments, a neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

[0113] Neutral lipids may be synthetic or naturally derived. Other neutral lipids suitable for use in a pharmaceutical composition are described in WO2021 / 026358, WO 2017 / 075531, and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference in their entirety.

[0114] In some embodiments, a pharmaceutical composition comprises about 1 to about 40 mol% of a neutral lipid (relative to the total lipids in the pharmaceutical composition). In some embodiments, a pharmaceutical composition comprises about 2 to about 25 mol% of a neutral lipid (relative to the total lipids in the pharmaceutical composition). In some embodiments, a pharmaceutical composition comprises about 5 to about 15 mol% of a neutral lipid (relative to thetotal lipids in the pharmaceutical composition). In some embodiments, a pharmaceutical composition comprises about 8 to about 12 mol% of a neutral lipid (relative to the total lipids in the pharmaceutical composition). In some embodiments, a pharmaceutical composition comprises about 10 mol% of a neutral lipid (relative to the total lipids in the pharmaceutical composition).

[0115] In some embodiments, a pharmaceutical composition comprises about 1 to about 40 mol% of a phospholipid. In some embodiments, a pharmaceutical composition comprises about 2 to about 25 mol% of a phospholipid. In some embodiments, a pharmaceutical composition comprises about 5 to about 15 mol% of a phospholipid. In some embodiments, a pharmaceutical composition comprises about 8 to about 12 mol% of a phospholipid. In some embodiments, a pharmaceutical composition comprises about 10 mol% of a phospholipid. In some embodiments, a pharmaceutical composition comprises about 5 to about 20 mol% of DSPC. In some embodiments, a pharmaceutical composition comprises about 8 to about 12 mol% of DSPC. In some embodiments, a pharmaceutical composition comprises about 10 mol% of DSPC.(ii) Polymer-conjugated lipids

[0116] As described herein, in some embodiments, a pharmaceutical composition described herein further comprises a polymer-conjugated lipid. In some embodiments, a polymer conjugated lipid is a lipid conjugated to polyethylene glycol (PEG-lipid). In some embodiments, a PEG lipid is selected from pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy- polyethyleneglycol)- 2,3 -dimyristoylglycerol (PEG-DMG) (e.g., 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG2000-DMG)), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'- di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly ethylene glycol)-2000] (DSPE- PEG2000 amine), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω- m ethoxy (polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, 2,3- di(tetradecanoxy)propy 1-N-(ω-methoxy(polyethoxy)ethyl)carbamate, and N-palmitoyl- sphingosine-1-{succinyl[methoxy(poly ethylene glyco 1)2000]} (C16-PEG2000 ceramide or C16Cer-PEG2K).

[0117] In some embodiments, a PEG-lipid is PEG2000-DMG:

[0118] In some embodiments, a PEG-lipid is DMG-PEG.

[0119] In some embodiments, a PEG-lipid is provided in WO2021 / 026358, WO 2017 / 075531, or WO 2018 / 081480, each of which is incorporated by reference in its entirety.

[0120] In some embodiments, a polymer conjugated lipid is C16 PEG2000:

[0121] In some embodiments, the PEG- lipid has the following structure:wherein n in the formula above is from 30 to 60, such as about 50. In one embodiment, the PEG- conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N-[(ω-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000).

[0122] In some embodiments, a PEG-lipid is selected from PEG-DAG, PEG-PE, PEG-S- DAG, PEG2000-DMG, PEG-S-DMG, PEG-cer, a PEG dialkoxypropylcarbamate (e.g., ω- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(ω- methoxy(polyethoxy)ethyl)carbamate), and combinations thereof. In some embodiments, a PEG-lipid is PEG2000-DMG. In some embodiments, a PEG- lipid is PEG-DAG. In some embodiments, a PEG-lipid is PEG-PE. In some embodiments, a PEG- lipid is PEG-S-DAG. In some embodiments, a PEG-lipid is PEG-cer. In some embodiments, a PEG-lipid is a PEG dialkoxypropylcarbamate.

[0123] In some embodiments, a PEG group that is part of a PEG-lipid has, on average in a composition comprising one or more PEG-lipid molecules, a weight average molecular weight (Mw) of about 2000 g / mol.

[0124] In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine) portion.

[0125] In some embodiments, a polymer-conjugated lipid is a polyoxazoline (POX)- conjugated and / or polyoxazine (POZ)-conjugated lipid, also referred to herein as a conjugate of a POX and / or POZ polymer and one or more hydrophobic chains or as oxazolinylated and / or oxazinylated lipid or POX- and / or POZ-lipid. The term "oxazolinylated lipid" or "POX-lipid" refers to a molecule comprising both a lipid portion and a polyoxazoline portion. The term "oxazinylated lipid" or "POZ-lipid" refers to a molecule comprising both a lipid portion and a polyoxazine portion. The term "oxazolinylated / oxazinylated lipid" or "POX / POZ-lipid" or "POXZ-lipid" refers to a molecule comprising both a lipid portion and a portion of a copolymer of polyoxazoline and polyoxazine.

[0126] In some embodiments, an LNP described herein may comprise a sarcosinylated lipid. In some embodiments, the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein comprise a sarcosinylated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid).

[0127] In some embodiments, the nucleic acid compositions (such as DNA or RNA compositions) described herein comprise a cationic / cationically ionizable lipid as described herein and a sarcosinylated lipid (pSAR-conjugated lipid). In some embodiments, the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein may further comprise a neutral lipid (e.g., a phospholipid, cholesterol or a derivative thereof) or a combination of neutral lipids (e.g., a phospholipid, and cholesterol or a derivative thereof). In some embodiments, the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein comprise a cationic / cationically ionizable lipid as described herein, a sarcosinylated lipid, a neutral lipid (e.g., a phospholipid), and cholesterol or a derivative thereof. In some embodiments, the phospholipid is DSPC.

[0128] In some embodiments of the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein which comprise a sarcosinylated lipid, said compositions are substantially free of a pegylated lipid (or do not contain a pegylated lipid).

[0129] In some embodiments, the sarcosinylated lipid comprises between 2 and 200 sarcosine units, such as between 5 and 100 sarcosine units, between 10 and 50 sarcosine units, between 15 and 40 sarcosine units, e.g., about 23 sarcosine units.

[0130] In some embodiments, the sarcosinylated lipid comprises the structure of the following general formula (XVII):wherein s is the number of sarcosine units.

[0131] In some embodiments, the sarcosinylated lipid comprises the structure of the following general formula (XVIII):wherein one of R21and R22comprises a hydrophobic group and the other is H, a hydrophilic group or a functional group optionally comprising a targeting moiety; and x is the number of sarcosine units.

[0132] In some embodiments, an LNP herein may comprise an oxazolinylated and / or / oxazinylated lipid. In some embodiments, the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein comprise an oxazolinylated and / or / oxazinylated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid).

[0133] In some embodiments, a polymer-conjugated lipid comprises monomers of 2-(2-(2- aminoethoxy)ethoxy)acetic acid. In some embodiments, a polymer-conjugated lipid comprises monomers of 2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the polymer of the polymer-conjugated lipid is or comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof, as defined herein.

[0134] In some embodiments, the polymer comprises the following general formula:wherein X11and X12taken together are optionally substituted amide, optionally substituted thioamide or ester; Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100.

[0135] In some embodiments, (i) when X11is -C(O)- then X12is -NR10-; (ii) when X11is - NR10- then X12is -C(O)-; (iii) when X11is -C(S)- then X12is -NR1-; (iv) when X11is -NR1- then X12is -C(S)-; (v) when X11is -C(O)- then X12is -O-; or (vi) when X11is -O- then X12is -C(O)-; wherein R10is hydrogen or C1-8alkyl. In some embodiments, X11is -C(O)- and X12is -NR10-, wherein R10is hydrogen or C1-8alkyl. In some embodiments, X11is -C(O)- and X12is -NR10-, wherein R10is hydrogen or methyl. In some embodiments, X11is -C(O)- and X12is -NR10-, wherein R10is hydrogen. In some embodiments, Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.

[0136] In some embodiments, the polymer comprises the following general formula:wherein R10is hydrogen or Cl -8 alkyl; z is 2 to 24; and n is 1 to 100. In some embodiments of the above formulas, z is 2 to 10. In some embodiments, z is 2 to 5. In some embodiments, z is 2. In some embodiments of the above formulas, R10is hydrogen or methyl. In some embodiments, R10is hydrogen.

[0137] In some embodiments, a polymer conjugated lipid comprises “n” monomers of the following structure:

[0138] In some embodiments of the above formulas, n is 5 to 50. In some embodiments, n is 5 to 25. In some embodiments, n is 7 to 14. In some embodiments, n is 10 to 25. In some embodiments, n is 14 to 17. In some embodiments, n is 8 or 14.

[0139] In some embodiments, a polymer conjugated lipid comprises monomers of the following structure:

[0140] In some embodiments, a polymer conjugated lipid is selected from:

[0141] In some embodiments, a polymer-conjugated lipid is about 0.5 to about 5 mol% relative to total lipids in the LNP. In some embodiments, an LNP comprises about 1.0 to about 2.5 mol% of a polymer-conjugated lipid. In some embodiments, an LNP comprises about 1.5 to about 2.0 mol% of a polymer-conjugated lipid. In some embodiments, an LNP comprises about 1.5 to about 1.8 mol% of a polymer-conjugated lipid.

