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 of related applications This application claims priority to International Patent Application PCT / CN2021 / 122693, filed on 8 October 2021, the entirety of which is incorporated herein by reference.

[0002] 2. Technical Fields This disclosure broadly relates to lipid compounds that can be used in combination with other lipid components such as neutral lipids, cholesterol, and polymer-compounded lipids to form lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acid molecules including nucleic acid mimetic compounds such as Loc nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino) both in vitro and in vivo for therapeutic or preventive 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 through the design of nucleic acid molecules and methods of their delivery. However, nucleic acid therapeutics still face several challenges, including low cell permeability and high sensitivity to the degradation of certain nucleic acid molecules, including RNA. Therefore, there is a need to develop new nucleic acid molecules, as well as related methods and compositions to facilitate their delivery in vitro or in vivo for therapeutic and / or preventive purposes. [Overview of the project]

[0004] 4. Outline of the Invention In one embodiment, as used herein, a lipid compound (including its pharmaceutically acceptable salts, prodrugs, or stereoisomers) is provided that can be used alone or in combination with other lipid components and / or polymers, such as neutral lipids, charged lipids, steroids (including all sterols), and / or their analogs, and / or polymer-conjugated lipids, to form lipid nanoparticles for the delivery of therapeutic agents (e.g., nucleic acid molecules including nucleic acid mimetics such as locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino). In some examples, the lipid nanoparticles are used to deliver nucleic acids such as antisense RNA and / or messenger RNA. Also provided are methods of using such lipid nanoparticles for the treatment of various diseases or disorders, such as those caused by deficiencies in infectious entities and / or proteins.

[0005] In one embodiment, the lipid compound provided herein is a substituted-2-(hydroxymethyl)propane-1,3-diol-based lipid compound.

[0006] In one embodiment, as used herein, formula (I):

Chemical formula

[0007] In one embodiment, as used herein, formula (II):

Chemical formula

[0008] In one embodiment, this specification provides 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.

[0009] Further features of this disclosure will become apparent to those skilled in the art upon consideration of the following detailed description of specific embodiments. 5. Brief description of the drawing [Brief explanation of the drawing]

[0010] [Figure 1] This graph shows the luminescence levels of specific lipid nanoparticles provided herein, which encapsulate luciferase-encoding (luciferase) mRNA. [Modes for carrying out the invention]

[0011] 6. Modes for Carrying Out the Invention 6.1 General techniques The techniques and procedures described or referenced herein are generally understood and / or commonly used by those skilled in the art, using conventional methodologies such as, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001) and Current Protocols in Molecular Biology (Ausubel et al. eds., 2003).

[0012] 6.2 Terminology Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. For the purpose of interpreting this specification, the following definitions of terms apply, and where appropriate, singular terms also include plural forms, and vice versa. All patents, applications, publications, and other publications are incorporated herein by reference. In the event of any conflict between a definition of a term provided herein and any document incorporated herein by reference, the definition provided below shall prevail.

[0013] As used herein, unless otherwise specified, the term “lipids” refers to a group of organic compounds including, but not limited to, esters of fatty acids, generally characterized by being poorly soluble in water but soluble in many nonpolar organic solvents. Lipids are generally poorly soluble in water, but possess limited water solubility, and there are certain categories of lipids (e.g., lipids modified with polar groups, e.g., DMG-PEG2000) that can dissolve in water under certain conditions. Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be classified into at least three categories: (1) “simple lipids” including fats and oils and waxes, (2) “complex lipids” including phospholipids and glycolipids (e.g., DMPE-PEG2000), and (3) “derivative lipids” such as steroids. Furthermore, as used herein, lipids also encompass lipidoid compounds. The term "lipidoid compound" (sometimes simply "lipidoid") refers to a lipid-like compound (for example, an amphiphilic compound that has lipid-like physical properties).

