Lipid Compounds and Lipid Nanoparticle Compositions

Abstract

Provided herein are lipid compounds that can be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer-conjugated lipids, to form lipid nanoparticles for delivery of therapeutic agents (e.g., nucleic acid molecules) for therapeutic or prophylactic purposes, including vaccination. Also provided herein are lipid nanoparticle compositions comprising the lipid compounds.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2022 / 072694, filed January 19, 2022, and International Patent Application No. PCT / CN2022 / 116960, filed September 5, 2022, which are incorporated herein by reference in their entireties.

[0002] 2.Technical Field The present disclosure generally relates to lipid compounds that can be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer-conjugated lipids, to form lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acid molecules, including nucleic acid mimetics such as locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos) both in vitro and in vivo for therapeutic or prophylactic purposes, including vaccination. [Background technology]

[0003] 3.Background technology Therapeutic nucleic acids have the potential to revolutionize vaccination, gene therapy, protein replacement therapy, and other treatments for genetic diseases. Since the first clinical studies on therapeutic nucleic acids began in the 2000s, significant progress has been made in the design of nucleic acid molecules and their delivery methods. However, nucleic acid therapeutics still face several challenges, including poor cell permeability and the high susceptibility of certain nucleic acid molecules, including RNA, to degradation. Therefore, there is a need to develop new nucleic acid molecules and related methods and compositions that facilitate their delivery in vitro or in vivo for therapeutic and / or prophylactic purposes. Lipid compounds that can be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer-conjugated lipids, to form lipid nanoparticles for the delivery of therapeutic agents. There is a need to develop new lipid compounds (e.g., cationic lipid compounds) that provide efficient delivery of therapeutic agents, sufficient activity of the therapeutic agent (e.g., mRNA expression after delivery), optimal pharmacokinetics, and / or other favorable physiological, biological, and / or therapeutic properties. Summary of the Invention

[0004] 4. Summary of the Invention In one embodiment, provided herein are lipid compounds comprising pharmaceutically acceptable salts, prodrugs, or stereoisomers thereof that can be used alone or in combination with other lipid components and / or polymers, such as neutral lipids, charged lipids, steroids (including, for example, all sterols) and / or their analogs, and / or polymer-conjugated lipids, to form lipid nanoparticles for delivery of therapeutic agents (e.g., nucleic acid molecules, including nucleic acid mimetics such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos). In some examples, the lipid nanoparticles are used to deliver nucleic acids, such as antisense RNA and / or messenger RNA. Methods for the use of such lipid nanoparticles for the treatment of various diseases or conditions, such as those caused by infectious agents and / or protein deficiencies, are also provided.

[0005] In one embodiment, provided herein is a compound of formula (I): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein G 1 , G 2 , G 3 , L 1 , L 2 , and R 3 is as defined herein and elsewhere.

[0006] In one embodiment, provided herein is a compound of formula (II): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein G 1 , G 2 , G 3 , L 1 , L 2 , and R 3 is as defined herein and elsewhere.

[0007] In one embodiment, provided herein is a nanoparticle composition comprising a compound provided herein and a therapeutic or prophylactic agent, in one embodiment, the therapeutic or prophylactic agent comprises at least one mRNA encoding an antigen, or a fragment or epitope thereof.

[0008] Further features of the present disclosure will become apparent to those skilled in the art upon consideration of the following detailed description of certain embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0009] 5. MODE FOR CARRYING OUT THE INVENTION 5.1 General techniques The techniques and procedures described or referenced herein generally include those understood and / or commonly used by those of skill in the art using conventional methodology, such as the widely used methodology described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (Ausubel et al. eds., 2003).

[0010] 5.2 Terminology Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For purposes of interpreting this specification, the following explanations of terms shall apply, and where appropriate, terms used in the singular shall also include the plural, and vice versa. All patents, applications, published applications and other publications are incorporated herein by reference. In the event that the explanation of any term set forth contradicts any document incorporated herein by reference, the explanation of the term set forth below shall prevail.

[0011] As used herein, unless otherwise specified, the term "lipid" refers to a group of organic compounds, including but not limited to, esters of fatty acids, and is generally characterized by being poorly soluble in water but soluble in many non-polar organic solvents. Lipids are generally poorly soluble in water, although there are specific categories of lipids (e.g., lipids modified with polar groups, e.g., DMG-PEG2000) that have limited water solubility and can be dissolved in water under certain conditions. Known types of lipids include fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and biomolecules such as phospholipids. Lipids can be divided into at least three classes: (1) "simple lipids," including fats and oils and waxes; (2) "complex lipids," including phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) "derived lipids," such as steroids. Furthermore, as used herein, lipids also encompass lipidoid compounds. The term "lipidoid compound," or simply "lipidoid," refers to a lipid-like compound (eg, an amphiphilic compound with lipid-like physical properties).

[0012] The term "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension on the nanometer (nm) order (e.g., 1-1,000 nm) that contains one or more lipid molecules. The LNPs provided herein may further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNPs comprise a non-lipid payload molecule that is either partially or completely encapsulated inside the lipid shell. In particular, in some embodiments, the payload is a negatively charged molecule (e.g., mRNA encoding a viral protein), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is believed that the cationic lipid can interact with the negatively charged payload molecule and facilitate the incorporation and / or encapsulation of the payload into the LNP during LNP formation. Other lipids that may form part of the LNPs provided herein include, but are not limited to, neutral and charged lipids such as steroids, polymer-conjugated lipids, and various zwitterionic lipids. In certain embodiments, LNPs according to the present disclosure comprise one or more lipids of formula (I) or (II) (and subformulas thereof) described herein.

[0013] The term "cationic lipid" refers to a lipid that is either positively charged at any pH value or hydrogen ion activity in its environment, or that can become positively charged in response to the pH value or hydrogen ion activity in its environment (e.g., the environment of its intended use). Thus, the term "cationic" encompasses both "permanently cationic" and "cationizable." In certain embodiments, the positive charge in a cationic lipid arises from the presence of a quaternary nitrogen atom. In certain embodiments, a cationic lipid includes a zwitterionic lipid that is positively charged in its intended use environment (e.g., at physiological pH). In certain embodiments, the cationic lipid is one or more lipids of formula (I) or (II) (and subformulas thereof) described herein.

[0014] The term "polymer-conjugated lipid" refers to a molecule that includes both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a pegylated lipid (PEG-lipid), in which the polymer portion includes polyethylene glycol.

[0015] The term "neutral lipid" encompasses any lipid molecule that exists in an uncharged or neutral zwitterionic form at a selected pH value or within a selected pH range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment of the lipid's intended use, such as physiological pH. By way of non-limiting example, neutral lipids that may be used in connection with the present disclosure include, but are not limited to, phosphotidylcholines such as 1,2-distearoyl-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), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP), sphingomyelin (SM), steroids such as ceramides, sterols, and derivatives thereof. The neutral lipids provided herein can be synthetic or derived (isolated or modified) from natural sources or compounds.

[0016] The term "charged lipid" encompasses any lipid molecule that exists in either a positively or negatively charged form at a selected pH or within a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH conditions in the environment of the lipid's intended use, such as physiological pH. Non-limiting examples of charged lipids that can be used in connection with the present disclosure include, but are not limited to, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DOPG-Na), and 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPA-Na). The charged lipids provided herein can be synthesized or derived (isolated or modified) from natural sources or compounds.

[0017] As used herein, unless otherwise specified, the term "alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of saturated carbon and hydrogen atoms. In one embodiment, an alkyl group is, for example, a group having 1 to 24 carbon atoms (C1-C 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (C1-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl), which are attached to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, alkyl groups are optionally substituted.

[0018] As used herein, unless otherwise specified, the term "alkenyl" refers to a straight or branched hydrocarbon chain radical, composed solely of carbon and hydrogen atoms, containing one or more carbon-carbon double bonds. The term "alkenyl" also encompasses radicals having "cis" and "trans" configurations, or alternatively, "E" and "Z" configurations, as understood by one of ordinary skill in the art. In one embodiment, an alkyl group is an alkyl group having, for example, 2 to 24 carbon atoms (C2-C6). 24 Alkenyl, 4 to 20 carbon atoms (C4-C 20 Alkenyl, 6 to 16 carbon atoms (C6-C 16 alkenyl), 6 to 9 carbon atoms (C6-C9 alkenyl), 2 to 15 carbon atoms (C2-C 15 alkenyl), 2 to 12 carbon atoms (C2-C 12 Alkenyl groups have from 2 to 8 carbon atoms (C-C alkenyl), 2 to 8 carbon atoms (C-C alkenyl), or 2 to 6 carbon atoms (C-C alkenyl), which are attached to the rest of the molecule by a single bond. Examples of alkenyl groups include, but are not limited to, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like. Unless otherwise specified, alkenyl groups are optionally substituted.

[0019] As used herein, unless otherwise specified, the term "alkynyl" refers to a straight or branched hydrocarbon chain radical, consisting solely of carbon and hydrogen atoms, containing one or more carbon-carbon triple bonds. In one embodiment, an alkyl group is an alkyl group having, for example, 2 to 24 carbon atoms (C2-C6). 24 Alkynyl, 4 to 20 carbon atoms (C4-C 20 Alkynyl, 6 to 16 carbon atoms (C6-C 16 alkynyl), 6 to 9 carbon atoms (C6-C9 alkynyl), 2 to 15 carbon atoms (C2-C 15 Alkynyl), 2 to 12 carbon atoms (C2-C 12 C-C alkynyl), 2 to 8 carbon atoms (C-C alkynyl), or 2 to 6 carbon atoms (C-C alkynyl), which are attached to the rest of the molecule by a single bond. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise specified, alkynyl groups are optionally substituted.

[0020] As used herein, unless otherwise specified, the term "alkylene" or "alkylene chain" refers to a linear or branched polyvalent (e.g., divalent or trivalent) hydrocarbon chain that is saturated and consists solely of carbon and hydrogen atoms, and that connects the remainder of the molecule to a radical group (or groups). In one embodiment, alkylene refers to, for example, an alkylene having 1 to 24 carbon atoms (C1-C6). 24 Alkylene, 1 to 15 carbon atoms (C1-C 15 Alkylene, 1 to 12 carbon atoms (C1-C 12Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group(s) can be through one carbon or any two (or more) carbons within the chain. Unless otherwise specified, alkylene chains are optionally substituted.

[0021] As used herein, unless otherwise specified, the term "alkenylene" refers to a linear or branched polyvalent (e.g., divalent or trivalent) hydrocarbon chain, consisting solely of carbon and hydrogen atoms, containing one or more carbon-carbon double bonds and connecting the remainder of the molecule to a radical group(s). In one embodiment, alkylene refers to an alkylene having, for example, 2 to 24 carbon atoms (C-C 24 Alkenylene, 2 to 15 carbon atoms (C2-C 15 Alkenylene, 2 to 12 carbon atoms (C2-C 12 Alkenylenes have 2 to 8 carbon atoms (C2-C8 alkenylene), 2 to 6 carbon atoms (C2-C6 alkenylene), or 2 to 4 carbon atoms (C2-C4 alkenylene). Examples of alkenylene include, but are not limited to, ethenylene, propenylene, n-butenylene, and the like. An alkenylene is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkenylene to the rest of the molecule and to the radical group(s) can be through one carbon or any two (or more) carbons within the chain. Unless otherwise specified, alkenylene is optionally substituted.

[0022] As used herein, unless otherwise specified, the term "cycloalkyl" refers to a saturated, non-aromatic, monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms. Cycloalkyl groups can include fused or bridged ring systems. In one embodiment, cycloalkyl refers to a group having, for example, 3 to 15 ring carbon atoms (C3-C4). 15 Cycloalkyl), 3 to 10 ring carbon atoms (C3-C 10 Cycloalkyl radicals have from 1 to 8 ring carbon atoms (C3-C8 cycloalkyl). The cycloalkyl is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl radicals include, but are not limited to, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, bicyclo[1.1.1]pentyl, and the like. Unless otherwise specified, cycloalkyl groups are optionally substituted.

[0023] As used herein, unless otherwise specified, the term "cycloalkylene" refers to a polyvalent (e.g., divalent or trivalent) cycloalkyl group. Unless otherwise specified, a cycloalkylene group is optionally substituted.

[0024] As used herein, unless otherwise specified, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms and containing one or more carbon-carbon double bonds. Cycloalkenyls can include fused or bridged ring systems. In one embodiment, cycloalkyls are substituted or unsubstituted cycloalkenyls, e.g., cycloalkenyls having from 3 to 15 ring carbon atoms (C3-C4). 15 Cycloalkenyl), 3 to 10 ring carbon atoms (C3-C 10Cycloalkenyls have from 1 to 8 ring carbon atoms (C3-C8 cycloalkenyl), or from 3 to 8 ring carbon atoms (C3-C8 cycloalkenyl). The cycloalkenyl is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkenyl radicals include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Unless otherwise specified, cycloalkenyl groups are optionally substituted.

[0025] As used herein, unless otherwise specified, the term "cycloalkenylene" refers to a polyvalent (e.g., divalent or trivalent) cycloalkenylene group. Unless otherwise specified, a cycloalkenylene group is optionally substituted.

[0026] As used herein, unless otherwise specified, the term "heterocyclyl" refers to a non-aromatic radical monocyclic or polycyclic moiety containing one or more (e.g., 1, 1 or 2, 1 to 3, or 1 to 4) heteroatoms independently selected from nitrogen, oxygen, phosphorus, and sulfur. The heterocyclyl can be attached to the main structure at any heteroatom or carbon atom. Heterocyclyl groups can be monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic ring systems, and polycyclic ring systems can be fused, bridged, or spirocyclic ring systems. Heterocyclyl polycyclic ring systems can contain one or more heteroatoms in one or more rings. Heterocyclyl groups can be saturated or partially unsaturated. Saturated heterocycloalkyl groups can be referred to as "heterocycloalkyl." Partially unsaturated heterocycloalkyl groups can be referred to as "heterocycloalkenyls" if the heterocyclyl contains at least one double bond, or "heterocycloalkynyls" if the heterocyclyl contains at least one triple bond. In one embodiment, the heterocyclyl has, for example, 3 to 18 ring atoms (3- to 18-membered heterocyclyls), 4 to 18 ring atoms (4- to 18-membered heterocyclyls), 5 to 18 ring atoms (5- to 18-membered heterocyclyls), 4 to 8 ring atoms (4- to 8-membered heterocyclyls), or 5 to 8 ring atoms (5- to 8-membered heterocyclyls). Whenever it appears herein, a numerical range such as "3 to 18" refers to each integer within the given range; for example, "3 to 18 ring atoms" means that the heterocyclyl group can contain 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, etc., up to a maximum of 18 ring atoms. Examples of heterocyclyl groups include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl. Unless otherwise specified, a heterocyclyl group is optionally substituted.

[0027] As used herein, unless otherwise specified, the term "heterocyclylene" refers to a polyvalent (e.g., divalent or trivalent) heterocyclyl group. Unless otherwise specified, a heterocyclylene group is optionally substituted.

[0028] As used herein, unless otherwise specified, the term "aryl" refers to a monocyclic aromatic group and / or a polycyclic monovalent aromatic group containing at least one aromatic hydrocarbon ring. In certain embodiments, aryl refers to a ring of 6 to 18 ring carbon atoms (C-C). 18 aryl), 6 to 14 ring carbon atoms (C6-C 14 aryl), or 6 to 10 ring carbon atoms (C6-C 10 aryl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. The term "aryl" also refers to bicyclic, tricyclic, or other polycyclic hydrocarbon rings in which at least one ring is aromatic and the others can be saturated, partially unsaturated, or aromatic, such as dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl). Unless otherwise specified, aryl groups are optionally substituted.

[0029] As used herein, unless otherwise specified, the term "arylene" refers to a polyvalent (e.g., divalent or trivalent) aryl group. Unless otherwise specified, an arylene group is optionally substituted.

[0030] As used herein, unless otherwise specified, the term "heteroaryl" refers to monocyclic and / or polycyclic aromatic groups containing at least one aromatic ring, wherein the at least one aromatic ring contains one or more (e.g., 1, 1 or 2, 1 to 3, or 1 to 4) heteroatoms independently selected from O, S, and N. A heteroaryl can be attached to the main structure at any heteroatom or carbon atom. In certain embodiments, a heteroaryl has 5 to 20, 5 to 15, or 5 to 10 ring atoms. The term "heteroaryl" also refers to bicyclic, tricyclic, or other polycyclic rings, wherein at least one of the rings is aromatic and the others can be saturated, partially unsaturated, or aromatic, and wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, and xanthenyl. Unless otherwise specified, heteroaryl groups are optionally substituted.

[0031] As used herein, unless otherwise specified, the term "heteroarylene" refers to a polyvalent (e.g., divalent or trivalent) heteroaryl group. Unless otherwise specified, a heteroarylene group is optionally substituted.

[0032] When groups described herein are referred to as "substituted," they may be substituted with any suitable substituent or substituents. Illustrative examples of substituents include those found in the exemplary compounds and embodiments provided herein, as well as halogen atoms such as F, Cl, Br, or I, cyano, oxo (=O), hydroxyl (-OH), alkyl, alkenyl, alkynyl, cycloalkyl, aryl, -(C=O)OR', -O(C=O)R', -C(=O)R', -OR', -S(O) x R', -S-SR', -C(=O)SR', -SC(=O)R', -NR'R', -NR'C(=O)R', -C(=O)NR'R', -NR'C(=O)NR'R', -OC(=O)NR'R', -NR'C(=O)OR', -NR'S(O) x NR'R', -NR'S(O) x R', and -S(O) x Examples include, but are not limited to, NR'R', where R', in each occurrence, is independently H, C-C 15 alkyl, or cycloalkyl, and x is 0, 1, or 2. In some embodiments, the substituent is C-C 12 In another embodiment, the substituent is an alkyl group. In another embodiment, the substituent is a cycloalkyl group. In another embodiment, the substituent is a halo group, such as fluoro. In another embodiment, the substituent is an oxo group. In another embodiment, the substituent is a hydroxyl group. In another embodiment, the substituent is an alkoxy group (-OR'). In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amino group (-NR'R').

[0033] As used herein, unless otherwise specified, the term "optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and that the description includes both substituted alkyl radicals and alkyl radicals that have no substitution.

[0034] As used herein, unless otherwise specified, the term "prodrug" of a biologically active compound refers to a compound that can be converted into a biologically active compound under physiological conditions or by dissolution. In one embodiment, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a biologically active compound. A prodrug may be inactive when administered to a subject in need thereof, but is converted into a biologically active compound in vivo. Prodrugs are typically rapidly converted in vivo to the original biologically active compound, for example, by hydrolysis in blood. Prodrug compounds often offer advantages of solubility, tissue compatibility, or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0035] In one embodiment, the term "prodrug" is also meant to include any covalently bonded carrier that releases an active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound can be prepared by modifying functional groups present in the compound in such a way that the modification is cleaved to the original compound, either in routine manipulation or in vivo. Prodrugs include compounds in which a hydroxyl group, an amino group, or a mercapto group is bonded to any group that cleaves to form a free hydroxyl group, a free amino group, or a free mercapto group, respectively, when a prodrug of the compound is administered to a mammalian subject.

[0036] Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol or amide derivatives of amine functional groups in the compounds provided herein.

[0037] As used herein, unless otherwise specified, the term "pharmaceutically acceptable salts" includes both acid and base addition salts.

[0038] Examples of pharmaceutically acceptable acid addition salts include those with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as, but not limited to, those with acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamine acid, and the like. Examples of organic acids include glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0039] Examples of pharmaceutically acceptable base addition salts include, but are not limited to, salts prepared from the addition of an inorganic or organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. In one embodiment, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, including ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins. In one embodiment, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0040] The compounds provided herein may contain one or more asymmetric centers and may therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or for amino acids, as (D)- or (L)-. Unless otherwise specified, the compounds provided herein are meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or racemate of a salt or derivative), using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0041] As used herein, unless otherwise specified, the term "isomer" refers to different compounds that have the same molecular formula. "Stereoisomers" are isomers that differ only in the way their atoms are arranged in space. "Atropisomers" are stereoisomers that result from hindrance of rotation about a single bond. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any proportions may be known as a "racemic" mixture. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0042] "Stereoisomer" can also include E and Z isomers, or mixtures thereof, as well as cis and trans isomers, or mixtures thereof. In certain embodiments, the compounds described herein are isolated as either the E or Z isomer. In other embodiments, the compounds described herein are a mixture of E and Z isomers.

[0043] "Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The concentrations of isomeric forms depend on the environment in which the compound is found, and can vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution.

[0044] It should also be noted that the compounds described herein may contain unnatural proportions of atomic isotopes at one or more of the atoms. For example, the compounds may contain, for example, tritium ( 3 H), iodine-125( 125 I), sulfur 35( 35 S), or carbon-14 ( 14 It can be radiolabeled with a radioisotope such as C, or deuterium ( 2 H), carbon-13 ( 13 C), or nitrogen-15( 15The term "isotopic species" as used herein refers to an isotopically enriched compound. The term "isotopically enriched" refers to an atom having an isotopic composition other than the natural isotopic composition of that atom. "Isotopically enriched" can also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom. The term "isotopic composition" refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, e.g., cancer therapeutic agents, research reagents, e.g., binding assay reagents, and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of the compounds described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, isotopic species of the compounds described herein are provided, e.g., isotopic species enriched in deuterium, carbon-13, and / or nitrogen-15. As used herein, "deuterated" means that at least one hydrogen (H) has been replaced with a deuterium (D or 2 H), meaning that the compound is enriched with deuterium at at least one position.

[0045] It should be noted that where there is a discrepancy between a depicted structure and the name for that structure, the depicted structure is given weight.

[0046] As used herein, unless otherwise specified, the term "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the United States Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0047] The term "composition" is intended to encompass products having specified components (e.g., mRNA molecules provided herein), optionally in specified amounts.

[0048] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymers of nucleotides of any length, including, for example, DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and their analogs. Nucleic acids can be in either single-stranded or double-stranded form. As used herein, unless otherwise specified, "nucleic acid" also includes nucleic acid mimetics such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos. "Oligonucleotides," as used herein, generally, but not necessarily, refer to short, synthetic polynucleotides that are less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is referred to as the transcription direction, and the region of sequence on the DNA strand that is at the 5' to 5' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "upstream sequence," and the region of sequence on the DNA strand that is at the 3' to 3' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "downstream sequence."

[0049] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the natural sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules present in the nucleic acid molecule's natural source. Furthermore, an "isolated" nucleic acid molecule, such as mRNA, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In certain embodiments, one or more nucleic acid molecules encoding the antigens described herein are isolated or purified. The term encompasses nucleic acid sequences removed from their naturally occurring environment, including recombinant or cloned DNA or RNA isolates, as well as chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule.

[0050] When used in reference to a nucleic acid molecule, the term "encoding nucleic acid" or its grammatical equivalents includes (a) a nucleic acid molecule that can be transcribed, in its natural state or when manipulated by methods well known to those skilled in the art, to produce mRNA that is then translated into a peptide and / or polypeptide, as well as (b) the mRNA molecule itself. The antisense strand is the complement of such a nucleic acid molecule, from which a coding sequence can be derived. The term "coding region" refers to the portion within a coding nucleic acid sequence that is translated into a peptide or polypeptide. The term "untranslated region" or "UTR" refers to the portion of a coding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of the UTR with respect to the coding region of a nucleic acid molecule, a UTR is referred to as a 5'-UTR if it is located at the 5' end of the coding region, and a 3'-UTR if it is located at the 3' end of the coding region.

[0051] As used herein, the term "mRNA" refers to a message RNA molecule that contains one or more open reading frames (ORFs) that can be translated by the cell or organism that contains the mRNA to produce one or more peptide or protein products. The region that contains one or more ORFs is referred to as the coding region of the mRNA molecule. In some embodiments, the mRNA molecule further contains one or more untranslated regions (UTRs).

[0052] In certain embodiments, the mRNA is a monocistronic mRNA containing only one ORF. In certain embodiments, the monocistronic mRNA encodes a peptide or protein containing at least one epitope of a selected antigen (e.g., a pathogenic antigen or a tumor-associated antigen). In other embodiments, the mRNA is a multicistronic mRNA containing two or more ORFs. In certain embodiments, the multicistronic mRNA encodes two or more peptides or proteins that may be the same or different from one another. In certain embodiments, each peptide or protein encoded by the multicistronic mRNA contains at least one epitope of a selected antigen. In certain embodiments, different peptides or proteins encoded by the multicistronic mRNA each contain at least one epitope of a different antigen. In any of the embodiments described herein, the at least one epitope can be at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten epitopes of an antigen.

[0053] The term "nucleobase" encompasses purines and pyrimidines, including the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and their natural or synthetic analogues or derivatives.

[0054] As used herein, the term "functional nucleotide analog" refers to a modified version of the standard nucleotides A, G, C, U, or T that (a) retains the base-pairing properties of the corresponding standard nucleotide and (b) contains at least one chemical modification to (i) the nucleobase, (ii) the sugar group, (iii) the phosphate group, or (iv) any combination of (i)-(iii) of the corresponding natural nucleotide. As used herein, base pairing encompasses not only standard Watson-Crick adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between a standard nucleotide and a functional nucleotide analog or between a pair of functional nucleotide analogs, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between a modified nucleobase and a standard nucleobase or between two complementary nucleobase structures. For example, a functional analog of guanosine (G) retains the ability to base pair with cytosine (C) or a functional analog of cytosine. An example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. As described herein, functional nucleotide analogs can be either naturally occurring or non-naturally occurring. Thus, nucleic acid molecules containing functional nucleotide analogs can have at least one modified nucleobase, sugar group, and / or internucleoside linkage. Exemplary chemical modifications to the nucleobase, sugar group, or internucleoside linkage of nucleic acid molecules are provided herein.

[0055] As used herein, the terms "translation enhancer element," "TEE," and "translation enhancer" refer to a region within a nucleic acid molecule that functions to promote translation of a coding sequence of the nucleic acid into a protein or peptide product, e.g., via cap-dependent or cap-independent translation. TEEs are typically located in the UTR region of a nucleic acid molecule (e.g., mRNA) and enhance the level of translation of a coding sequence located either upstream or downstream. For example, a TEE in the 5' UTR of a nucleic acid molecule can be located between the promoter and start codon of the nucleic acid molecule. Various TEE sequences are known in the art (Wellensiek et al., Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug;10(8):747-750; Chappell et al., PNAS June 29, 2004 101(26)9590-9594). Some TEEs are known to be conserved across multiple species (Panek et al. Nucleic Acids Research, Volume 41, Issue 16, 1 September 2013, Pages 7625-7634).

[0056] As used herein, the term "stem-loop sequence" refers to a single-stranded polynucleotide sequence having at least two regions that are complementary or substantially complementary to each other when read in opposite directions, and are therefore capable of base-pairing with each other to form at least one double helix and an unpaired loop. The resulting structure is known as a stem-loop structure, hairpin, or hairpin loop, which are secondary structures found in many RNA molecules.

[0057] As used herein, the term "peptide" refers to a polymer containing 2 to 50 amino acid residues linked by one or more covalent peptide bond(s). The term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs or unnatural amino acids).

[0058] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of more than 50 amino acid residues linked by covalent peptide bonds. That is, a description of a polypeptide applies equally to a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers and to amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). As used herein, the terms encompass amino acid chains of any length, including full-length proteins (e.g., antigens).

[0059] The term "antigen" refers to a substance that can be recognized by a subject's immune system (including the adaptive immune system) and that can elicit an immune response (including an antigen-specific immune response) after a subject comes into contact with the antigen. In certain embodiments, an antigen is a protein associated with a diseased cell, such as a cell infected by a pathogen or a neoplastic cell (e.g., a tumor-associated antigen (TAA)).

[0060] The term "fragment," as used herein in the context of a peptide or polypeptide, refers to a peptide or polypeptide that comprises less than the full-length amino acid sequence. Such fragments can result, for example, from truncation at the amino terminus, truncation at the carboxy terminus, and / or internal deletion of a residue(s) from the amino acid sequence. Fragments can result, for example, from alternative RNA splicing or in vivo protease activity. In certain embodiments, a fragment comprises at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 10 ...0 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 100 contiguous amino acid residues, "Fragment" refers to a polypeptide comprising an amino acid sequence of contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, or at least 950 contiguous amino acid residues. In certain embodiments, a fragment of a polypeptide retains at least one, at least two, at least three, or more functions of the polypeptide.

[0061] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds, such as a site on the surface of an antigen that has antigenic or immunogenic activity in an animal, such as a mammal (e.g., a human), that can bind to one or more antigen-binding regions of an antibody and elicit an immune response. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide to which an antibody binds, as determined by any method known in the art, including, for example, immunoassays. An antigenic epitope is not necessarily immunogenic. Epitopes often consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a contiguous sequence of amino acids in a protein. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure. An induced epitope is formed when the three-dimensional structure of a protein is in an altered conformation, such as following activation or binding of another protein or ligand. In certain embodiments, an epitope is a three-dimensional surface feature of a polypeptide. In other embodiments, an epitope is a linear feature of a polypeptide. Generally, an antigen may have several or many different epitopes and react with many different antibodies.