[0142] In some embodiments, a molar ratio of total cationic lipid to total polymer-conjugated lipid is from about 100: 1 to about 20: 1. In some embodiments, a molar ratio of total cationic lipid to total polymer-conjugated lipid is from about 50: 1 to about 20: 1. In some embodiments, a molarratio of total cationic lipid to total polymer-conjugated lipid is from about 40:1 to about 20: 1. In some embodiments, a molar ratio of total cationic lipid to total polymer-conjugated lipid (e.g., PEG-conjugated lipid) is from about 35: 1 to about 25: 1.(iii) Steroid

[0143] As described herein, in some embodiments, a nucleic acid particle further comprises a steroid. In some embodiments, a steroid is a sterol. In some embodiments, a sterol is β-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, cycloartenol, lanosterol, or α-tocopherol. In some embodiments, a sterol is β-sitosterol. In some embodiments, a sterol is stigmasterol. In some embodiments, a sterol is cholesterol. In some embodiments, a sterol is cholecalciferol. In some embodiments, a sterol is ergocalciferol. In some embodiments, a sterol is calcipotriol. In some embodiments, a sterol is botulin. In some embodiments, a sterol is lupeol. In some embodiments, a sterol is cycloartenol. In some embodiments, a sterol is lanosterol. In some embodiments, a sterol is α-tocopherol.

[0144] In some embodiments, a pharmaceutical composition comprises about 30 to about 50 mol% of a steroid. In some embodiments, a pharmaceutical composition comprises about 35 to about 45 mol% of a steroid. In some embodiments, a pharmaceutical composition comprises about 38 to about 40 mol% of a steroid. In some embodiments, a pharmaceutical composition comprises about 38.5 mol% of a steroid. In some embodiments, a pharmaceutical composition comprises about 40 mol% of a steroid.

[0145] In some embodiments, a pharmaceutical composition comprises about 30 to about 50 mol% of cholesterol. In some embodiments, a pharmaceutical composition comprises about 35 to about 45 mol% of cholesterol. In some embodiments, a pharmaceutical composition comprises about 38 to about 41 mol% of cholesterol. In some embodiments, a pharmaceutical composition comprises about 38.5 mol% of cholesterol. In some embodiments, a pharmaceutical composition comprises about 40.7 mol% of cholesterol.(iv) Anionic Amphiphiles

[0146] As described herein, in some embodiments, a pharmaceutical composition comprises an anionic amphiphile. An “amphiphile” is understood to be a molecule having both hydrophilic and lipophilic moieties. A “lipophilic” moiety is one that is soluble in nonpolar solvents (e.g.,hexane, tetrahydrofuran (THF), and / or chloroform). A “hydrophilic” moiety is one that is soluble in aqueous solution, e.g., water.

[0147] In some embodiments, an anionic amphiphile comprises a negative charge at particular pH (e.g., physiological pH) in the hydrophilic portion of the amphiphile. In some embodiments, an anionic amphiphile is a hemiester of a dicarboxylic acid with diacylglycerol. In some embodiments, the hydrocarbyl portion of the acyl moieties of the diacylglycerol portion is an alkyl group (as defined above) having 6 to 40, preferably 8 to 18, carbon atoms or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 18, carbon atoms. In some embodiments, acyl moieties are present on the 1- and 2-positions of the glycerol moiety. In some embodiments, the acyl parts of the diacylglycerol moiety are same. In some embodiments, the acyl parts of the diacylglycerol moiety are different. In some embodiments, the acyl moieties are saturated fatty acid moieties, preferably selected from the group consisting of stearoyl, palmitoyl, myristoyl, lauroyl, decanoyl and octanoyl moieties.

[0148] In some embodiments, the acyl moieties are unsaturated fatty acid moieties, preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties. In some embodiments, the dicarboxylic acid moiety has 2 to 20 carbon atoms, 2 to 10 carbon atoms, or 2 to 8 carbon atoms. Examples of the dicarboxylic acid moiety include oxalate, malonate, succinate, glutarate, adipate, pimelate and suberate.

[0149] In some embodiments, the negatively charged amphiphile is a hemiester of succinic acid with diacylglycerol (i.e., the dicarboxylic acid moiety is a succinic acid moiety) - also referred to herein as a “diacylglycerol hemisuccinate”.

[0150] In some embodiments, an anionic amphiphile is selected from 1,2-dilauroylgly ceryl hemisuccinate (DLGS), 1,2-dimyristoylgly ceryl hemisuccinate (DMGS), 1,2-dipalmitoylgly ceryl hemisuccinate (DPGS), 1-palmitoyl-2-stearoylglyceryl hemisuccinate (PSGS), distearoylglyceryl hemisuccinate (DSGS), 1,2-dioleoylglyceryl hemisuccinate (DOGS), 1-stearoyl, 2-myristoyl- gly cerylhemisuccinate (SMGS), 1-palmitoyl-2-oleoylgly ceryl hemisuccinate (POGS) and analogues of any of the above thereof wherein the dicarboxylic acid portion is oxalate, malonate, succinate, glutarate, adipate, pimelate or suberate In some embodiments, an anionic amphiphile is dimyristoylglyceryl hemisuccinate, dipalmitoylglyceryl hemisuccinate or distearoylglyceryl hemisuccinate. . In some embodiments, an anionic amphiphile is ursolic acid, oleanolic acid.

[0151] In some embodiments, an anionic amphiphile is a hemi ester of a dicarboxylic acid with cholesterol. In some embodiments, an anionic amphiphile is selected from:

[0152] In some embodiments, a lipid nanoparticle described herein comprises about 0.5 to about 10 mol% of the anionic amphiphile.

[0153] In some embodiments, a pharmaceutical composition described herein comprises: about 30 to about 60 mol% of the compound (e.g., a cationic ionizable lipid of formula I, 11-1, 11-2, II-3, or II-4); about 20 to about 60 mol% of the steroid; about 1 to about 2 mol% of the polymer-conjugated lipid; and about 5 to about 20 mol% of the neutral lipid.

[0154] In some embodiments, a pharmaceutical composition described herein comprises: about 30 to about 60 mol% of the compound (e.g., a cationic ionizable lipid of formula I, 11-1, 11-2, II-3, or II-4); about 20 to about 60 mol% of the steroid; about 0.5 to about 10 mol% of the anionic amphiphile; and about 5 to about 20 mol% of the neutral lipid.

[0155] In some embodiments, a pharmaceutical composition described herein comprises: about 30 to about 60 mol% of the compound (e.g., a cationic ionizable lipid of formula I, 11-1, 11-2, II-3, or II-4); about 20 to about 60 mol% of the steroid; about 0.5 to about 10 mol% of the anionic amphiphile; and about 5 to about 20 mol% of the neutral lipid, and further wherein the pharmaceutical composition does not include a polymer-conjugated lipid.

[0156] The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0157] The “polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).

[0158] Different types of nucleic acid particles have been described previously to be suitable for delivery of nucleic acid in particulate form (e.g. Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

[0159] Some embodiments described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species. The nucleic acid species may be RNA and / or DNA. For example, the particles described herein may contain one species of RNA (e.g., one species of mRNA) and one species of DNA.In a nucleic acid particle composition, it is possible that each nucleic acid species is separately formulated as an individual nucleic acid particle formulation. In that case, each individual nucleic acid particle formulation will comprise one nucleic acid species. The individual nucleic acid particle formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations).(v) Methods of Making Lipid Nanoparticles

[0160] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos. 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, 2013 / 0022649, 2013 / 0017223, 2012 / 0295832, 2012 / 0183581, 2012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741, WO 2018 / 081480, WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO2013 / 016058, WO 2013 / 086373, W02011 / 141705, and WO 2001 / 07548, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein.

[0161] For example, in some embodiments, lipids that are useful for delivery of nucleic acids are solubilized in ethanol at a pre- determined weight or molar ratios / percentages (e.g., ones described herein). In some embodiments, lipid nanoparticles (LNP) are prepared at a total lipid to RNA or DNA molar ratio of approximately 6: 1 to 30: 1. In some embodiments, such RNA or DNA can be diluted to 0.2 mg / mL in acetate buffer.

[0162] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising nucleic acids (e.g., RNA or DNA) can be formed as follows: an ethanol solution comprising lipids, such as cyclic ionizable lipids described herein, neutral lipids, steroids, and, optionally polymer-conjugated lipids or anionic amphiphiles, is injected into an aqueous solution comprising nucleic acids (e.g., RNA or DNA).

[0163] In some embodiments, lipid and nucleic acid solutions can be mixed at room temperature by pumping each solution (e.g., a lipid solution comprising a cyclic ionizable lipid compound described herein, a neutral lipid, steroids, and the optional polymer conjugated lipids or anionic amphiphile or any other additives) at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a nucleic acid solution into a mixing unit are maintained at a ratio of 1 :3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous RNAs. The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged nucleic acid.

[0164] In some embodiments, a solution comprising nucleic acid (e.g., RNA)-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and / or filtration.

[0165] In some embodiments, a composition or complex described herein further comprises a pharmaceutically acceptable surfactant. In some embodiments, a pharmaceutically acceptable surfactant is selected from a polysorbate (e.g., polysorbate 20 (Tween20), polysorbate 40 (Tween40), polysorbate 60 (Tween60), and polysorbate 80 (Tween80)), poloxamers, and an amphiphilic group comprising a moiety selected from polyalkylene glycols (e.g., polyethylene glycol), poly(2-oxazoline), poly(2-methyl-2-oxazoline), polysarcosine, polyvinylpyrrolidone, andpoly[N-(2-hydroxypropyl)methacrylamide, wherein the moiety is bound to one or more C12-C20aliphatic groups.RNA

[0166] In some embodiments, a particle described herein comprises one or more oligosaccharide compositions and a nucleic acid. In some embodiments, a nucleic acid is RNA.

[0167] In some embodiments, an RNA amenable to technologies described herein is a single- stranded RNA. In some embodiments, an RNA as disclosed herein is a linear RNA. In some embodiments, a single-stranded RNA is a non-coding RNA in that its nucleotide sequence does not include an open reading frame (or complement thereof). In some embodiments, a single- stranded RNA has a nucleotide sequence that encodes (or is the complement of a sequence that encodes) a polypeptide or a plurality of polypeptides (e.g., epitopes) of the present disclosure.

[0168] In some embodiments, an RNA is or comprises an siRNA, an miRNA, or other non- coding RNA.