[0014] The terms “lipid nanoparticles” or “LNPs” refer to particles having at least one dimension on the order of nanometers (nm) (e.g., 1 to 1,000 nm) that contain one or more lipid molecules. LNPs provided herein may further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, LNPs include a non-lipid payload molecule partially or completely encapsulated inside a lipid shell. In particular, in some embodiments, the payload is a negatively charged molecule (e.g., mRNA encoding a viral protein), and the lipid components of the LNP include at least one cationic lipid. While not bound by theory, it is thought that cationic lipids can interact with negatively charged payload molecules to 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-complexed lipids, and various zwitterionic lipids. In certain embodiments, the LNP relating to this disclosure comprises one or more lipids of formula (I) or formula (II) (and its subformulas) as described herein.

[0015] The term “cationic lipid” refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or that can be positively charged in response to the pH value or hydrogen ion activity of its environment (e.g., the environment of its intended use). Thus, the term “cationic” encompasses both “permanently cationic” and “cationically catalyzable.” In certain embodiments, the positive charge in a cationic lipid arises from the presence of a quaternary nitrogen atom. In certain embodiments, cationic lipids include amphoteric lipids that are positively charged in their intended use environment (e.g., at physiological pH). In certain embodiments, cationic lipids are one or more lipids of formula (I) or formula (II) (and its subformulas) as described herein.

[0016] The term "polymer-complexed lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-complexed lipid is PEG-lipid, in which the polymer portion contains polyethylene glycol.

[0017] The term "neutral lipid" encompasses any lipid molecules that exist in an uncharged or neutral amphoteric form within a selected pH value or range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment of the intended use of the lipid, such as physiological pH. Examples of neutral lipids that may be used in connection with this 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), and 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), and sphingomyelin (SM); steroids such as ceramides and sterols, and their derivatives. The neutral lipids provided herein may be synthesized or derived (isolated or modified) from natural sources or compounds.

[0018] The term "charged lipid" encompasses any lipid molecule that exists in either a positively charged or uncharged 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 intended use of the lipid, such as physiological pH. Non-limiting examples of charged lipids that may be used in connection with this disclosure include, but are not limited to, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylaluminium-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-phosphorate sodium salt (DOPA-Na). The charged lipids provided herein may be synthesized or derived (isolated or modified) from natural sources or compounds.

[0019] As used herein, unless otherwise specified, the term “alkyl” refers to a linear or branched hydrocarbon chain radical consisting only of saturated carbon and hydrogen atoms. In one embodiment, an alkyl group is, for example, a group of 1 to 24 carbon atoms (C1 to C24). 24 Alkyl), 4 to 20 carbon atoms (C4~C 20 Alkyl), 6-16 carbon atoms (C6-C 16 Alkyl), 6-9 carbon atoms (C6-C9 alkyl), 1-15 carbon atoms (C1-C 15 Alkyl), 1 to 12 carbon atoms (C1 to C 12Alkyl groups have 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl), which are bonded to the rest of the molecule by single bonds. 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, and 2-methylhexyl. Unless otherwise specified, alkyl groups are substituted by choice.

[0020] As used herein, unless otherwise specified, the term “alkenyl” refers to a linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms and 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 those skilled in the art. In one embodiment, the alkyl group is, for example, 2 to 24 carbon atoms (C2 to C2). 24 Alkenyl), 4-20 carbon atoms (C4-C 20 Alkenyl), 6-16 carbon atoms (C6-C6) 16 Alkenyls), 6-9 carbon atoms (C6-C9 alkenyls), 2-15 carbon atoms (C2-C9 alkenyls), 15 Alkenyl), 2 to 12 carbon atoms (C2 to C2) 12 Alkenyl groups have 2 to 8 carbon atoms (C2-C8 alkenyls) or 2 to 6 carbon atoms (C2-C6 alkenyls), which are bonded to the rest of the molecule by single bonds. Examples of alkenyl groups include, but are not limited to, ethenyl, prop-1-enyl, buto-1-enyl, pento-1-enyl, and penta-1,4-dienyl. Unless otherwise specified, alkenyl groups are optionally substituted.