[0062] As used herein, the term "genetic vaccine" refers to a therapeutic or prophylactic composition comprising at least one nucleic acid molecule encoding an antigen associated with a target disease (e.g., an infectious disease or a neoplastic disease). Administration of the vaccine to a subject ("vaccination") allows for the production of the encoded peptide or protein, thereby eliciting an immune response against the target disease in the subject. In certain embodiments, the immune response includes an adaptive immune response, such as the production of antibodies against the encoded antigen and / or the activation and proliferation of immune cells capable of specifically eliminating disease cells expressing the antigen. In certain embodiments, the immune response further includes an innate immune response. According to the present disclosure, a vaccine can be administered to a subject either before or after the onset of clinical symptoms of the target disease. In some embodiments, vaccination of a healthy or asymptomatic subject immunizes or reduces the susceptibility of the vaccinated subject to the development of the target disease. In some embodiments, vaccination of a subject exhibiting symptoms of the disease ameliorates or treats the disease state in the vaccinated subject.

[0063] The terms "innate immune response" and "innate immunity" are art-recognized and refer to nonspecific defense mechanisms initiated by the body's immune system upon recognition of pathogen-associated molecular patterns, involving different forms of cellular activity, including cytokine production and cell death via various pathways. As used herein, innate immune responses include, but are not limited to, increased production of inflammatory cytokines (e.g., type I interferon or IL-10 production), activation of the NFκB pathway, increased proliferation, maturation, differentiation, and / or survival of immune cells, and, in some cases, induction of cellular apoptosis. Activation of innate immunity can be detected using methods known in the art, such as measuring (NF)-κB activation.

[0064] The terms "adaptive immune response" and "adaptive immunity" are art-recognized and refer to antigen-specific defense mechanisms initiated by the body's immune system upon recognition of a particular antigen, including both humoral and cell-mediated responses. As used herein, an adaptive immune response includes a cellular response elicited and / or augmented by a vaccine composition, such as the genetic compositions described herein. In some embodiments, a vaccine composition comprises an antigen that is the target of an antigen-specific adaptive immune response. In other embodiments, a vaccine composition, upon administration, enables the production in an immunized subject of an antigen that is the target of an antigen-specific adaptive immune response. Activation of an adaptive immune response can be detected using methods known in the art, such as measuring the level of antigen-specific antibody production or antigen-specific cell-mediated cytotoxicity.

[0065] The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides capable of binding to a specific molecular antigen and consisting of two identical pairs of polypeptide chains, each pair having one heavy chain (approximately 50-70 kDa) and one light chain (approximately 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 amino acids or more, and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). In certain embodiments, specific molecular antigens can be bound by the antibodies provided herein, including polypeptides, fragments thereof, or epitopes. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above, where a functional fragment refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments include single-chain Fvs (scFvs) (e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules containing an antigen-binding domain or site (e.g., one or more CDRs of an antibody).Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0066] The term "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance (e.g., a lipid nanoparticle composition described herein) to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease, disorder, condition, or symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease, disorder, condition, or symptom thereof. When a disease, disorder, condition, or symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease, disorder, condition, or symptom thereof.

[0067] "Chronic" administration refers to the administration of an agent(s) in a continuous mode (e.g., over a period of days, weeks, months, or years) to maintain the initial therapeutic effect (activity) over an extended period, as opposed to an acute mode. "Intermittent" administration is cyclical rather than continuous without interruption.

[0068] As used herein, the term "targeted delivery" or the verb form "target" refers to a process that facilitates the delivery of a delivery agent (such as a therapeutic payload molecule in a lipid nanoparticle composition described herein) to a specific organ, tissue, cell, and / or subcellular component (referred to as a targeted location) rather than any other organ, tissue, cell, or subcellular component (referred to as a non-targeted location). Targeted delivery can be detected using methods known in the art, for example, by comparing the concentration of the delivery agent in a targeted cell population after systemic administration with the concentration of the delivery agent in a non-targeted cell population. In certain embodiments, targeted delivery results in at least a two-fold higher concentration in the targeted location compared to non-targeted locations.

[0069] An "effective amount" generally is an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, prevent the occurrence of symptoms and / or their underlying causes, and / or ameliorate or correct damage caused by or associated with a disease, disorder, or condition, including, for example, infections and tumors. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0070] As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., a vaccine composition) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder, or condition, and / or its associated symptoms (e.g., an infectious disease, such as that caused by a viral infection, or a neoplastic disease, such as cancer). A "therapeutically effective amount" of a substance / molecule / agent of the present disclosure (e.g., a lipid nanoparticle composition described herein) may vary according to factors such as the individual's condition, age, sex, and weight, as well as the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the substance / molecule / agent. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a lipid nanoparticle composition described herein or a therapeutic or prophylactic agent (e.g., a therapeutic mRNA) contained therein that is effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0071] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., prevent, delay, or reduce the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., an infectious disease such as caused by a viral infection, or a neoplastic disease such as cancer). Typically, a prophylactic dose is used in a subject prior to or at an early stage of a disease, disorder, or condition, so the prophylactically effective amount may, but need not, be less than the therapeutically effective amount. A complete therapeutic or prophylactic effect does not necessarily occur by administration of a single dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations.

[0072] As used herein, unless otherwise specified, the terms "treat," "treating," and "treatment" refer to the total or partial alleviation of a disorder, disease, or condition, or the total or partial alleviation of one or more symptoms associated with a disorder, disease, or condition, or the slowing or halting of further progression or worsening of these symptoms, or the amelioration or eradication of the cause(s) of the disorder, disease, or condition itself.

[0073] As used herein, unless otherwise specified, the terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., an infectious disease, such as caused by a viral infection, or a neoplastic disease, such as cancer).

[0074] As used herein, unless otherwise specified, the terms "manage," "managing," and "management" refer to the beneficial effects a subject derives from a therapy (e.g., a prophylactic or therapeutic agent) that does not result in a cure of the disease. In certain embodiments, a subject is administered one or more therapies (e.g., a prophylactic or therapeutic agent, such as a lipid nanoparticle composition described herein) to prevent progression or worsening of the disease, thereby "managing" an infectious or neoplastic disease, or one or more symptoms thereof.

[0075] The term "prophylactic agent" refers to any agent that can completely or partially inhibit the onset, recurrence, development, or spread of a disease and / or its associated symptoms in a subject.

[0076] The term "therapeutic agent" refers to any agent that can be used to treat, prevent, or alleviate a disease, disorder, or condition, including treating, preventing, or alleviating one or more symptoms of the disease, disorder, or condition and / or symptoms associated therewith.

[0077] The term "therapy" refers to any protocol, method, and / or agent that can be used in the prevention, management, treatment, and / or amelioration of a disease, disorder, or condition. In certain embodiments, the terms "therapies" and "therapy" refer to biological, supportive, and / or other therapies known to those of skill in the art, such as health care professionals, that are useful in the prevention, management, treatment, and / or amelioration of a disease, disorder, or condition.

[0078] As used herein, a "prophylactically effective serum titer" is a serum titer of antibodies in a subject (e.g., a human) that completely or partially inhibits the onset, recurrence, progression, or spread of a disease, disorder, or condition, and / or symptoms associated therewith, in the subject.

[0079] In certain embodiments, a "therapeutically effective serum titer" is a serum titer of an antibody in a subject (e.g., a human) that reduces the severity, duration, and / or symptoms associated with a disease, disorder, or condition in the subject.

[0080] The term "serum titer" refers to the average serum titer from multiple samples (e.g., at multiple time points) in a subject, or in a population of at least 10, at least 20, at least 40 subjects, up to about 100, 1000, or more.

[0081] The term "side effect" encompasses unwanted and / or adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Unwanted effects are not necessarily harmful. Side effects from a therapy (e.g., a prophylactic or therapeutic agent) can be harmful, uncomfortable, or dangerous. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, hair loss, difficulty breathing, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, loss of appetite, rash or swelling at the site of administration, flu-like symptoms such as fever, chills, fatigue, digestive problems, and allergic reactions. Additional unwanted effects experienced by patients are numerous and known in the art. Many are described in the Physician's Desk Reference (68th ed. 2014).

[0082] The terms "subject" and "patient" may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, a subject is a human. In one embodiment, a subject is a mammal (e.g., a human) with an infectious disease or a neoplastic disease. In another embodiment, a subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0083] The term "detectable probe" refers to a composition that provides a detectable signal. The term includes, but is not limited to, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, etc. that provides a detectable signal through its activity.

[0084] The term "detectable agent" refers to a substance that can be used to confirm the existence or presence of a desired molecule, such as an antigen encoded by an mRNA molecule described herein, in a sample or subject. A detectable agent can be a substance that can be visualized or otherwise determined and / or measured (e.g., by quantification).

[0085] "Substantially all" refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0086] As used herein, unless otherwise indicated, the term "about" or "approximately" refers to the tolerance of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0087] As used herein, the singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0088] All publications, patent applications, accession numbers, and other references cited herein are incorporated by reference in their entirety, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0089] Numerous embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the descriptions in the experimental section and examples are intended to be illustrative, but not limiting, of the scope of the invention as set forth in the claims.

[0090] 5.3 Lipid compounds Unless otherwise specified, the descriptions provided herein apply to all formulas provided herein (e.g., Formula (I) and Formula (II), including subformulas thereof), to the extent applicable.

[0091] In one embodiment, provided herein is a compound of formula (I): [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 are each independently C2-C 25 Alkylene or C2-C 25 alkenylene, where G 1 and G 2 wherein one or more -CH2- are optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L 1is, independently, -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ), -NR a P(=O)(OR b )(OR c ), and each L 2 is, independently, -OC(=O)R 2 , -C(=O)OR 2 , -OC(=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NRd C(=O)OR 2 , -SC(=S)R 2 , -C(=S)SR 2 , -C(=S)R 2 , -CH(OH)R 2 , -P(=O)(OR e )(OR f ), -NR d P(=O)(OR e )(OR f ) and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 alkenyl, where R 1 and R 2 wherein one or more -CH2- are optionally replaced by -SS-; R a , R b , R d , and R e are each independently H, C1-C 24 Alkyl, or C2-C 24 is alkenyl, R c and R f are each independently C1-C 24 Alkyl or C2-C 24 alkenyl, where R c and R f wherein one or more -CH2- are optionally replaced by -SS; G 3 is C2-C 12 Alkylene or C2-C 12 alkenylene, wherein some or all of the alkylene or alkenylene are optionally replaced by C3-C8 cycloalkylene or C3-C8 cycloalkenylene; R 3 is -N(R 4 )R 5 , -OR 6 , or -SR 6 and R 4 is C1-C12 Alkyl, C2-C 12 Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 5 is H, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

[0092] In one embodiment, provided herein is a compound of formula (I): [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene, where G 1 and G 2 wherein one or more -CH2- are optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L 1 are independently -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O)x R 1 、 -S-SR 1 、 -C(=O)SR 1 、 -SC(=O)R 1 、 -NR a C(=O)R 1 、 -C(=O)NR b R c 、 -NR a C(=O)NR b R c 、 -OC(=O)NR b R c 、 -NR a C(=O)OR 1 、 -SC(=S)R 1 、 -C(=S)SR 1 、 -C(=S)R 1 、 -CH(OH)R 1 、 -P(=O)(OR b )(OR c )、 -NR a P(=O)(OR b )(OR c ) and each L 2 is independently -OC(=O)R 2 、 -C(=O)OR 2 、 -OC(=O)OR 2 、 -C(=O)R 2 、 -OR 2 、 -S(O) x R 2 、 -S-SR 2 、 -C(=O)SR 2 、 -SC(=O)R 2 、 -NR d C(=O)R 2 、 -C(=O)NR e R f 、 -NR d C(=O)NR e R f 、 -OC(=O)NR e R f 、 -NR d C(=O)OR 2 、 -SC(=S)R 2 、 -C(=S)SR 2 、 -C(=S)R 2 、 -CH(OH)R 2, -P(=O)(OR e )(OR f ), -NR d P(=O)(OR e )(OR f ) and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 is alkenyl, R a , R b , R d , and R e are each independently H, C1-C 24 Alkyl, or C2-C 24 is alkenyl, R c and R f are each independently C1-C 24 Alkyl or C2-C 24 is alkenyl, G 3 is C2-C 12 Alkylene or C2-C 12 alkenylene, wherein some or all of the alkylene or alkenylene are optionally replaced by C3-C8 cycloalkylene or C3-C8 cycloalkenylene; R 3 is -N(R 4 )R 5 , -OR 6 , or -SR 6 and R 4 is C1-C 12 Alkyl, C2-C 12 Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 5 is H, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 6is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

[0093] In one embodiment, provided herein is a compound of formula (I): [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene, where G 1 and G 2 wherein one or more -CH2- are optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L 1 are independently -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NRa C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ), -NR a P(=O)(OR b )(OR c ) and Each L 2 are independently -OC(=O)R 2 , -C(=O)OR 2 , -OC(=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2 , -SC(=S)R 2 , -C(=S)SR 2 , -C(=S)R 2 , -CH(OH)R 2 , -P(=O)(OR e )(OR f ), -NR d P(=O)(OR e )(OR f ) and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 is alkenyl, R a , R b , R d, and R e are each independently H, C1-C 24 Alkyl, or C2-C 24 is alkenyl, R c and R f are each independently C1-C 24 Alkyl or C2-C 24 is alkenyl, G 3 is C2-C 24 Alkylene, C2-C 24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; R 3 is -N(R 4 )R 5 -OR 6 and R 4 is C1-C 12 Alkyl, C2-C 12 Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 5 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

[0094] In one embodiment, provided herein is a compound of formula (I): [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene, where G 1 and G 2 wherein one or more -CH2- are optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L 1 are independently -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ), -NR a P(=O)(OR b )(OR c ) and Each L 2 are independently -OC(=O)R 2 , -C(=O)OR 2 , -OC(=O)OR 2 , -C(=O)R2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2 , -SC(=S)R 2 , -C(=S)SR 2 , -C(=S)R 2 , -CH(OH)R 2 , -P(=O)(OR e )(OR f ), -NR d P(=O)(OR e )(OR f ) and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 is alkenyl, R a , R b , R d , and R e are each independently H, C1-C 24 Alkyl, or C2-C 24 is alkenyl, R c and R f are each independently C1-C 24 Alkyl or C2-C 24 is alkenyl, G 3 is C2-C 12 Alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; R 3 is -N(R4 )R 5 -OR 6 and R 4 is C1-C 12 Alkyl, C2-C 12 Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 5 is H, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

[0095] In one embodiment, provided herein is a compound of formula (II): [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 are each independently C2-C 25 Alkylene or C2-C 25 alkenylene, where G 1 and G 2 wherein one or more -CH2- are optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L 1 are independently -OC(=O)R 1, -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ), -NR a P(=O)(OR b )(OR c ), and each L 2 is independently -OC(=O)R 2 , -C(=O)OR 2 , -OC(=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2, -SC(=S)R 2 , -C(=S)SR 2 , -C(=S)R 2 , -CH(OH)R 2 , -P(=O)(OR e )(OR f ), -NR d P(=O)(OR e )(OR f ) and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 alkenyl, where R 1 and R 2 wherein one or more -CH2- are optionally replaced by -SS-; R a , R b , R d , and R e are each independently H, C1-C 24 Alkyl, or C2-C 24 is alkenyl, R c and R f are each independently C1-C 24 Alkyl or C2-C 24 alkenyl, where R c and R f wherein one or more -CH2- are optionally replaced by -SS-; G 3 is C2-C 12 Alkylene or C2-C 12 alkenylene, wherein some or all of the alkylene or alkenylene are optionally replaced by C3-C8 cycloalkylene or C3-C8 cycloalkenylene; R 3 is -N(R 4 )R 5 , -OR 6 , or -SR 6 and R 4 is C1-C 12 Alkyl, C2-C 12Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 5 is H, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted; However, the compound is [ka] (not).

[0096] In one embodiment, provided herein is a compound of formula (II): [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene, where G 1 and G 2 wherein one or more -CH2- are optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L 1 are independently -OC(=O)R 1 , -C(=O)OR 1、 -OC(=O)OR 1 、 -C(=O)R 1 、 -OR 1 、 -S(O) x R 1 、 -S-SR 1 、 -C(=O)SR 1 、 -SC(=O)R 1 、 -NR a C(=O)R 1 、 -C(=O)NR b R c 、 -NR a C(=O)NR b R c 、 -OC(=O)NR b R c 、 -NR a C(=O)OR 1 、 -SC(=S)R 1 、 -C(=S)SR 1 、 -C(=S)R 1 、 -CH(OH)R 1 、 -P(=O)(OR b )(OR c )、 -NR a P(=O)(OR b )(OR c ) and each L 2 is independently -OC(=O)R 2 、 -C(=O)OR 2 、 -OC(=O)OR 2 、 -C(=O)R 2 、 -OR 2 、 -S(O) x 2 、 -S-SR 2 、 -C(=O)SR 2 、 -SC(=O)R 2 、 -NR d C(=O)R 2 、 -C(=O)NR e R f 、 -NR d C(=O)NR e R f 、 -OC(=O)NR e R f 、 -NR d C(=O)OR 2 、 -SC(=S)R 2 ​, -C(=S)SR 2 , -C(=S)R 2 , -CH(OH)R 2 , -P(=O)(OR e )(OR f ), -NR d P(=O)(OR e )(OR f ) and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 is alkenyl, R a , R b , R d , and R e are each independently H, C1-C 24 Alkyl, or C2-C 24 is alkenyl, R c and R f are each independently C1-C 24 Alkyl or C2-C 24 is alkenyl, G 3 is C2-C 12 Alkylene or C2-C 12 alkenylene, wherein some or all of the alkylene or alkenylene are optionally replaced by C3-C8 cycloalkylene or C3-C8 cycloalkenylene; R 3 is -N(R 4 )R 5 , -OR 6 , or -SR 6 and R 4 is C1-C 12 Alkyl, C2-C 12 Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 5 is H, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4- to 8-membered heterocycloalkyl; R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted; However, the compound is [ka] (not).

[0097] In one embodiment, provided herein is a compound of formula (II): [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene, where G 1 and G 2 wherein one or more -CH2- are optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L 1 are independently -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NRa C(=O)R 1 、 -C(=O)NR b R c 、 -NR a C(=O)NR b R c 、 -OC(=O)NR b R c 、 -NR a C(=O)OR 1 、 -SC(=S)R 1 、 -C(=S)SR 1 、 -C(=S)R 1 、 -CH(OH)R 1 、 -P(=O)(OR b )(OR c )、 -NR a P(=O)(OR b )(OR c ) and each L 2 is independently -OC(=O)R 2 、 -C(=O)OR 2 、 -OC(=O)OR 2 、 -C(=O)R 2 、 -OR 2 、 -S(O) x R 2 、 -S - SR 2 、 -C(=O)SR 2 、 -SC(=O)R 2 、 -NR d C(=O)R 2 、 -C(=O)NR e R f 、 -NR d C(=O)NR e R f 、 -OC(=O)NR e R f 、 -NR d C(=O)OR 2 、 -SC(=S)R 2 、 -C(=S)SR 2 、 -C(=S)R 2 、 -CH(OH)R 2 、 -P(=O)(OR e )(OR f )、 -NR d P(=O)(OR e )(OR f ) and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 is alkenyl, R a , R b , R d , and R e are each independently H, C1-C 24 Alkyl, or C2-C 24 is alkenyl, R c and R f are each independently C1-C 24 Alkyl or C2-C 24 is alkenyl, G 3 is C2-C 24 Alkylene, C2-C 24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; R 3 is -N(R 4 )R 5 -OR 6 and R 4 is C1-C 12 Alkyl, C2-C 12 Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 5 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted; However, the compound is [ka] (not).

[0098] In one embodiment, provided herein is a compound of formula (II): [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene, where G 1 and G 2 wherein one or more -CH2- are optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L 1 are independently -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1, -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ), -NR a P(=O)(OR b )(OR c ) and Each L 2 are independently -OC(=O)R 2 , -C(=O)OR 2 , -OC(=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2 , -SC(=S)R 2 , -C(=S)SR 2 , -C(=S)R 2 , -CH(OH)R 2 , -P(=O)(OR e )(OR f ), -NR d P(=O)(OR e )(OR f ) and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 is alkenyl, R a , R b , R d , and R e are each independently H, C1-C24 Alkyl, or C2-C 24 is alkenyl, R c and R f are each independently C1-C 24 Alkyl or C2-C 24 is alkenyl, G 3 is C2-C 12 Alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; R 3 is -N(R 4 )R 5 -OR 6 and R 4 is C1-C 12 Alkyl, C2-C 12 Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 5 is H, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl; R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted; However, the compound is [ka] (not).

[0099] In one embodiment, the compound has the formula [ka] In one embodiment, the compound is not a compound of the formula [ka] In one embodiment, the compound is not a compound of 1 teeth, [ka] provided that it is not.

[0100] In one embodiment, G 3 is C2-C 12 In one embodiment, G is an alkylene. 3 is a C2-C8 alkylene. In one embodiment, G 3 is a C2-C6 alkylene. In one embodiment, G 3 is a C2-C4 alkylene. In one embodiment, G 3 is a C alkylene. In one embodiment, G 3 is a C alkylene. In one embodiment, G 3 is a C4 alkylene. In one embodiment, G 3 is a C5 alkylene. In one embodiment, G 3 is a C alkylene. In one embodiment, G 3 is -CH2CH2-.

[0101] In one embodiment, G 3 is C2-C 12 In one embodiment, G is an alkenylene. 3 is C2-C8 alkenylene. In one embodiment, G 3 is C-C alkenylene. In one embodiment, G 3 is C2-C4 alkenylene. In one embodiment, G 3 is C2 alkenylene. In one embodiment, G 3is C alkenylene. In one embodiment, G 3 is C4 alkenylene. In one embodiment, G 3 is C5 alkenylene. In one embodiment, G 3 is C6 alkenylene.

[0102] In one embodiment, G 3 is C2-C 12 Alkylene or C2-C 12 alkenylene, where some or all of the alkylene or alkenylene are replaced by C-C cycloalkylene. 3 is (C-C alkylene)-(C-C cycloalkylene)-(C-C alkylene). In one embodiment, G 3 is (C1-C2 alkylene)-(C3-C8 cycloalkylene)-(C1-C2 alkylene). In one embodiment, G 3 is (C1-C2 alkylene)-(C3-C8 cycloalkylene). In one embodiment, G 3 is (C-C cycloalkylene)-(C-C alkylene). In one embodiment, G 3 is C2-C 12 Alkylene or C2-C 12 alkenylene, where every alkylene or alkenylene is replaced by C3-C8 cycloalkylene, i.e., G 3 is a C3-C8 cycloalkylene.

[0103] G 3 The cycloalkylene moiety in can be monocyclic or polycyclic (e.g., fused, spirocyclic, or bridged rings). In one embodiment, the cycloalkylene moiety is cyclopropylene. In one embodiment, the cycloalkylene moiety is cyclobutylene. In one embodiment, the cycloalkylene moiety is cyclopentylene. In one embodiment, the cycloalkylene moiety is cyclohexylene. In one embodiment, the cycloalkylene moiety is cycloheptylene. In one embodiment, the cycloalkylene moiety is cyclooctylene.

[0104] In one embodiment, G 3 is C2-C 12 Alkylene or C2-C 12 alkenylene, where some or all of the alkylene or alkenylene are replaced by C-C cycloalkenylene. 3 is (C-C alkylene)-(C-C cycloalkenylene)-(C-C alkylene). In one embodiment, G 3 is (C1-C2 alkylene)-(C3-C8 cycloalkenylene)-(C1-C2 alkylene). In one embodiment, G 3 is (C1-C2 alkylene)-(C3-C8 cycloalkenylene). In one embodiment, G 3 is (C-C cycloalkenylene)-(C-C alkylene). In one embodiment, G 3 is C2-C 12 Alkylene or C2-C 12 alkenylene, where every alkylene or alkenylene is replaced by C3-C8 cycloalkenylene, i.e., G 3 is a C3-C8 cycloalkenylene.

[0105] G 3 The cycloalkenylene moiety in can be monocyclic or polycyclic (e.g., fused, spirocyclic, or bridged rings). In one embodiment, the cycloalkenylene moiety is cyclopropenylene. In one embodiment, the cycloalkenylene moiety is cyclobutenylene. In one embodiment, the cycloalkenylene moiety is cyclopentenylene. In one embodiment, the cycloalkenylene moiety is cyclohexenylene. In one embodiment, the cycloalkenylene moiety is cycloheptenylene. In one embodiment, the cycloalkenylene moiety is cyclooctenylene.

[0106] In one embodiment, G 3 is unsubstituted. In one embodiment, G 3is substituted with one or more C1-C6 alkyl. 3 is substituted with two C1-C6 alkyl. In one embodiment, the C1-C6 alkyl is optionally substituted with hydroxy.

[0107] In one embodiment, G 3 teeth, [ka] is.

[0108] Unless otherwise specified, the G provided herein 3 The left-to-right direction of the group is from the nitrogen atom to the R 3 The left attachment point is to the nitrogen atom, and the right attachment point is to the R 3 In one embodiment, the two points of attachment are G 3 In one embodiment, the two points of attachment are on the same atom of G 3 In one embodiment, G 3 The cycloalkylene or cycloalkenylene moiety in has a cis configuration relative to the two points of attachment. 3 The cycloalkylene or cycloalkenylene moiety in has a trans configuration relative to the two points of attachment.

[0109] In one embodiment, the compound is a compound of formula (III): [ka] (wherein z is an integer of 2 to 12) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0110] In one embodiment, the compound is a compound of formula (III-A) or (III-B): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0111] In one embodiment, the compound is a compound of formula (IV): [ka] (wherein z is an integer of 2 to 12) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0112] In one embodiment, the compound is a compound of formula (IV-A) or (IV-B): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0113] In one embodiment, z is an integer from 2 to 12. In one embodiment, z is an integer from 2 to 8. In one embodiment, z is an integer from 2 to 6. In one embodiment, z is an integer from 2 to 4. In one embodiment, z is 2. In one embodiment, z is 3. In one embodiment, z is 4. In one embodiment, z is 5. In one embodiment, z is 6.

[0114] In one embodiment, G 1 is C2-C 25 In one embodiment, G is an alkylene. 1 is C2-C 20 In one embodiment, G is an alkylene. 1 is C2-C 16 In one embodiment, G is an alkylene. 1 is C2-C 14 In one embodiment, G is an alkylene. 1 is C2-C 12 In one embodiment, G is an alkylene. 1 is a C4-C8 alkylene. In one embodiment, G 1 is C4-C 10 In one embodiment, G is an alkylene.1 is a C5-C7 alkylene. In one embodiment, G 1 is a C alkylene. In one embodiment, G 1 is a C alkylene. In one embodiment, G 1 is a C4 alkylene. In one embodiment, G 1 is a C5 alkylene. In one embodiment, G 1 is a C alkylene. In one embodiment, G 1 is a C7 alkylene. In one embodiment, G 1 is C 12 In one embodiment, G is an alkylene. 1 is C 14 In one embodiment, G is an alkylene. 1 is C 16 In one embodiment, G is an alkylene. 1 is C 18 In one embodiment, G is an alkylene. 1 is C 20 In one embodiment, G is an alkylene. 1 is C 22 In one embodiment, G is an alkylene. 1 is C 24 In one embodiment, G is an alkylene. 1 is C2-C 14 In one embodiment, G is an alkenylene. 1 is C2-C 12 In one embodiment, G is an alkenylene. 1 is C-C alkenylene. In one embodiment, G 1 is C5-C7 alkenylene. In one embodiment, G 1 is C5 alkenylene. In one embodiment, G 1 is C7 alkenylene. In one embodiment, G 1 is C 10 In one embodiment, G is an alkenylene. 1 is linear. In one embodiment, G 1 is branched. In one embodiment, G 1 is divalent. In one embodiment, G 1 is trivalent.

[0115] In one embodiment, G 2 is a bond. In one embodiment, G 2 is C2-C 25 In one embodiment, G is an alkylene. 2 is C2-C 20 In one embodiment, G is an alkylene. 2 is C2-C 16 In one embodiment, G is an alkylene. 2 is C2-C 14 In one embodiment, G is an alkylene. 2 is C2-C 12 In one embodiment, G is an alkylene. 2 is C4-C 10 In one embodiment, G is an alkylene. 2 is a C4-C8 alkylene. In one embodiment, G 2 is a C5-C7 alkylene. In one embodiment, G 2 is a C alkylene. In one embodiment, G 2 is a C alkylene. In one embodiment, G 2 is a C4 alkylene. In one embodiment, G 2 is a C5 alkylene. In one embodiment, G 2 is a C alkylene. In one embodiment, G 2 is a C7 alkylene. In one embodiment, G 2 is C 12 In one embodiment, G is an alkylene. 2 is C 14 In one embodiment, G is an alkylene. 2 is C 16 In one embodiment, G is an alkylene. 2 is C 18 In one embodiment, G is an alkylene. 2 is C 20 In one embodiment, G is an alkylene. 2 is C 22 In one embodiment, G is an alkylene. 2 is C 24 In one embodiment, G is an alkylene. 2 is C2-C14 In one embodiment, G is an alkenylene. 2 is C2-C 12 In one embodiment, G is an alkenylene. 2 is C-C alkenylene. In one embodiment, G 2 is C5-C7 alkenylene. In one embodiment, G 2 is C5 alkenylene. In one embodiment, G 2 is C7 alkenylene. In one embodiment, G 2 is C 10 In one embodiment, G is an alkenylene. 2 is linear. In one embodiment, G 2 is branched. In one embodiment, G 2 is divalent. In one embodiment, G 2 is trivalent.