[0169] In many embodiments, a relevant RNA includes at least one open reading frame (ORF) (e.g., is an mRNA); in some embodiments, a relevant RNA includes a single ORF; in some embodiments, a relevant RNA includes more than one ORF.

[0170] In some embodiments, an RNA comprises an ORF, e.g., encoding a polypeptide of interest or encoding a plurality of polypeptides of interest. In some embodiments, an RNA produced in accordance with technologies provided herein comprises a plurality of ORFs (e.g., encoding a plurality of polypeptides). In some embodiments, an RNA produced in accordance with technologies herein comprises a single ORF that encodes a plurality of polypeptides. In some such embodiments, polypeptides are or comprise antigens or epitopes thereof (e.g., relevant antigens).

[0171] In some embodiments, an ORF for use in accordance with the present disclosure encodes a polypeptide that includes a signal sequence, e.g., that is functional in mammalian cells, such as an intrinsic signal sequence or a heterologous signal sequence. In some embodiments, a signal sequence directs secretion of an encoded polypeptide, in some embodiments, a signal sequence directs transport of an encoded polypeptide into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.

[0172] In some embodiments, an ORF encodes a polypeptide that includes a multimerization element (e.g, an intrinsic or heterologous multimerization element). In some embodiments, anORF that encodes a surface polypeptide (e.g., that includes a signal sequence directing surface localization) includes a multimerization element.

[0173] In some embodiments, an ORF encodes a polypeptide that includes a transmembrane element or domain.

[0174] In some embodiments, an ORF is codon-optimized for expression in a cells of a particular host, e.g., a mammalian host, e.g., a human.

[0175] In some embodiments, an RNA includes unmodified uridine residues; an RNA that includes only unmodified uridine residues may be referred to as a “uRNA”. In some embodiments, an RNA includes one or more modified uridine residues; in some embodiments, such an RNA (e.g., an RNA including entirely modified uridine residues) is referred to as a “modRNA”. In some embodiments, an RNA may be a self-amplifying RNA (saRNA). In some embodiments, an RNA may be a trans-amplifying RNA (taRNA) (see, for example, WO2017 / 162461).

[0176] In some embodiments, a relevant RNA includes a polypeptide-encoding portion or a plurality of polypeptide-encoding portions. In some particular embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or comprises a biologically active polypeptide or portion thereof (e.g., an enzyme or cytokine or therapeutic protein such as a replacement protein or antibody or portion thereof). In some particular embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or comprises an antigen (or an epitope thereof), a cytokine, an enzyme, etc. In some embodiments, an encoded polypeptide or polypeptides may be or include one or more neoantigens or neoepitopes associated with a tumor. In some embodiments, an encoded polypeptide or polypeptides may be or include one or more antigens (or epitopes thereof) of an infectious agent (e.g., a bacterium, fungus, virus, etc.). In certain embodiments, an encoded polypeptide may be a variant of a wild type polypeptide.

[0177] In some embodiments, a single-stranded RNA (e.g., mRNA) may comprise a secretion signal-encoding region (e.g., a secretion signal-encoding region that allows an encoded target entity or entities to be secreted upon translation by cells). In some embodiments, such a secretion signal-encoding region may be or comprise a non-human secretion signal. In some embodiments, such a secretion signal-encoding region may be or comprise a human secretion signal.

[0178] In some embodiments, a single-stranded RNA (e.g., mRNA) may comprise at least one non-coding element (e.g., to enhance RNA stability and / or translation efficiency). Examples of non-coding elements include but are not limited to a 3’ untranslated region (UTR), a 5’ UTR, acap structure (e.g., in some embodiments, an enzymatically-added cap; in some embodiments, a co-transcriptional cap), a poly adenine (poly A) tail (e.g., that, in some embodiments, may be or comprise 100 A residues or more, and / or in some embodiments may include one or more “interrupting” [i.e., non- A] sequence elements), and any combinations thereof. Exemplary embodiments of such non-coding elements may be found, for example, in WO2011015347, WO2017053297, US 10519189, US 10494399, W02007024708, W02007036366, W02017060314, W02016005324, W02005038030, WO2017036889, WO2017162266, and WO2017162461, each of which is incorporated herein by referenced in its entirety.Formats

[0179] At least four formats useful for RNA pharmaceutical compositions (e.g., immunogenic compositions or vaccines) have been developed, namely non-modified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA), self-amplifying mRNA (saRNA), and trans- amplifying RNAs.

[0180] Features of a non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, good tolerability and safety, and strong antibody and T cell responses.

[0181] Features of modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus augmented antigen expression, good tolerability and safety, and strong antibody and CD4-T cell responses. As noted herein, the present disclosure provides an insight that such strong antibody and CD4 T cell responses may be particularly useful for vaccination.

[0182] Features of self-amplifying platform may include, for example, long duration of polypeptide (e.g., protein) expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.

[0183] In some embodiments, a self-amplifying platform (e.g., .saRNA) comprises a nucleic acid molecule, encoding both a replicase (e.g., a viral replicase) and a gene of interest, wherein the nucleic acid molecule is capable of being replicated by said replicase in cis ( cis-replication system). In some embodiments, a trans-amplifying platform (e.g., taRNA) comprises two nucleic acid molecules, wherein one nucleic acid molecule encodes a replicase (e.g., a viral replicase) and the other nucleic acid molecule is capable of being replicated (e.g., a replicon) by said replicase in trans ( trans-replication system). In some embodiments, a self / trans-amplifying platform (e.g.,RNA) comprises a plurality of nucleic acid molecules, wherein said nucleic acids encode a plurality of replicases and / or replicons.

[0184] In some embodiments, a trans-replication system comprises the presence of both nucleic acid molecules in a single host cell.

[0185] In some such embodiments, a nucleic acid encoding a replicase (e.g., a viral replicase) is not capable of self-replication in a target cell and / or target organism. In some such embodiments, a nucleic acid encoding a replicase (e.g., a viral replicase) lacks at least one conserved sequence element important for (-) strand synthesis based on a (+) strand template and / or for (+) strand synthesis based on a (-) strand template.

[0186] In some embodiments, a self-amplifying RNA comprises a 5’-cap; in some trans- replication systems, at least an RNA encoding a replicase is capped. Without wishing to be bound by any one theory, it has been found that a 5’-cap can be important for high level expression of a gene of interest in trans.

[0187] In some embodiments, a self / trans-amplifying platform does not require propagation of virus particles (e.g., is not associated with undesired virus-particle formation). In some embodiments, a self / trans-amplifying platform is not capable of forming virus particles.

[0188] In some embodiments, an RNA may comprise an Internal Ribosomal Entry Site (IRES) element. In some embodiments, an RNA does not comprise an IRES site; in particular, in some embodiments, an saRNA does not comprise an IRES site. In some such embodiments, translation of a gene of interest and / or replicase is not driven by an IRES element. In some embodiments, an IRES element is substituted by a 5’-cap. In some such embodiments, substitution by a 5’-cap does not affect the sequence of a polypeptide encoded by an RNA.

[0189] In some embodiments, a complex described herein comprises modRNA, saRNA, taRNA, or uRNA. In some embodiments, a complex comprises modRNA. In some embodiments, a complex comprises saRNA. In some embodiments, a complex comprises taRNA. In some embodiments, a complex comprises uRNA.Methods of Use

[0190] Particles described herein are useful in the treatment and prophylaxis in a subject of diseases, disorders, and conditions described herein. In some embodiments, the present disclosure provides a method of treating a disease, disorder or condition comprising administering to a patient a composition comprising particles described herein. In some embodiments, the present disclosureprovides use of a composition comprising particles described herein for the treatment of a disease, disorder, or condition. In some embodiments, a disease, disorder, or condition is an infectious disease, cancer, an autoimmune disease, or a rare disease.

[0191] In some embodiments, an infectious disease is caused by or associated with a viral pathogen. In some embodiments, a viral pathogen is of a family selected from poxviridae, rhabdoviridae, filoviridae, paramyxoviridae, hepadnaviridae, coronaviridae, caliciviridae, picornaviridae, reoviridae, retroviridae, and orthomyxoviridae. In some embodiments, an infectious disease is caused by or associated with a virus selected from SARS-CoV-2, influenza, Crimean-Congo Hemorhhagic Fever (CCHF), Ebola virus, Lassa virus, Marburg virus, HIV, Nipah virus, and MERS-CoV.

[0192] In some embodiments, an infectious disease is caused by or associated with a bacterial pathogen. In some embodiments, a bacterial pathogen is of a species selected from Actinomyces israelii, bacillus antracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campolobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium idphteriae, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroids, Rickettsia ricektssii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

[0193] In some embodiments, an infectious disease is caused by or associated with a parasite. In some embodiments, a parasite is of a family selected from Plasmodium, Leishmania, Cryptosporidium, Entamoeba, Trypanosomas, Schistosomes, Ascaris, Echinococcus and Taeniidae.

[0194] In some embodiments, a disease, disorder, or condition is a cancer. In some embodiments, a cancer is selected from bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral / oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

[0195] In some embodiments, a disease, disorder, or condition is a genetic disorder. In some embodiments, a genetic disorder is associated with a gain-of-function mutation or a loss-of- function mutation.

[0196] In some embodiments, a disease, disorder, or condition is an autoimmune disease. In some embodiments, an autoimmune disease is selected from Addison disease, celiac disease, rheumatoid arthritis, lupus, inflammatory bowel disease, dermatomyositis, multiple sclerosis, diabetes, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, pernicious anemia, graves’ disease, Hashimoto’s thyroiditis, myasthenia gravis, and vasculitis Sjogren syndrome.

[0197] In some embodiments, a disease, disorder, or condition is a rare disease. As described herein, a rare disease refers to a life-threatening or chronically debilitating diseases which are of such low prevalence (e.g., fewer than 1 / 2000 people) that special combined efforts are needed to address them.