[0021] As used herein, unless otherwise specified, the term "alkynyl" refers to a linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms and containing one or more carbon-carbon triple bonds. In one embodiment, the alkyl group is, for example, 2 to 24 carbon atoms (C2 to C2). 24 Alkynyl), 4 to 20 carbon atoms (C4~C 20 Alkynyl), 6-16 carbon atoms (C6-C6) 16 Alkynyl), 6-9 carbon atoms (C6-C9 alkynyl), 2-15 carbon atoms (C2-C 15 Alkynyl), 2 to 12 carbon atoms (C2 to C2) 12 Alkynyl groups have 2 to 8 carbon atoms (C2-C8 alkynyl) or 2 to 6 carbon atoms (C2-C6 alkynyl), which are bonded to the rest of the molecule by single bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, and pentynyl. Unless otherwise specified, alkynyl groups are optionally substituted.

[0022] As used herein, unless otherwise specified, the terms “alkylene” or “alkylene chain” refer to a linear or branched polyvalent (e.g., divalent or trivalent) hydrocarbon chain in which the rest of the molecule is linked to a radical group (or more groups) consisting only of saturated carbon and hydrogen atoms. In one embodiment, the alkylene is, for example, a chain of 1 to 24 carbon atoms (C1 to C24). 24 Alkylene), 1 to 15 carbon atoms (C1 to C) 15 Alkylene), 1 to 12 carbon atoms (C1 to C 12Alkylenes have 1 to 8 carbon atoms (C1-C8 alkylenes), 1 to 6 carbon atoms (C1-C6 alkylenes), 2 to 4 carbon atoms (C2-C4 alkylenes), or 1 to 2 carbon atoms (C1-C2 alkylenes). Examples of alkylene groups include, but are not limited to, methylene, ethylene, and propylene. Alkylene chains are bonded to the rest of the molecule via single bonds and to radical groups via single bonds. The bonding sites of the alkylene chain to the rest of the molecule and radical groups may be via one carbon or any two (or more) carbon atoms in the chain. Unless otherwise specified, alkylene chains are optionally substituted.

[0023] As used herein, unless otherwise specified, the term “alkenylene” refers to a linear or branched polyvalent (e.g., divalent or trivalent) hydrocarbon chain consisting only of carbon and hydrogen atoms, containing one or more carbon-carbon double bonds, with the rest of the molecule linked to a radical group (or more groups). In one embodiment, the alkyl group is, for example, 2 to 24 carbon atoms (C2 to C2). 24 Alkenylenes), 2 to 15 carbon atoms (C2 to C2) 15 Alkenylenes), 2 to 12 carbon atoms (C2 to C2) 12 Alkenylenes have 2 to 8 carbon atoms (C2-C8 alkenylenes), 2 to 6 carbon atoms (C2-C6 alkenylenes), and 2 to 4 carbon atoms (C2-C4 alkenylenes). Examples of alkenylenes include, but are not limited to, etenylene, propenylene, and n-butenylene. Alkenylenes are bonded to the rest of the molecule via single or double bonds and to radical groups via single or double bonds. The bonding sites of alkenylenes to the rest of the molecule and radical groups may be via one or any two (or more) carbon atoms in the chain. Unless otherwise specified, alkenylenes are optionally substituted.

[0024] As used herein, unless otherwise specified, the term “cycloalkyl” refers to a saturated, non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms. Cycloalkyl groups may include condensed or crosslinked ring systems. In one embodiment, a cycloalkyl group may, for example, consist of 3 to 15 ring carbon atoms (C3 to C3). 15 Cycloalkyl), 3-10 ring carbon atoms (C3-C 10 A cycloalkyl group has 3 to 8 ring carbon atoms (C3-C8 cycloalkyl). The cycloalkyl group 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, dekalinyl, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless otherwise specified, the cycloalkyl group is optionally substituted.

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

[0026] As used herein, unless otherwise specified, the term “cycloalkenyl” refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms and containing one or more carbon-carbon double bonds. Cycloalkenyls may include condensed or bridging ring systems. In one embodiment, a cycloalkenyl is, for example, a ring carbon atom (C3-C) with 3 to 15 carbon atoms. 15 Cycloalkenyl), 3-10 ring carbon atoms (C3-C 10A monocyclic cycloalkenyl radical has 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, and cyclooctenyl. Unless otherwise specified, the cycloalkenyl group is optionally substituted.