[0116] In one embodiment, G 1 and G 2 are each independently C2-C 25 In one embodiment, G is an alkylene. 1 and G 2 are each independently C2-C 20 In one embodiment, G is an alkylene. 1 and G 2 are each independently C2-C 16 In one embodiment, G is an alkylene. 1 and G 2 are each independently C2-C 12 In one embodiment, G is an alkylene. 1 and G 2 are each independently a C alkylene. 1 and G 2 are each independently a C alkylene. 1 and G 2 are each independently a C alkylene. 1 and G 2 are each independently C 10 In one embodiment, G is an alkylene. 1 and G2 are each independently C 12 In one embodiment, G is an alkylene. 1 and G 2 are each independently C 14 In one embodiment, G is an alkylene. 1 and G 2 are each independently C 16 In one embodiment, G is an alkylene. 1 and G 2 are each independently C 18 In one embodiment, G is an alkylene. 1 and G 2 are each independently C 20 In one embodiment, G is an alkylene. 1 and G 2 are each independently C 22 In one embodiment, G is an alkylene. 1 and G 2 are each independently C 24 It is alkylene.

[0117] In one embodiment, G 1 In one embodiment, one or more -CH2- in G is replaced by -O-. 1 In one embodiment, one or more non-terminal -CH2- in G is replaced by -O-. 1 In one embodiment, one non-terminal -CH2- in G is replaced by -O-. 1 is (C2-C 12 alkylene)-O-(C2-C 12 In one embodiment, G 1 is (C2-C5 alkylene)-O-(C2-C6 alkylene).

[0118] In one embodiment, G 1 In one embodiment, one or more -CH2- in G is replaced by -C(=O)O-. 1 In one embodiment, one or more non-terminal -CH2- in G is replaced by -C(=O)O-. 1is replaced by -C(=O)O-. 1 is (C2-C 12 alkylene)-C(=O)O-(C2-C 12 In one embodiment, G 1 is (C2-C5 alkylene)-C(=O)O-(C2-C6 alkylene).

[0119] In one embodiment, G 1 In one embodiment, one or more -CH2- in G is replaced by -OC(=O)-. 1 In one embodiment, one or more non-terminal -CH2- in G is replaced by -OC(=O)-. 1 In one embodiment, one non-terminal -CH2- in G is replaced by -OC(=O)-. 1 is (C2-C 12 alkylene)-OC(=O)-(C2-C 12 In one embodiment, G 1 is (C2-C5 alkylene)-OC(=O)-(C2-C6 alkylene).

[0120] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] In one embodiment, G 1 teeth, [ka] is.

[0121] Unless otherwise specified, the G provided herein 1 The direction from left to right of the group is from the nitrogen atom to the L 1 The left-hand point of attachment is to the nitrogen atom, and the right-hand point of attachment is to the L 1 It is the point of attachment to the group(s).

[0122] In one embodiment, G 2 In one embodiment, one or more -CH2- in G is replaced by -O-. 2 In one embodiment, one or more non-terminal -CH2- in G is replaced by -O-. 2 In one embodiment, one non-terminal -CH2- in G is replaced by -O-. 2is (C2-C 12 alkylene)-O-(C2-C 12 In one embodiment, G 2 is (C2-C5 alkylene)-O-(C2-C6 alkylene).

[0123] In one embodiment, G 2 In one embodiment, one or more -CH2- in G is replaced by -C(=O)O-. 2 In one embodiment, one or more non-terminal -CH2- in G is replaced by -C(=O)O-. 2 is replaced by -C(=O)O-. 2 is (C2-C 12 alkylene)-C(=O)O-(C2-C 12 In one embodiment, G 2 is (C2-C5 alkylene)-C(=O)O-(C2-C6 alkylene).

[0124] In one embodiment, G 2 In one embodiment, one or more -CH2- in G is replaced by -OC(=O)-. 2 In one embodiment, one or more non-terminal -CH2- in G is replaced by -OC(=O)-. 2 In one embodiment, one non-terminal -CH2- in G is replaced by -OC(=O)-. 2 is (C2-C 12 alkylene)-OC(=O)-(C2-C 12 In one embodiment, G 2 is (C2-C5 alkylene)-OC(=O)-(C2-C6 alkylene).

[0125] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] is.

[0126] Unless otherwise specified, the G provided herein 2 The direction from left to right of the group is from the nitrogen atom to the L 2 The left-hand point of attachment is to the nitrogen atom, and the right-hand point of attachment is to the L 2 It is the point of attachment to the group(s).

[0127] In one embodiment, the compound is a compound of formula (V): [ka] (In the formula, X 1 and X 2 are each independently a bond, —O—, —C(═O)O—, or —OC(═O)—; z is an integer from 2 to 12, x2 is X 1 is -O-, -C(=O)O-, or -OC(=O)-, it is an integer of 2 to 5, and x2 is X 1 is an integer from 2 to 6 when is a bond; y2 is X 2 is -O-, -C(=O)O-, or -OC(=O)-, it is an integer of 2 to 5, and y2 is X 2 is an integer from 2 to 6 when is a bond; G 4 and G 5 are each independently a C2-C6 alkylene. or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0128] In one embodiment, the compound is a compound of formula (V): [ka] (In the formula, X 1 and X 2 are each independently a bond, —O—, —C(═O)O—, or —OC(═O)—; z is an integer from 2 to 12, x2 is X 1 is -O-, -C(=O)O-, or -OC(=O)-, it is an integer of 2 to 12, and x2 is X 1 is an integer from 2 to 13 when is a bond; y2 is X 2 is -O-, -C(=O)O-, or -OC(=O)-, it is an integer of 2 to 12, and y2 is X 2 is an integer from 2 to 13 when is a bond; G 4 and G 5 are each independently C2-C 12 alkylene) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0129] In one embodiment, z is an integer from 2 to 12. In one embodiment, z is an integer from 2 to 8. In one embodiment, z is an integer from 2 to 6. In one embodiment, z is an integer from 2 to 4. In one embodiment, z is 2. In one embodiment, z is 3. In one embodiment, z is 4. In one embodiment, z is 5. In one embodiment, z is 6.

[0130] In one embodiment, x2 is an integer from 2 to 12. In one embodiment, x2 is an integer from 2 to 8. In one embodiment, x2 is an integer from 2 to 6. In one embodiment, x2 is an integer from 2 to 4. In one embodiment, x2 is 2. In one embodiment, x2 is 3. In one embodiment, x2 is 4. In one embodiment, x2 is 5. In one embodiment, x2 is 6. In one embodiment, x2 is 7.

[0131] In one embodiment, y2 is an integer from 2 to 12. In one embodiment, y2 is an integer from 2 to 8. In one embodiment, y2 is an integer from 2 to 6. In one embodiment, y2 is an integer from 2 to 4. In one embodiment, y2 is 2. In one embodiment, y2 is 3. In one embodiment, y2 is 4. In one embodiment, y2 is 5. In one embodiment, y2 is 6. In one embodiment, y2 is 7.

[0132] In one embodiment, x2 and y2 are independently an integer from 2 to 12. In one embodiment, x2 and y2 are independently an integer from 2 to 8. In one embodiment, x2 and y2 are independently an integer from 2 to 6. In one embodiment, x2 and y2 are independently an integer from 2 to 4. In one embodiment, x2 and y2 are both 2. In one embodiment, x2 and y2 are both 3. In one embodiment, x2 and y2 are both 4. In one embodiment, x2 and y2 are both 5. In one embodiment, x2 and y2 are both 6. In one embodiment, x2 and y2 are both 7.

[0133] In one embodiment, the compound is a compound of formula (VA), (VB), (VC), (VD), (VE), or (VF): [ka] (wherein z is an integer of 2 to 12, x0 is an integer from 1 to 11, y0 is an integer from 1 to 11, x1 is an integer from 0 to 9, y1 is an integer from 0 to 9, x2 is an integer from 2 to 5, x3 is an integer from 1 to 5, x4 is an integer from 0 to 3, y2 is an integer from 2 to 5, y3 is an integer from 1 to 5, y4 is an integer from 0 to 3. or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0134] In one embodiment, the compound is a compound of formula (VA), (VB), (VC), (VD), (VE), (VF), or (VG): [ka] (wherein z is an integer of 2 to 12, x0 is an integer from 1 to 11, y0 is an integer from 1 to 11, x1 is an integer from 0 to 9, y1 is an integer from 0 to 9, x2 is an integer from 2 to 12, x3 is an integer from 1 to 5, x4 is an integer from 0 to 3, x5 is an integer from 0 to 5, y2 is an integer from 2 to 12, y3 is an integer from 1 to 5, y4 is an integer from 0 to 3, y5 is an integer from 0 to 5. or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0135] In one embodiment, x0 is an integer from 2 to 10. In one embodiment, x0 is an integer from 2 to 8. In one embodiment, x0 is an integer from 2 to 6. In one embodiment, x0 is an integer from 4 to 6. In one embodiment, x0 is 2. In one embodiment, x0 is 3. In one embodiment, x0 is 4. In one embodiment, x0 is 5. In one embodiment, x0 is 6.

[0136] In one embodiment, y0 is an integer from 2 to 10. In one embodiment, y0 is an integer from 2 to 8. In one embodiment, y0 is an integer from 2 to 6. In one embodiment, y0 is an integer from 4 to 6. In one embodiment, y0 is 2. In one embodiment, y0 is 3. In one embodiment, y0 is 4. In one embodiment, y0 is 5. In one embodiment, y0 is 6.

[0137] In one embodiment, x0 and y0 are independently an integer from 2 to 10. In one embodiment, x0 and y0 are independently an integer from 2 to 8. In one embodiment, x0 and y0 are independently an integer from 2 to 6. In one embodiment, x0 and y0 are independently an integer from 4 to 6. In one embodiment, x0 and y0 are both 4. In one embodiment, x0 and y0 are both 5.

[0138] In one embodiment, x1 is an integer from 0 to 9. In one embodiment, x1 is an integer from 2 to 8. In one embodiment, x1 is an integer from 2 to 6. In one embodiment, x1 is an integer from 4 to 6. In one embodiment, x1 is 2. In one embodiment, x1 is 3. In one embodiment, x1 is 4. In one embodiment, x1 is 5. In one embodiment, x1 is 6.

[0139] In one embodiment, y1 is an integer from 0 to 9. In one embodiment, y1 is an integer from 2 to 8. In one embodiment, y1 is an integer from 2 to 6. In one embodiment, y1 is an integer from 4 to 6. In one embodiment, y1 is 2. In one embodiment, y1 is 3. In one embodiment, y1 is 4. In one embodiment, y1 is 5. In one embodiment, y1 is 6.

[0140] In one embodiment, x1 and y1 are independently an integer from 0 to 9. In one embodiment, x1 and y1 are independently an integer from 2 to 8. In one embodiment, x1 and y1 are independently an integer from 2 to 6. In one embodiment, x1 and y1 are independently an integer from 4 to 6. In one embodiment, x1 and y1 are both 4. In one embodiment, x1 and y1 are both 5.

[0141] In one embodiment, x2 is an integer from 2 to 12. In one embodiment, x2 is an integer from 2 to 7. In one embodiment, x2 is an integer from 2 to 5. In one embodiment, x2 is an integer from 2 to 4. In one embodiment, x2 is 2. In one embodiment, x2 is 3. In one embodiment, x2 is 4. In one embodiment, x2 is 5. In one embodiment, x2 is 7. In one embodiment, x2 is 12.

[0142] In one embodiment, y2 is an integer from 2 to 12. In one embodiment, y2 is an integer from 2 to 7. In one embodiment, y2 is an integer from 2 to 5. In one embodiment, y2 is an integer from 2 to 4. In one embodiment, y2 is 2. In one embodiment, y2 is 3. In one embodiment, y2 is 4. In one embodiment, y2 is 5. In one embodiment, y2 is 7. In one embodiment, y2 is 12.

[0143] In one embodiment, x2 and y2 are independently an integer from 2 to 12. In one embodiment, x2 and y2 are independently an integer from 2 to 7. In one embodiment, x2 and y2 are independently an integer from 2 to 5. In one embodiment, x2 and y2 are independently an integer from 2 to 4. In one embodiment, x2 and y2 are both 2. In one embodiment, x2 and y2 are both 3. In one embodiment, x2 and y2 are both 4. In one embodiment, x2 and y2 are both 5. In one embodiment, x2 and y2 are both 7. In one embodiment, x2 and y2 are both 12.

[0144] In one embodiment, x3 is an integer from 1 to 5. In one embodiment, x3 is 1. In one embodiment, x3 is 2. In one embodiment, x3 is 3. In one embodiment, x3 is 4. In one embodiment, x3 is 5.

[0145] In one embodiment, y3 is an integer from 1 to 5. In one embodiment, y3 is 1. In one embodiment, y3 is 2. In one embodiment, y3 is 3. In one embodiment, y3 is 4. In one embodiment, y3 is 5.

[0146] In one embodiment, x3 and y3 are independently an integer from 1 to 5. In one embodiment, x3 and y3 are both 1. In one embodiment, x3 and y3 are both 2. In one embodiment, x3 and y3 are both 3. In one embodiment, x3 and y3 are both 4. In one embodiment, x3 and y3 are both 5.

[0147] In one embodiment, x4 is an integer from 0 to 3. In one embodiment, x4 is 0. In one embodiment, x4 is 1. In one embodiment, x4 is 2. In one embodiment, x4 is 3.

[0148] In one embodiment, y4 is an integer from 0 to 3. In one embodiment, y4 is 0. In one embodiment, y4 is 1. In one embodiment, y4 is 2. In one embodiment, y4 is 3.

[0149] In one embodiment, x4 and y4 are independently an integer from 0 to 3. In one embodiment, x4 and y4 are both 0. In one embodiment, x4 and y4 are both 1. In one embodiment, x4 and y4 are both 2. In one embodiment, x4 and y4 are both 3.

[0150] In one embodiment, x5 is an integer from 0 to 5. In one embodiment, x5 is 0. In one embodiment, x5 is 1. In one embodiment, x5 is 2. In one embodiment, x5 is 3. In one embodiment, x5 is 4. In one embodiment, x5 is 5. In one embodiment, y5 is an integer from 0 to 5. In one embodiment, y5 is 0. In one embodiment, y5 is 1. In one embodiment, y5 is 2. In one embodiment, y5 is 3. In one embodiment, y5 is 4. In one embodiment, y5 is 5. In one embodiment, x4 and y4 are independently integers from 0 to 5. In one embodiment, x5 and y5 are both 0. In one embodiment, x5 and y5 are both 1. In one embodiment, x5 and y5 are both 2. In one embodiment, x5 and y5 are both 3. In one embodiment, x5 and y5 are both 4. In one embodiment, x5 and y5 are both 5.

[0151] In one embodiment, the compound is a compound of formula (VI): [ka] (In the formula, X 1 and X 2 are each independently a bond, —O—, —C(═O)O—, or —OC(═O)—; z is an integer from 2 to 12, x2 is X 1is -O-, -C(=O)O-, or -OC(=O)-, it is an integer of 2 to 5, and x2 is X 1 is an integer from 2 to 6 when is a bond; y2 is X 2 is -O-, -C(=O)O-, or -OC(=O)-, it is an integer of 2 to 5, and y2 is X 2 is an integer from 2 to 6 when is a bond; G 4 and G 5 are each independently a C2-C6 alkylene. or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0152] In one embodiment, the compound is a compound of formula (VI): [ka] (In the formula, X 1 and X 2 are each independently a bond, —O—, —C(═O)O—, or —OC(═O)—; z is an integer from 2 to 12, x2 is X 1 is -O-, -C(=O)O-, or -OC(=O)-, it is an integer of 2 to 12, and x2 is X 1 is an integer from 2 to 13 when is a bond; y2 is X 2 is -O-, -C(=O)O-, or -OC(=O)-, it is an integer of 2 to 12, and y2 is X 2 is an integer from 2 to 13 when is a bond; G 4 and G 5 are each independently C2-C 12 alkylene) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0153] In one embodiment, z is an integer from 2 to 12. In one embodiment, z is an integer from 2 to 8. In one embodiment, z is an integer from 2 to 6. In one embodiment, z is an integer from 2 to 4. In one embodiment, z is 2. In one embodiment, z is 3. In one embodiment, z is 4. In one embodiment, z is 5. In one embodiment, z is 6.

[0154] In one embodiment, x2 is an integer from 2 to 12. In one embodiment, x2 is an integer from 2 to 8. In one embodiment, x2 is an integer from 2 to 6. In one embodiment, x2 is an integer from 2 to 4. In one embodiment, x2 is 2. In one embodiment, x2 is 3. In one embodiment, x2 is 4. In one embodiment, x2 is 5. In one embodiment, x2 is 6. In one embodiment, x2 is 7. In one embodiment, x2 is 12.

[0155] In one embodiment, y2 is an integer from 2 to 12. In one embodiment, y2 is an integer from 2 to 8. In one embodiment, y2 is an integer from 2 to 6. In one embodiment, y2 is an integer from 2 to 4. In one embodiment, y2 is 2. In one embodiment, y2 is 3. In one embodiment, y2 is 4. In one embodiment, y2 is 5. In one embodiment, y2 is 6. In one embodiment, y2 is 7. In one embodiment, y2 is 12.

[0156] In one embodiment, x2 and y2 are independently an integer from 2 to 12. In one embodiment, x2 and y2 are independently an integer from 2 to 8. In one embodiment, x2 and y2 are independently an integer from 2 to 6. In one embodiment, x2 and y2 are independently an integer from 2 to 4. In one embodiment, x2 and y2 are both 2. In one embodiment, x2 and y2 are both 3. In one embodiment, x2 and y2 are both 4. In one embodiment, x2 and y2 are both 5. In one embodiment, x2 and y2 are both 6. In one embodiment, x2 and y2 are both 7. In one embodiment, x2 and y2 are both 12.

[0157] In one embodiment, the compound is a compound of formula (VI-A), (VI-B), (VI-C), (VI-D), (VI-E), or (VI-F): [ka] (wherein z is an integer of 2 to 12, y is an integer from 2 to 12, x0 is an integer from 1 to 11, x1 is an integer from 0 to 9, x2 is an integer from 2 to 5, x3 is an integer from 1 to 5, x4 is an integer between 0 and 3) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0158] In one embodiment, the compound is a compound of formula (VI-A), (VI-B), (VI-C), (VI-D), (VI-E), (VI-F) or (VI-G): [ka] (wherein z is an integer of 2 to 12, y is an integer from 2 to 12, x0 is an integer from 1 to 11, x1 is an integer from 0 to 9, x2 is an integer from 2 to 12, x3 is an integer from 1 to 5, x4 is an integer from 0 to 3, x5 is an integer between 0 and 5) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0159] In one embodiment, x0 is an integer from 2 to 10. In one embodiment, x0 is an integer from 2 to 8. In one embodiment, x0 is an integer from 2 to 6. In one embodiment, x0 is an integer from 4 to 6. In one embodiment, x0 is 2. In one embodiment, x0 is 3. In one embodiment, x0 is 4. In one embodiment, x0 is 5. In one embodiment, x0 is 6.

[0160] In one embodiment, x1 is an integer from 0 to 9. In one embodiment, x1 is an integer from 2 to 8. In one embodiment, x1 is an integer from 2 to 6. In one embodiment, x1 is an integer from 4 to 6. In one embodiment, x1 is 2. In one embodiment, x1 is 3. In one embodiment, x1 is 4. In one embodiment, x1 is 5. In one embodiment, x1 is 6.

[0161] In one embodiment, x2 is an integer from 2 to 12. In one embodiment, x2 is an integer from 2 to 7. In one embodiment, x2 is an integer from 2 to 5. In one embodiment, x2 is an integer from 2 to 4. In one embodiment, x2 is 2. In one embodiment, x2 is 3. In one embodiment, x2 is 4. In one embodiment, x2 is 5. In one embodiment, x2 is 8. In one embodiment, x2 is 12.

[0162] In one embodiment, x3 is an integer from 1 to 5. In one embodiment, x3 is 1. In one embodiment, x3 is 2. In one embodiment, x3 is 3. In one embodiment, x3 is 4. In one embodiment, x3 is 5.

[0163] In one embodiment, x4 is an integer from 0 to 3. In one embodiment, x4 is 0. In one embodiment, x4 is 1. In one embodiment, x4 is 2. In one embodiment, x4 is 3.

[0164] In one embodiment, x5 is 0. In one embodiment, x5 is 1. In one embodiment, x5 is 2. In one embodiment, x5 is 3. In one embodiment, x5 is 4. In one embodiment, x5 is 5.

[0165] In one embodiment, y is an integer from 2 to 10. In one embodiment, y is an integer from 2 to 8. In one embodiment, y is an integer from 2 to 6. In one embodiment, y is 2. In one embodiment, y is 3. In one embodiment, y is 4. In one embodiment, y is 5. In one embodiment, y is 6.

[0166] In one embodiment of Formula (VB), (VD), (VF), (VI-B), (VI-D), or (VI-F), [ka] The part is [ka] wherein x5 is 0 or 5. In one embodiment, x5 is 0 (i.e., [ka] In one embodiment, x5 is 1 (i.e., [ka] is replaced by ).

[0167] In one embodiment of Formula (VB), (VD), or (VF), [ka] The part is [ka] wherein y5 is 0 or 5. In one embodiment, y5 is 0 (i.e., [ka] In one embodiment, y5 is 1 (i.e., replaced by [ka] is replaced by ).

[0168] In one embodiment of formula (VA), (VC), (VE), (VI-A), (VI-C), or (VI-E), [ka] The part is [ka] wherein x5 is 0 or 5. In one embodiment, x5 is 0 (i.e., [ka] In one embodiment, x5 is 1 (i.e., [ka] is replaced by ).

[0169] In one embodiment of Formula (VA), (VC), or (VE), [ka] The part is [ka] wherein y5 is 0 or 5. In one embodiment, y5 is 0 (i.e., [ka] In one embodiment, y5 is 1 (i.e., replaced by [ka] is replaced by ).

[0170] In one embodiment, L 1 -OR 1 , -OC(=O)R 1 , -C(=O)OR 1 , -NR a C(=O)R 1 , or -C(=O)NR b R c In one embodiment, L 2 -OR 2 , -OC(=O)R 2 , -C(=O)OR 2 , -NR d C(=O)R 2 , or -C(=O)NR e R f In one embodiment, each L is independently -OR 1 or -OC(=O)R 1 and each L2 is independently -OR 2 or -OC(=O)R 2 is.

[0171] In one embodiment, two L 1 If there is, each L 1 are independently -OR 1 In one embodiment, two L 1 If there is, each L 1 are independently -OC(=O)R 1 In one embodiment, two L 2 If there is, each L 2 are independently -OR 2 In one embodiment, two L 2 If there is, each L 2 are independently -OC(=O)R 2 In one embodiment, R 1 or R 2are independently linear C6-C 10 In one embodiment, R 1 or R 2 are both straight chain C7 alkyl. In one embodiment, R 1 or R 2 are both straight chain C alkyl. In one embodiment, R 1 or R 2 and R are both 2-(octyldisulfanyl)ethyl. 1 or R 2 independently, -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10 Alkyl or C2-C 10 In one embodiment, R is an alkenyl. 1 or R 2 independently, -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 is independently C-C alkyl. In one embodiment, R 1 or R 2 -R 14 -SSR 15 where R 14 is C2-C 10 alkylene, and R 15 is C2-C 10 In one embodiment, R 1 or R 2 is -CH2CH2-SSR 16 where R 16 is C2-C 18 In one embodiment, R 16 is C4-C 16 In one embodiment, R 16 is C6-C 12In one embodiment, R 16 is C alkyl. In one embodiment, R 16 is a straight chain C8 alkyl.

[0172] In one embodiment, L 2 If there is only one, L 2 is -C(=O)OR 2 In one embodiment, L 2 If there is only one, L 2 is -OC(=O)R 2 In another embodiment, L 2 If there is only one, L 2 is -C(=O)NR e R f In one embodiment, R 2 or R f -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 is independently C-C alkyl. In one embodiment, R 2 or R f is C 10 -C 18 In one embodiment, R 2 or R f is C 18 In one embodiment, R 2 or R f is 2-(octyldisulfanyl)ethyl. In one embodiment, R 2 or R f is C 10 -C 18 In one embodiment, R is an alkenyl. 2 or R f is C 15 In one embodiment, R 2 or R f is C 17 In one embodiment, R is an alkenyl. 2 or R f -R 14-SSR 15 where R 14 is C2-C 10 alkylene, and R 15 is C2-C 10 In one embodiment, R 2 or R f is -CH2CH2-SSR 16 where R 16 is C2-C 18 In one embodiment, R 16 is C4-C 16 In one embodiment, R 16 is C6-C 12 In one embodiment, R 16 is C alkyl. In one embodiment, R 16 is a straight chain C8 alkyl. In one specific embodiment of any one of formulas (VA)-(VF) and (VI-A)-(VI-F), [ka] If part is present, each L 1 are independently -OC(=O)R 1 In one embodiment, each R 1 are independently linear C7-C 10 In one embodiment, one or more -CH2- in the alkyl is optionally replaced by -SS-.

[0173] In one specific embodiment of any one of formulas (VA) to (VF) and (VI-A) to (VI-F), [ka] If part is present, each L 2 are independently -OC(=O)R 2 In one embodiment, each R 2 are independently linear C7-C 10 In one embodiment, one or more -CH2- in the alkyl is optionally replaced by -SS-.

[0174] In one specific embodiment of any one of formulas (VA) to (VF) and (VI-A) to (VI-F), [ka] If part is present, each L 1 are independently -OR 1 In another embodiment, each L 1 are independently -OC(=O)R 1 In one embodiment, each R 1 are independently linear C7-C 10 It is alkyl.

[0175] In one specific embodiment of any one of formulas (VA) to (VF) and (VI-A) to (VI-F), [ka] If part is present, each L 2 are independently -OR 2 In another embodiment, each L 2 are independently -OC(=O)R 2 In one embodiment, each R 2 are independently linear C7-C 10 It is alkyl.

[0176] In one embodiment, R 3 is -N(R 4 )R 5 is.

[0177] In one embodiment, R 4 is C1-C 12 In one embodiment, R 4 is C1-C8 alkyl. In one embodiment, R 4 is C1-C6 alkyl. In one embodiment, R 4 is C1-C4 alkyl. In one embodiment, R 4 is methyl. In one embodiment, R 4is ethyl. In one embodiment, R 4 is n-propyl. In one embodiment, R 4 is isopropyl. In one embodiment, R 4 is n-butyl. In one embodiment, R 4 is n-pentyl. In one embodiment, R 4 is n-hexyl. In one embodiment, R 4 is n-octyl. In one embodiment, R 4 is n-nonyl.

[0178] In one embodiment, R 4 is C2-C 12 In one embodiment, R is an alkenyl. 4 is C-C alkenyl. In one embodiment, R 4 is C-C alkenyl. In one embodiment, R 4 is a C2-C4 alkenyl. In one embodiment, the alkenyl is a straight chain alkenyl. In one embodiment, the alkenyl is a branched alkenyl. In one embodiment, R 4 is ethenyl. In one embodiment, R 4 is an allyl.

[0179] In one embodiment, R 4 is C-C cycloalkyl. In one embodiment, R 4 is cyclopropyl. In one embodiment, R 4 is cyclobutyl. In one embodiment, R 4 is cyclopentyl. In one embodiment, R 4 is cyclohexyl. In one embodiment, R 4 is cycloheptyl.

[0180] In one embodiment, R 4 is C-C cycloalkenyl. In one embodiment, R 4 is cyclopropenyl. In one embodiment, R 4 is cyclobutenyl. In one embodiment, R 4is cyclopentenyl. In one embodiment, R 4 is cyclohexenyl. In one embodiment, R 4 is cycloheptenyl. In one embodiment, R 4 is cyclooctenyl. In one embodiment, the cycloalkenyl is substituted with one or more of hydroxyl, oxo, -NH, -NH(C-C alkyl), or -N(C-C alkyl). In one embodiment, the C-C alkyl is substituted with hydroxy.

[0181] In one embodiment, R 3 teeth, [ka] In one embodiment, R 3 teeth, [ka] In one embodiment, R 3 teeth, [ka] is.