[0198] In some embodiments, the present disclosure provides complexes that can selectively target particular systems within a body. As used herein, reference to “targeting” a particular system refers to causing increased expression of RNA derived from cargo in the complex in the desired system. For example, in some embodiments, complexes described herein can selectively target the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas. As described herein, a complex “selectively targets” an organ when a single target expresses mRNA in an amount that is 65% or greater than expression in other organs post administration (e.g., 65% or more of mRNA throughout the body is expressed from a single organ, with the remaining 35% distributed between one or more different organs). In some embodiments, a complex described herein selectively targets the lungs. In some embodiments, a complex described herein selectively targets the liver. In some embodiments, a complex described herein selectively targets the spleen. In some embodiments, a complex described herein selectively targets the heart.Methods of Delivery

[0199] The present disclosure provides, among other things, a particle that is incorporated into a composition (e.g., a pharmaceutical composition or a pharmaceutical formulation, as referred to herein) to be administered to a subject. For example, in some embodiments, a composition comprising particles described herein is administered as a monotherapy. In some embodiments, a composition comprising particles described herein is administered as part of a combination therapy In some embodiments, a concentration of total RNA (e.g., a total concentration of all of the one or more RNA molecules) in a composition described herein is of about 0.01 mg / mL to about 1.0 mg / mL, or about 0.03 mg / mL to about 0.3, or about 0.05 to about 0.15 mg / mL.

[0200] Compositions (also referred to as pharmaceutical compositions) may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0201] In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0202] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included inpharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0203] General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0204] In some embodiments, pharmaceutical compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0205] Pharmaceutical complexes and compositions described herein can be administered by appropriate methods known in the art. As will be appreciated by a skilled artisan, the route and / or mode of administration may depend on a number of factors, including, e.g., but not limited to stability and / or pharmacokinetics and / or pharmacodynamics of pharmaceutical compositions described herein.

[0206] In some embodiments, pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0207] In some embodiments, pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable carriers that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.

[0208] In some particular embodiments, pharmaceutical compositions described herein are formulated for subcutaneous (s.c) administration. In some particular embodiments, pharmaceutical compositions described herein are formulated for intramuscular (i.m) administration.

[0209] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, dispersion, powder(e.g., lyophilized powder), microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0210] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

[0211] In some embodiments, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0212] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0213] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into pharmaceutical compositions described herein. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0214] Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active ingredient(s) into association with a diluent or another excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0215] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using a system and / or method described herein.

[0216] In some embodiments, an active agent that may be included in a pharmaceutical composition described herein is or comprises a therapeutic agent administered in a combination therapy described herein. Pharmaceutical compositions described herein can be administered in combination therapy, i.e., combined with other agents. In some embodiments, such therapeutic agents may include agents leading to depletion or functional inactivation of regulatory T cells. For example, in some embodiments, a combination therapy can include a provided pharmaceutical composition with at least one immune checkpoint inhibitor.

[0217] In some embodiments, pharmaceutical composition described herein may be administered in conjunction with radiotherapy and / or autologous peripheral stem cell or bone marrow transplantation.

[0218] In some embodiments, a pharmaceutical composition described herein can be frozen to allow long-term storage.

[0219] The pharmaceutical compositions of the present disclosure may be in in a frozen form or in a "ready-to-use form" (i.e., in a form, in particular a liquid form, which can be immediately administered to a subject, e.g., without any processing such as thawing, reconstituting, or diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form. E.g., a frozen pharmaceutical composition has to be thawed. Ready to use injectables can be presented incontainers such as vials, ampoules, or syringes wherein the container may contain one or more doses.

[0220] In one embodiment, the pharmaceutical composition is lyophilized. In one embodiment, the pharmaceutical composition is spray dried. These techniques are well known to those skilled in the art.

[0221] In some embodiments, the pharmaceutical composition is in frozen form and can be stored at a temperature of about -90°C or higher, such as about -90°C to about -10°C. For example, the frozen pharmaceutical compositions described herein can be stored at a temperature ranging from about -90°C to about -10°C, such as from about -90°C to about -40°C or from about -40°C to about -25°C, or from about -25°C to about -10°C, or a temperature of about -20°C.

[0222] In some embodiments of the pharmaceutical compositions in frozen form, the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks. For example, the frozen pharmaceutical composition can be stored for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months at -20°C.

[0223] In some embodiments of the pharmaceutical compositions in frozen form, when the nucleic acid is mRNA, the mRNA integrity after thawing the frozen pharmaceutical composition is at least 90%, at least 95%, at least 97%, at least 98%, or substantially 100% of the initial mRNA integrity, e.g., after thawing the frozen composition which has been stored (for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks) at -20°C.

[0224] In some embodiments of the pharmaceutical compositions in frozen form, the size (Zaverage) and / or size distribution and / or PDI of the particles after thawing the frozen pharmaceutical composition is essentially equal to the size (Zaverage) and / or size distribution and / or PDI of the particles of the initial pharmaceutical composition before freezing. For example, if a ready-to-use pharmaceutical composition is prepared from a frozen pharmaceutical composition as described herein, it is preferred that the size (Zaverage) and / or size distribution and / or PDI of the particles contained in the ready-to-use pharmaceutical composition is essentially equal to the initialsize (Zaverage) and / or size distribution and / or PDI of the particles contained in the frozen pharmaceutical composition before freezing.

[0225] In some embodiments, when the nucleic acid is mRNA, the size of the mRNA particles and the mRNA integrity of the pharmaceutical composition after one freeze / thaw cycle, preferably after two freeze / thaw cycles, more preferably after three freeze / thaw cycles, more preferably after four freeze / thaw cycles, more preferably after five freeze / thaw cycles or more, are essentially equal to the size of the mRNA particles and the mRNA integrity of the initial pharmaceutical composition (i.e., before the pharmaceutical composition has been frozen for the first time).

[0226] In some embodiments, the pharmaceutical composition is in liquid form and can be stored at a temperature ranging from about 0°C to about 20°C. For example, the liquid pharmaceutical compositions described herein can be stored at a temperature ranging from about 1°C to about 15°C, such as from about 2°C to about 10°C, or from about 2°C to about 8°C, or at a temperature of about 5°C.

[0227] In some embodiments, when the nucleic acid is mRNA, the mRNA integrity of the pharmaceutical composition when stored is at least 70%, preferably at least 80%, more preferably at least 90%, of the initial mRNA integrity (i.e., the mRNA integrity of the initial pharmaceutical composition).

[0228] In some embodiments of the pharmaceutical compositions in liquid form, the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, or at least 24 months, preferably at least 4 weeks. For example, the liquid pharmaceutical composition can be stored for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months at 5°C.

[0229] In some embodiments of the pharmaceutical composition in liquid form, when the nucleic acid is mRNA, the mRNA integrity of the liquid composition, when stored, e.g., at 0°C or higher for at least one week, is such that the desired effect, e.g., to induce an immune response, can be achieved. For example, the mRNA integrity of the liquid composition, when stored, e.g., at 0°C or higher for at least one week (such as for at least 2 weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least 4 months, or at least 6 months), may be at least 90%, compared to the mRNA integrity of the initial composition, i.e.,the mRNA integrity before the composition has been stored. In some embodiments, the mRNA integrity of the composition after storage for at least four weeks (e.g., for at least three months), preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, is at least 90%, compared to the mRNA integrity before storage.

[0230] In some embodiments, when the nucleic acid is mRNA, the initial mRNA integrity of the pharmaceutical composition (i.e., after its preparation but before storage) is at least 50% and the mRNA integrity of the pharmaceutical composition after storage for at least one week (such as for at least 2 weeks, at least three weeks, at least four weeks, at least one month, at least two months, or at least 3 months), preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, is at least 90% of the initial mRNA integrity.

[0231] In some embodiments of the pharmaceutical composition in liquid form, the size (Zaverage) (and / or size distribution and / or polydispersity index (PDI)) of the particles of the pharmaceutical composition, when stored, e.g., at 0°C or higher for at least one week, is such that the desired effect, e.g., to induce an immune response, can be achieved. For example, the size (Zaverage) (and / or size distribution and / or polydispersity index (PDI)) of the particles of the pharmaceutical composition, when stored, e.g., at 0°C or higher for at least one week, is essentially equal to the size (Zaverage) (and / or size distribution and / or PDI) of the particles of the initial pharmaceutical composition, i.e., before storage.

[0232] In some embodiments, the size (Zaverage) of the particles after storage of the pharmaceutical composition, e.g., at 0°C or higher for at least one week is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm. In some embodiments, the PDI of the particles after storage of the pharmaceutical composition, e.g., at 0°C or higher for at least one week is less than 0.3, preferably less than 0.2, more preferably less than 0.1.

[0233] In some embodiments, the size (Zaverage) of the particles after storage of the pharmaceutical composition, e.g., at 0°C or higher for at least one week is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the size (Zaverage) (and / or size distribution and / or PDI) of the particles after storage of the pharmaceutical composition, e.g., at 0°C or higher for at least one week is essentially equal to the size (Zaverage) (and / or size distribution and / or PDI) of the particles before storage. In some embodiments, the size (Zaverage) of the particles after storage of thepharmaceutical composition, e.g., at 0°C or higher for at least one week is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the PDI of the particles after storage of the pharmaceutical composition, e.g., at 0°C or higher for at least one week is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0234] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation.Process for Preparing Lipids

[0235] As described herein, the present disclosure provides a method of preparing a cationic or ionizable lipid such as those described herein. Methods of preparing cationic or ionizable lipids described herein are not limited solely to those having symmetric aliphatic tails, and can be applied to a variety of lipid structures.