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

[0028] As used herein, unless otherwise specified, the term “heterocyclyl” refers to a non-aromatic radical monocyclic or polycyclic moiety containing one or more heteroatoms (e.g., one, one or two, one to three, or one to four) independently selected from nitrogen, oxygen, phosphorus, and sulfur. Heterocyclyls may be bonded to the main structure with 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, bridging, or spirocyclic. Heterocyclyl polycyclic ring systems may contain one or more heteroatoms in one or more rings. Heterocyclyl groups may be saturated or partially unsaturated. Saturated heterocycloalkyl groups may be referred to as “heterocycloalkyl.” A partially unsaturated heterocycloalkyl group may be referred to as a "heterocycloalkenyl" if the heterocyclyl contains at least one double bond, or as a "heterocycloalkynyl" 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 heterocyclyl), 4 to 18 ring atoms (4 to 18-membered heterocyclyl), 5 to 18 ring atoms (3 to 18-membered heterocyclyl), 4 to 8 ring atoms (4 to 8-membered heterocyclyl), or 5 to 8 ring atoms (5 to 8-membered heterocyclyl). Wherever it appears herein, numerical ranges such as "3 to 18" refer to each integer within a given range. For example, "3 to 18 ring atoms" means that a heterocyclyl group may consist of 3, 4, 5, 6, 7, 8, 9, 10 ring atoms, and may contain up to 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, heterocyclyl groups are substituted at the discretion of the user.

[0029] As used herein, unless otherwise specified, the term “heterocyclylene” refers to a polyvalent (e.g., divalent or trivalent) heterocyclyl group. Unless otherwise specified, the heterocyclylene group is optionally substituted.

[0030] 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, the aryl group contains 6 to 18 ring carbon atoms (C6 to C6). 18 Aryl), 6-14 ring carbon atoms (C6-C 14 Aryl), or 6-10 ring carbon atoms (C6-C 10 It contains an aryl group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azlenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. The term “aryl” also refers to a bicyclic, tricyclic, or other polycyclic hydrocarbon ring in which at least one ring is aromatic and the others may be saturated, partially unsaturated, or aromatic, such as dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl). Unless otherwise specified, the aryl group is optionally substituted.

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

[0032] As used herein, unless otherwise specified, the term “heteroaryl” means a monocyclic and / or polycyclic aromatic group containing at least one aromatic ring, the at least one aromatic ring independently containing one or more heteroatoms (e.g., one, one or two, one to three, or one to four) selected from O, S, and N. A heteroaryl can be bonded to the main structure by 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 means a bicyclic, tricyclic, or other polycyclic ring, the at least one of which is aromatic and the others may be saturated, partially unsaturated, or aromatic, and the 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, pyridadinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolidinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumalinyl, sinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, flupyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridine, and xanthenyl. Unless otherwise specified, heteroaryl groups are optionally substituted.

[0033] As used herein, unless otherwise specified, the term “heteroarylene” refers to a polyvalent (e.g., divalent or trivalent) heteroaryl group. Unless otherwise specified, the heteroarylene group is optionally substituted.

[0034] Where a group described herein is said to be “substituted,” it may be substituted with any suitable substituent or a combination of substituents. Exemplary examples of substituents are 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 NR'R' is one example, but it is not limited to these. In each existence, R' is independently H, C1~C 15 It is alkyl or cycloalkyl, where x is 0, 1, or 2. In some embodiments, the substituents are C1-C 12 In other embodiments, the substituent is an alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group such as a fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group (-NR'R').

[0035] Where used herein, unless otherwise specified, the terms “optional” or “optionally” (e.g., optionally substituted) mean that the events or circumstances described therein may or may not occur, and the description includes both cases in which such events or circumstances occur and cases in which they do not occur. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted, and the description includes both substituted alkyl radicals and unsubstituted alkyl radicals.

[0036] As used herein, unless otherwise specified, the term “prodrug” of a biologically active compound refers to a compound that can be converted to a biologically active compound under physiological conditions or by dissolution. In one embodiment, the term “prodrug” refers to a metabolic precursor of a pharmaceutically acceptable biologically active compound. A prodrug may be inactive when administered to a target that requires it, but is converted to a biologically active compound in vivo. Prodrugs are typically rapidly converted in vivo, for example, by hydrolysis in the blood, to produce the original biologically active compound. Prodrug compounds often offer advantages in solubility, histocompatibility, 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 in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0037] In one embodiment, the term “prodrug” also means that such a prodrug includes any covalent carrier that releases the active compound in vivo when administered to a mammalian subject. A prodrug of a compound can be prepared by modifying a functional group present in the compound in such a manner that the modification is cleaved either by conventional means or in vivo to become the original compound. A prodrug includes a compound to which a hydroxyl group, an amino group, or a mercapto group is bonded, which is cleaved when the prodrug of a compound is administered to a mammalian subject to form a free hydroxyl group, a free amino group, or a free mercapto group, respectively.