[0182] In one embodiment, R 4 is C6-C 10 In one embodiment, R 4 is phenyl.

[0183] In one embodiment, R 4 is a 4-8 membered heterocycloalkyl. In one embodiment, R 4 is oxetanyl. In one embodiment, R 4 is tetrahydrofuranyl. In one embodiment, R 4 is tetrahydropyranyl. In one embodiment, R 4 is tetrahydrothiopyranyl. In one embodiment, R 4 is N-methylpiperidinyl.

[0184] In one embodiment, R 4 is non-substituted.

[0185] In one embodiment, R 4 is oxo, -OR g , -NR g C(=O)R h , -C(=O)NR g R h , -C(=O)R h , -OC(=O)R h , -C(=O)OR h , -OR i -OH and -N(R 10 )R 11 and wherein the substituent is one or more selected from the group consisting of: R g is independently at each occurrence H or C1-C6 alkyl; R h is independently at each occurrence C1-C6 alkyl; R i is independently at each occurrence C1-C6 alkylene; R 10 is hydrogen or C1-C6 alkyl, R 11 is C1-C6 alkyl, C3-C8 cycloalkyl, or C3-C8 cycloalkenyl; or R 10 and R 11 form a cyclic moiety together with the nitrogen to which they are attached, R 11 Or the cyclic moiety is optionally substituted with one or more of hydroxyl, oxo, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2.

[0186] In one embodiment, R 4 is substituted with one or more hydroxyls. In one embodiment, R 4 is substituted with one hydroxyl.

[0187] In one embodiment, R 4 is the substitution C1-C12 In one embodiment, R 4 is -(CH2) p Q, -(CH2) p CHQR, -CHQR, or -CQ(R)2, where Q is C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4- to 8-membered heterocyclyl, C6-C 10 Aryl, 5-10 membered heteroaryl, -OR, -O(CH2) p N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R 22 , -O(CH2) p OR, -N(R)C(=NR 23 )N(R)2, -N(R)C(=CHR 23 )N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR 23 )N(R)2, -N(OR)C(=CHR 23 )N(R)2, -C(=NR 23 )N(R)2, -C(=NR 23 )R, —C(O)N(R)OR, or —C(R)N(R)C(O)OR, and each p is independently 1, 2, 3, 4, or 5; R 22 is C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4- to 8-membered heterocyclyl, C6-C 10 aryl, or 5- to 10-membered heteroaryl; R 23 is H, -CN, -NO2, C1-C6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4- to 8-membered heterocyclyl, C6-C 10aryl, or 5- to 10-membered heteroaryl; each R is independently H, C1-C3 alkyl, or C2-C3 alkenyl, or two R in an N(R)2 moiety together with the nitrogen to which they are attached form a cyclic moiety; Each X is independently F, CI, Br, or I.

[0188] In one embodiment, R 4 is —CHCHOH. In one embodiment, R 4 is —CHCHCHOH. In one embodiment, R 4 is —CH2CH2CH2CH2OH. In one embodiment, R 4 is -CH2CH2OCH2CH2OH.

[0189] In one embodiment, R 4 is one or more -N(R 10 )R 11 In one embodiment, R 4 is one -N(R 10 )R 11 is replaced by .

[0190] In one embodiment, R 10 is hydrogen.

[0191] In one embodiment, R 11 is C-C cycloalkenyl. In one embodiment, R 11 is cyclobutenyl. In one embodiment, R 11 is substituted with one or more of oxo, —NH2, —NH(C1-C6 alkyl), or —N(C1-C6 alkyl)2.

[0192] In one embodiment, R 10 and R 11together with the nitrogen to which they are attached form a cyclic moiety. In one embodiment, the cyclic moiety is a 5- to 10-membered heteroaryl. In one embodiment, the cyclic moiety is pyrimidin-1-yl. In one embodiment, the cyclic moiety is purin-9-yl. In one embodiment, the cyclic moiety is substituted with one or more of oxo, —NH, —NH(C-C alkyl), or —N(C-C alkyl). In one embodiment, the C-C alkyl is substituted with hydroxy.

[0193] In one embodiment, R 4 teeth, [ka] In one embodiment, R 4 teeth, [ka] In one embodiment, R 4 teeth, [ka] is replaced by .

[0194] In one embodiment, R 5 is hydrogen. In one embodiment, R 5 is C1-C 12 In one embodiment, R 5 is C1-C8 alkyl. In one embodiment, R 5 is C1-C6 alkyl. In one embodiment, R 5 is C1-C4 alkyl. In one embodiment, R 5 is methyl. In one embodiment, R 5 is ethyl. In one embodiment, R 5 is n-propyl. In one embodiment, R 5 is isopropyl. In one embodiment, R 5 is n-butyl. In one embodiment, R 5 is n-pentyl. In one embodiment, R5 is n-hexyl. In one embodiment, R 5 is n-octyl. In one embodiment, R 5 is n-nonyl.

[0195] In one embodiment, R 5 is C-C cycloalkyl. In one embodiment, R 5 is cyclopropyl. In one embodiment, R 5 is cyclobutyl. In one embodiment, R 5 is cyclopentyl. In one embodiment, R 5 is cyclohexyl. In one embodiment, R 5 is cycloheptyl. In one embodiment, R 5 is cyclooctyl.

[0196] In one embodiment, R 5 is C-C cycloalkenyl. In one embodiment, R 5 is cyclopropenyl. In one embodiment, R 5 is cyclobutenyl. In one embodiment, R 5 is cyclopentenyl. In one embodiment, R 5 is cyclohexenyl. In one embodiment, R 5 is cycloheptenyl. In one embodiment, R 5 is cyclooctenyl.

[0197] In one embodiment, R 5 is C6-C 10 In one embodiment, R 5 is phenyl.

[0198] In one embodiment, R 5 is a 4-8 membered heterocycloalkyl. In one embodiment, R 5 is oxetanyl. In one embodiment, R 5 is tetrahydrofuranyl. In one embodiment, R 5 is tetrahydropyranyl. In one embodiment, R5 is tetrahydrothiopyranyl.

[0199] In one embodiment, R 5 is non-substituted.

[0200] In one embodiment, R 5 is oxo, -OR g , -NR g C(=O)R h , -C(=O)NR g R h , -C(=O)R h , -OC(=O)R h , -C(=O)OR h , -OR i -OH and -N(R 10 )R 11 and wherein the substituent is one or more selected from the group consisting of: R g is independently at each occurrence H or C1-C6 alkyl; R h is independently at each occurrence C1-C6 alkyl; R i is independently at each occurrence C1-C6 alkylene; R 10 is hydrogen or C1-C6 alkyl, R 11 is C1-C6 alkyl, C3-C8 cycloalkyl, or C3-C8 cycloalkenyl; or R 10 and R 11 form a cyclic moiety together with the nitrogen to which they are attached, R 11 Or the cyclic moiety is optionally substituted with one or more of hydroxyl, oxo, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2.

[0201] In one embodiment, R 5 is one or more hydroxyl or -N(R 10 )R 11 is replaced by .

[0202] In one embodiment, R 5 is substituted with one or more hydroxyls. In one embodiment, R 5 is substituted with one hydroxyl.

[0203] In one embodiment, R 5 is one or more -N(R 10 )R 11 In one embodiment, R 5 is one -N(R 10 )R 11 is replaced by .

[0204] In one embodiment, R 10 is hydrogen.

[0205] In one embodiment, R 11 is C-C cycloalkenyl. In one embodiment, R 11 is cyclobutenyl. In one embodiment, R 11 is substituted with one or more of oxo, —NH2, —NH(C1-C6 alkyl), or —N(C1-C6 alkyl)2.

[0206] In one embodiment, R 10 and R 11 together with the nitrogen to which they are attached form a cyclic moiety. In one embodiment, the cyclic moiety is a 5- to 10-membered heteroaryl. In one embodiment, the cyclic moiety is pyrimidin-1-yl. In one embodiment, the cyclic moiety is purin-9-yl. In one embodiment, the cyclic moiety is substituted with one or more of oxo, —NH2, —NH(C1-C6 alkyl), or —N(C1-C6 alkyl)2.

[0207] In one embodiment, R 3 -OR 6 In one embodiment, R 3 -SR 6 is.

[0208] In one embodiment, R6 is hydrogen (i.e., R 3 is -OH or R 3 is -SH). In one embodiment, R 6 is C1-C 12 In one embodiment, R 6 is C1-C8 alkyl. In one embodiment, R 6 is C1-C6 alkyl. In one embodiment, R 6 is C1-C4 alkyl. In one embodiment, R 6 is methyl. In one embodiment, R 6 is ethyl. In one embodiment, R 6 is C-C cycloalkyl. In one embodiment, R 6 is C-C cycloalkenyl. In one embodiment, R 6 is C6-C 10 In one embodiment, R 6 is phenyl.

[0209] In one embodiment, R 6 is non-substituted.

[0210] In one embodiment, R 6 has been substituted.

[0211] In one embodiment, R 6 is substituted with one or more hydroxyls. In one embodiment, R 6 is substituted with one hydroxyl.

[0212] In one embodiment, R 1 is a straight chain C6-C 24 In one embodiment, R 1 is a straight chain C7-C 15 In one embodiment, R 1 is a straight chain C7 alkyl. In one embodiment, R 1 is a straight chain C alkyl. In one embodiment, R 1 is a straight chain C alkyl. In one embodiment, R 1 is a linear C10 In one embodiment, R 1 is a linear C 11 In one embodiment, R 1 is a linear C 12 In one embodiment, R 1 is a linear C 13 In one embodiment, R 1 is a linear C 14 In one embodiment, R 1 is a linear C 15 It is alkyl.

[0213] In one embodiment, R 1 is a straight chain C6-C 24 In one embodiment, R is an alkenyl. 1 is a straight chain C7-C 17 In one embodiment, R is an alkenyl. 1 is a straight chain C7 alkenyl. In one embodiment, R 1 is a straight chain C alkenyl. In one embodiment, R 1 is a straight chain C9 alkenyl. In one embodiment, R 1 is a linear C 10 In one embodiment, R is an alkenyl. 1 is a linear C 11 In one embodiment, R is an alkenyl. 1 is a linear C 12 In one embodiment, R is an alkenyl. 1 is a linear C 13 In one embodiment, R is an alkenyl. 1 is a linear C 14 In one embodiment, R is an alkenyl. 1 is a linear C 15 In one embodiment, R is an alkenyl. 1 is a linear C 16 In one embodiment, R is an alkenyl. 1 is a linear C 17 It is alkenyl.

[0214] In one embodiment, R 1 is branched C6-C 24In one embodiment, R 1 -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10 In one embodiment, R 1 -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 is independently C4-C8 alkyl.

[0215] In one embodiment, R 1 is branched C6-C 24 In one embodiment, R is an alkenyl. 1 -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10 In one embodiment, R is an alkenyl. 1 -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 independently, C6-C 10 It is alkenyl.

[0216] In one embodiment, R 1 In one embodiment, one or more -CH- in R 1 is replaced by -SS-. 1 has one or more disulfide bonds. In one embodiment, R 1has one disulfide bond. In one embodiment, R 1 has two disulfide bonds. In one embodiment, R 1 -R 14 -SSR 15 where R 14 is C2-C 10 alkylene, and R 15 is C2-C 10 In one embodiment, R 1 is -CH2CH2-SSR 16 where R 16 is C2-C 18 In one embodiment, R 16 is C4-C 16 In one embodiment, R 16 is C6-C 12 In one embodiment, R 16 is C alkyl. In one embodiment, R 16 is a straight chain C alkyl. In one embodiment, R 1 is 2-(octyldisulfanyl)ethyl.

[0217] In one embodiment, R 2 is a straight chain C6-C 24 In one embodiment, R 2 is a straight chain C7-C 15 In one embodiment, R 2 is a straight chain C7 alkyl. In one embodiment, R 2 is a straight chain C alkyl. In one embodiment, R 2 is a straight chain C alkyl. In one embodiment, R 2 is a linear C 10 In one embodiment, R 2 is a linear C 11 In one embodiment, R 2 is a linear C 12 In one embodiment, R 2 is a linear C 13 In one embodiment, R 2 is a linear C 14In one embodiment, R 2 is a linear C 15 It is alkyl.

[0218] In one embodiment, R 2 is a straight chain C6-C 24 In one embodiment, R is an alkenyl. 2 is a straight chain C7-C 17 In one embodiment, R is an alkenyl. 2 is a straight chain C7 alkenyl. In one embodiment, R 2 is a straight chain C alkenyl. In one embodiment, R 2 is a straight chain C9 alkenyl. In one embodiment, R 2 is a linear C 10 In one embodiment, R is an alkenyl. 2 is a linear C 11 In one embodiment, R is an alkenyl. 2 is a linear C 12 In one embodiment, R is an alkenyl. 2 is a linear C 13 In one embodiment, R is an alkenyl. 2 is a linear C 14 In one embodiment, R is an alkenyl. 2 is a linear C 15 In one embodiment, R is an alkenyl. 2 is a linear C 16 In one embodiment, R is an alkenyl. 2 is a linear C 17 It is alkenyl.

[0219] In one embodiment, R 2 is branched C6-C 24 In one embodiment, R 2 -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10 In one embodiment, R 2 -R7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 is independently C4-C8 alkyl.

[0220] In one embodiment, R 2 is branched C6-C 24 In one embodiment, R is an alkenyl. 2 -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10 In one embodiment, R is an alkenyl. 2 -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 independently, C6-C 10 It is alkenyl.

[0221] In one embodiment, R 2 In one embodiment, one or more -CH- in R 2 is replaced by -SS-. 2 has one or more disulfide bonds. In one embodiment, R 2 has one disulfide bond. In one embodiment, R 2 has two disulfide bonds. In one embodiment, R 2 -R 14 -SSR 15 where R 14 is C2-C 10 alkylene, and R 15 is C2-C 10In one embodiment, R 2 is -CH2CH2-SSR 16 where R 16 is C2-C 18 In one embodiment, R 16 is C4-C 16 In one embodiment, R 16 is C6-C 12 In one embodiment, R 16 is C alkyl. In one embodiment, R 16 is a straight chain C alkyl. In one embodiment, R 2 is 2-(octyldisulfanyl)ethyl.

[0222] In one embodiment, R c is a straight chain C6-C 24 In one embodiment, R c is a straight chain C7-C 15 In one embodiment, R c is a straight chain C7 alkyl. In one embodiment, R c is a straight chain C alkyl. In one embodiment, R c is a straight chain C alkyl. In one embodiment, R c is a linear C 10 In one embodiment, R c is a linear C 11 In one embodiment, R c is a linear C 12 In one embodiment, R c is a linear C 13 In one embodiment, R c is a linear C 14 In one embodiment, R c is a linear C 15 It is alkyl.

[0223] In one embodiment, R c is a straight chain C6-C 24 In one embodiment, R is an alkenyl. c is a straight chain C7-C 17In one embodiment, R is an alkenyl. c is a straight chain C7 alkenyl. In one embodiment, R c is a straight chain C alkenyl. In one embodiment, R c is a straight chain C9 alkenyl. In one embodiment, R c is a linear C 10 In one embodiment, R is an alkenyl. c is a linear C 11 In one embodiment, R is an alkenyl. c is a linear C 12 In one embodiment, R is an alkenyl. c is a linear C 13 In one embodiment, R is an alkenyl. c is a linear C 14 In one embodiment, R is an alkenyl. c is a linear C 15 In one embodiment, R is an alkenyl. c is a linear C 16 In one embodiment, R is an alkenyl. c is a linear C 17 It is alkenyl.

[0224] In one embodiment, R c is branched C6-C 24 In one embodiment, R c -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10 In one embodiment, R c -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 is independently C4-C8 alkyl.

[0225] In one embodiment, R cis branched C6-C 24 In one embodiment, R is an alkenyl. c -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10 In one embodiment, R is an alkenyl. c -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 independently, C6-C 10 It is alkenyl.

[0226] In one embodiment, R c In one embodiment, one or more -CH- in R c is replaced by -SS-. c has one or more disulfide bonds. In one embodiment, R c has one disulfide bond. In one embodiment, R c has two disulfide bonds. In one embodiment, R c -R 14 -SSR 15 where R 14 is C2-C 10 alkylene, and R 15 is C2-C 10 In one embodiment, R c is -CH2CH2-SSR 16 where R 16 is C2-C 18 In one embodiment, R 16 is C4-C 16 In one embodiment, R 16 is C6-C 12In one embodiment, R 16 is C alkyl. In one embodiment, R 16 is a straight chain C alkyl. In one embodiment, R c is 2-(octyldisulfanyl)ethyl.

[0227] In one embodiment, R f is a straight chain C6-C 24 In one embodiment, R f is a straight chain C7-C 15 In one embodiment, R f is a straight chain C7 alkyl. In one embodiment, R f is a straight chain C alkyl. In one embodiment, R f is a straight chain C alkyl. In one embodiment, R f is a linear C 10 In one embodiment, R f is a linear C 11 In one embodiment, R f is a linear C 12 In one embodiment, R f is a linear C 13 In one embodiment, R f is a linear C 14 In one embodiment, R f is a linear C 15 It is alkyl.

[0228] In one embodiment, R f is a straight chain C6-C 24 In one embodiment, R is an alkenyl. f is a straight chain C7-C 17 In one embodiment, R is an alkenyl. f is a straight chain C7 alkenyl. In one embodiment, R f is a straight chain C alkenyl. In one embodiment, R f is a straight chain C9 alkenyl. In one embodiment, R f is a linear C 10 In one embodiment, R is an alkenyl. f is a linear C11 In one embodiment, R is an alkenyl. f is a linear C 12 In one embodiment, R is an alkenyl. f is a linear C 13 In one embodiment, R is an alkenyl. f is a linear C 14 In one embodiment, R is an alkenyl. f is a linear C 15 In one embodiment, R is an alkenyl. f is a linear C 16 In one embodiment, R is an alkenyl. f is a linear C 17 It is alkenyl.

[0229] In one embodiment, R f is branched C6-C 24 In one embodiment, R f -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10 In one embodiment, R f -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 is independently C4-C8 alkyl.

[0230] In one embodiment, R f is branched C6-C 24 In one embodiment, R is an alkenyl. f -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10In one embodiment, R is an alkenyl. f -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 independently, C6-C 10 It is alkenyl.

[0231] In one embodiment, R f In one embodiment, one or more -CH- in R f is replaced by -SS-. f has one or more disulfide bonds. In one embodiment, R f has one disulfide bond. In one embodiment, R f has two disulfide bonds. In one embodiment, R f -R 14 -SSR 15 where R 14 is C2-C 10 alkylene, and R 15 is C2-C 10 In one embodiment, R f is -CH2CH2-SSR 16 where R 16 is C2-C 18 In one embodiment, R 16 is C4-C 16 In one embodiment, R 16 is C6-C 12 In one embodiment, R 16 is C alkyl. In one embodiment, R 16 is a straight chain C alkyl. In one embodiment, R f is 2-(octyldisulfanyl)ethyl.

[0232] In one embodiment, R 1 , R 2 , Rc , and R f are each independently a straight chain C6-C 18 Alkyl, straight chain C6-C 18 alkenyl, or -R 7 -CH(R 8 )(R 9 ), where R 7 is a C0-C5 alkylene, and R 8 and R 9 independently, C2-C 10 Alkyl or C2-C 10 It is alkenyl.

[0233] In one embodiment, R 1 , R 2 , R c , and R f are each independently a straight chain C7-C 15 Alkyl, straight chain C7-C 15 alkenyl, or -R 7 -CH(R 8 )(R 9 ), where R 7 is C-C alkylene, and R 8 and R 9 are independently C4-C8 alkyl or C6-C 10 It is alkenyl.

[0234] In one embodiment, R 1 , R 2 , R c , and R f each independently contains two or more sulfurs. In one embodiment, R 1 , R 2 , R c , and R f each independently contains two sulfurs. In one embodiment, R 1 , R 2 , R c , and R f each independently contains one or more disulfide bonds. In one embodiment, R 1 , R 2 , R c , and R feach independently contains one disulfide bond. In one embodiment, R 1 , R 2 , R c , and R f each independently contains two disulfide bonds.

[0235] In one embodiment, R 1 , R 2 , R c , and R f is each independently one of the following structures: [ka]

[0236] In one embodiment, R a is H. In one embodiment, R d is H. In one embodiment, R a , R b , R d , and R e are each independently H. In one embodiment, R b is C1-C 24 In one embodiment, R b is C1-C 12 In one embodiment, R b is C2-C 24 In one embodiment, R is an alkenyl. b is C2-C 12 In one embodiment, R is an alkenyl. e is C1-C 24 In one embodiment, R e is C1-C 12 In one embodiment, R e is C2-C 24 In one embodiment, R is an alkenyl. e is C2-C 12 It is alkenyl.

[0237] In one embodiment, the compound is a compound of Table 1 or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0238] In one embodiment, the compound is a compound of Table 1A or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof. [Table 2]

[0239] It is understood that any of the embodiments of the compounds provided herein above, and any particular substituents and / or variables in the compounds provided herein above, can be independently combined with substituents and / or variables of other embodiments and / or compounds to form embodiments not specifically set forth above. Additionally, when a list of substituents and / or variables is recited for any particular group or variable, it is understood that each individual substituent and / or variable can be deleted from a particular embodiment and / or claim, and the remaining list of substituents and / or variables would be considered within the scope of the embodiments provided herein.

[0240] It is understood that combinations of substituents and / or variables of the depicted formulae herein are permissible only if such combinations result in stable compounds.

[0241] 5.4 Nanoparticle Composition In one aspect, described herein are nanoparticle compositions comprising a lipid compound described herein. In certain embodiments, the nanoparticle composition comprises a compound according to Formula (I) or (II) (and subformulas thereof) described herein.

[0242] In some embodiments, the largest dimension of the nanoparticle compositions provided herein is 1 μm or less (e.g., ≦1 μm, ≦900 nm, ≦800 nm, ≦700 nm, ≦600 nm, ≦500 nm, ≦400 nm, ≦300 nm, ≦200 nm, ≦175 nm, ≦150 nm, ≦125 nm, ≦100 nm, ≦75 nm, ≦50 nm, or less), such as when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. In one embodiment, the lipid nanoparticles provided herein have at least one dimension in the range of about 40 to about 200 nm. In one embodiment, at least one dimension is in the range of about 40 to about 100 nm.

[0243] Nanoparticle compositions that can be used in connection with the present disclosure include, for example, lipid nanoparticles (LNPs), nanolipoprotein particles, liposomes, lipid vesicles, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayers can comprise one or more ligands, proteins, or channels.

[0244] The properties of a nanoparticle composition may depend on its components. For example, a nanoparticle composition containing cholesterol as a structural lipid may have different properties from a nanoparticle composition containing a different structural lipid. Similarly, the properties of a nanoparticle composition may depend on the absolute or relative amounts of its components. For example, a nanoparticle composition containing a higher molar fraction of phospholipids may have different properties from a nanoparticle composition containing a lower molar fraction of phospholipids. The properties may also vary depending on the preparation method and conditions of the nanoparticle composition.

[0245] Nanoparticle compositions can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure several properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0246] Dh (size): The average size of the nanoparticle composition can be tens of nanometers to hundreds of nanometers. For example, the average size can be about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the nanoparticle composition can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In certain embodiments, the average size of the nanoparticle composition can be about 70 nm to about 100 nm. In some embodiments, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0247] PDI: The nanoparticle composition can be relatively uniform. The polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20.

[0248] Encapsulation efficiency: The efficiency of encapsulation of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. A high encapsulation efficiency is desirable (e.g., approaching 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the nanoparticle composition before and after dissolving the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0249] Apparent pKa: The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with a relatively low positive or negative charge are generally desirable because more highly charged species can cause undesirable interactions with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0250] In another embodiment, the self-replicating RNA can be formulated into liposomes. As a non-limiting example, the self-replicating RNA can be formulated into liposomes as described in International Publication No. WO20120067378, which is incorporated herein by reference in its entirety. In one aspect, the liposome can comprise lipids with pKa values ​​that can be advantageous for the delivery of mRNA. In another aspect, the liposome can have an essentially neutral surface charge at physiological pH, and therefore can be effective for immunization (see, for example, the liposomes described in International Publication No. WO20120067378, which is incorporated herein by reference in its entirety).

[0251] In some embodiments, the nanoparticle compositions described include a lipid component comprising at least one lipid, such as a compound according to one of Formulas (I) or (II) (and subformulas thereof) described herein. For example, in some embodiments, the nanoparticle composition can include a lipid component comprising one of the compounds provided herein. The nanoparticle composition can also include one or more other lipid or non-lipid components described below.

[0252] In one embodiment, a nanoparticle composition comprising a compound provided herein and mRNA exhibits improved mRNA expression levels (e.g., compared to standard cationic lipid compounds known in the art, such as MC3). In one embodiment, after a nanoparticle composition comprising a compound provided herein is administered to a subject, the compound exhibits rapid tissue clearance (e.g., hepatic clearance).

[0253] 5.4.1 Cationic / Ionizable Lipids As described herein, in some embodiments, the nanoparticle compositions provided herein comprise one or more charged or ionizable lipids in addition to a lipid according to Formula (I) or (II) (and subformulas thereof). Without being bound by theory, it is believed that certain charged or zwitterionic lipid components of the nanoparticle compositions resemble lipid components in cell membranes, thereby improving cellular uptake of the nanoparticles.Exemplary charged or ionizable lipids that can form part of the nanoparticle compositions include 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL26), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL27), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL28), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL29), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL30), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL31), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL32), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL33), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL34), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL35), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL36), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL37), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL38), 1,2-dilinoleyloxy-N,N-dimethylaminopropanol (KL39), N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3[(9Z,12Z)-octadeca-9,12-dien-1-[yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA( 2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-den-1-amine, and N,N-dimethyl-1-{(1S,2R)-2-octylcyclopropyl}heptadecan-8-amine.Further exemplary charged or ionizable lipids that can form part of the nanoparticle compositions include those described in Sabnis et al. "A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates," Molecular Therapy Vol. 26 No. 6, 2018 (e.g., lipid 5), the entire contents of which are incorporated herein by reference.

[0254] In some embodiments, suitable cationic lipids include N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino] ]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamido-glycylspermine (DOGS), 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), SAINT-2, N-methyl-4-(dioleyl)methylpyridinium, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI), dialkylated amino acids (DILA) 2) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC), (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-Cl), (R)-5-guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G), (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride (DOTAPen). Primary amines (e.g., DODAG Also suitable are cationic lipids with head groups that are charged at physiological pH, such as N',N'-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycinamide) and guanidinium head groups (e.g., bis-guanidinium-spermidine-cholesterol (BGSC), bis-guanidiniumtren-cholesterol (BGTC), PONA, and (R)-5-guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G)). Yet another suitable cationic lipid is (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DOPen-G). In certain embodiments, cationic lipid is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-Cl) or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium sulfate (DOTAP-sulfate).In certain embodiments, cationic lipid is specific enantiomer or racemic form, and includes various salt forms (such as chloride or sulfate) of cationic lipid as described above.For example, in some embodiments, cationic lipid is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-Cl) or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium sulfate (DOTAP-sulfate).In some embodiments, the cationic lipid is an ionizable cationic lipid, such as, for example, dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), and morpholinocholesterol (Mo-CHOL). In certain embodiments, the lipid nanoparticles comprise a combination or two or more cationic lipids (e.g., two or more of the cationic lipids described above).

[0255] Additionally, in some embodiments, the charged or ionizable lipid that can form part of the nanoparticle composition is a lipid that contains a cyclic amine group. Additional cationic lipids suitable for the formulations and methods disclosed herein include those described in WO2015199952, WO2016176330, and WO2015011633, the entire contents of each of which are incorporated herein by reference in their entirety.

[0256] 5.4.2 Polymer-conjugated lipids In some embodiments, the lipid component of the nanoparticle composition may include one or more polymer-conjugated lipids, such as PEGylated lipids (PEG lipids). Without being bound by theory, it is believed that the polymer-conjugated lipid component in the nanoparticle composition may improve colloidal stability and / or reduce protein absorption of the nanoparticles. Exemplary cationic lipids that may be used in connection with the present disclosure include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, or Chol-PEG2000.

[0257] In one embodiment, the polymer-conjugated lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), 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), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0258] In one embodiment, the polymer-conjugated lipid is present at a concentration ranging from 1.0 to 2.5 mole percent. In one embodiment, the polymer-conjugated lipid is present at a concentration of about 1.7 mole percent. In one embodiment, the polymer-conjugated lipid is present at a concentration of about 1.5 mole percent.

[0259] In one embodiment, the molar ratio of cationic lipid to polymer-conjugated lipid ranges from about 35:1 to about 25:1. In one embodiment, the molar ratio of cationic lipid to polymer-conjugated lipid ranges from about 100:1 to about 20:1.

[0260] In one embodiment, the PEGylated lipid has the formula: [ka] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R 12 and R 13 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester linkages; w has an average value in the range of 30 to 60.