[0236] For example, in some embodiments, the present disclosure provides a method of preparing a compound of formula III:or a pharmaceutically acceptable salt thereof, the method comprising contacting a compound of formula IV- 1 with a compound of formula IV-2 in the presence of heat:to provide a first intermediate product, and wherein the first intermediate product is contacted with a platinum catalyst and a first acid in the presence of hydrogen gas to provide a second intermediate product, and wherein the second intermediate product is contacted with a second acid to provide the compound of formula III, whereinM1and M2are independently selected from a bond or optionally substituted C2-C10aliphatic;L1and L2are each independently selected from a bond, -OC(O)- and -C(O)O-;T1and T2are each independently selected from optionally substituted C2-C20aliphatic;L3is optionally substituted C1-C6aliphatic;LG1is a suitable leaving group; andPG is a suitable alcohol protecting group.

[0237] In some embodiments, a compound of formula IV-2 is prepared by contacting a compound of formula V-1a and formula V-1b with a compound of formula V-2in the presence of a palladium coupling catalyst, a copper(I) cocatalyst, and an amine base to provide the compound of formula IV-2, wherein LG2is a suitable leaving group.

[0238] In some embodiments, the present disclosure provides a method of preparing a compound of formula III- 1 :or a pharmaceutically acceptable salt thereof, the method comprising contacting a compound of formula IV- 1 with a compound of formula IV-3 in the presence of heat:to provide a first intermediate product, and wherein the first intermediate product is contacted with an acid to provide the compound of formula III- 1 , whereinM1and M2are each independently selected from a bond or optionally substituted C2-C10aliphatic;T1and T2are each independently selected from optionally substituted C2-C20aliphatic;L3is optionally substituted C1-C6aliphatic;LG1is a suitable leaving group; andPG is a suitable alcohol protecting group.

[0239] In some embodiments, the compound of formula IV-3 is prepared by contacting a compound of formula IV-4:with one or more equivalents of each of a compound of formula IV-5 and IV-6:in the presence of 4-(Dimethylamino)pyridine (DMAP) and 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDO) to provide the compound of formula IV-3.

[0240] In some embodiments, the compound of formula IV-4 is prepared by contacting a compound of formula IV-7with hydrogen gas in the presence of a platinum or palladium reducing catalyst to provide a compound of formula IV-4.

[0241] In some embodiments, the compound of formula IV-7 is prepared by contacting a compound of V-2 with one equivalent each of a compound of formula V-lc and V-ldin the presence of a palladium coupling catalyst, a copper(I) cocatalyst, and an amine base to provide the compound of formula IV-2, wherein LG2is a suitable leaving group

[0242] As described herein, M1and M2are independently selected from a bond or optionally substituted C2-C10aliphatic. In some embodiments, M1and M2are the same. In some embodiments, M1and M2are different. In some embodiments, M1and M2are each independently selected from optionally substituted C2-C10alkylene. In some embodiments, M1and M2are each independently selected from -(CH2)4-6-.

[0243] As described herein, L1and L2are each independently selected from a bond, -OC(O)- and -C(O)O-. In some embodiments, L1and L2are the same. In some embodiments, L1and L2are different. In some embodiments, L1is a bond. In some embodiments, L1is -OC(O)-. In some embodiments, L1is -C(O)O-. In some embodiments, L2is a bond. In some embodiments, L2is -OC(O)-. In some embodiments, L2is -C(O)O-. In some embodiments, L1and L2are each a bond. In some embodiments, L1and L2are each -OC(O)-. In some embodiments, L1and L2are each -C(O)O-.

[0244] As described herein, L3is optionally substituted ci-C6 aliphatic. In some embodiments, L3is optionally substituted C3-C5aliphatic. In some embodiments, L3is optionally substituted C1-C6alkylene. In some embodiments, L3is C3-C5alkylene. In some embodiments, L3is -(CH2)1-6-. In some embodiments, L3is -(CH2)3-, -(CH2)4-, or -(CH2)5- In some embodiments, L3is -(CH2)3-. In some embodiments, L3is -(CH2)4-. In some embodiments, L3' is -(CH2)5-.

[0245] As described herein, T1and T2are each independently selected from optionally substituted C2-C20aliphatic. In some embodiments, T1and T2are the same. In some embodiments, T1and T2are different. In some embodiments, T1and T2are each branched C3- C20aliphatic. In some embodiments, T1and T2are each linear C2-C20aliphatic. In some embodiments, one of T1and T2is branched C3-C20aliphatic, and the other of T1and T2is linear C2-C20aliphatic.

[0246] In some embodiments, T1and T2are each independently selected from

[0247] In some embodiments, -L1-T1is selected from:wherein: each nl is independently 0, 1, 2, 3, 4, 5, or 6; and each n2 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0248] As described herein, LG1is a suitable leaving group. In some embodiments, LG1is halogen or -OTf. In some embodiments, LG1is selected from bromo, chloro, or iodo. In some embodiments, LG1is bromo.

[0249] As described herein, PG is a suitable alcohol protecting group. A person of skill in the art will readily appreciate suitable protecting groups for an alcohol moiety. In some embodiments, PG is an acetyl group, a benzoyl, group, a benzyl group, a methoxyethoxymethyl ether (MEM) group, a dimethoxytrityl (DMT) group, a methoxymethyl ether (MOM) group, a methoxytrityl (MMT) group, a p-methoxybenzyl ether (PMB) group, a pivaloyl (Piv) group, a tert-butyl ether group, a tetrahydropyranyl group, a tetrahydrofuran group, a trityl group, or a silylether, including a tert-butyldimethylsilyl ether (TBS). In some embodiments, PG is a TBS group.

[0250] As described herein LG2is a suitable leaving group. In some embodiments, each LG2is independently selected from halogen and -OTf. In some embodiments, each LG2is independently selected from bromo, chloro, and iodo. In some embodiments, each LG2is bromo.

[0251] In some embodiments, a platinum catalyst is PtO2.

[0252] In some embodiments, a palladium reducing catalyst is palladium on carbon.

[0253] In some embodiments, a first acid is an organic acid. In some embodiments, an organic acid is acetic acid.

[0254] In some embodiments, a second acid is hydrochloric acid.

[0255] In some embodiments, a palladium coupling catalyst is Pd(PPh3)4.

[0256] In some embodiments, a copper (I) cocatalyst is Cui.

[0257] In some embodiments an amine base is triethylamine.

[0258] In some embodiments, a compound described herein (e.g., a compound of formula I) is prepared according to a method described herein.Exemplary Embodiments