[0038] Examples of prodrugs include, but are not limited to, acetic acid, formic acid, and benzoic acid derivatives of alcohols, or amide derivatives of amine functional groups, in the compounds provided herein.

[0039] As used herein, unless otherwise specified, the term “pharmaceutically acceptable salt” includes both acid and base addition salts.

[0040] Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, 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, carbonate, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, Examples include salts of organic acids such as glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucinic 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.

[0041] Examples of pharmaceutically acceptable base addition salts include, but are not limited to, salts prepared by adding an inorganic or organic base to a free acid compound. Examples of salts derived from inorganic bases include, but are not limited to, salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. In one embodiment, the inorganic salts are salts of ammonium, sodium, potassium, calcium, and magnesium. Examples of salts derived from organic bases include, but are not limited to, salts of ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamin, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, primary, secondary and tertiary amines such as polyamine resins, substituted amines including naturally substituted amines, cyclic amines, and basic ion exchange resins. In one embodiment, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0042] The compounds provided herein may contain one or more chiral centers and thus may give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined from the viewpoint of absolute stereochemistry as (R)- or (S)-, or for amino acids as (D)- or (L)-. Unless otherwise specified, the compounds provided herein 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 they can be 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 racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). Where a compound described herein contains an olefin double bond or other geometrically asymmetric center, unless otherwise specified, the compound is intended to include both E and Z geometric isomers. Similarly, all tautomer forms are also intended to be included.

[0043] As used herein, unless otherwise specified, the term “isomer” refers to different compounds having the same molecular formula. “Stereoisomers” are isomers that differ only in the way their atoms are arranged in space. “Atropisomers” are stereoisomers that derive from rotational impediments around a single bond. “Enantiomers” are a pair of stereoisomers that are mirror images of each other and cannot be superimposed. A mixture of any proportion of a pair of enantiomers may be known as a “racemic” mixture. “Diastereoisomers” are stereoisomers that have at least two chiral atoms but are not mirror images of each other.

[0044] "Stereoisomers" may 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 mixtures of the E and Z isomers.

[0045] A "tautomer" refers to an isomer of a compound that exists in equilibrium with it. The concentration of the isomer depends on the environment in which the compound is found, and may differ depending on whether the compound is in solid form or in an organic solution or aqueous solution.

[0046] It should also be noted that the compounds described herein may contain one or more atomic isotopes in unnatural proportions. For example, a compound may contain tritium ( 3 H), Iodine-125 ( 125 I), Sulfur 35 ( 35 S), or carbon-14 ( 14 Can it be radiolabeled with radioactive isotopes such as C), or deuterium ( 2 H), carbon-13 ( 13 C), or nitrogen 15 ( 15They can be isotope-enriched, for example, with N). As used herein, “isotope species” refers to an isotope-enriched compound. The term “isotope-enriched” refers to an atom that has an isotope composition other than the natural isotope composition of that atom. “Isotope-enriched” may also refer to a compound containing at least one atom that has an isotope composition other than the natural isotope composition of that atom. The term “isotope composition” refers to the amount of each isotope present for a given atom. Radiolabeled compounds and isotope-enriched compounds are useful as therapeutic agents, e.g., cancer treatments, research reagents, e.g., binding assay reagents, and diagnostic agents, e.g., in vivo contrast agents. All isotope variations of the compounds described herein, whether radioactive or not, are intended to be included within the scope of the embodiments provided herein. In some embodiments, isotope species of the compounds described herein are provided, for example, isotope species enriched with deuterium, carbon 13, and / or nitrogen 15. As used herein, “deuterated” means that at least one hydrogen (H) is deuterized (D or 2 A compound in which deuterium is replaced (indicated by H) means that the compound is concentrated at at least one position.