[0261] In one embodiment, R 12 and R 13 are each independently a linear, saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, the average w ranges from 42 to 55, e.g., the average w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In certain embodiments, the average w is about 49.

[0262] In one embodiment, the PEGylated lipid has the formula: [ka] where the average w is about 49.

[0263] 5.4.3 Structural lipids In some embodiments, the lipid component of the nanoparticle composition may comprise one or more structural lipids. Without being bound by theory, it is believed that the structural lipids can stabilize the amphiphilic structure of the nanoparticles, such as, but not limited to, the lipid bilayer structure of the nanoparticles. Exemplary structural lipids that can be used in connection with the present disclosure include, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0264] In one embodiment, the lipid nanoparticles provided herein comprise a steroid or steroid analog. In one embodiment, the steroid or steroid analog is cholesterol. In one embodiment, the steroid is present at a concentration ranging from 39 to 49 molar percent, 40 to 46 molar percent, 40 to 44 molar percent, 40 to 42 molar percent, 42 to 44 molar percent, or 44 to 46 molar percent. In one embodiment, the steroid is present at a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent.

[0265] In one embodiment, the molar ratio of cationic lipid to steroid ranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol ranges from about 5:1 to 1:1. In one embodiment, the steroid is present at a concentration ranging from 32 to 40 molar percent of the steroid.

[0266] 5.4.4 Phospholipids In some embodiments, the lipid component of the nanoparticle composition may comprise one or more phospholipids, such as one or more (poly)unsaturated lipids. Without being bound by theory, it is believed that the phospholipids assemble into one or more lipid bilayer structures. Exemplary phospholipids that may form part of the nanoparticle composition include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DL ... Glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine Examples of suitable nanoparticles include, but are not limited to, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. In certain embodiments, the nanoparticle composition comprises DSPC. In certain embodiments, the nanoparticle composition comprises DOPE.In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

[0267] Further exemplary neutral lipids include, for example, dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleoyl phosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0268] In one embodiment, the neutral lipid is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), or phosphatidylglycerol (PG).

[0269] Additionally, phospholipids that may form part of the nanoparticle compositions include those described in WO 2017 / 112865, the entire contents of which are incorporated herein by reference in their entirety.

[0270] 5.4.5 Therapeutic payloads In accordance with the present disclosure, the nanoparticle compositions described herein can further comprise one or more therapeutic and / or prophylactic agents. These therapeutic and / or prophylactic agents may be referred to in the present disclosure as "therapeutic payloads" or "payloads." In some embodiments, the therapeutic payloads can be administered in vivo or in vitro using nanoparticles as delivery vehicles.

[0271] In some embodiments, the nanoparticle compositions contain, as a therapeutic payload, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents) (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (diphenhydramine, chlorpheniramine, promethazine), antibiotics / antibacterial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, anesthetics, and contrast media.

[0272] In some embodiments, the therapeutic payload comprises a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that may be harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, such as maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium.

[0273] In other embodiments, the therapeutic payload of the nanoparticle composition is an antimetabolite (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), an alkylating agent (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0274] In some embodiments, the nanoparticle compositions include biological molecules, such as peptides and polypeptides, as therapeutic payloads. The biological molecules forming part of the nanoparticle compositions can be either naturally occurring or synthetic. For example, in some embodiments, the therapeutic payload of the nanoparticle compositions can include, but is not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), VIR factors, luteinizing hormone-releasing hormone (LHRH) analogs, interferons, heparin, hepatitis B surface antigen, typhoid vaccine, cholera vaccine, and peptides and polypeptides.

[0275] 5.4.5.1 Nucleic acids In some embodiments, the nanoparticle compositions comprise one or more nucleic acid molecules (e.g., DNA or RNA molecules) as a therapeutic payload. Exemplary forms of nucleic acid molecules that can be included in the nanoparticle compositions as a therapeutic payload include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger RNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, etc. In certain embodiments, the therapeutic payload comprises RNA. RNA molecules that can be included in the nanoparticle compositions as therapeutic payloads include, but are not limited to, shortomers, agomils, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and other forms of RNA molecules known in the art. In certain embodiments, the RNA is mRNA.

[0276] In other embodiments, the nanoparticle composition comprises an siRNA molecule as a therapeutic payload. In particular, in some embodiments, the siRNA molecule can selectively interfere with and downregulate the expression of a gene of interest. For example, in some embodiments, the siRNA payload selectively silences a gene associated with a particular disease, disorder, or condition when administered to a subject in need of the nanoparticle composition comprising the siRNA. In some embodiments, the siRNA molecule comprises a sequence complementary to an mRNA sequence encoding a protein product of interest. In some embodiments, the siRNA molecule is an immunomodulatory siRNA.

[0277] In some embodiments, the nanoparticle composition comprises an shRNA molecule or a vector encoding an shRNA molecule as a therapeutic payload. In particular, in some embodiments, the therapeutic payload produces shRNA in target cells when administered to the target cells. The structures and mechanisms of shRNA are well known in the relevant technical field.

[0278] In some embodiments, the nanoparticle composition comprises an mRNA molecule as a therapeutic payload. In particular, in some embodiments, the mRNA molecule encodes a polypeptide of interest, including any natural or non-natural or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA payload can have a therapeutic effect when expressed in cells.

[0279] In some embodiments, a nucleic acid molecule of the present disclosure comprises an mRNA molecule. In certain embodiments, a nucleic acid molecule comprises at least one coding region (e.g., an open reading frame (ORF)) encoding a peptide or polypeptide of interest. In some embodiments, a nucleic acid molecule further comprises at least one untranslated region (UTR). In certain embodiments, the untranslated region (UTR) is located upstream (towards the 5' end) of the coding region and is referred to herein as a 5'-UTR. In certain embodiments, the untranslated region (UTR) is located downstream (towards the 3' end) of the coding region and is referred to herein as a 3'-UTR. In certain embodiments, a nucleic acid molecule comprises both a 5'-UTR and a 3'-UTR. In some embodiments, the 5'-UTR comprises a 5'-cap structure. In some embodiments, a nucleic acid molecule comprises a Kozak sequence (e.g., in the 5'-UTR). In some embodiments, a nucleic acid molecule comprises a polyA region (e.g., in the 3'-UTR). In some embodiments, a nucleic acid molecule comprises a polyadenylation signal (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecule comprises a stabilizing region (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecule comprises a secondary structure. In some embodiments, the secondary structure is a stem-loop. In some embodiments, the nucleic acid molecule comprises a stem-loop sequence (e.g., in the 5'-UTR and / or 3'-UTR). In some embodiments, the nucleic acid molecule comprises one or more intron regions that can be excised during splicing. In certain embodiments, the nucleic acid molecule comprises one or more regions selected from a 5'-UTR and a coding region. In certain embodiments, the nucleic acid molecule comprises one or more regions selected from a coding region and a 3'-UTR. In certain embodiments, the nucleic acid molecule comprises one or more regions selected from a 5'-UTR, a coding region, and a 3'-UTR.

[0280] Code Region In some embodiments, a nucleic acid molecule of the present disclosure comprises at least one coding region. In some embodiments, the coding region is an open reading frame (ORF) that encodes a single peptide or protein. In some embodiments, the coding region comprises at least two ORFs, each encoding a peptide or protein. In those embodiments in which the coding region comprises two or more ORFs, the encoded peptides and / or proteins may be the same as or different from one another. In some embodiments, the multiple ORFs in the coding region are separated by non-coding sequences. In certain embodiments, the non-coding sequences separating the two ORFs comprise an internal ribosome entry site (IRES).

[0281] Without being bound by theory, it is believed that an internal ribosome entry site (IRES) can act as the sole ribosome binding site or function as one of multiple ribosome binding sites of an mRNA. An mRNA molecule containing two or more functional ribosome binding sites can encode several peptides or polypeptides that are independently translated by the ribosome (e.g., a multicistronic mRNA). Thus, in some embodiments, a nucleic acid molecule (e.g., an mRNA) of the present disclosure comprises one or more internal ribosome entry sites (IRES). Examples of IRES sequences that can be used in connection with the present disclosure include, but are not limited to, those derived from picomaviruses (e.g., FMDV), plague viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SIV), or cricket paralysis viruses (CrPV).

[0282] In various embodiments, nucleic acid molecules of the present disclosure encode at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 peptides or proteins. The peptides and proteins encoded by the nucleic acid molecules can be the same or different. In some embodiments, nucleic acid molecules of the present disclosure encode dipeptides (e.g., chamocin and anserine). In some embodiments, nucleic acid molecules encode tripeptides. In some embodiments, nucleic acid molecules encode tetrapeptides. In some embodiments, nucleic acid molecules encode pentapeptides. In some embodiments, nucleic acid molecules encode hexapeptides. In some embodiments, nucleic acid molecules encode heptapeptides. In some embodiments, nucleic acid molecules encode octapeptides. In some embodiments, nucleic acid molecules encode nonapeptides. In some embodiments, nucleic acid molecules encode decapeptides. In some embodiments, nucleic acid molecules encode peptides or polypeptides having at least about 15 amino acids. In some embodiments, nucleic acid molecules encode peptides or polypeptides having at least about 50 amino acids. In some embodiments, nucleic acid molecules encode peptides or polypeptides having at least about 100 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 150 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 300 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 500 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 1000 amino acids.

[0283] In some embodiments, nucleic acid molecules of the present disclosure are at least about 30 nucleotides (nt) in length. In some embodiments, nucleic acid molecules are at least about 35 nt in length. In some embodiments, nucleic acid molecules are at least about 40 nt in length. In some embodiments, nucleic acid molecules are at least about 45 nt in length. In some embodiments, nucleic acid molecules are at least about 50 nt in length. In some embodiments, nucleic acid molecules are at least about 55 nt in length. In some embodiments, nucleic acid molecules are at least about 60 nt in length. In some embodiments, nucleic acid molecules are at least about 65 nt in length. In some embodiments, nucleic acid molecules are at least about 70 nt in length. In some embodiments, nucleic acid molecules are at least about 75 nt in length. In some embodiments, nucleic acid molecules are at least about 80 nt in length. In some embodiments, nucleic acid molecules are at least about 85 nt in length. In some embodiments, nucleic acid molecules are at least about 90 nt in length. In some embodiments, nucleic acid molecules are at least about 95 nt in length. In some embodiments, nucleic acid molecules are at least about 100 nt in length. In some embodiments, the nucleic acid molecule is at least about 120 nt in length. In some embodiments, the nucleic acid molecule is at least about 140 nt in length. In some embodiments, the nucleic acid molecule is at least about 160 nt in length. In some embodiments, the nucleic acid molecule is at least about 180 nt in length. In some embodiments, the nucleic acid molecule is at least about 200 nt in length. In some embodiments, the nucleic acid molecule is at least about 250 nt in length. In some embodiments, the nucleic acid molecule is at least about 300 nt in length. In some embodiments, the nucleic acid molecule is at least about 400 nt in length. In some embodiments, the nucleic acid molecule is at least about 500 nt in length. In some embodiments, the nucleic acid molecule is at least about 600 nt in length. In some embodiments, the nucleic acid molecule is at least about 700 nt in length. In some embodiments, the nucleic acid molecule is at least about 800 nt in length. In some embodiments, the nucleic acid molecule is at least about 900 nt in length. In some embodiments, the nucleic acid molecule is at least about 1000 nt in length.In some embodiments, the nucleic acid molecule is at least about 1100 nt in length. In some embodiments, the nucleic acid molecule is at least about 1200 nt in length. In some embodiments, the nucleic acid molecule is at least about 1300 nt in length. In some embodiments, the nucleic acid molecule is at least about 1400 nt in length. In some embodiments, the nucleic acid molecule is at least about 1500 nt in length. In some embodiments, the nucleic acid molecule is at least about 1600 nt in length. In some embodiments, the nucleic acid molecule is at least about 1700 nt in length. In some embodiments, the nucleic acid molecule is at least about 1800 nt in length. In some embodiments, the nucleic acid molecule is at least about 1900 nt in length. In some embodiments, the nucleic acid molecule is at least about 2000 nt in length. In some embodiments, the nucleic acid molecule is at least about 2500 nt in length. In some embodiments, the nucleic acid molecule is at least about 3000 nt in length. In some embodiments, the nucleic acid molecule is at least about 3500 nt in length. In some embodiments, the nucleic acid molecule is at least about 4000 nt in length. In some embodiments, the nucleic acid molecule is at least about 4500 nt in length. In some embodiments, the nucleic acid molecule is at least about 5000 nt in length.

[0284] In certain embodiments, the therapeutic payload comprises a vaccine composition (e.g., a genetic vaccine) described herein. In some embodiments, the therapeutic payload comprises a compound capable of inducing immunity against one or more target conditions or diseases. In some embodiments, the target condition is associated with or caused by infection by a pathogen, such as coronavirus (e.g., 2019-nCoV), influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis. In some embodiments, the therapeutic payload comprises a nucleic acid sequence (e.g., mRNA) encoding a pathogenic protein, or an antigenic fragment or epitope thereof, characteristic of the pathogen. When administered to a vaccinated subject, the vaccine allows for expression of the encoded pathogenic protein (or antigenic fragment or epitope thereof), thereby inducing immunity in the subject against the pathogen.

[0285] In some embodiments, the target condition is associated with or caused by neoplastic proliferation of cells, such as cancer. In some embodiments, the therapeutic payload comprises a nucleic acid sequence (e.g., mRNA) encoding a tumor-associated antigen (TAA) characteristic of the cancer, or an antigenic fragment or epitope thereof. When administered to a vaccinated subject, the vaccine allows expression of the encoded TAA (or antigenic fragment or epitope thereof), thereby eliciting immunity in the subject against tumor cells expressing the TAA.

[0286] 5' cap structure Without being bound by theory, it is believed that the 5' cap structure of a polynucleotide is involved in nuclear transport and increased polynucleotide stability, and binds to mRNA cap-binding protein (CBP), which is involved in polynucleotide stability and translation competence in cells through the association of CBP with polyA-binding protein to form mature circular mRNA species. The 5' cap structure further assists in 5'-proximal intron removal during mRNA splicing. Thus, in some embodiments, nucleic acid molecules of the present disclosure comprise a 5' cap structure.

[0287] Nucleic acid molecules can be capped at the 5' end by a cell's endogenous transcription machinery, generating a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue of the polynucleotide and the 5'-terminal transcribed sense nucleotide. This 5' guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugar of the terminal and / or anteterminal transcribed nucleotide at the 5' end of the polynucleotide can also optionally be 2'-O-methylated. 5' decapping and cleavage of the guanylate cap structure via hydrolysis can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0288] In some embodiments, the nucleic acid molecules of the present disclosure contain one or more modifications to the native 5' cap structure produced by endogenous processes. Without being bound by theory, modifications to the 5' cap may increase the stability of the polynucleotide, increase the half-life of the polynucleotide, or increase the translation efficiency of the polynucleotide.

[0289] Exemplary modifications to the native 5' cap structure include the creation of a non-hydrolyzable cap structure, which prevents decapping and therefore increases polynucleotide half-life. In some embodiments, modified nucleotides may be used during the capping reaction, since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester linkage. For example, in some embodiments, vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) may be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to create phosphorothioate linkages in the 5'-ppp-5' cap. Additional modified guanosine nucleotides, such as α-methyl-phosphonate and seleno-phosphate nucleotides, may be used.

[0290] Further exemplary modifications to the native 5' cap structure include modifications at the 2' and / or 3' positions of the capped guanosine triphosphate (GTP), replacement of the sugar ring oxygen (which produced a carbocyclic ring) with a methylene moiety (CH), modifications in the triphosphate bridge portion of the cap structure, or modifications in the nucleobase (G) portion.

[0291] Further exemplary modifications to the native 5' cap structure include, but are not limited to, 2'-O-methylation of the ribose sugar of the 5'-terminal and / or 5'-preterminal nucleotide of a polynucleotide (as described above) on the 2' hydroxy group of the sugar. Multiple distinct 5' cap structures may be used to generate the 5' cap of a polynucleotide, such as an mRNA molecule. Additional exemplary 5' cap structures that may be used in connection with the present disclosure further include those described in International Patent Publication Nos. WO2008127688, WO2008016473, and WO2011015347, the entire contents of each of which are incorporated herein by reference.

[0292] In various embodiments, the 5'-end cap can comprise a cap analog. Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ in their chemical structure from the native (i.e., endogenous, wild-type, or physical) 5' cap while retaining cap function. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or linked to a polynucleotide.

[0293] For example, the anti-reverse cap analog (ARCA) cap contains two guanosines linked by a 5'-5'-triphosphate group, with one guanosine containing an N7-methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7(G-3'mppp-G, which may equivalently be designated 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the otherwise unmodified guanosine is linked to the 5'-terminal nucleotide of the capped polynucleotide (e.g., mRNA). The N7- and 3'-O-methylated guanosine provides the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary cap structure is mCAP, which is similar to ARCA but has a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0294] In some embodiments, the cap analog can be a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog can be modified at different phosphate positions with boranophosphate or phosphoroselenoate groups, such as the dinucleotide cap analogs described in U.S. Patent No. 8,519,110, the entire contents of which are incorporated herein by reference in their entirety.

[0295] In some embodiments, the cap analog can be an N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, e.g., Kore et al., Bioorganic & Medicinal Chemistry 2013 21:4570-4574 for various cap analogs and methods for synthesizing cap analogs, the entire contents of which are incorporated herein by reference). In other embodiments, a cap analog useful in connection with the nucleic acid molecules of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0296] In various embodiments, the cap analog can comprise a guanosine analog. Useful guanosine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0297] Without being bound by theory, it is believed that cap analogs allow for concomitant capping of polynucleotides in in vitro transcription reactions, but that up to 20% of the transcripts remain uncapped. This, as well as the structural differences of cap analogs from the native 5' cap structure of polynucleotides produced by the cell's endogenous transcription machinery, can lead to reduced translational capacity and reduced cellular stability.

[0298] Thus, in some embodiments, nucleic acid molecules of the present disclosure can be post-transcriptionally capped using enzymes to generate more authentic 5' cap structures. As used herein, the phrase "more authentic" refers to features that closely reflect or mimic, either structurally or functionally, endogenous or wild-type features. That is, a "more authentic" feature better represents an endogenous wild-type, natural, or physiological cellular function and / or structure compared to prior art synthetic features or analogs, or is superior in one or more respects to the corresponding endogenous wild-type, natural, or physiological feature. Non-limiting examples of more authentic 5' cap structures useful in connection with nucleic acid molecules of the present disclosure include those that have, among others, enhanced cap-binding protein binding, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art. For example, in some embodiments, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase enzyme can create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, where the cap guanosine contains an N7 methylation and the 5'-terminal nucleotide of the polynucleotide contains a 2'-O-methyl. Such a structure is referred to as a Cap 1 structure. This cap results in higher translational competence, cellular stability, and reduced activation of cellular pro-inflammatory cytokines, for example, compared to other 5'-cap analog structures known in the art. Other exemplary cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')-ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).

[0299] Without being bound by theory, it is believed that the nucleic acid molecules of the present disclosure may be post-transcriptionally capped, and that this process is more efficient so that nearly 100% of the nucleic acid molecules may be capped.

[0300] Untranslated Regions (UTRs) In some embodiments, nucleic acid molecules of the present disclosure include one or more untranslated regions (UTRs). In some embodiments, a UTR is located upstream of a coding region in a nucleic acid molecule and is referred to as a 5'-UTR. In some embodiments, a UTR is located downstream of a coding region in a nucleic acid molecule and is referred to as a 3'-UTR. The sequence of a UTR can be homologous or heterologous to the sequence of the coding region found in the nucleic acid molecule. Multiple UTRs can be included in a nucleic acid molecule and can be of the same or different sequence and / or genetic origin. According to the present disclosure, any portion (including none) of a UTR in a nucleic acid molecule can be codon-optimized, and any can independently contain one or more different structural or chemical modifications before and / or after codon optimization.

[0301] In some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise UTRs and coding regions that are homologous with respect to one another. In other embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise UTRs and coding regions that are heterologous with respect to one another. In some embodiments, to monitor the activity of a UTR sequence, a nucleic acid molecule comprising the coding sequence of a UTR and a detectable probe can be administered in vitro (e.g., to a cell or tissue culture) or in vivo (e.g., to a subject), and the effect of the UTR sequence (e.g., modulation of expression levels, cellular localization of the encoded product, or half-life of the encoded product) can be measured using methods known in the art.

[0302] In some embodiments, the UTR of a nucleic acid molecule (e.g., mRNA) of the present disclosure contains at least one translational enhancer element (TEE), which functions to increase the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the TEE is located in the 5'-UTR of the nucleic acid molecule. In other embodiments, the TEE is located in the 3'-UTR of the nucleic acid molecule. In still other embodiments, at least two TEEs are located in the 5'-UTR and 3'-UTR of the nucleic acid molecule, respectively. In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure may contain one or more copies of a TEE sequence, or may contain two or more different TEE sequences. In some embodiments, different TEE sequences present in a nucleic acid molecule of the present disclosure may be homologous or heterologous with respect to each other.

[0303] Various TEE sequences known in the art can be used in connection with the present disclosure.For example, in some embodiments, the TEE can be an internal ribosome entry site (IRES), an HCV-IRES, or an IRES element.Chappell et al.Proc.Natl.Acad.Sci.USA 101:9590-9594,2004; Zhou et al.Proc.Natl.Acad.Sci.102:6273-6278,2005. Additional internal ribosome entry sites (IRES) that may be used in connection with the present disclosure include, but are not limited to, those described in U.S. Patent No. 7,468,275, U.S. Patent Publication Nos. 2007 / 0048776 and 2011 / 0124100, and International Patent Publication Nos. WO2007 / 025008 and WO2001 / 055369, the contents of each of which are incorporated by reference in their entirety. In some embodiments, the TEE may be those described in Supplementary Table 1 and Supplementary Table 2 of Wellensiek et al., "Genome-wide profiling of human cap-independent translation-enhancing elements," Nature Methods, 2013 Aug;10(8):747-750, the contents of which are incorporated by reference in their entirety.

[0304] Further exemplary TEEs that may be used in connection with the present disclosure include those disclosed in U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395, U.S. Patent Publication No. 2009 / 0226470, U.S. Patent Publication No. 2013 / 0177581, U.S. Patent Publication No. 2007 / 0048776, U.S. Patent Publication No. 2011 / 0124100, U.S. Patent Publication No. 2009 / 0093049, International Patent Publication No. WO2009 / 075886, International Patent Publication No. WO2012 / 009644, and These include, but are not limited to, the TEE sequences disclosed in International Patent Publication No. WO1999 / 024595, International Patent Publication No. WO2007 / 025008, International Patent Publication No. WO2001 / 055371, European Patent No. 2610341, and European Patent No. 2610340, the contents of each of which are incorporated herein by reference in their entirety.

[0305] In various embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise at least one UTR comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or more than 60 TEE sequences. In some embodiments, the TEE sequences in the UTR of the nucleic acid molecule are copies of the same TEE sequence. In other embodiments, at least two TEE sequences in the UTR of the nucleic acid molecule are different TEE sequences. In some embodiments, multiple different TEE sequences are arranged in one or more repeating patterns in the UTR region of a nucleic acid molecule. For illustrative purposes only, the repeating pattern can be, for example, ABABAB, AABBAABBAABB, ABCABCABC, etc., where each capital letter (A, B, or C) in these exemplary patterns represents a different TEE sequence. In some embodiments, at least two TEE sequences are contiguous with each other (i.e., no spacer sequence between them) in the UTR of a nucleic acid molecule. In other embodiments, at least two TEE sequences are separated by a spacer sequence. In some embodiments, a UTR can include a TEE sequence-spacer sequence module that is repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or more than nine times in the UTR. In any of the embodiments described in this paragraph, the UTR can be the 5'-UTR, the 3'-UTR, or both the 5'-UTR and the 3'-UTR of a nucleic acid molecule.

[0306] In some embodiments, the UTR of a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises at least one translational repression element that functions to reduce the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the UTR of the nucleic acid molecule comprises one or more miR sequences or fragments thereof (e.g., miR seed sequences) recognized by one or more microRNAs. In some embodiments, the UTR of the nucleic acid molecule comprises one or more stem-loop structures that downregulate the translational activity of the nucleic acid molecule. Other mechanisms for repressing translational activity associated with a nucleic acid molecule are known in the art. In any of the embodiments described in this paragraph, the UTR can be the 5'-UTR, 3'-UTR, or both the 5'-UTR and 3'-UTR of the nucleic acid molecule.

[0307] Polyadenylation (polyA) region During natural RNA processing, long chains of adenosine nucleotides (polyA tracts) are typically added to messenger RNA (mRNA) molecules to increase the molecule's stability. Immediately after transcription, the 3' end of the transcript is cleaved to release a 3'-hydroxyl group. PolyA polymerase then adds chains of adenosine nucleotides to the RNA. This process, called polyadenylation, adds polyA tracts between 100 and 250 residues in length. Without being bound by theory, it is believed that polyA tracts can confer various advantages to the nucleic acid molecules of the present disclosure.

[0308] Thus, in some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a polyadenylation signal. In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises one or more polyadenylation (polyA) regions. In some embodiments, the polyA region consists entirely of adenine nucleotides or functional analogs thereof. In some embodiments, a nucleic acid molecule comprises at least one polyA region at its 3' end. In some embodiments, a nucleic acid molecule comprises at least one polyA region at its 5' end. In some embodiments, a nucleic acid molecule comprises at least one polyA region at its 5' end and at least one polyA region at its 3' end.

[0309] According to the present disclosure, the poly A region can vary in length in different embodiments. In particular, in some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 30 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 35 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 40 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 45 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 50 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 55 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 60 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 65 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 70 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 75 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 80 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 85 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 90 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 95 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 100 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 110 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 120 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 130 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 140 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 150 nucleotides in length.In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 160 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 170 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 180 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 190 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 200 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 225 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 250 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 275 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 300 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 350 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 400 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 450 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 500 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 600 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 700 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 800 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 900 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1000 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1100 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1200 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1300 nucleotides in length.In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1400 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1500 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1600 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1700 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1800 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 1900 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 2000 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 2250 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 2500 nucleotides in length. In some embodiments, the poly A region of a nucleic acid molecule of this disclosure is at least 2750 nucleotides in length. In some embodiments, the polyA region of a nucleic acid molecule of the present disclosure is at least 3000 nucleotides in length.

[0310] In some embodiments, the length of the polyA region in a nucleic acid molecule can be selected based on the total length of the nucleic acid molecule or a portion thereof (e.g., the length of the coding region or the length of the open reading frame of the nucleic acid molecule, etc.) For example, in some embodiments, the polyA region comprises about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the total length of the nucleic acid molecule including the polyA region.

[0311] Without being bound by theory, it is believed that certain RNA-binding proteins can bind to the polyA region located at the 3' end of mRNA molecules. These polyA-binding proteins (PABPs) can regulate mRNA expression, such as by interacting with the translation initiation machinery in cells and / or protecting the 3' polyA tail from degradation. Thus, in some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure contains at least one binding site for a polyA-binding protein (PABP). In other embodiments, the nucleic acid molecule is conjugated or complexed with a PABP before being loaded into a delivery vehicle (e.g., lipid nanoparticle).

[0312] In some embodiments, the nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a poly-AG quartet. A G-quartet is a cyclic hydrogen-bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of a poly-A tract. The resulting polynucleotide (e.g., mRNA) can be assayed at various time points for stability, protein production, and other parameters, including half-life. It has been discovered that a poly-AG quartet structure results in protein production equivalent to at least 75% of that seen using only a 120-nucleotide poly-A tract.

[0313] In some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure may contain a polyA region and may be stabilized by the addition of a 3' stabilization region. In some embodiments, a 3' stabilization region that can be used to stabilize a nucleic acid molecule (e.g., mRNA) comprises a polyA or polyAG quartet structure as described in International Patent Publication No. WO2013 / 103659, the contents of which are incorporated herein by reference in their entirety.

[0314] In other embodiments, 3' stabilizing regions that may be used in connection with nucleic acid molecules of the present disclosure include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, etc. This includes any 2',3'-dideoxynucleoside, 2'-deoxynucleoside, or chain-terminating nucleoside such as, but not limited to, O-methylnucleosides, 3'-deoxynucleosides, 2',3'-dideoxynucleosides, 3'-O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and / or described herein.

[0315] secondary structure Without being bound by theory, it is believed that the stem-loop structure may direct RNA folding, protect the structural stability of nucleic acid molecules (e.g., mRNA), provide recognition sites for RNA-binding proteins, and serve as a substrate for enzymatic reactions. For example, the incorporation of miR and / or TEE sequences alters the shape of the stem-loop region, which may increase and / or decrease translation (Kedde et al. A Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221 and miR-222 accessibility. Nat Cell Biol., 2010 Oct;12(10):1014-20, the contents of which are incorporated herein by reference in their entirety).