[0259] The following numbered embodiments, while non-limiting, are exemplary of certain aspects of the present disclosure:1. A compound of formula I:or a pharmaceutically acceptable salt thereof, whereinM1and M2are each -(CH2)p1-;L1and L2are each selected from a bond, -OC(O)-, and -C(O)O-;T1and T2are each:L3is optionally substituted C1-C6aliphatic;G1is -OH, or -N(Ra)2, andRais H, optionally substituted C1-C6aliphatic, or optionally substituted C3-C6cycloaliphatic; p1 is an integer selected from 2-20, inclusive;p2 and p3 each an integer selected from 1-20, inclusive, and p2 and p3 are the same. The compound of Embodiment 1, wherein p1 is an integer selected from 2-10, inclusive. The compound of Embodiments 1 or 2, wherein M1is -(CH2)4-6- and M2is -(CH2)4-6-. The compound of any one of Embodiments 1 to 3, wherein M1and M2are the same. The compound of any one of Embodiments 1 to 4, wherein T1and T2are the same. The compound of any one of Embodiments 1 to 5 wherein L1and L2are the same. The compound of any one of Embodiments 1 to 6, wherein p2 and p3 are each an integer selected from 3, 4, 5, 6, 7, 8 or 9. The compound of Embodiment 7, wherein p2 and p3 are each an integer selected from 4, 5, or 6. The compound of Embodiment 1 , wherein T1and T2are each selected from:The compound of Embodiment 1, wherein M1-L1-T1is represented by:wherein p2 and p3 are each 4, 5, or 6. The compound of Embodiments 1 or 10, wherein M2-L2-T2is represented by:wherein p2 and p3 are each 4, 5, or 6.12. The compound of Embodiment 1, wherein M1-L1-T1and M2-L2-T2are each selected from:13. The compound of any one of Embodiments 1-12, wherein -L3-G1is -(CH2)3-OH or - (CH2)4-OH.14. The compound of Embodiment 1, wherein the compound is represented by formula II- 1 :II- 1 where * represents a stereopure carbon atom.15. The compound of any one of Embodiments 1-14, wherein the compound is characterized by having a Wiener path of between about 15,000 and 30,000 and an MMFF94 energy between -15 and 0.The compound of any one of Embodiments 1-15, wherein the compound is characterized by having a k between 0.1 and 0.2. The compound of Embodiment 1, wherein the compound is selected from Table 1. A pharmaceutical composition comprising the compound of any one of Embodiments 1- 17, and a nucleic acid. The pharmaceutical composition of Embodiment 18, further comprising one or more of: a neutral lipid, a polymer- conjugated lipid, a steroid, or an anionic amphiphile. The pharmaceutical composition of Embodiment 19, further comprising a neutral lipid. The pharmaceutical composition of Embodiments 19 or 20, wherein the neutral lipid is a phospholipid. The pharmaceutical composition of Embodiment 21, wherein the phospholipid is or comprises distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphocholine (DOPG), 1 ,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)(DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), or N- palmitoyl-D-erythro-sphingosylphosphorylcholine (SM). The pharmaceutical composition of any one of Embodiments 19 to 22, further comprising a polymer conjugated lipid. The pharmaceutical composition of Embodiment 23, wherein the polymer-conjugated lipid is a lipid comprising a polymer that is polyethylene glycol. The pharmaceutical composition of Embodiment 24, wherein the polymer-conjugated lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, PEG-S-DMG, PEG-cer, a PEG dialkoxypropylcarbamate (e.g., ω-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω- methoxy(polyethoxy)ethyl)carbamate), and combinations thereof. The pharmaceutical composition of any one of Embodiments 19 to 25, further comprising a steroid. The pharmaceutical composition of Embodiment 26, wherein the steroid is a sterol. The pharmaceutical composition of Embodiment 27, wherein the sterol is cholesterol. The pharmaceutical composition of any one of Embodiments 19-28, further comprising an anionic amphiphile. The pharmaceutical composition of Embodiment 29, wherein the anionic amphiphile is selected from DMGS, DPGS, and DMGS. The pharmaceutical composition of any one of Embodiments 19 to 22, and 26 to 29, wherein the pharmaceutical composition comprises a neutral lipid, a steroid, and ananionic amphiphile, and the pharmaceutical composition does not include a polymer- conjugated lipid. The pharmaceutical composition of any one of Embodiments 18-31, wherein the nucleic acid is RNA. The pharmaceutical composition of Embodiment 32, wherein the RNA is modRNA, saRNA, taRNA, or uRNA. The pharmaceutical composition of any of Embodiments 18-31, wherein the nucleic acid is DNA. The pharmaceutical composition of any one of Embodiments 18-31, wherein the nucleic acid is a mixture of RNA and DNA. The pharmaceutical composition of any one of Embodiments 19-35, wherein the pharmaceutical composition comprises: about 30 to about 60 mol% of the compound; about 20 to about 60 mol% of the steroid; about 1 to about 2 mol% of the polymer-conjugated lipid; and about 5 to about 20 mol% of the neutral lipid. The pharmaceutical composition of any one of Embodiments 19-35, wherein the pharmaceutical composition comprises: about 30 to about 60 mol% of the compound; about 20 to about 60 mol% of the steroid; about 0.5 to about 10 mol% of the anionic amphiphile; and about 5 to about 20 mol% of the neutral lipid. The pharmaceutical composition of Embodiment 37, wherein the pharmaceutical composition does not include a polymer-conjugated lipid.A suspension comprising a dispersed phase and an aqueous phase, and wherein the dispersed phase comprises the pharmaceutical composition of any one of Embodiments 18-38 in the form of particles. The suspension of Embodiment 39, wherein the particles have an average diameter of between about 50 nm to about 200 nm. The suspension of Embodiment 40, wherein the aqueous phase comprises less than 20% of the total concentration of nucleic acid in the suspension. The suspension of any one of Embodiments 39-41, wherein the aqueous phase is substantially free of the nucleic acid. A method of treating a disease, disorder, or condition comprising administering to a subject the suspension of any one of Embodiments 39-42. The method of Embodiment 43, wherein the disease, disorder, or condition is selected from an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease. A method of increasing or causing increased expression of RNA in a target in a subject comprising administering to the subject the suspension of any one of Embodiments 39- 42. The method of Embodiment 45, wherein the target is selected from the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas. The method of any one of Embodiments 43-46, wherein the suspension is administered intramuscularly, intranasally, intravenously, subcutaneously, or intratumorally.A method of preparing a compound of formula III:or a pharmaceutically acceptable salt thereof, the method comprising contacting a compound of formula IV- 1 with a compound of formula IV-2 in the presence of heat:to provide a first intermediate product, and wherein the first intermediate product is contacted with a platinum catalyst and a first acid in the presence of hydrogen gas to provide a second intermediate product, and wherein the second intermediate product is contacted with a second acid to provide the compound of formula III, whereinM1and M2are independently selected from a bond or optionally substituted C2-C10aliphatic;L1and L2are each independently selected from a bond, -OC(O)- and -C(O)O-;T1and T2are each independently selected from optionally substituted C2-C20aliphatic;L3is optionally substituted C1-C6aliphatic;LG1is a suitable leaving group; andPG is a suitable alcohol protecting group.49. The method of Embodiment 48, wherein a compound of formula IV-2 is prepared by contacting a compound of formula V-la and formula V-lb with a compound of formula V-2in the presence of a palladium coupling catalyst, a copper(I) cocatalyst, and an amine base to provide the compound of formula IV-2, wherein LG2is a suitable leaving group.50. A method of preparing a compound of formula III- 1 :or a pharmaceutically acceptable salt thereof, the method comprising contacting a compound of formula IV- 1 with a compound of formula IV-3 in the presence of heat:to provide a first intermediate product, and wherein the first intermediate product is contacted with an acid to provide the compound of formula III- 1 , whereinM1and M2are each independently selected from a bond or optionally substituted C2-C10aliphatic;T1and T2are each independently selected from optionally substituted C2-C20aliphatic;L3is optionally substituted C1-C6aliphatic;LG1is a suitable leaving group; andPG is a suitable alcohol protecting group.EXAMPLES

[0260] 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 Abbreviations aq. = aqueousDMGS = dimyristoylglycerol hemisuccinateDPGS = dipalmitoylglycerol hemisuccinateDSGS = distearoylglyceryl hemisuccinateDSPC = distearoylphosphatidylcholine equiv. = equivalent h or hr = hour or hoursHRMS = high resolution mass spectrometryLNP = lipid nanoparticleNMR = nuclear magnetic resonanceOtf = trifluoromethanesulfonatePEG = polyethylene glycol rt = room temperature sat. = saturatedTBS = tert-butyldimethylsilyl etherTHF = tetrahydrofuranTLC = thin layer chromatographyGeneral Synthetic Route for the Synthesis of the Lipids:

[0261] The general synthetic route for the synthesis of the compounds described herein comprises two linear steps, a Sonogashira coupling followed by alkylation, reduction and TBS deprotection. A scheme illustrating an example synthesis method is provided below.wherein A is a suitable leaving group (e.g., a halogen such as I, Br, Cl, or triflate, i.e., -O-S(O)- CF3), and each of X, Y, and Z, are independently, at each instance, a bond, or -(CH2)1-4-.

[0262] In some embodiments, compounds described herein can also be prepared according to the following process:wherein A is a suitable leaving group (e.g., a halogen such as I, Br, Cl, or triflate, i.e., -O-S(O)- CF3), and each instance of X, Y, and Z, are independently, at each instance, a bond, or -(CH2)1-4-Example 1: pyridine-3,5-diylbis(but-3-yne-4,1-diyl) bis(2-heptylnonanoate):

[0263] A 25 mL flame dried Schlenk flask with magnetic stir bar was charged with 2,5- dibromopyridine (473.0 mg, 2.0 mmol, 1.0 equiv.), copper(I)iodide (38.0 mg, 0.2 mmol, 0.1 equiv.) and tetrakis-(triphenylphosphin)-palladium (116.0 mg, 0.1 mmol, 0.05 equiv.). The flask was then carefully evacuated under high vacuum and put under argon. Triethylamine (10.0 mL) was added to the solids and then but-3-yn-1-yl 2-heptylnonanoate (1.8 g, 6 mmol, 3.0 equiv.) was added dropwise. The reaction mixture was heated at 60 °C for 18 h. The progress of the reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure and the crude mixture was purified crude mixture was purified by column chromatography on silica gelusing hexanes / ethyl acetate (95:5, v / v) as eluent. The product was obtained as brown color liquid (0.83 g, 60% yield).1H NMR (501 MHz, CDCI3) δ 8.49 (d, J = 2.0 Hz, 2H), 7.64 (t, J= 2.0 Hz, 1H), 4.26 (t, J = 6.7 Hz, 4H), 2.76 (t, J = 6.7 Hz, 4H), 2.35 (tt, J= 8.9, 5.3 Hz, 2H), 1.66-1.55 (m, 4H), 1.45 (ddt, J = 14.7, 11.4, 5.4 Hz, 4H), 1.33-1.12 (m, 40H), 0.86 (t, J = 6.9 Hz, 12H).13C NMR (126 MHz, CDCI3) δ 176.48, 150.92, 140.91, 120.19, 90.08, 78.14, 61.73, 45.89, 32.63, 31.94, 29.67, 29.27, 27.61, 27.12, 22.76, 20.21, 14.22.Example 2: pyridine-3,5-diylbis(hex-5-yne-6,1-diyl) bis(2-hexyldecanoate)

[0264] The compound of the present example was prepared in an analogous method to the method described in Example 1, instead using hex-5-yn-1-yl 2-hexyldecanoate to provide the title compound as a brown color liquid (1.1 g, 92% yield).1H NMR (501 MHz, CDCI3) δ 8.43 (d, J = 2.0 Hz, 2H), 7.59 (t, J= 2.1 Hz, 1H), 4.09 (t, J= 6.4 Hz, 4H), 2.42 (t, J= 7.0 Hz, 4H), 2.28 (tt, J = 9.1, 5.3 Hz, 2H), 1.76 (dq, J= 8.9, 6.5 Hz, 4H), 1.69-1.60 (m, 4H), 1.60-1.49 (m, 4H), 1.39 (dp, J= 14.3, 5.0 Hz, 4H), 1.22 (dd, J= 13.1, 6.1 Hz, 40H), 0.82 (td, J= 6.9, 2.3 Hz, 12H).13C NMR (126 MHz, CDCI3) δ 176.56, 150.49, 140.69, 120.44, 93.57, 77.32, 63.39, 45.84, 32.58, 31.89, 31.74, 29.60, 29.49, 29.29, 29.26, 28.01, 27.53, 27.49, 25.13, 22.70, 22.63, 19.13, 14.13, 14.08. HRMS m / z (ESI): calculated for C49H82O4N [M+H]: 748.62384; found: 748.62334.Example 3: pyridine-3,5-diylbis(hex-5-yne-6,1-diyl) bis(2-heptylnonanoate):

[0265] The compound of the present example was prepared in an analogous method to the method described in Example 1, instead using hex-5-yn-1-yl 2-heptylnonanoate to provide the title compound as a brown color liquid (1.4 g, 94% yield).Example 4: (1-(4-hydroxybutyl)piperidinee-3,5-diyl)bis(hexane-6,1-diyl) bis(2- hexyldecanoate): PIL4677

[0266] A 25 mL flame dried round-botom flask with magnetic stir bar was charged with pyridine-3,5-diylbis(hex-5-yne-6,1-diyl) bis(2-hexyldecanoate) (1.0 g, 1.45 mmol, 1.0 equiv.) and 1-Brom-4-(tert-butyldimethylsilyloxy)-butane (1.1 g, 3.9 mmol, 2.7 equiv.) at rt. The reaction mixture was then heated at 110 °C for 16 h. The progress of the reaction was monitored by TLC.