[0047] Please note that if there is a discrepancy between the structure shown and the name of that structure, the structure shown will be given more weight.

[0048] As used herein, unless otherwise specified, the term “pharmaceutically acceptable carrier, diluent or excipient” includes, but is not limited to, any adjuvants, carriers, excipients, lubricants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are approved by the United States Food and Drug Administration as acceptable for use in human or animal husbandry.

[0049] The term “composition” is intended to optionally encompass a product containing a specified amount of a specified component (e.g., the mRNA molecule provided herein).

[0050] As used interchangeably herein, the terms “polynucleotide” and “nucleic acid” refer to polymers of nucleotides of any length, including, for example, DNA and RNA. A nucleotide may be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or analogues thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogues. Nucleic acids may be single-stranded or double-stranded. As used herein, unless otherwise specified, “nucleic acid” also includes nucleic acid mimics such as loc nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino. “Oligonilocyte” as used herein refers to short synthetic polynucleotides, generally less than about 200 nucleotides in length, though not necessarily so. 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 nascent RNA transcription is referred to as the transcription direction, the sequence region on the DNA strand having the same sequence as the RNA transcript at the 5'-to-5' end of the RNA transcript is referred to as the "upstream sequence," and the sequence region on the DNA strand having the same sequence as the RNA transcript at the 3'-to-3' end of the RNA transcript is referred to as the "downstream sequence."

[0051] "Isolated nucleic acid" refers to nucleic acids, such as RNA, DNA, or mixed nucleic acids, that are substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany natural sequences. "Isolated" nucleic acid molecules are nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Furthermore, "isolated" nucleic acid molecules, such as mRNA, if produced by recombinant techniques, substantially contain no other cellular material or culture medium, or if chemically synthesized, substantially contain no chemical precursors or other chemical substances. In certain embodiments, one or more nucleic acid molecules encoding the antigens described herein are isolated or purified. The term encompasses nucleic acid sequences taken from their naturally occurring environment and includes recombinant or cloned DNA or RNA isolates, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule may include the isolated form of the molecule.

[0052] When used in reference to nucleic acid molecules, the term “coding nucleic acid” or its grammatical equivalent encompasses (a) nucleic acid molecules capable of producing mRNA, which is transcribed and subsequently translated into peptides and / or polypeptides, either in its natural state or when manipulated by methods well known to those skilled in the art, and (b) the mRNA molecule itself. The antisense strand is the complement of such nucleic acid molecule from which the coding sequence can be derived. The term “coding region” refers to the portion of the coding nucleic acid sequence that is translated into peptides or polypeptides. The term “untranslated region” or “UTR” refers to the portion of the coding nucleic acid that is not translated into peptides or polypeptides. Depending on the orientation of the UTR with respect to the coding region of the nucleic acid molecule, the UTR is referred to as the 5'-UTR if it is located at the 5' end of the coding region, and as the UTR is referred to as the 3'-UTR if it is located at the 3' end of the coding region.

[0053] As used herein, the term “mRNA” refers to a message RNA molecule comprising one or more open reading frames (ORFs) that can be translated by a cell or organism provided with the mRNA to produce one or more peptide or protein products. The region comprising one or more ORFs is referred to as the coding region of the mRNA molecule. In some embodiments, the mRNA molecule further comprises one or more untranslated regions (UTRs).

[0054] 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 each other. 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 may 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 the antigen.

[0055] The term "nucleic acid bases" encompasses purines and pyrimidines, including the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and their natural or synthetic analogs or derivatives.

[0056] As used herein, the term “functional nucleotide analog” refers to a modified version of a standard nucleotide 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) a nucleic acid base, (ii) a sugar group, (iii) a phosphate group, or (iv) any combination of (i) to (iii) of the corresponding native nucleotide. As used herein, base pairing includes not only the 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 enables hydrogen bonding between a modified nucleic acid base and a standard nucleic acid base or between two complementary nucleic acid base 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 may be either naturally occurring or unnaturally occurring. Thus, nucleic acid molecules containing functional nucleotide analogs may have at least one modified nucleic acid base, sugar group, and / or nucleoside linkage. Exemplary chemical modifications to nucleic acid bases, sugar groups, or nucleoside linkages of nucleic acid molecules are provided herein.