[0316] Thus, in some embodiments, a nucleic acid molecule (e.g., mRNA) described herein, or a portion thereof, can adopt a stem-loop structure, such as, but not limited to, a histone stem-loop. In some embodiments, the stem-loop structure is formed from a stem-loop sequence that is about 25 or about 26 nucleotides in length, such as, but not limited to, those described in International Patent Publication No. WO2013 / 103659, the contents of which are incorporated herein by reference in their entireties. Further examples of stem-loop sequences include those described in International Patent Publication Nos. WO2012 / 019780 and WO201502667, the contents of which are incorporated herein by reference. In some embodiments, the stem-loop sequence comprises a TEE described herein. In some embodiments, the stem-loop sequence comprises a miR sequence described herein. In certain embodiments, the stem-loop sequence can comprise a miR-122 seed sequence. In certain embodiments, the nucleic acid molecule comprises two stem-loop sequences as described in WO2021204175, the contents of which are incorporated herein by reference.

[0317] In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a stem-loop sequence located upstream (towards the 5' end) of a coding region in the nucleic acid molecule. In some embodiments, the stem-loop sequence is located within the 5'-UTR of the nucleic acid molecule. In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a stem-loop sequence located downstream (towards the 3' end) of a coding region in the nucleic acid molecule. In some embodiments, the stem-loop sequence is located within the 3'-UTR of the nucleic acid molecule. In some cases, a nucleic acid molecule may contain more than one stem-loop sequence. In some embodiments, a nucleic acid molecule comprises at least one stem-loop sequence in the 5'-UTR and at least one stem-loop sequence in the 3'-UTR.

[0318] In some embodiments, the nucleic acid molecule comprising the stem-loop structure further comprises a stabilizing region, which in some embodiments comprises at least one chain-terminating nucleoside, which functions to slow degradation and thus increase the half-life of the nucleic acid molecule. Exemplary chain-terminating nucleosides that may be used in connection with the present disclosure include 2',3'-dideoxynucleosides, such as 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, 2'-deoxynucleosides or O-methylnucleosides, 3'-deoxynucleosides, 2',3'-dideoxynucleosides, 3'-O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and / or described herein. In other embodiments, the stem-loop structure may be stabilized by modifications to the 3' region of the polynucleotide that can prevent and / or inhibit the addition of oligo(U) (International Patent Publication No. WO2013 / 103659, incorporated herein by reference in its entirety).

[0319] In some embodiments, a nucleic acid molecule of the present disclosure comprises at least one stem-loop sequence and a polyA tract or polyadenylation signal. Non-limiting examples of polynucleotide sequences comprising at least one stem-loop sequence and a polyA tract or polyadenylation signal include those described in International Patent Publication Nos. WO2013 / 120497, WO2013 / 120629, WO2013 / 120500, WO2013 / 120627, WO2013 / 120498, WO2013 / 120626, WO2013 / 120499, and WO2013 / 120628, the contents of each of which are incorporated herein by reference in their entirety.

[0320] In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a polyA tract or polyadenylation signal can encode a pathogenic antigen or a fragment thereof, such as the polynucleotide sequences described in International Patent Publication Nos. WO2013 / 120499 and WO2013 / 120628, the contents of each of which are incorporated herein by reference in their entirety.

[0321] In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a polyA tract or polyadenylation signal can encode a therapeutic protein, such as the polynucleotide sequences described in International Patent Publication Nos. WO2013 / 120497 and WO2013 / 120629, the contents of each of which are incorporated herein by reference in their entirety.

[0322] In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a polyA tract or polyadenylation signal can encode a tumor antigen or a fragment thereof, such as the polynucleotide sequences described in International Patent Publication Nos. WO2013 / 120500 and WO2013 / 120627, the contents of each of which are incorporated herein by reference in their entirety.

[0323] In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a polyA tract or polyadenylation signal can encode an allergic antigen or an autoimmune autoantigen, such as the polynucleotide sequences described in International Patent Publication Nos. WO2013 / 120498 and WO2013 / 120626, the contents of each of which are incorporated herein by reference in their entirety.

[0324] Functional Nucleotide Analogues In some embodiments, the payload nucleic acid molecules described herein contain only standard nucleotides selected from A (adenosine), G (guanosine), C (cytosine), U (uridine), and T (thymidine). Without being bound by theory, it is believed that certain functional nucleotide analogs can confer useful properties to nucleic acid molecules. Examples of such useful properties in the context of the present disclosure include, but are not limited to, increased stability of the nucleic acid molecule, reduced immunogenicity of the nucleic acid molecule in inducing an innate immune response, enhanced production of the protein encoded by the nucleic acid molecule, increased intracellular delivery and / or retention of the nucleic acid molecule, and / or reduced cytotoxicity of the nucleic acid molecule.

[0325] Thus, in some embodiments, a payload nucleic acid molecule comprises at least one functional nucleotide analog described herein. In some embodiments, the functional nucleotide analog comprises at least one chemical modification to a nucleobase, sugar group, and / or phosphate group. Thus, a payload nucleic acid molecule comprising at least one functional nucleotide analog contains at least one chemical modification to a nucleobase, sugar group, and / or internucleoside linkage. Exemplary chemical modifications to a nucleobase, sugar group, or internucleoside linkage of a nucleic acid molecule are provided herein.

[0326] As described herein, a range of 0% to 100% of all nucleotides in a payload nucleic acid molecule can be a functional nucleotide analog as described herein. For example, in various embodiments, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, or about 20% to about 70% of all nucleotides in a nucleic acid molecule. , about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% are functional nucleotide analogs as described herein. In any of these embodiments, the functional nucleotide analog can be present at any position(s) of the nucleic acid molecule, including at the 5' end, the 3' end, and / or one or more internal positions. In some embodiments, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and / or different types of internucleoside linkages (e.g., backbone structures).

[0327] As described herein, a range of 0% to 100% of all nucleotides of a certain type (e.g., all purine-containing nucleotides of a certain type, or all pyrimidine-containing nucleotides of a certain type, or all A, G, C, T, or U of a certain type) in a payload nucleic acid molecule can be a functional nucleotide analog as described herein. For example, in various embodiments, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70% , about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% are functional nucleotide analogs as described herein. In any of these embodiments, the functional nucleotide analog can be present at any position(s) of the nucleic acid molecule, including at the 5' end, the 3' end, and / or one or more internal positions. In some embodiments, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and / or different types of internucleoside linkages (e.g., backbone structures).

[0328] Nucleobase Modifications In some embodiments, functional nucleotide analogs contain non-standard nucleobases. In some embodiments, standard nucleobases in nucleotides (e.g., adenine, guanine, uracil, thymine, and cytosine) can be modified or substituted to provide one or more functional analogs of the nucleotide. Exemplary modifications to nucleobases include one or more substitutions or modifications, including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions, one or more condensation or ring opening, oxidation, and / or reduction.

[0329] In some embodiments, the non-standard nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s 2 U), 4-thio-uracil (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho 5 U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m 3 U), 5-methoxy-uracil (mo 5 U), uracil 5-hydroxyacetic acid (cmo 5 U), uracil 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uracil (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm 5 U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uracil (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouracil (mcm 5 s 2 U), 5-aminomethyl-2-thio-uracil (nm 5 s2 U), 5-methylaminomethyl-uracil (mnm 5 U), 5-methylaminomethyl-2-thiouracil (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uracil (mnm 5 se 2 U), 5-carbamoylmethyl-uracil (ncm 5 U), 5-carboxymethylaminomethyl-uracil (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm 5 s 2 U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil (τm 5 5s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m 5 U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m 1 ψ), 1-ethyl-pseudouridine (Et 1 ψ), 5-methyl-2-thiouracil (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m 5D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uracil (m 5 U), 5-(isopentenylaminomethyl)-2-thiouracil (m 5 s 2 U), 5,2'-O-dimethyl-uridine (m 5 Um), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil, and 5-[3-(1-E-propenylamino)]uracil.

[0330] In some embodiments, the non-standard nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5- Hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Pseudoisocytidine, Zebularine, 5-Aza-Zebularine, 5-Methyl-Zebularine, 5-Aza-2-Thio-Zebularine, 2-Thio-Zebularine, 2-Methoxy-Cytosine, 2-Methoxy-5-Methyl-Cytosine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, Lysidine (k2C), 5,2'-O-Dimethyl-Cytidine (m5Cm), N4-Aza- These include cetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (fSCm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine.

[0331] In some embodiments, the non-standard nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2-amino-purine, Aza-2,6-diaminopurine, 1-methyl-adenine (m1A), 2-methyl-adenine (m2A), N6-methy-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarboxamide N6-threonylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl -adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0332] In some embodiments, the non-standard nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OHyW), hypomodified hydroxywyobutosine (OHyW*), 7-deaza-guanine, queuosine (Q), Epoxyqueousine (oQ), galactosyl-queousine (galQ), mannosyl-queousine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl-guanine Thio-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethylguanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl Examples of methyl-2'-O-methyl-guanosine include 1-methyl-6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 1-thio-guanine, and O-6-methyl-guanine.

[0333] In some embodiments, the non-standard nucleobase of a functional nucleotide analog can independently be a purine, a pyrimidine, a purine or pyrimidine analog. For example, in some embodiments, the non-standard nucleobase can be a modified adenine, cytosine, guanine, uracil, or hypoxanthine. In other embodiments, non-standard nucleobases include, for example, pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkoxy, 8-hydroxy and other 8- Naturally occurring and synthetic derivatives of bases including substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine, or 1,3,5 triazine may also be included.

[0334] Sugar modifications In some embodiments, the functional nucleotide analog contains a non-standard sugar group. In various embodiments, the non-standard sugar group can be a 5- or 6-carbon sugar (e.g., pentose, ribose, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof) with one or more substitutions such as halo, hydroxy, thiol, alkyl, alkoxy, alkenyloxy, alkynyloxy, cycloalkyl, aminoalkoxy, alkoxyalkoxy, hydroxyalkoxy, amino, azido, aryl, aminoalkyl, aminoalkenyl, or aminoalkynyl groups.

[0335] Generally, RNA molecules contain a ribose sugar group, which is a five-membered ring with oxygen. Exemplary, non-limiting alternative nucleotides include replacing oxygen in ribose (e.g., with S, Se, or alkylenes such as methylene or ethylene), adding double bonds (e.g., replacing ribose with cyclopentenyl or cyclohexenyl), ring contraction of ribose (e.g., forming a four-membered ring of cyclobutane or oxetane), ring expansion of ribose (e.g., anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino (also having a phosphoramidate backbone)), etc. These include "unlocked" forms such as tricyclic and glycol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, in which the ribose is replaced by a glycol unit linked to a phosphodiester bond), threose nucleic acids (TNA, in which the ribose is replaced by α-L-threofuranosyl-(3'→2'), and peptide nucleic acids (PNA, in which 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone).

[0336] In some embodiments, the sugar group contains one or more carbons that have the opposite stereochemical configuration of the corresponding carbon in ribose. Thus, nucleic acid molecules can contain, for example, nucleotides that contain arabinose or L-ribose as the sugar. In some embodiments, nucleic acid molecules contain at least one nucleoside, and the sugar is L-ribose, 2'-O-methylribose, 2'-fluororibose, arabinose, hexitol, LNA, or PNA.

[0337] Internucleoside linkage modification In some embodiments, payload nucleic acid molecules of the present disclosure can include one or more modified internucleoside linkages (e.g., phosphate backbones). The backbone phosphate groups can be modified by replacing one or more oxygen atoms with different substituents.

[0338] In some embodiments, functional nucleotide analogs can include replacing the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates replace both non-linking oxygens with sulfur. Phosphate linkers can also be modified by replacing the linked oxygens with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0339] Alternative nucleosides and nucleotides may include replacing one or more of the non-bridging oxygens with a borane moiety (BH3), sulfur (thio), methyl, ethyl, and / or methoxy. As a non-limiting example, two non-bridging oxygens at the same position (e.g., alpha (α), beta (β), or gamma (γ) position) may be replaced with sulfur (thio) and methoxy. Replacement of one or more oxygen atoms at the phosphate moiety position (e.g., α-thiophosphate) provides stability (e.g., against exonucleases and endonucleases) to RNA and DNA via the unnatural phosphorothioate backbone linkage. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in the cellular environment.

[0340] Other internucleoside linkages that can be used in accordance with the present disclosure, including internucleoside linkages that do not contain a phosphorus atom, are described herein.

[0341] Further examples of nucleic acid molecules (e.g., mRNA), compositions, formulations, and / or methods related thereto that may be used in connection with the present disclosure include WO2002 / 098443, WO2003 / 051401, WO2008 / 052770, WO2009127230, WO2006122828, WO2008 / 083949, WO2010088927, WO2010 / 037539, WO2004 / 004743, WO2 005 / 016376, WO2006 / 024518, WO2007 / 095976, WO2008 / 014979, WO2008 / 077592, WO2009 / 030481, WO2009 / 09522 6, WO2011069586, WO2011026641, WO2011 / 144358, WO2012019780, WO2012013326, WO2012089338, WO2012113513, WO2012116811, WO2012116810, WO2013113502, WO2013113501, WO2013113736, WO2013143698, WO2013143699, WO 2013143700, WO2013 / 120626, WO2013120627, WO2013120628, WO2013120629, WO2013174409, WO2014127917, WO20 WO2015 / 024669, WO2015 / 024668, WO2015 / 024667, WO2015 / 024665, WO2015 / 024666, WO2015 / 024664, WO2015101415, WO2015101414, WO2015024667, WO2015062738, WO2015101416, the contents of each of which are incorporated herein in their entirety.

[0342] 5.5 Formulation According to the present disclosure, the nanoparticle compositions described herein can include at least one lipid component and one or more additional components, such as a therapeutic agent and / or a prophylactic agent. The nanoparticle compositions can be designed for one or more specific applications or targets. The components of the nanoparticle composition can be selected based on the specific application or target and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, a particular formulation of the nanoparticle composition can be selected for a particular application or target, depending, for example, on the efficacy and toxicity of a particular combination of components.

[0343] The lipid component of the nanoparticle composition can include, for example, lipids according to one of formulas (I) or (II) (and subformulas thereof) described herein, phospholipids (unsaturated lipids such as DOPE or DSPC), PEG lipids, and structural lipids. Elements of the lipid component can be provided in specific fractions.

[0344] In one embodiment, provided herein is a nanoparticle composition comprising a cationic or ionizable lipid compound provided herein, a therapeutic agent, and one or more excipients. In one embodiment, the cationic or ionizable lipid compound comprises a compound according to one of formulas (I) or (II) (and subformulas thereof) described herein, and optionally one or more additional ionizable lipid compounds. In one embodiment, the one or more excipients are selected from neutral lipids, steroids, and polymer-conjugated lipids. In one embodiment, the therapeutic agent is encapsulated within or associated with the lipid nanoparticles.

[0345] In one embodiment, provided herein is a nanoparticle composition (lipid nanoparticle) comprising: i) 40 to 50 mole percent cationic lipids; ii) neutral lipids, iii) steroids, iv) polymer-conjugated lipids, and v) Therapeutic agents.

[0346] As used herein, "mole percent" refers to the ratio of moles of a component to the total moles of all lipid components (i.e., the total moles of cationic lipid(s), neutral lipids, steroids, and polymer-conjugated lipids) in the LNP.

[0347] In one embodiment, the lipid nanoparticles comprise 41-49 mole percent, 41-48 mole percent, 42-48 mole percent, 43-48 mole percent, 44-48 mole percent, 45-48 mole percent, 46-48 mole percent, or 47.2-47.8 mole percent cationic lipid. In one embodiment, the lipid nanoparticles comprise about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mole percent cationic lipid.

[0348] In one embodiment, the neutral lipid is present at a concentration ranging from 5 to 15 molar percent, 7 to 13 molar percent, or 9 to 11 molar percent. In one embodiment, the neutral lipid is present at a concentration of about 9.5, 10, or 10.5 molar percent. In one embodiment, the molar ratio of cationic lipid to neutral lipid is in the range of about 4.1:1.0 to about 4.9:1.0, about 4.5:1.0 to about 4.8:1.0, or about 4.7:1.0 to 4.8:1.0.

[0349] In one embodiment, the steroid is present at a concentration ranging from 39-49 molar percent, 40-46 molar percent, 40-44 molar percent, 40-42 molar percent, 42-44 molar percent, or 44-46 molar percent. In one embodiment, the steroid is present at a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent. In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2. In one embodiment, the steroid is cholesterol.

[0350] In one embodiment, the ratio of therapeutic agent to lipid in the LNP (i.e., N / P, where N represents the moles of cationic lipid and P represents the moles of phosphate present as part of the nucleic acid backbone) ranges from 2:1 to 30:1, e.g., 3:1 to 22:1. In one embodiment, N / P ranges from 6:1 to 20:1 or 2:1 to 12:1. Exemplary N / P ranges include about 3:1, about 6:1, about 12:1, and about 22:1.

[0351] In one embodiment, provided herein is a lipid nanoparticle comprising: i) a cationic lipid having an effective pKa greater than 6.0, ii) 5 to 15 mole percent neutral lipid; iii) 1 to 15 mole percent of anionic lipids; iv) 30 to 45 mole percent steroids; v) polymer-conjugated lipids, and vi) a therapeutic agent, or a pharmaceutically acceptable salt or prodrug thereof; The mole percentage is determined based on the total moles of lipid present in the lipid nanoparticle.

[0352] In one embodiment, the cationic lipid can be any of several lipid species that carry a net positive charge at a selected pH, such as physiological pH.Exemplary cationic lipids are described herein below.In one embodiment, the cationic lipid has a pKa greater than 6.25.In one embodiment, the cationic lipid has a pKa greater than 6.5.In one embodiment, the cationic lipid has a pKa greater than 6.1, greater than 6.2, greater than 6.3, greater than 6.35, greater than 6.4, greater than 6.45, greater than 6.55, greater than 6.6, greater than 6.65, or greater than 6.7.

[0353] In one embodiment, the lipid nanoparticles comprise 40-45 mole percent cationic lipids. In one embodiment, the lipid nanoparticles comprise 45-50 mole percent cationic lipids.

[0354] In one embodiment, the molar ratio of cationic lipid to neutral lipid ranges from about 2: 1 to about 8: 1. In one embodiment, the lipid nanoparticles comprise 5 to 10 mole percent neutral lipid.

[0355] Exemplary anionic lipids include, but are not limited to, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG) or 1,2-distearoyl-sn-glycero-3-phospho-(1'-lac-glycerol) (DSPG).

[0356] In one embodiment, the lipid nanoparticles comprise 1 to 10 mole percent anionic lipids. In one embodiment, the lipid nanoparticles comprise 1 to 5 mole percent anionic lipids. In one embodiment, the lipid nanoparticles comprise 1 to 9 mole percent, 1 to 8 mole percent, 1 to 7 mole percent, or 1 to 6 mole percent anionic lipids. In one embodiment, the molar ratio of anionic lipid to neutral lipid is in the range of 1:1 to 1:10.

[0357] In one embodiment, the steroid is cholesterol. In one embodiment, the molar ratio of cationic lipid to cholesterol ranges from about 5:1 to 1:1. In one embodiment, the lipid nanoparticles comprise 32 to 40 mole percent of steroid.

[0358] In one embodiment, the sum of the mole percent of neutral lipids and the mole percent of anionic lipids ranges from 5 to 15 mole percent, hi one embodiment, the sum of the mole percent of neutral lipids and the mole percent of anionic lipids ranges from 7 to 12 mole percent.

[0359] In one embodiment, the molar ratio of anionic lipid to neutral lipid ranges from 1:1 to 1:10. In one embodiment, the sum of the molar percentage of neutral lipid and the molar percentage of steroid ranges from 35 to 45 molar percent.

[0360] In one embodiment, the lipid nanoparticle comprises: i) 45 to 55 mole percent cationic lipid; ii) 5 to 10 mole percent neutral lipids; iii) 1 to 5 mole percent of an anionic lipid, and iv) 32-40 mole percent steroids.

[0361] In one embodiment, the lipid nanoparticles comprise 1.0 to 2.5 mole percent of conjugated lipid. In one embodiment, the polymer-conjugated lipid is present at a concentration of about 1.5 mole percent.

[0362] In one embodiment, the neutral lipid is present at a concentration ranging from 5 to 15 molar percent, 7 to 13 molar percent, or 9 to 11 molar percent. In one embodiment, the neutral lipid is present at a concentration of about 9.5, 10, or 10.5 molar percent. In one embodiment, the molar ratio of cationic lipid to neutral lipid is in the range of about 4.1:1.0 to about 4.9:1.0, about 4.5:1.0 to about 4.8:1.0, or about 4.7:1.0 to 4.8:1.0.

[0363] In one embodiment, the steroid is cholesterol. In some embodiments, the steroid is present at a concentration ranging from 39 to 49 molar percent, 40 to 46 molar percent, 40 to 44 molar percent, 40 to 42 molar percent, 42 to 44 molar percent, or 44 to 46 molar percent. In one embodiment, the steroid is present at a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent. In some embodiments, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2.

[0364] In one embodiment, the molar ratio of cationic lipid to steroid ranges from 5:1 to 1:1.

[0365] In one embodiment, the lipid nanoparticles comprise 1.0 to 2.5 mole percent of conjugated lipid. In one embodiment, the polymer-conjugated lipid is present at a concentration of about 1.5 mole percent.

[0366] In one embodiment, the molar ratio of cationic lipid to polymer-conjugated lipid ranges from about 100:1 to about 20:1. In one embodiment, the molar ratio of cationic lipid to polymer-conjugated lipid ranges from about 35:1 to about 25:1.

[0367] In one embodiment, the lipid nanoparticles have an average diameter in the range of 50 nm to 100 nm, or 60 nm to 85 nm.

[0368] In one embodiment, the composition comprises a cationic lipid provided herein, DSPC, cholesterol, and PEG lipid, and mRNA. In one embodiment, the cationic lipid provided herein, DSPC, cholesterol, and PEG lipid are in a molar ratio of about 50:10:38.5:1.5.

[0369] Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof within a mammalian body. The physiochemical properties of the nanoparticle composition can be modified to increase selectivity for specific bodily targets. For example, particle size can be adjusted based on the fenestration size of various organs. The therapeutic and / or prophylactic agents included in the nanoparticle composition can be selected based on the desired delivery target(s). For example, therapeutic and / or prophylactic agents can be selected for a specific indication, condition, disease, or disorder and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., localized or specific delivery). In certain embodiments, nanoparticle compositions can contain mRNA encoding a polypeptide of interest that can be translated intracellularly to produce the polypeptide of interest. Such compositions can be designed for specific delivery to a specific organ. In certain embodiments, compositions can be designed for specific delivery to the mammalian liver.

[0370] The amount of therapeutic and / or prophylactic agent in a nanoparticle composition can depend on the size, composition, desired target and / or use, or other properties of the nanoparticle composition, as well as the nature of the therapeutic and / or prophylactic agent. For example, the amount of RNA useful in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agent and other components (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the weight / weight ratio of lipid component to therapeutic and / or prophylactic agent in the nanoparticle composition can be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the weight / weight ratio of lipid component to therapeutic and / or prophylactic agent can be from about 10:1 to about 40:1. In certain embodiments, the weight / weight ratio is about 20:1. The amount of therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0371] In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and their amounts can be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N:P ratio is selected. The one or more RNAs, lipids, and their amounts can be selected to provide an N:P ratio of about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio can be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio can be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. For example, the N:P ratio can be about 5.67:1.

[0372] The physical properties of nanoparticle compositions can depend on their components. For example, nanoparticle compositions containing cholesterol as a structural lipid can have different properties compared to nanoparticle compositions containing different structural lipids. Similarly, the properties of nanoparticle compositions can depend on the absolute or relative amounts of their components. For example, nanoparticle compositions containing a higher molar fraction of phospholipids can have different properties than nanoparticle compositions containing a lower molar fraction of phospholipids. Properties can also vary depending on the preparation method and conditions of the nanoparticle composition.

[0373] Nanoparticle compositions can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure several properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0374] In various embodiments, the average size of the nanoparticle composition can be tens to hundreds of nanometers, for example, about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the nanoparticle composition can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In certain embodiments, the average size of the nanoparticle composition can be about 70 nm to about 100 nm. In some embodiments, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0375] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20.

[0376] The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Nanoparticle compositions with a relatively low positive or negative charge are generally desirable because more highly charged species can cause undesirable interactions with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0377] The encapsulation efficiency of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. A high encapsulation efficiency is desirable (e.g., approaching 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the nanoparticle composition before and after dissolving the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0378] Nanoparticle composition can optionally comprise one or more coatings.For example, nanoparticle composition can be formulated into capsules, films or tablets with coatings.The capsules, films or tablets comprising the compositions described herein can have any useful size, tensile strength, hardness or density.

[0379] 5.6 Pharmaceutical Compositions In accordance with the present disclosure, nanoparticle compositions can be formulated in whole or in part as pharmaceutical compositions. A pharmaceutical composition can include one or more nanoparticle compositions. For example, a pharmaceutical composition can include one or more nanoparticle compositions containing one or more different therapeutic and / or prophylactic agents. A pharmaceutical composition can further include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and medicaments can be found, for example, in Remington's The Science and Practice of Pharmacy, 21 st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and auxiliary ingredients can be used in any pharmaceutical composition, except insofar as any conventional excipient or auxiliary ingredient may be incompatible with one or more components of the nanoparticle composition. An excipient or auxiliary ingredient may be incompatible with a component of the nanoparticle composition if its combination with the component could result in any undesirable biological or other adverse effect.

[0380] In some embodiments, one or more excipients or auxiliary ingredients can comprise more than 50% of the total mass or volume of a pharmaceutical composition, including a nanoparticle composition. For example, one or more excipients or auxiliary ingredients can comprise 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the United States Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and / or International Pharmacopoeia.

[0381] The relative amounts of one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional components in a pharmaceutical composition according to the invention may vary depending on the identity, size, and / or condition of the subject being treated, and / or further depending on the route by which the composition is administered. By way of example, the pharmaceutical composition may contain from 0.1% to 100% (w / w) of one or more nanoparticle compositions.

[0382] In certain embodiments, the nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or shipping (e.g., stored at a temperature of 4°C or less, such as from about -150°C to about 0°C, or from about -80°C to about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C)). For example, a pharmaceutical composition comprising a compound of any of Formula (I) or (II) (and subformulas thereof) is a solution that is refrigerated for storage and / or shipping, e.g., at about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. In certain embodiments, the present disclosure also relates to methods of increasing the stability of nanoparticle compositions and / or pharmaceutical compositions comprising compounds of any of Formula (I) or (II) (and subformulas thereof) by storing the nanoparticle compositions and / or pharmaceutical compositions at temperatures below 4°C, such as from about -150°C to about 0°C, or from about -80°C to about -20°C, e.g., at temperatures of about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C. For example, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein are stable, e.g., at temperatures below 4°C (e.g., about 4°C to -20°C), for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In one embodiment, the formulation is stabilized at about 4°C for at least 4 weeks. In certain embodiments, the pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose.In certain embodiments, a pharmaceutical composition of the present disclosure has a pH of about 7-8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or 7.5-8, or 7-7.8). For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5-8, suitable for storage and / or shipping at, for example, about -20°C. For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and PBS, and has a pH of about 7-7.8, suitable for storage and / or shipping at, for example, about 4°C or below. "Stability," "stabilized," and "stable," in the context of this disclosure, refer to the resistance of the nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given conditions of manufacture, preparation, transportation, storage, and / or use, e.g., when subjected to stresses such as shear forces, freeze / thaw stresses, etc.

[0383] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including those who can benefit from a therapeutic effect provided by delivery of a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof, such as the renal system. While the description of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein is primarily directed to compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Modifications of compositions suitable for administration to humans to provide compositions suitable for administration to a variety of animals are well understood, and an ordinarily skilled veterinary pharmacologist can design and / or implement such modifications, if any, with no more than routine experimentation. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0384] Pharmaceutical compositions containing one or more nanoparticle compositions may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparative methods involve bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients, and then dividing, shaping, and / or packaging the product into desired single- or multi-dose units, as desired or necessary.

[0385] Pharmaceutical compositions according to 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 a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0386] Pharmaceutical compositions can be prepared in a variety of forms suitable for various routes and methods of administration. For example, pharmaceutical compositions can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0387] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, cyclodextrin, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid ester sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may include additional agents such as additional therapeutic and / or prophylactic agents, wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents, etc. In certain embodiments for parenteral administration, the compositions are mixed with solubilizing agents such as Cremophor™, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0388] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0389] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other injectable sterile media before use.

[0390] The disclosure features methods for delivering a therapeutic and / or prophylactic agent to a mammalian cell or organ, producing a polypeptide of interest in the mammalian cell, and treating a disease or disorder in the mammal in need thereof, the methods comprising administering to the mammal and / or contacting the mammalian cell with a nanoparticle composition comprising the therapeutic and / or prophylactic agent. [Example]

[0391] 6. Working Example The examples in this section are offered by way of illustration and not by way of limitation.

[0392] Common method. General Preparative HPLC Method: HPLC purification is performed on a Waters 2767 equipped with a diode array detector (DAD) on an Inertsil Pre-C8 OBD column, typically using water containing 0.1% TFA as solvent A and acetonitrile as solvent B.