[0267] After 16 h, the reaction mixture was cooled at rt and was obtained the pyridinium salt in quantitative yield. To this crude pyridinium salt, acetic acid (1.5 mL) and PtO2(22.5 mg, 0.1 mmol, 0.1 equiv.) were added. The reaction mixture was heated at 40 °C under H2atmosphere (1 atm) for 18 h. The progress of the reaction was monitored by TLC.

[0268] After 18 h, the reaction mixture was cooled at rt. To this reaction mixture, THF (5 mL) and 5 mL of aq. HCl (10%) were added. The mixture was stirred at rt for 30 min. Then the reaction mixture was diluted with Et2O (25 mL) and the organic phase was collected. The aq. phase was extracted with Et2O (3x20 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude mixture was purified on silica gel column chromatography using ethyl acetate / EtOH (95:5 -> 85:15, v / v) as eluent. The product was isolated as an HCl-salt. The HCl-salt was treated with sat. aq. NaHCO3and the lipid was obtained as a light yellow color oil (0.36 g, 44% yield over three steps).1H NMR (501 MHz, CDCI3) δ 4.02 (t, J = 6.6 Hz, 4H), 3.43 (q, J= 7.0 Hz, 1H), 3.03-2.96 (m, 2H), 2.80 (dd, J= 12.1, 3.7 Hz, 1H), 2.45 (dd, J= 12.2, 6.4 Hz, 1H), 2.26 (t, J= 9.0, 5.3 Hz, 2H), 2.21-2.09 (m, 1H), 2.06 (t, J= 11.5 Hz, 2H), 1.80 (d, J = 12.8 Hz, 1H), 1.55 (dp, J = 16.0, 7.2 Hz, 8H), 1.45-1.00 (m, 65H), 0.93-0.71 (m, 12H), 0.55 (q, J= 12.1 Hz, 1H).13C NMR (126 MHz, CDCI3) δ 176.68, 65.89, 64.11, 60.39, 53.06, 51.98, 45.91, 38.96, 37.44, 36.15, 34.76, 32.96, 32.87, 32.63, 31.94, 31.78, 29.63, 29.60, 29.52, 29.45, 29.32, 29.30, 28.81, 28.79, 27.54, 27.50, 27.29, 26.71, 26.07, 26.02, 22.73, 22.66, 15.33, 14.26, 14.16, 14.13. HRMS m / z (ESI): calculated for C53H104NO5[M+H]: 834.79090; found: 834.79011.Example 5: (1-(3-hydroxypropyl)piperidinee-3,5-diyl)bis(hexane-6,1-diyl) bis(2- heptylnonanoate): PIL 3677

[0269] The compound of the present example was prepared according to the method described in Example 4, where pyridine-3,5-diylbis(hex-5-yne-6,1-diyl) bis(2-heptylnonanoate) was reacted with 1-bromo-3-(tert-butyldimethylsilyloxy)-propane to provide the title compound as a light yellow color oil (0.17 g, 48% yield over three steps).1H NMR (501 MHz, CDCI3) δ 4.06 (t, J = 6.6 Hz, 4H), 3.82-3.75 (m, 2H), 3.02 (d, J = 10.1 Hz, 2H), 2.58 (t, J = 5.5 Hz, 2H), 2.31-2.30 (m, 2H), 1.85-1.77 (m, 1H), 1.72 (p, J= 5.3 Hz, 2H), 1.66-1.01 (m, 73H), 0.87 (t, J= 6.9 Hz, 12H), 0.47 (q, J = 11.8 Hz, 1H).13C NMR (126 MHz, CDCI3) δ 176.86, 64.24, 60.72, 60.53, 59.43, 46.01, 38.11, 36.24, 34.73, 32.70, 31.96, 29.75, 29.67, 29.31, 28.85, 27.62, 27.23, 26.87, 26.09, 22.79, 21.19, 14.35, 14.24.Example 6: (1-(3-hydroxypropyl)piperidinee-3,5-diyl)bis(butane-4,1-diyl) bis(2- heptylnonanoate): CPIL 3477

[0270] The compound of the present example was prepared according the method described in Example 4, where pyridine-3,5-diylbis(but-3-yne-4,1-diyl) bis(2-heptylnonanoate) was reacted with 1-bromo-3-(tert-butyldimethylsilyloxy)-propane to provide the title compound as a light yellow color oil (0.15 g, 43% yield over three steps).1H NMR (501 MHz, CDCI3) δ 4.06 (t, J = 6.6 Hz, 4H), 3.79 (dt, J = 11.8, 5.4 Hz, 2H), 3.67-3.45 (m, 1H), 3.02 (d, J = 10.3 Hz, 2H), 2.58 (s, 2H), 2.31 (tt, J = 8.9, 5.3 Hz, 2H), 1.94-1.76 (m, 1H), 1.71 (s, 2H), 1.59 (dt, J = 13.2, 6.5 Hz, 10H), 1.50-1.07 (m, 54H), 0.87 (t, J = 6.9 Hz, 12H), 0.49 (q, J = 11.9 Hz, 1H).13C NMR (126 MHz, CDCI3) δ 176.83, 64.04, 45.99, 34.37, 32.67, 31.97, 29.68, 29.32, 29.17, 27.63, 23.34, 22.79, 14.25.Example 7: (1-(4-hydroxybutyl)piperidine-3,5-diyl)bis(hexane-6,1-diyl) bis(2-octyldecanoate)PIL4668Example 8: Biological Activity of Cyclic Ionizable Lipids

[0271] The present example describes the preparation of lipid nanoparticle (LNP) compositions from piperidine ionizable lipid as described herein, cholesterol, DSPC and a polymer-conjugated lipid.Study design

[0272] The present example reports a range of compositions that were tested wherein the piperidine ionizable lipid was between 29.9 and 66.2 mol%, cholesterol was between 17.7 and 57.1 mol %, DSPC was between 2.6 and 23.8 mol% and PEG-Lipid was 1.8 mol%.Generation of LNP

[0273] Stock solution concentrations were prepared with the following concentrations: mRNA: 0.22 mg / mL in deionized water, piperidine ionizable lipid: 50 mM (acidified with 75 mM acetic acid), cholesterol: 50 mM, DSPC: 33.33 mM, PEG-Lipid: 8 mM, wherein ethanol was used as solvent for all lipids. Drop mixing process: piperidine ionizable lipid was added to the well plate, followed by cholesterol, DSPC, and PEG-lipid (used for the reference formulations) in that order. Ethanol was added q.s. to a final volume of 33 μL ethanol. The mRNA solution was added to a final concentration of 0.055 mg / mL, followed by buffer (35 mM Tris:Hac @ pH = 5.5). Both the mRNA and buffer solutions completed the final volume of 200 μL. Aliquots were taken and further diluted stepwise using buffer (35 mM Tris:Hac @ pH = 7.5). Particle size was monitored at this point using a Wyatt Dynapro III Dynamic Light Scattering Instrument.

[0274] Materials having a very small size below 40nm or a large size above 250nm were de- selected in further analysis.

[0275] For in vitro expression, 10 μL samples were transferred to a new well plate, and 10 μL of human serum were added. The mixture was incubated at room temperature for 30 minutes beforetransfection. A second 10 μL sample was transferred into another well plate, and 10 μL of water were added. Cells were then transfected with a total mRNA content of 50 ng per well for both the serum-naive and serum-containing nanoparticles. The final ethanol volume in cell culture was 0.125%.

[0276] In vitro expression was measured in HEK-293 and HEPG2 cells.

[0277] The expression data were clustered (n=5) and the cluster with the highest expression was selected per lipid. The table below shows the mean expression level.

[0278] The data demonstrate that materials having a particle size <=250nm can be obtained for materials comprising piperidine ionizable lipids described herein, cholesterol, DSPC and PEG- lipid.Example 9: Composition of LNP made from Piperidine ionizable lipids

[0279] For piperidine ionizable lipids having the highest activity, the lipid composition was optimized. Ranges of compositions were tested wherein the piperidine ionizable lipids was between 30 and 66 mol%, cholesterol was between 18 and 570mol%, DSPC was between 2.6 and 24 mol % and PEG-Lipid was 1.8 mol%.

[0280] The particles were prepared and analyzed as outlined in Example 8.

[0281] FIG. 1 shows in vitro expression for LNP made from piperidine ionizable lipid, cholesterol, DSPC and PEG-Lipid in a fully combinatorial matrix when tested in HepG2 cells with pre-incubation of lipid nanoparticles in human serum.

[0282] LNP were made as described above and particle size was monitored. LNP larger than 250nm are denoted with “x” and excluded from further analysis. Signals were normalized to referenced and categorized to “low activity” in light grey having an expression less than 10-0.3ofthe reference, “good activity” in grey having an expression between 10-0.3and 10-0of the reference and “high activity” in dark grey having an expression higher than reference.

[0283] The data demonstrate that materials having a particle size <=250nm can be obtained for materials comprising piperidine ionizable lipid, cholesterol, DSPC and PEG-lipid. For PIL3677, colloidal properties and high activity of the lipid nanoparticles was observed for almost the entire range of lipid compositions, e.g. having between 30 and 65mol% of ionizable lipid and 20 and 58mol% cholesterol and 2.5 and 24mol% DSPC. For PIL4677, compositions having between 30 and 50 mol% of ionizable lipid and 35 to 50 mol% cholesterol are preferred. See FIG. 1.Example 10: LNPs comprising piperidine ionizable lipids that lack a stealth lipid

[0284] In this Example, lipid nanoparticle (LNP) compositions were generated from piperidine ionizable lipids (e.g., compounds described herein), cholesterol, DSPC and an anionic amphiphile.Study design

[0285] As the piperidine ionizable lipids are novel and the existence of LNP not having a stealth lipid is unknown, an a priori lipid composition may not result in the formation of lipid nanoparticles or those formed may not have a high activity. As such a range of compositions was tested wherein the piperidine ionizable lipid PIL3677was between 25 and 55 mol%, cholesterol was between 17.5 and 59.0 mol %, DSPC was between 2.5 and 25.0 mol% and DMGS, DPGS or SMGS were used as an anionic amphiphile between 0.5 and 2.5 mol%.The particles were prepared and analyzed as described in example 8.