[0393] General LCMS Method: LCMS analysis is performed on a Shimadzu (LC-MS2020) system. Generally, chromatography is performed on a SunFire C18 using water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B.

[0394] 6.1 Example 1: Preparation of Compound 1. [ka] Step 1: Preparation of Compound 1-2 To a solution of compound 1-1 (1.0 g, 2.5 mmol, 1.0 equiv) and DIPEA (0.65 g, 4.99 mmol, 2.0 equiv) in DCM (30 mL) was added MsCl (350 mg, 3.0 mmol, 1.2 equiv). The mixture was stirred at room temperature for 2 hours. The mixture was poured into water and extracted with DCM. The combined organic layer was washed with brine, dried over NaSO, and concentrated to give the title compound 1-2 (1.0 g, 84% yield) as a yellow oil, which was used in the next step without further purification.

[0395] Step 2: Preparation of Compound 1 To a solution of compound 1-2 (1.0 g, 1.96 mmol, 3.0 equiv.) and compound S (40 mg, 0.65 mmol, 1.0 equiv.) in ACN (30 mL) was added K2CO3 (270 mg, 1.96 mmol, 3.0 equiv.), Cs2CO3 (65 mg, 0.19 mmol, 0.3 equiv.), and NaI (30 mg, 0.19 mmol, 0.3 equiv.). The mixture was stirred at 80 °C for 10 h. LCMS showed the reaction was complete. The mixture was concentrated and purified by preparative HPLC to give the title compound 1 (35 mg, 6% yield) as a yellow oil.

[0396] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H), 1.27-1.63 (m, 53H), 1.97-2.01 (m, 2H), 2.28-2.64 (m, 14H), 3.52-3.58 (m, 2H), 4.00-4.10 (m, 8H). LCMS: Rt: 1.080 min; MS m / z (ESI): 826.0 [M+H] + .

[0397] The following compounds were prepared in a similar manner to compound 1 using the corresponding starting materials. [Table 3-1] [Table 3-2] [Table 3-3]

[0398] 6.2 Example 2: Preparation of Compound 2. [ka] Step 1: Preparation of Compound 2-1 To a solution of compound 1 (250 mg, 0.3 mmol, 1.0 equiv) in DCM (10 mL) was added SOCl (108 mg, 0.91 mmol, 3.0 equiv). The mixture was stirred at 35 °C for 10 h. The mixture was concentrated to give the title compound 2-1 (255 mg, 100% yield) as a yellow oil. LCMS: Rt: 0.613 min; MS m / z (ESI): 843.9 [M+H] + .

[0399] Step 2: Preparation of Compound 2 To a solution of compound 2-1 (200 mg, 0.24 mmol, 1.0 equiv.) and compound B (55 mg, 0.47 mmol, 2.0 equiv.) in THF (10 mL) was added DIPEA (95 mg, 0.71 mmol, 3.0 equiv.) and NaI (36 mg, 0.24 mmol, 1.0 equiv.). The mixture was stirred at 70° C. for 10 hours. LCMS showed that the reaction was complete. The mixture was concentrated and purified by preparative HPLC to give the title compound 2 (51 mg, 23% yield) as a yellow oil.

[0400] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H), 1.28-1.67 (m, 54H), 1.88-2.01 (m, 7H), 2.28-2.56 (m, 18H), 3.16-3.20 (m, 1H), 3.52-3.54 (m, 2H), 4.00-4.10 (m, 8H). LCMS: Rt: 1.060 min; MS m / z (ESI): 923.0 [M+H] + .

[0401] 6.3 Example 3: Preparation of Compound 4. [ka] Step 1: Preparation of compound 4-2 A solution of compound 4-1 (900 mg, 4.45 mmol, 1.0 equiv), TsOH.HO (423 mg, 2.23 mmol, 0.5 equiv), and compound W (931 mg, 4.77 mmol, 1.1 equiv) in toluene (30 mL) was stirred at 180 °C for 2 h. The mixture was cooled to room temperature and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 100 / 1) to give the title compound 4-2 (1.43 g, 88.6% yield) as a pale yellow oil.

[0402] Step 2: Preparation of compound 4-3 To a solution of compound 4-2 (500 mg, 1.38 mmol, 1.0 equiv.) and compound S (336 mg, 5.5 mmol, 4.0 equiv.) in ACN (20 mL) was added K2CO3 (570 mg, 4.13 mmol, 3.0 equiv.), Cs2CO3 (13 mg, 0.04 mmol, 0.03 equiv.), and NaI (103 mg, 0.69 mmol, 0.5 equiv.). The mixture was stirred at 80 °C for 16 h. The mixture was concentrated. The residue was purified by column chromatography on silica gel (MeOH / DCM = 1 / 10) to give the title compound 4-3 (265 mg, 56.0% yield) as a pale yellow oil. LCMS: Rt: 0.840 min; MS m / z (ESI): 344.1 [M+H] + .

[0403] Step 3: Preparation of Compound 4 To a solution of compound 4-3 (198 mg, 0.58 mmol, 1.0 equiv) and compound 6-3 (300 mg, 0.58 mmol, 1.0 equiv) in THF (10 mL) was added DIPEA (223 mg, 1.73 mmol, 3.0 equiv) and NaI (43 mg, 0.29 mmol, 0.5 equiv). The mixture was stirred at 70 °C for 16 h. LCMS showed the reaction was complete. The mixture was concentrated and purified by preparative HPLC to give the title compound 4 (27 mg, 5.91% yield) as a colorless oil.

[0404] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.26-1.32 (m, 34H), 1.41-1.49 (m, 4H), 1.61-1.66 (m, 15H), 2.00-2.03 (m, 1H), 2.21-2.38 (m, 8H), 2.43-2.47 (m, 4H), 2.56-2.60 (m, 2H), 3.50-3.54 (m, 2H), 4.03-4.14 (m, 8H). LCMS: Rt: 1.030 min; MS m / z (ESI): 798.0 [M+H] + .

[0405] 6.4 Example 4: Preparation of Compound 6. [ka] Step 1: Preparation of compound 6-2 To a solution of compound 6-1 (1.0 g, 9.43 mmol, 1.0 equiv) and compound W (1.6 g, 8.48 mmol, 0.9 equiv) in DCM (30 mL) was added EDCI (2.17 g, 11.31 mmol, 1.2 equiv), DIPEA (3.65 g, 28.28 mmol, 3.0 equiv), and DMAP (230 mg, 1.89 mmol, 0.2 equiv). The mixture was stirred at 35 °C for 16 h. The mixture was poured into water and extracted with DCM. The combined organic layers were washed with brine, dried over NaSO, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 1 / 0 to 0 / 1) to give the title compound 6-2 (641 mg, 24.0% yield) as a colorless oil.

[0406] Step 2: Preparation of compound 6-3 To a solution of compound 6-2 (641 mg, 2.26 mmol, 0.9 equiv) and compound R (815 mg, 5.65 mmol, 2.5 equiv) in DCM (10 mL) was added EDCI (1.03 g, 5.65 mmol, 2.5 equiv), DIPEA (1.5 g, 11.30 mmol, 5.0 equiv), and DMAP (83 mg, 0.68 mmol, 0.3 equiv). The mixture was stirred at 35 °C for 16 h. The mixture was poured into water and extracted with DCM. The combined organic layers were washed with brine, dried over NaSO, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 1 / 0 to 5 / 1) to give the title compound 6-3 (510 mg, 42.07% yield) as a colorless oil.

[0407] Step 3: Preparation of Compound 6 To a solution of compound 6-3 (501 mg, 0.94 mmol, 1.0 equiv) in ACN (10 mL) was added compound S (23 mg, 0.37 mmol, 0.4 equiv), K2CO3 (387 mg, 0.81 mmol, 3.0 equiv), Cs2CO3 (9 mg, 0.03 mmol, 0.03 equiv), and NaI (70 mg, 0.47 mmol, 0.5 equiv) at room temperature. The mixture was stirred at 70 °C overnight. The mixture was washed with water and brine, and the organic layer was concentrated and purified by preparative HPLC to give the title compound 6 (112 mg, 12.34% yield) as a colorless oil.

[0408] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H), 1.28-1.32 (m, 42H), 1.42-1.47 (m, 4H), 1.62-1.65 (m, 6H), 1.97-2.01 (m, 1H), 2.29-2.48 (m, 18H), 2.53-2.58 (m, 2H), 3.50-3.54 (m, 2H), 4.12-4.14 (m, 12H). LCMS: Rt: 1.055 min; MS m / z (ESI): 970.8 [M+H] + .

[0409] 6.5 Example 5: Preparation of Compound 7. [ka] Step 1: Preparation of compound 7-2 To a suspension of compound 7-1 (9.0 g, 69.15 mmol, 1.0 equiv) in THF (300 mL) was added LiAlH (2.62 g, 69.15 mmol, 1.0 equiv). The resulting mixture was stirred at room temperature for 2 h. The mixture was poured into water and extracted with EA. The combined organic layers were washed with brine, dried over NaSO, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA=1 / 1) to give the title compound 7-2 (6.0 g, 65% yield) as a yellow oil.

[0410] Step 2: Preparation of compound 7-3 To a suspension of compound 7-2 (6.0 g, 45.4 mmol, 1.0 equiv) in THF (300 mL) was added NaH (3.63 g, 90.8 mmol, 2.0 equiv). The resulting mixture was stirred at room temperature for 10 h. Then, compound M1 (20.8 g, 90.8 mmol, 2.0 equiv) was added dropwise, and the resulting mixture was stirred at 60 °C for 10 h. After cooling to room temperature, the mixture was poured into water and extracted with EA. The combined organic layers were washed with brine, dried over Na SO and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 4 / 1) to give the title compound 7-3 (3.0 g, 23% yield) as a yellow oil.

[0411] Step 3: Preparation of compound 7-4 To a solution of compound 7-3 (3.0 g, 10.7 mmol, 1.0 equiv) in THF (20 mL) was added HCl (aq) (20 mL, 60 mmol, 5.6 equiv). The mixture was stirred at room temperature for 3 h. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over NaSO, and concentrated to give the title compound 7-4 (2.2 g, 86% yield) as a yellow oil.

[0412] Step 4: Preparation of compound 7-5 To a solution of compound 7-4 (2.2 g, 9.16 mmol, 1.0 equiv) and compound R (3.96 g, 27.47 mmol, 3.0 equiv) in DCM (150 mL) was added DIEA (5.92 g, 45.783 mmol, 5.0 equiv), EDCI (5.27 g, 27.47 mmol, 3.0 equiv), and DMAP (1.12 g, 9.16 mmol, 1.0 equiv). The mixture was stirred at 45 °C for 10 h. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 10:1) to give the title compound 7-5 (4.0 g, 88% yield) as a colorless oil.

[0413] Step 5: Preparation of compound 7-6 To a solution of compound 7-5 (3.8 g, 7.71 mmol, 1.0 equiv) in EtOAc (150 mL) was added Pd / C (1.0 g). The mixture was stirred at room temperature under H for 2 h. The mixture was filtered through a pad of Celite and washed with MeOH. The filtrate was concentrated to give the title compound 7-6 (5.1 g, 58% yield) as a yellow oil.

[0414] Step 6: Preparation of compound 7-7 To a solution of compound 7-6 (2.0 g, 4.97 mmol, 1.0 equiv) and DIPEA (1.28 g, 9.94 mmol, 2.0 equiv) in DCM (50 mL) was added MsCl (680 mg, 5.96 mmol, 1.2 equiv). The mixture was stirred at room temperature for 2 h. The mixture was poured into water and extracted with DCM. The combined organic layer was washed with brine, dried over NaSO, and concentrated to give the title compound 7-7 (2.39 g, 100% yield) as a yellow oil, which was used in the next step without further purification.

[0415] Step 7: Preparation of Compound 7 To a solution of compound 7-7 (2.36 g, 4.91 mmol, 3.0 equiv.) and compound S (100 mg, 1.64 mmol, 1.0 equiv.) in ACN (30 mL) was added K2CO3 (680 mg, 4.91 mmol, 3.0 equiv.), Cs2CO3 (160 mg, 0.49 mmol, 0.3 equiv.), and NaI (75 mg, 0.49 mmol, 0.3 equiv.). The mixture was stirred at 80 °C for 10 h. LCMS showed the reaction was complete. The reaction mixture was concentrated and purified by column chromatography on silica gel (DCM / MeOH = 20 / 1) to give the title compound 7 (600 mg, 48% yield) as a yellow oil. LCMS: Rt: 1.087 min; MS m / z (ESI): 830.6 [M+H] + Compound 7 (150 mg) was then purified by preparative HPLC to give the desired product as a yellow oil (13 mg, yield: 9%).

[0416] 1H NMR (400 MHz, CDCl3) δ: 0.77-0.83 (m, 12H), 1.21-1.36 (m, 33H), 1.51-1.71 (m, 14H), 2.24-2.27 (m, 8H), 2.54-2.56 (m, 4H), 3.53-3.63 (m, 8H), , 4.01-4.15 (m, 8H). LCMS: Rt: 0.970 min; MS m / z (ESI): 830.6 [M+H] + .

[0417] The following compounds were prepared in a similar manner to compound 7 using the corresponding starting materials. [Table 4]

[0418] 6.6 Example 6: Preparation of Compound 8. [ka] Step 1: Preparation of compound 8-1 To a solution of compound 7-7 (1.19 g, 2.48 mmol, 1.0 equiv.) and compound S (450 mg, 7.43 mmol, 3.0 equiv.) in ACN (30 mL) was added K2CO3 (1.03 g, 7.43 mmol, 3.0 equiv.), Cs2CO3 (242 mg, 0.74 mmol, 0.3 equiv.), and NaI (115 mg, 0.74 mmol, 0.3 equiv.). The mixture was stirred at 80 °C for 10 h. LCMS showed that the reaction was complete. The reaction mixture was concentrated and purified by column chromatography on silica gel (DCM / MeOH = 20 / 1) to give the title compound 8-1 (650 mg, 59% yield) as a yellow oil. LCMS: Rt: 0.787 min; MS m / z (ESI): 446.4 [M+H] + .

[0419] Step 2: Preparation of Compound 8 To a solution of compound 8-1 (300 mg, 0.67 mmol, 1.0 equiv) and compound 8-2 (240 mg, 0.67 mmol, 1.0 equiv) in ACN (10 mL) was added K2CO3 (280 mg, 2.20 mmol, 3.2 equiv), Cs2CO3 (70 mg, 0.22 mmol, 0.3 equiv), and NaI (30 mg, 0.22 mmol, 0.3 equiv). The mixture was stirred at 80 °C for 10 h. LCMS showed the reaction was complete. The mixture was concentrated and purified by preparative HPLC to give the title compound 8 (30 mg, 14% yield) as a yellow oil.

[0420] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.26-1.74 (m, 47H), 2.28-2.34 (m, 6H), 2.46-2.61 (m, 6H), 3.54-3.69 (m, 5H), 4.04-4.21 (m, 6H). LCMS: Rt: 0.967 min; MS m / z (ESI): 714.5 [M+H] + .

[0421] 6.7 Example 7: Preparation of Compound 9. [ka] Step 1: Preparation of compound 9-1 To a solution of compound 7 (250 mg, 0.3 mmol, 1.0 equiv) in DCM (10 mL) was added SOCl (110 mg, 0.90 mmol, 3.0 equiv). The mixture was stirred at 35 °C for 10 h. The mixture was concentrated to give the title compound 9-1 (255 mg, 100% yield) as a yellow oil. LCMS: Rt: 0.560 min; MS m / z (ESI): 847.9 [M+H] + .

[0422] Step 2: Preparation of Compound 9 To a solution of compound 9-1 (200 mg, 0.23 mmol, 1.0 equiv.) and compound B (55 mg, 0.47 mmol, 2.0 equiv.) in THF (10 mL) was added DIPEA (95 mg, 0.71 mmol, 3.0 equiv.) and NaI (36 mg, 0.23 mmol, 1.0 equiv.). The mixture was stirred at 70° C. for 10 hours. LCMS showed that the reaction was complete. The mixture was concentrated and purified by preparative HPLC to give the title compound 9 (30 mg, 14% yield) as a yellow oil.

[0423] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H), 1.28-1.30 (m, 33H), 1.58-2.01 (m, 18H), 2.30-2.54 (m, 18H), 3.10-3.19 (m, 1H), 3.52-3.68 (m, 8H), 4.09-4.20 (m, 8H). LCMS: Rt: 1.677 min; MS m / z (ESI): 927.7 [M+H] + .

[0424] The following compounds were prepared in a similar manner to compound 9 using the corresponding starting materials. [Table 5]

[0425] 6.8 Example 8: Preparation of Compound 10. [ka] To a solution of compound 1-2 (310 mg, 0.65 mmol, 1.0 equiv) and compound M2 (260 mg, 0.65 mmol, 1.0 equiv) in ACN (10 mL) was added K2CO3 (269 mg, 1.95 mmol, 3.0 equiv), Cs2CO3 (65 mg, 0.20 mmol, 0.3 equiv), and NaI (30 mg, 0.20 mmol, 0.3 equiv). The mixture was stirred at 70 °C for 16 h. LCMS showed that the reaction was complete. The mixture was poured into water and extracted with EA. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC to give the title compound 10 (60 mg, 12% yield) as a yellow oil.

[0426] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H), 1.26-1.41 (m, 48H), 1.51-1.72 (m, 11H), 1.94-2.03 (m, 1H), 2.29-2.32 (m, 6H), 2.41-2.91 (m, 5H), 3.51-3.76(m, 2H), 3.96-4.10 (m, 6H). LCMS: Rt: 1.327 min; MS m / z (ESI): 782.6 [M+H] + .

[0427] The following compounds were prepared in a similar manner to compound 10 using the corresponding starting materials. [Table 6]

[0428] 6.9 Example 9: Preparation of Compound 12. [ka] Step 1: Preparation of compound 12-2 To a solution of compound 12-1 (3 g, 12.59 mmol, 1.0 equiv.) and compound M3 (7.2 g, 26.44 mmol, 2.1 equiv.) in DCM (70 mL) was added EDCI (7.2 g, 37.77 mmol, 3.0 equiv.), DMAP (0.46 g, 3.777 mmol, 0.3 equiv.), and DIEA (8.1 g, 62.95 mmol, 5.0 equiv.). The mixture was stirred at 55 °C for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated and purified by column chromatography on silica gel to give the title compound 12-2 (5.3 g, 56.64% yield) as a yellow oil.

[0429] Step 2: Preparation of compound 12-3 To a solution of compound 12-2 (5.3 g, 7.131 mmol, 1.0 equiv) in EA (100 mL) was added Pd / C (600 mg) and concentrated HCl (3 drops). The mixture was stirred at room temperature under H for 16 h. The mixture was filtered through a pad of Celite and washed with EA. The filtrate was concentrated and purified by column chromatography on silica gel (PE / EA = 3 / 1) to give the title compound 12-3 (3.7 g, 79.45% yield) as a yellow oil.

[0430] Step 3: Preparation of compound 12-4 To a solution of compound 12-3 (3.7 g, 5.67 mmol, 1.0 equiv) and DIPEA (1.5 g, 11.3 mmol, 2.0 equiv) in DCM (50 mL) at 0 °C was added MsCl (0.78 g, 6.8 mmol, 1.2 equiv). The mixture was stirred for an additional 2 h. The mixture was poured into water and extracted with DCM. The combined organic layers were washed with brine, dried over Na SO and concentrated to give the title compound 12-4 (4 g, 96%) as a yellow oil.

[0431] Step 4: Preparation of Compound 12 To a solution of compound 12-4 (300 mg, 0.41 mmol, 1.0 equiv) and compound SM16 (200 mg, 0.45 mmol, 1.1 equiv) in ACN (15 mL) was added K2CO3 (170 mg, 1.23 mmol, 3.0 equiv), Cs2CO3 (39 mg, 0.12 mmol, 0.3 equiv), and NaI (18 mg, 0.12 mmol, 0.3 equiv). The mixture was stirred at 80 °C for 16 h. LCMS showed the reaction was complete. The reaction mixture was concentrated and purified by preparative HPLC to give the title compound 12 (130 mg, 30.18%) as a yellow oil.

[0432] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.89 (m, 18H), 1.25-1.35 (m, 53H), 1.41-1.48 (m, 8H), 1.56-1.61 (m, 20H), 1.95-2.01 (m, 2H), 2.28-2.35 (m, 6H), 2.43-2.46 (m, 4H), 2.56-2.58 (m, 2H), 3.51-3.54 (m, 2H), 4.00-4.10 (m, 8H). LCMS: Rt: 0.080 min;MS m / z (ESI): 1050.8 [M+H] + .

[0433] 6.10 Example 10: Preparation of Compound 15. [ka] To a solution of compound 15-1 (600 mg, 1.47 mmol, 3.0 equiv) in ACN (10 mL) was added compound S (30 mg, 0.49 mmol, 1.0 equiv), K2CO3 (203 mg, 1.47 mmol, 3.0 equiv), Cs2CO3 (47 mg, 0.15 mmol, 0.3 equiv), and NaI (21 mg, 0.15 mmol, 0.3 equiv). The mixture was stirred at 80 °C for 10 h. LCMS showed the reaction was complete. The mixture was poured into water and extracted with EA. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC to give the title compound 15 (30 mg, 2.7% yield) as a colorless oil.

[0434] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H), 1.28-1.63 (m, 67H), 2.66-2.84 (m, 5H), 3.40-3.43 (m, 8H), 3.53-3.66 (m, 1H), 4.43-4.46 (m, 2H). LCMS: Rt: 1.590 min; MS m / z (ESI): 742.7 [M+H] + .

[0435] The following compounds were prepared in a similar manner to compound 15 using the corresponding starting materials. [Table 7]

[0436] 6.11 Example 11: Preparation of Compound 20. [ka] Step 1: Preparation of compound 20-3 To a solution of compound 20-1 (10 g, 55.2 mmol, 1.0 equiv.) and compound 20-2 (14.1 g, 55.2 mmol, 1.0 equiv.) in toluene (100 mL) was added TsOH (2 g). The mixture was stirred at reflux in a separatory oven for 2 h. The mixture was concentrated and purified by FCC to give the title compound (18 g, 77.73%) as a colorless oil.

[0437] Step 2: Preparation of Compound 20 To a solution of compound 20-3 (570 mg, 1.35 mmol, 3.0 equiv) and compound SM16 (200 mg, 0.45 mmol, 1.0 equiv) in ACN (10 mL) was added K2CO3 (190 mg, 1.35 mmol, 3.0 equiv), Cs2CO3 (46 mg, 0.14 mmol, 0.3 equiv), and NaI (21 mg, 0.14 mmol, 0.3 equiv). The mixture was stirred at 80 °C for 16 hours. LCMS showed that the reaction was complete. The reaction mixture was concentrated and purified by preparative HPLC to give the title compound (80 mg, 22.73%) as a yellow oil.

[0438] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.25-1.36 (m, 48H), 1.41-1.48 (m, 5H), 1.60-1.62 (m, 8H), 1.97-2.00 (m, 1H), 2.27-2.32 (m, 6H), 2.43-2.46 (m, 4H), 2.56-2.59 (m, 2H), 3.52-3.54 (m, 2H), 4.01-4.10 (m, 6H). LCMS: Rt: 0.093 min; MS m / z (ESI): 782.6 [M+H] + .

[0439] The following compounds were prepared in a similar manner to compound 20 using the corresponding starting materials. [Table 8-1] [Table 8-2] [Table 8-3]

[0440] 6.12 Example 12: Preparation of Compound 26. [ka] To a solution of compound 12-4 (330 mg, 0.48 mmol, 1.0 equiv) and compound SM17 (190 mg, 0.48 mmol, 1.0 equiv) in ACN (10 mL) was added K2CO3 (200 mg, 1.44 mmol, 3.0 equiv), Cs2CO3 (46 mg, 0.14 mmol, 0.3 equiv), and NaI (21 mg, 0.14 mmol, 0.3 equiv). The mixture was stirred at 80 °C for 16 h. LCMS showed the reaction was complete. The reaction mixture was concentrated and purified by preparative HPLC to give the title compound (55 mg, 11.39%) as a yellow oil.

[0441] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.89 (m, 18H), 1.26-1.36 (m, 70H), 1.43-1.46 (m, 9H), 1.56-1.67 (m, 7H), 1.97-2.00 (m, 1H), 2.15-2.19 (m, 2H), 2.29-2.36 (m, 2H), 2.43-2.46 (m, 4H), 2.56-2.58 (m, 2H), 3.17-3.19 (m, 2H), 3.51-3.54 (m, 2H), 4.02-4.10 (m, 4H), 5.48 (s, 1H). LCMS: Rt: 0.093 min;MS m / z (ESI): 1005.9 [M+H] + .

[0442] 6.13 Example 13: Preparation of Compound 27. [ka] Step 1: Preparation of compound 27-1 To a solution of compound 1-2 (1.3 g, 2.72 mmol, 3.0 equiv.) and compound M4 (160 mg, 0.91 mmol, 1.0 equiv.) in ACN (15 mL) was added K2CO3 (380 mg, 2.72 mmol, 3.0 equiv.), Cs2CO3 (90 mg, 0.27 mmol, 0.3 equiv.), and NaI (40 mg, 1.04 mmol, 0.3 equiv.). The mixture was stirred at 80 °C for 16 h. The reaction mixture was concentrated and purified by column chromatography on silica gel to give the title compound 27-1 (700 mg, 81.89% yield) as a yellow oil.

[0443] Step 2: Preparation of compound 27-2 To a solution of compound 27-1 (700 mg, 0.75 mmol, 1.0 equiv) in DCM (10 mL) was added TFA (3 mL) at room temperature. The mixture was stirred for 16 hours. The mixture was concentrated to give the title compound 27-2 (700 mg, crude) as a yellow oil.

[0444] Step 3: Preparation of compound 27-3 A mixture of compound 27-2 (700 mg, 0.75 mmol, 1.0 equiv) and compound M5 (640 mg, 3.75 mmol, 5.0 equiv) in EtOH (15 mL) was stirred at 40° C. for 16 hours. LCMS showed the reaction was complete. The mixture was used in the next step without further workup. LCMS: Rt: 0.827 min; MS m / z (ESI): 963.7 [M+H] + .

[0445] Step 4: Preparation of compound 27 To a stirred solution of compound 27-3 (700 mg, 0.75 mmol, 1.0 equiv.) was added compound M6 (5 mL, 10 mmol, 13.0 equiv.). The mixture was stirred at 40° C. for 16 hours. LCMS showed that the reaction was complete. The reaction mixture was concentrated and purified by preparative HPLC to give the title compound 27 (120 mg, 18.36% yield) as a yellow oil.

[0446] 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H), 1.28-1.42 (m, 42H), 1.59-1.74 (m, 12H), 1.95-2.00 (m, 2H), 2.29-2.32 (m, 8H), 2.41-2.44 (m, 4H), 2.59-2.61 (m, 2H), 3.21 (s, 6H), 3.86-3.90 (m, 2H), 4.00-4.11 (m, 8H), 7.44-7.46 (s, 1H). LCMS: Rt:0.093min;MS m / z (ESI): 962.6 [M+H] + .

[0447] The following compounds were prepared in a similar manner to compound 27 using the corresponding starting materials. [Table 9-1] [Table 9-2]

[0448] 6.14 Example 14: Preparation of Compound 64 [ka] Step 1: Preparation of compound 64-2 To a solution of 64-1 (1.0 g, 8.3 mmol, 1.0 equiv) in pyridine (10 mL) was added octanoyl chloride (2.97 g, 18.26 mmol, 2.2 equiv). The mixture was stirred at 0 °C for 1 h. TLC showed the reaction was complete. The reaction mixture was poured into water and extracted with EA. The combined organic layers were washed with brine, dried over Na SO and concentrated. The residue was purified by silica gel column chromatography (PE / EA=9 / 1) to give the title compound 64-2 (2.4 g, 77.77% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ: 0.80-0.85 (m, 6H), 0.90-0.95 (s, 3H), 1.20-1.40 (m, 16H), 1.50-1.72 (m, 4H), 2.25-2.38 (m, 4H), 2.40-2.65 (m, 1H), 3.31-3.41 (m, 2H), 3.90-4.10 (m, 4H).

[0449] Step 2: Preparation of compound 64-3 To a solution of 64-2 (1.0 g, 2.6 mmol, 1.0 equiv) and TEA (0.4 g, 4.0 mmol, 1.5 equiv) in DCM (50 mL) was added 6-bromohexanoyl chloride (0.85 g, 4.02 mmol, 1.5 equiv). The mixture was stirred at 0 °C for 1 h. TLC showed the reaction was complete. The reaction mixture was poured into water and extracted with EA. The combined organic layers were washed with brine, dried over Na SO and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 9 / 1) to give the title compound 64-3 (1.2 g, 83.91% yield) as a yellow oil.

[0450] Step 3: Preparation of compound 64 To a solution of 64-3 (462 mg, 0.84 mmol, 2.5 equiv) and 2-aminoethanol (21 mg, 0.34 mmol, 1.0 equiv) in ACN (20 mL) was added K2CO3 (116 mg, 0.84 mmol, 2.5 equiv) and NaI (26 mg, 0.17 mmol, 0.5 equiv). The reaction mixture was stirred at 80 °C for 18 h. LCMS showed the reaction was complete. The reaction mixture was poured into water and extracted with EA. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC to give the title compound 64 (20 mg, 5.97% yield) as a yellow oil.

[0451] 1H NMR (400 MHz, CDCl3) δ: 0.80-0.90 (m, 13H), 1.014 (s, 6H), 1.20-1.30 (m, 37H), 1.50-1.72 (m, 16H), 2.25-2.30 (m, 12H), 2.40-2.65 (m,4H), 3.61-3.64 (m, 2H), 3.90-4.10 (m, 12H). LCMS: Rt: 1.059 min;MS m / z (ESI): 998.8 [M+H] + .

[0452] The following compounds were prepared in a similar manner to compound 64 using the corresponding starting materials. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0453] 6.15 Example 15: Preparation of Compound 76 [ka] Step 1: Preparation of compound 76-2 A mixture of 76-1 (2.7 g, 16.8 mmol, 1.0 equiv.) and 1,2-di(pyridin-2-yl)disulfane (7.4 g, 33.7 mmol, 2.0 equiv.) in MeOH (50 ml) was stirred at room temperature overnight. TLC showed that 76-1 had been consumed. The mixture was concentrated, and the residue was diluted with DCM, washed with water and brine, dried, and concentrated. The residue was purified by FCC to give the title product (3.2 g, 74.6% yield) as a colorless oil.

[0454] Step 2: Preparation of compound 76-3 A mixture of 76-2 (3.1 g, 11.5 mmol, 1.0 equiv.) and 2-mercaptoethanol (1.8 g, 23.0 mmol, 2.0 equiv.) in MeOH (50 ml) was stirred at room temperature overnight. TLC showed that 76-2 had been consumed. The mixture was concentrated, and the residue was diluted with DCM, washed with water and brine, dried, and concentrated. The residue was purified by FCC to give the title product (2.3 g, 89.9% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ: 0.87-0.90 (m, 3H), 1.28-1.40 (m, 10H), 1.64-1.72 (m, 2H), 2.01 (s, 1H), 2.69-2.73 (m, 2H), 2.84-2.87 (m, 2H), 3.87-3.91 (m, 2H).

[0455] Step 3: Preparation of compound 76-4 To a mixture of 76-3 (1.2 g, 5.4 mmol, 1.0 equiv.) and TEA (1.1 g, 10.8 mmol, 2.0 equiv.) in DCM (30 ml), 6-bromohexanoyl chloride (1.4 g, 6.5 mmol, 1.2 equiv.) was added and stirred at room temperature for 2 h. TLC showed that 76-3 had been consumed. The mixture was diluted with DCM, washed with water and brine, dried, and concentrated. The residue was purified by FCC to give the title product (2.0 g, 94% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ: 0.87-0.90 (m, 3H), 1.25-1.40 (m, 12H), 1.47-1.54 (m, 2H), 1.67-1.72 (m, 4H), 1.85-1.92 (m, 2H), 2.33-2.37 (m, 2H), 2.68-2.72 (m, 2H), 2.88-2.92 (m, 2H), 3.39-3.43 (m, 2H), 4.32-4.35 (m, 2H).

[0456] Step 4: Preparation of compound 76-5 To a solution of 5-aminopentan-1-ol (7.76 g, 75.2 mmol, 3.0 equiv) in ACN (50 mL) was added 1-2 (12 g, 25.06 mmol, 1.0 equiv), K2CO3 (10.39 g, 75.2 mmol, 3.0 equiv), Cs2CO3 (2.45 g, 7.52 mmol, 0.3 equiv), and NaI (1.13 g, 7.52 mmol, 0.3 equiv). The mixture was stirred at 80 °C for 16 h. TLC showed the reaction was complete. The mixture was poured into water and extracted with EA. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography (DCM / MeOH = 30 / 1) to give the title compound 76-5 (8.0 g, 22.3% yield) as a yellow oil.

[0457] Step 5: Preparation of Compound 76 A mixture of 76-4 (300 mg, 0.8 mmol, 1.0 equiv), 76-5 (364 mg, 0.8 mmol, 1.0 equiv), KCO (210 mg, 1.5 mmol, 2.0 equiv), and NaI (60 mg, 0.4 mmol, 0.5 equiv) in ACN (20 ml) was stirred at 80 °C overnight. LCMS showed the title product. The mixture was diluted with EA, washed with water and brine, dried, and concentrated. The residue was purified by preparative HPLC to give the title product (45 mg, 9.2% yield) as a colorless oil.

[0458] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.28-1.54 (m, 42H), 1.62-1.70 (m, 13H), 1.94-2.03 (m, 1H), 2.28-2.41 (m, 12H), 2.68-2.73 (m, 2H), 2.2.88-2.91 (m, 2H), 3.63-3.65 (m, 2H), 4.00-4.10 (m, 4H), 4.31-4.35 (m, 2H). LCMS: Rt:0.910 min;MS m / z (ESI): 804.6 [M+H] + .

[0459] 6.16 Example 16: Preparation and Characterization of Lipid Nanoparticles Briefly, the cationic lipids provided herein, DSPC, cholesterol, and PEG lipids were solubilized in ethanol at a molar ratio of 50:10:38.5:1.5, and mRNA was diluted with 10-50 mM citrate buffer (pH = 4). Using a microfluidic device at a total flow rate of 9-30 mL / min, LNPs with total lipid to mRNA weight ratios of approximately 10:1-30:1 were prepared by mixing the ethanolic lipid solution with the aqueous mRNA solution at a volume ratio of 1:3. Dialysis was used to remove the ethanol and replace it with DPBS. Finally, the lipid nanoparticles were filtered through a 0.2 µm sterile filter.

[0460] The size of lipid nanoparticles was determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK) using backscatter detection mode at 173°. The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification assay kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions.

[0461] As reported in the literature, the apparent pKa of an LNP formulation correlates with the in vivo delivery efficiency of the LNP for nucleic acids. The apparent pKa of each formulation was determined using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). LNP formulations consisting of cationic lipid / DSPC / cholesterol / DMG-PEG (50 / 10 / 38.5 / 1.5 mol%) in PBS were prepared as described above. TNS was prepared as a 300 μM stock solution in distilled water. The LNP formulation was diluted to 0.1 mg / mL total lipid in 3 mL of buffer solution containing 50 mM sodium citrate, 50 mM sodium phosphate, 50 mM sodium borate, and 30 mM sodium chloride, with a pH ranging from 3 to 9. Aliquots of TNS solution were added to give a final concentration of 0.1 mg / mL, and after subsequent vortex mixing, the fluorescence intensity was measured at room temperature in a Molecular Devices Spectramax iD3 spectrometer using excitation and emission wavelengths of 325 nm and 435 nm. A sigmoidal best-fit analysis was applied to the fluorescence data, and the pKa value was determined as the pH resulting in half-maximal fluorescence intensity.

[0462] 6.17 Example 17: Animal Study 1 Lipid nanoparticles containing the compounds listed in the table below encapsulating human erythropoietin (hEPO) mRNA were administered systemically to 6-8 week-old female ICR mice (Xipuer-Bikai, Shanghai) via tail vein injection at a dose of 0.5 mg / kg, and blood samples were collected from the mice at specific time points (e.g., 6 hours) after administration. In addition to the test groups described above, lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, commonly abbreviated as MC3) encapsulating hEPO mRNA were similarly administered at the same dose to a group of mice of comparable age and sex as a positive control.

[0463] After the final sampling time point, mice were euthanized by CO2 overdose. Serum was separated from whole blood by centrifugation at 5000g for 10 minutes at 4°C, flash-frozen, and stored at -80°C for analysis. ELISA assays were performed using a commercially available kit (DEP00, R&D systems) according to the manufacturer's instructions.

[0464] The properties of the lipid nanoparticles tested, including the EPO expression levels for MC3 measured from the test groups, are listed in the table below. [Table 11-1] [Table 11-2]

[0465] 6.18 Example 18: Animal Study 2 Lipid nanoparticles containing the compounds listed in the table below encapsulating luciferase-encoding mRNA were administered systemically to 6-8-week-old female Balb / c mice (Charles River Lab, Zhejiang) via tail vein injection at a dose of 0.25 mg / kg. Blood samples were collected from the mice at specific time points (e.g., 6 hours) after administration. Optical imaging was performed using an IVIS Spectrum CT system (PerkinElmer Inc., Paris, France). Luminescence levels were assessed by applying an ROI to the injection site area (Living Image software, PerkinElmer Inc., Paris, France). In addition to the test groups described above, lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (also known as DLin-MC3-DMA or MC3) encapsulating luciferase mRNA were administered at the same dose to a group of mice of similar age and gender as a positive control.

[0466] Six hours after administration, the brightness of the heart, liver, spleen, lungs, kidneys, brain, and bone marrow was measured. The dissected organs were placed on a black sheet and imaged using an IVIS Spectrum CT (PerkinElmer, Hopkinton, MA). To quantify the bioluminescence signal, identical regions of interest (ROIs) were placed around each organ area, and the imaging signal was quantified as total flux [p / s]. Organ data for nanoparticles containing compound 76 are summarized in Table 4. As shown in Table 4, significant gene transfer and luciferase expression were observed in the liver and spleen, and LNPs also showed high absolute expression in the heart, spleen, lungs, kidneys, and brain.

[0467] The properties of the lipid nanoparticles tested, including the luciferase expression levels for MC3 measured from the test groups, are listed in the table below. [Table 12]

[0468] Organ bioluminescence data for nanoparticles containing compound 76 are summarized in the table below. [Table 13]

[0469] 6.19 Example 19: Lipid Clearance Studies LNP was injected into mice (ICR females, IV, 0.5 mg mRNA / kg) via the tail vein. Mice were then anesthetized under carbon dioxide at different times (e.g., 6 h, 24 h, and 48 h) after administration and sacrificed via cardiac puncture. Liver tissue was immediately collected and then washed with ice-cold saline. Liver samples were weighed and homogenized in an ice-water bath by adding pre-cooled 20% methanol-water (v / v) at a ratio of 1:5 (w / v) at 2-8 °C. Homogenized tissue samples were stored in a freezer at -90 to -60 °C before analysis.

[0470] Sample processing. All liver tissue homogenate samples were thawed at room temperature. A 50 μL aliquot of the sample was added with 50 μL of MgCl2 (2 M), followed by verapamil, 5 ng mL, for protein precipitation. -1 and glibenclamide, 50 ng·mL -1 and diclofenac, 200 ng·mL -1 and tolbutamide, 200 ng·mL -1 The mixture was added with ACN containing 100 μL of HCl and centrifuged at 13,000 rpm for 8 minutes. 100 μL of the supernatant was then added with 100 μL of water and vortexed thoroughly. 5 μL of the mixture was injected into the LC-MS / MS system.

[0471] The results for MC3 and selected lipid compounds provided herein are listed in the table below. [Table 14]

Claims

1. Compounds of formula (I): 【Chemistry 1】 or a pharma- ceutically acceptable salt, prodrug, or stereoisomer thereof, G １ and G ２ are each independently ２ -C ２５ Alkylene or C ２ -C ２５ alkenylene, where G １ and G ２ At least one -CH ２ - is optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L １ is independently -OC(=O)R １ , -C(=O)OR １ , -OC(=O)OR １ , -C(=O)R １ , -OR １ , -S(O) ｘ R １ , -S-SR １ , -C(=O)SR １ , -SC(=O)R １ , -NR ａ C(=O)R １ , -C(=O)NR ｂ R ｃ , -NR ａ C(=O)NR ｂ R ｃ , -OC(=O)NR ｂ R ｃ , -NR ａ C(=O)OR １ , -SC(=S)R １ , -C(=S)SR １ , -C(=S)R １ , -CH(OH)R １ , -P(=O)(OR ｂ )(OR ｃ ), -NR ａ P(=O)(OR ｂ )(OR ｃ ) and Each L ２ is, independently, -OC(=O)R ２ , -C(=O)OR ２ , -OC(=O)OR ２ , -C(=O)R ２ , -OR ２ , -S(O) ｘ R ２ , -S-SR ２ , -C(=O)SR ２ , -SC(=O)R ２ , -NR ｄ C(=O)R ２ , -C(=O)NR ｅ R ｆ , -NR ｄ C(=O)NR ｅ R ｆ , -OC(=O)NR ｅ R ｆ , -NR ｄ C(=O)OR ２ , -SC(=S)R ２ , -C(=S)SR ２ , -C(=S)R ２ , -CH(OH)R ２ , -P(=O)(OR ｅ )(OR ｆ ), -NR ｄ P(=O)(OR ｅ )(OR ｆ ) and is R １ and R ２ are each independently ６ -C ２４ Alkyl or C ６ -C ２４ alkenyl, where R １ and R ２ At least one -CH ２ - is optionally replaced by -S-S-, R ａ , R ｂ , R ｄ , and R ｅ are each independently H, C １ -C ２４ Alkyl, or C ２ -C ２４ alkenyl, R ｃ and R ｆ are each independently １ -C ２４ Alkyl or C ２ -C ２４ alkenyl, where R ｃ and R ｆ At least one -CH ２ - is optionally replaced by -S-S-, G ３ is C ２ -C １２ Alkylene or C ２ -C １２ alkenylene, where some or all of the alkylene or alkenylene are C ３ -C ８ Cycloalkylene or C ３ -C ８ optionally replaced by cycloalkenylene; R ３ is -N(R ４ ) R ５ , -OR ６ , or -SR ６ and R ４ is C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ３ -C ８ Cycloalkyl, C ３ -C ８ Cycloalkenyl, C ６ -C １０ aryl, or 4- to 8-membered heterocycloalkyl; R ５ is H, C １ -C １２ Alkyl, C ３ -C ８ Cycloalkyl, C ３ -C ８ Cycloalkenyl, C ６ -C １０ aryl, or 4- to 8-membered heterocycloalkyl; R ６ is hydrogen, C １ -C １２ Alkyl, C ３ -C ８ Cycloalkyl, C ３ -C ８ Cycloalkenyl, or C ６ -C １０ is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

2. Compound of formula (II): 【Chemistry 2】 or a pharma- ceutically acceptable salt, prodrug, or stereoisomer thereof, G １ and G ２ are each independently ２ -C ２５ Alkylene or C ２ -C ２５ alkenylene, where G １ and G ２ At least one -CH ２ - is optionally replaced by -O-, -C(=O)O-, or -OC(=O)-; Each L １ is, independently, -OC(=O)R １ , -C(=O)OR １ , -OC(=O)OR １ , -C(=O)R １ , -OR １ , -S(O) ｘ R １ , -S-SR １ , -C(=O)SR １ , -SC(=O)R １ , -NR ａ C(=O)R １ , -C(=O)NR ｂ R ｃ , -NR ａ C(=O)NR ｂ R ｃ , -OC(=O)NR ｂ R ｃ , -NR ａ C(=O)OR １ , -SC(=S)R １ , -C(=S)SR １ , -C(=S)R １ , -CH(OH)R １ , -P(=O)(OR ｂ )(OR ｃ , -NR ａ P(=O)(OR ｂ )(OR ｃ ) and Each L ２ is, independently, -OC(=O)R ２ , -C(=O)OR ２ , -OC(=O)OR ２ , -C(=O)R ２ , -OR ２ , -S(O) ｘ R ２ , -S-SR ２ , -C(=O)SR ２ , -SC(=O)R ２ , -NR ｄ C(=O)R ２ , -C(=O)NR ｅ R ｆ , -NR ｄ C(=O)NR ｅ R ｆ , -OC(=O)NR ｅ R ｆ , -NR ｄ C(=O)OR ２ , -SC(=S)R ２ , -C(=S)SR ２ , -C(=S)R ２ , -CH(OH)R ２ , -P(=O)(OR ｅ )(OR ｆ ), -NR ｄ P(=O)(OR ｅ )(OR ｆ ) and R １ and R ２ are each independently ６ -C ２４ Alkyl or C ６ -C ２４ alkenyl, where R １ and R ２ At least one -CH ２ - is optionally replaced by -S-S-, R ａ , R ｂ , R ｄ , and R ｅ are each independently H, C １ -C ２４ Alkyl, or C ２ -C ２４ alkenyl, R ｃ and R ｆ are each independently １ -C ２４ Alkyl or C ２ -C ２４ alkenyl, where R ｃ and R ｆ At least one -CH ２ - is optionally replaced by -S-S-, G ３ is C ２ -C １２ Alkylene or C ２ -C １２ alkenylene, where some or all of the alkylene or alkenylene are C ３ -C ８ Cycloalkylene or C ３ -C ８ optionally replaced by cycloalkenylene; R ３ is -N(R ４ ) R ５ , -OR ６ , or -SR ６ and R ４ is C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ３ -C ８ Cycloalkyl, C ３ -C ８ Cycloalkenyl, C ６ -C １０ aryl, or 4- to 8-membered heterocycloalkyl; R ５ is H, C １ -C １２ Alkyl, C ３ -C ８ Cycloalkyl, C ３ -C ８ Cycloalkenyl, C ６ -C １０ aryl, or 4- to 8-membered heterocycloalkyl; R ６ is hydrogen, C １ -C １２ Alkyl, C ３ -C ８ Cycloalkyl, C ３ -C ８ Cycloalkenyl, or C ６ -C １０ is aryl, x is 0, 1, or 2; wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted; However, the compound is 【Chemistry 3】 isn't it).

3. The compound is 【Chemistry 4】 3. The compound of claim 2, further provided that it is not:

4. G ３ But, C ２ -C ４ The compound according to any one of claims 1 to 3, which is an alkylene.

5. G ３ However, (C ０ -C ２ alkylene)-(C ３ -C ８ Cycloalkylene)-(C ０ -C ２ alkylene) or (C ０ -C ２ alkylene)-(C ３ -C ８ Cycloalkenylene)-(C ０ -C ２ The compound according to any one of claims 1 to 3, wherein the aryl group is aryl, ...

6. Compounds of formula (III), (III-A), or (III-B): 【Chemistry 5】 (wherein z is an integer from 2 to 12). or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof.

7. Compounds of formula (IV), (IV-A), or (IV-B): 【Chemistry 6】 (wherein z is an integer from 2 to 12). or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof.

8. 8. The compound of claim 6 or 7, wherein z is 2.

9. G １ and G ２ But independently, C ４ -C １０ The compound according to any one of claims 1 to 8, which is an alkylene.

10. G １ One -CH in ２ The compound according to any one of claims 1 to 9, wherein only - is replaced by -O-, -C(=O)O-, or -OC(=O)-.

11. G ２ One -CH in ２ The compound according to any one of claims 1 to 10, wherein only - is replaced by -O-, -C(=O)O-, or -OC(=O)-.

12. Compound of formula (V): 【Chemistry 7】 (In the formula, X １ and X ２ each independently is a bond, —O—, —C(═O)O—, or —OC(═O)—; z is an integer from 2 to 12; x2 is X １ is an integer from 2 to 12, and x2 is X １ is an integer from 2 to 13 when is a bond; y2 is X ２ is an integer from 2 to 12 when X is -O-, -C(=O)O-, or -OC(=O)-; ２ is an integer from 2 to 13 when is a bond; G ４ and G ５ are each independently ２ -C １２ alkylene) or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof.

13. Compound of formula (VA), (V-B), (V-C), (V-D), (V-E), (V-F), or (V-G): 【Chemistry 8】 (wherein z is an integer from 2 to 12; x0 is an integer from 1 to 11; y0 is an integer from 1 to 11; x1 is an integer from 0 to 9, y1 is an integer from 0 to 9, x2 is an integer from 2 to 12; x3 is an integer from 1 to 5, x4 is an integer from 0 to 3, x5 is an integer from 0 to 5, y2 is an integer from 2 to 12; y3 is an integer from 1 to 5, y4 is an integer from 0 to 3, y5 is an integer from 0 to 5. or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof.

14. The compound according to claim 12 or 13, wherein z is an integer from 2 to 6.

15. 15. The compound of claim 14, wherein z is 2, 4, or 5.

16. The compound according to any one of claims 13 to 15, wherein x0 and y0 are independently 2 to 6.

17. 17. The compound of claim 16, wherein x0 and y0 are independently 4 or 5.

18. The compound according to any one of claims 13 to 15, wherein x1 and y1 are independently 2 to 6.

19. 19. The compound of claim 18, wherein x1 and y1 are independently 4 or 5.

20. The compound according to any one of claims 13 to 15, wherein x2 and y2 are independently an integer from 2 to 12.

21. 21. The compound of claim 20, wherein x2 and y2 are independently 3 or 5.

22. The compound according to any one of claims 13 to 15, 20 and 21, wherein x3 and y3 are both 1.

23. The compound according to any one of claims 13 to 15, 20 and 21, wherein x4 and y4 are independently 0 or 1.

24. Each L １ is independently -OR １ , -OC(=O)R １ Or -C(=O)OR １ and each L ２ are independently -OR ２ , -OC(=O)R ２ Or -C(=O)OR ２ The compound according to any one of claims 1 to 23,

25. 【Chemical 9】 When moieties are present, each L １ are independently -OC(=O)R １ The compound according to any one of claims 13 to 24,

26. [Chemical 10] When moieties are present, each L １ are independently -OR １ The compound according to any one of claims 13 to 24,

27. 【Chemical 11】 When moieties are present, each L ２ are independently -OC(=O)R ２ The compound according to any one of claims 13 to 26,

28. 【Chemical 12】 When moieties are present, each L ２ are independently -OR ２ The compound according to any one of claims 13 to 26,

29. R １ and R ２ Independently, linear C ６ -C １０ Alkyl, or -R ７ -CH(R ８ ) (R ９ ), where R ７ is C ０ -C ５ alkylene, R ８ and R ９ is independently ２ -C １０ Alkyl or C ２ -C １０ The compound according to any one of claims 24 to 28, which is alkenyl.

30. Compound of formula (VI): 【Chemistry 13】 (In the formula, X １ and X ２ each independently is a bond, —O—, —C(═O)O—, or —OC(═O)—; z is an integer from 2 to 12; x2 is X １ is an integer from 2 to 12, and x2 is X １ is an integer from 2 to 13 when is a bond; y2 is X ２ is an integer from 2 to 12 when X is -O-, -C(=O)O-, or -OC(=O)-; ２ is an integer from 2 to 13 when is a bond; G ４ and G ５ are each independently ２ -C １２ alkylene) or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof.

31. Compound of formula (VI-A), (VI-B), (VI-C), (VI-D), (VI-E), (VI-F), or (VI-G): 【Chemistry 14】 (wherein z is an integer from 2 to 12; y is an integer from 2 to 12; x0 is an integer from 1 to 11; x1 is an integer from 0 to 9, x2 is an integer from 2 to 12; x3 is an integer from 1 to 5, x4 is an integer from 0 to 3, x5 is an integer from 0 to 5. or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof.

32. 32. The compound according to claim 30 or 31, wherein z is an integer from 2 to 6.

33. 33. The compound of claim 32, wherein z is 2, 4 or 5.

34. The compound according to any one of claims 31 to 33, wherein x0 is 4 or 5.

35. The compound according to any one of claims 31 to 33, wherein x1 is 4 or 5.

36. The compound according to any one of claims 31 to 33, wherein x2 is an integer from 2 to 12.

37. 37. The compound of claim 36, wherein x2 is 3 or 5.

38. The compound according to any one of claims 31 to 33, 36 and 37, wherein x3 is 0 or 1.

39. The compound according to any one of claims 31 to 38, wherein y is an integer from 2 to 6.

40. 40. The compound of claim 39, wherein y is 5.

41. Each L １ is independently -OR １ , -OC(=O)R １ Or -C(=O)OR １ And L ２ But -OC(=O)R ２ Or -C(=O)OR ２ , -NR ｄ C(=O)R ２ or -C(=O)NR ｅ R ｆ The compound according to any one of claims 30 to 40,

42. 【Catalogue 15】 When moieties are present, each L １ are independently -OC(=O)R １ The compound according to any one of claims 31 to 41,

43. 【Catalog 16】 When moieties are present, each L １ are independently -OR １ The compound according to any one of claims 31 to 41,

44. R １ But linear C ６ -C １０ Alkyl or -R ７ -CH(R ８ ) (R ９ ), where R ７ is C ０ -C ５ alkylene, R ８ and R ９ is independently ２ -C １０ Alkyl or C ２ -C １０ The compound according to any one of claims 41 to 43, which is alkenyl.

45. R ２ and R ｆ each independently represents a linear C ６ -C １８ Alkyl, C ６ -C １８ alkenyl, or -R ７ -CH(R ８ ) (R ９ ), where R ７ is C ０ -C ５ alkylene, R ８ and R ９ is independently ２ -C １０ Alkyl or C ２ -C １０ The compound according to any one of claims 41 to 44, which is alkenyl.

46. R ｄ and R ｅ The compound of any one of claims 41 to 45, wherein each is independently H.

47. R ３ But, -OR ６ The compound according to any one of claims 1 to 46,

48. R ６ 48. The compound of claim 47, wherein is hydrogen.

49. R ６ But, C １ -C ６ 48. The compound of claim 47, which is alkyl.

50. R ６ 50. The compound of claim 49, wherein is substituted with one or more hydroxyls.

51. R ３ But -N(R ４ ) R ５ The compound according to any one of claims 1 to 46,

52. R ４ But, C ３ -C ８ Cycloalkyl or C ３ -C ８ 52. The compound of claim 51 which is a cycloalkenyl.

53. R ４ 53. The compound of claim 51 or 52, wherein is unsubstituted.

54. R ４ is hydroxyl, oxo, -NH ２ , -NH(C １ -C ６ alkyl), or -N(C １ -C ６ Alkyl) ２ 53. The compound of claim 51 or 52, substituted with one or more of:

55. R ５ But, C １ -C ６ The compound according to any one of claims 51 to 54, which is alkyl.

56. R ５ 56. The compound of claim 55, wherein is methyl, ethyl, n-propyl, isopropyl, n-butyl, n-pentyl, or n-hexyl.

57. R ５ 57. The compound of claim 55 or 56, wherein is unsubstituted.

58. R ５ 57. The compound of claim 55 or 56, wherein is substituted with one or more hydroxyls.

59. A compound of Table 1 or Table 1A, or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof.

60. A composition comprising a compound according to any one of claims 1 to 59 and a therapeutic or prophylactic agent.

61. 61. The composition of claim 60, further comprising one or more structured lipids.

62. 61. The composition of claim 60, wherein the one or more structured lipids is DSPC.

63. 62. The composition of claim 60 or 61, wherein the molar ratio of said compound to said structured lipid ranges from about 2:1 to about 8:

1.

64. 64. The composition of any one of claims 60 to 63, further comprising a steroid.

65. 65. The composition of claim 64, wherein the steroid is cholesterol.

66. 66. The composition of claim 64 or 65, wherein the molar ratio of the compound to the steroid ranges from about 5:1 to about 1:

1.

67. 67. The composition of any one of claims 60 to 66, wherein the composition further comprises one or more polymer-conjugated lipids.

68. 68. The composition of claim 67, wherein the polymer-conjugated lipid is DMG-PEG2000 or DMPE-PEG2000.

69. 69. The composition of claim 67 or 68, wherein the molar ratio of said compound to said polymer-conjugated lipid ranges from about 100:1 to about 20:

1.

70. The composition of any one of claims 60 to 69, wherein the therapeutic or prophylactic agent comprises at least one mRNA encoding an antigen or a fragment or epitope thereof.

71. 71. The composition of claim 70, wherein the mRNA is a monocistronic mRNA.

72. 71. The composition of claim 70, wherein the mRNA is a multicistronic mRNA.

73. The composition of any one of claims 70 to 72, wherein the antigen is a pathogenic antigen.

74. The composition according to any one of claims 70 to 72, wherein the antigen is a tumor-associated antigen.

75. 75. The composition of any one of claims 70 to 74, wherein the mRNA comprises one or more functional nucleotide analogues.

76. 76. The composition of claim 75, wherein the functional nucleotide analog is one or more selected from pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

77. The composition of any one of claims 60 to 76, wherein the composition is a nanoparticle.

78. A lipid nanoparticle comprising a compound according to any one of claims 1 to 59 or a composition according to any one of claims 60 to 76.

79. A pharmaceutical composition comprising a compound according to any one of claims 1 to 59, a composition according to any one of claims 60 to 76, or a lipid nanoparticle according to claim 78, and a pharma- ceutically acceptable excipient or diluent.

80. 80. A method of delivering a therapeutic or prophylactic agent to a mammalian cell or organ, comprising contacting said mammalian cell or organ with a composition according to any one of claims 60 to 77, a lipid nanoparticle according to claim 78, wherein said lipid nanoparticle comprises said therapeutic or prophylactic agent, or a pharmaceutical composition according to claim 79, wherein said pharmaceutical composition comprises said therapeutic or prophylactic agent.

Images