[0286] FIG. 2 shows in vitro expression for LNPs made from piperidine ionizable lipid, cholesterol, DSPC and one of DMGS (dimyristoylglycerol hemisuccinate), DPGS (dipalmitoylglycerol hemisuccinate) or SMGS (stearoylmyristoylglycerol hemisuccinate) as an anionic amphiphile in a fully combinatorial matrix.

[0287] LNP were made as described above and particle size was monitored. LNP larger than 200nm are denoted with “x” and excluded from further analysis. Levels of in vitro expression were obtained without prior incubation with serum on HEK293 cells. Signals were normalized to referenced and categorized to “low activity” in light grey having an expression less than 10-0.3ofthe reference, “good activity” in grey having an expression between 10-0.3and 10° of the reference and “high activity” in dark grey having an expression higher than reference.

[0288] The data demonstrate that materials having a particle size <=250nm can be obtained for materials comprising piperidine ionizable lipid, cholesterol, DSPC and one of DMGS, DPGS and DSGS as an anionic amphiphile. When selecting PIL3677, lipid nanoparticles comprising anionic amphiphiles frequently display an activity comparable to benchmark. Preferred compositions had between 30 and 60 mol% of cholesterol.

[0289] The embodiments of the disclosure described above are intended to be merely exemplary, numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

CLAIMS1. A compound of formula I:or a pharmaceutically acceptable salt thereof, whereinM1and M2are each -(CH2)p1-;L1and L2are each selected from a bond, -OC(O)-, and -C(O)O-;T1and T2are each:L3is optionally substituted C1-C6aliphatic;G1is -OH, or -N(Ra)2, andRais H, optionally substituted C1-C6aliphatic, or optionally substituted C3-C6cycloaliphatic; p1 is an integer selected from 2-20, inclusive; p2 and p3 each an integer selected from 1-20, inclusive, and p2 and p3 are the same.

2. The compound of claim 1, wherein p1 is an integer selected from 2-10, inclusive.

3. The compound of claims 1 or 2, wherein M1is -(CH2)4-6- and M2is -(CH2)4-6-.

4. The compound of any one of claims 1 to 3, wherein M1and M2are the same.

5. The compound of any one of claims 1 to 4, wherein T1and T2are the same.

6. The compound of any one of claims 1 to 5 wherein L1and L2are the same.

7. The compound of any one of claims 1 to 6, wherein p2 and p3 are each an integer selected from 3, 4, 5, 6, 7, 8 or 9.

8. The compound of claim 7, wherein p2 and p3 are each an integer selected from 4, 5, or 6.

9. The compound of claim 1, wherein T1and T2are each selected from:

10. The compound of claim 1, wherein M1-L1-T1is represented by:wherein p2 and p3 are each 4, 5, or 6.

11. The compound of claims 1 or 10, wherein M2-L2-T2is represented by:wherein p2 and p3 are each 4, 5, or 6.

12. The compound of claim 1, wherein M1-L1-T1and M2-L2-T2are each selected from:

13. The compound of any one of claims 1-12, wherein -L3-G1is -(CH2)3-OH or -(CH2)4-OH.

14. The compound of claim 1 , wherein the compound is represented by formula II- 1 :where * represents a stereopure carbon atom.

15. The compound of any one of claims 1-14, wherein the compound is characterized by having a Wiener path of between about 15,000 and 30,000 and an MMFF94 energy between -15 and 0.

16. The compound of any one of claims 1-15, wherein the compound is characterized by having a k between 0.1 and 0.2.

17. The compound of claim 1, wherein the compound is selected from Table 1.

18. A pharmaceutical composition comprising the compound of any one of claims 1-17, and a nucleic acid.

19. The pharmaceutical composition of claim 18, further comprising one or more of: a neutral lipid, a polymer- conjugated lipid, a steroid, or an anionic amphiphile.

20. The pharmaceutical composition of claim 19, further comprising a neutral lipid.

21. The pharmaceutical composition of claims 19 or 20, wherein the neutral lipid is a phospholipid.

22. The pharmaceutical composition of claim 21, wherein the phospholipid is or comprises distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphocholine (DOPG), 1 ,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), or N- palmitoyl-D-erythro-sphingosylphosphorylcholine (SM).

23. The pharmaceutical composition of any one of claims 19 to 22, further comprising a polymer conjugated lipid.

24. The pharmaceutical composition of claim 23, wherein the polymer-conjugated lipid is a lipid comprising a polymer that is polyethylene glycol.

25. The pharmaceutical composition of claim 24, wherein the polymer-conjugated lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, PEG-S-DMG, PEG- cer, a PEG dialkoxypropylcarbamate (e.g., ω-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy )pr opy 1) carbamate or 2, 3 -di(tetradecanoxy)propy 1-N - (ω- methoxy(polyethoxy)ethyl)carbamate), and combinations thereof.

26. The pharmaceutical composition of any one of claims 19 to 25, further comprising a steroid.

27. The pharmaceutical composition of claim 26, wherein the steroid is a sterol.

28. The pharmaceutical composition of claim 27, wherein the sterol is cholesterol.

29. The pharmaceutical composition of any one of claims 19-28, further comprising an anionic amphiphile.

30. The pharmaceutical composition of claim 29, wherein the anionic amphiphile is selected from DMGS, DPGS, and DMGS.

31. The pharmaceutical composition of any one of claims 19 to 22, and 26 to 29, wherein the pharmaceutical composition comprises a neutral lipid, a steroid, and an anionic amphiphile, and the pharmaceutical composition does not include a polymer-conjugated lipid.

32. The pharmaceutical composition of any one of claims 18-31, wherein the nucleic acid is RNA.

33. The pharmaceutical composition of claim 32, wherein the RNA is modRNA, saRNA, taRNA, or uRNA.

34. The pharmaceutical composition of any of claims 18-31, wherein the nucleic acid is DNA.

35. The pharmaceutical composition of any one of claims 18-31, wherein the nucleic acid is a mixture of RNA and DNA.

36. The pharmaceutical composition of any one of claims 19-35, wherein the pharmaceutical composition comprises: about 30 to about 60 mol% of the compound; about 20 to about 60 mol% of the steroid; about 1 to about 2 mol% of the polymer-conjugated lipid; and about 5 to about 20 mol% of the neutral lipid.

37. The pharmaceutical composition of any one of claims 19-35, wherein the pharmaceutical composition comprises: about 30 to about 60 mol% of the compound; about 20 to about 60 mol% of the steroid; about 0.5 to about 10 mol% of the anionic amphiphile; and about 5 to about 20 mol% of the neutral lipid.

38. The pharmaceutical composition of claim 37, wherein the pharmaceutical composition does not include a polymer-conjugated lipid.

39. A suspension comprising a dispersed phase and an aqueous phase, and wherein the dispersed phase comprises the pharmaceutical composition of any one of claims 18-38 in the form of particles.

40. The suspension of claim 39, wherein the particles have an average diameter of between about 50 nm to about 200 nm.

41. The suspension of claim 40, wherein the aqueous phase comprises less than 20% of the total concentration of nucleic acid in the suspension.

42. The suspension of any one of claims 39-41, wherein the aqueous phase is substantially free of the nucleic acid.

43. A method of treating a disease, disorder, or condition comprising administering to a subject the suspension of any one of claims 39-42.

44. The method of claim 43, wherein the disease, disorder, or condition is selected from an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease.

45. A method of increasing or causing increased expression of RNA in a target in a subject comprising administering to the subject the suspension of any one of claims 39-42.

46. The method of claim 45, wherein the target is selected from the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas.

47. The method of any one of claims 43-46, wherein the suspension is administered intramuscularly, intranasally, intravenously, subcutaneously, or intratumorally.

48. A method of preparing a compound of formula III:or a pharmaceutically acceptable salt thereof, the method comprising contacting a compound of formula IV- 1 with a compound of formula IV-2 in the presence of heat:to provide a first intermediate product, and wherein the first intermediate product is contacted with a platinum catalyst and a first acid in the presence of hydrogen gas to provide a second intermediate product, and wherein the second intermediate product is contacted with a second acid to provide the compound of formula III, whereinM1and M2are independently selected from a bond or optionally substituted C2-C10aliphatic;L1and L2are each independently selected from a bond, -OC(O)- and -C(O)O-;T1' and T2are each independently selected from optionally substituted C2-C20aliphatic;L3is optionally substituted C1-C6aliphatic;LG1is a suitable leaving group; andPG is a suitable alcohol protecting group.

49. The method of claim 48, wherein a compound of formula IV-2 is prepared by contacting a compound of formula V-la and formula V-lb with a compound of formula V-2in the presence of a palladium coupling catalyst, a copper(I) cocatalyst, and an amine base to provide the compound of formula IV-2, wherein LG2is a suitable leaving group.

50. A method of preparing a compound of formula III- 1 :or a pharmaceutically acceptable salt thereof, the method comprising contacting a compound of formula IV- 1 with a compound of formula IV-3 in the presence of heat:to provide a first intermediate product, and wherein the first intermediate product is contacted with an acid to provide the compound of formula III- 1 , whereinM1and M2are each independently selected from a bond or optionally substituted C2-C10aliphatic;T1and T2are each independently selected from optionally substituted C2-C20aliphatic;L3is optionally substituted C1-C6aliphatic;LG1is a suitable leaving group; andPG is a suitable alcohol protecting group.

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