[0057] As used herein, the terms “translation-enhancing element,” “TEE,” and “translation-enhancer” refer to regions within a nucleic acid molecule that function to facilitate the translation of a nucleic acid coding sequence into a protein or peptide product, for example, through 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 translation level of coding sequences 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 the 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).

[0058] 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 each other to form at least one double helix and one unpaired loop. The resulting structure is known as a stem-loop structure, hairpin, or hairpin loop, a secondary structure found in many RNA molecules.

[0059] As used herein, the term “peptide” refers to a polymer containing 2 to 50 amino acid residues linked by one or more covalent peptide bonds. The term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are unnaturally occurring amino acids (e.g., amino acid analogs or unnatural amino acids).

[0060] 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, descriptions relating to polypeptides are equally applicable to descriptions relating to proteins, and vice versa. The terms apply to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are unnaturally 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).

[0061] The term "antigen" refers to a substance that can be recognized by the immune system of a target (including the adaptive immune system) and can trigger an immune response (including an antigen-specific immune response) after the target comes into contact with the antigen. In certain embodiments, an antigen is a protein associated with disease cells, such as cells infected by a pathogen or neoplastic cells (e.g., tumor-associated antigens (TAAs)).

[0062] In the context of peptides or polypeptides, the term “fragment” as used herein refers to a peptide or polypeptide comprising an amino acid sequence less than the full length. Such fragments may arise, for example, from cleavage at the amino terminus, cleavage at the carboxy terminus, and / or internal deletion of residues(s) from the amino acid sequence. Fragments may also arise, for example, from alternative RNA splicing or in vivo protease activity. In certain embodiments, the fragment may be at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 30 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, or at least 100 consecutive amino acid residues from the amino acid sequence of the polypeptide. This refers to a polypeptide comprising an amino acid sequence of consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive 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 consecutive amino acid residues. In certain embodiments, a polypeptide fragment retains at least one, at least two, at least three, or more functions of the polypeptide.

[0063] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule can bind, such as a site on the surface of an antigen or an immunogenic antigen in an animal such as a mammal (e.g., a human), which can bind to one or more antigen-binding regions of an antibody and induce an immune response. An immunogenic epitope is a portion of a polypeptide that induces an antibody response in an animal. An antigenic epitope is a portion of a polypeptide to which an antibody binds, determined by any method known in the art, including, for example, immunoassays. Antigenic epitopes do not necessarily have to be 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 properties and specific charge properties. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure. Inducible epitopes are formed when the three-dimensional structure of a protein is in an altered conformation, such as following the activation or binding of another protein or ligand. In certain embodiments, epitopes are three-dimensional surface features of a polypeptide. In other embodiments, epitopes are linear features of a polypeptide. Generally, antigens have several or many different epitopes and can react with many different antibodies.

[0064] As used herein, the term “gene 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”) enables the production of the encoded peptide or protein, thereby inducing an immune response to the target disease in the subject. In certain embodiments, the immune response includes adaptive immune responses such as the production of antibodies against the encoded antigen and / or the activation and proliferation of immune cells that can specifically eliminate disease cells expressing the antigen. In certain embodiments, the immune response further includes innate immune responses. According to this disclosure, the vaccine may 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 makes the vaccinated subject immune to or reduces susceptibility to the development of the target disease. In some embodiments, vaccination of a subject exhibiting symptoms of the disease improves or treats the disease condition in the vaccinated subject.

[0065] "Innate immune response" and "innate immunity" are recognized in the art 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, an innate immune response includes, but is 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 the measurement of (NF)-κB activation.

[0066] The terms “adaptive immune response” and “adaptive immunity” are recognized in the art 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 induced and / or amplified by a vaccine composition, such as a gene composition described herein. In some embodiments, the vaccine composition contains an antigen that is the target of an antigen-specific adaptive immune response. In other embodiments, the vaccine composition, upon administration, enables the production of the antigen that is the target of an antigen-specific adaptive immune response in an immunized subject. 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.

[0067] The term “antibody” is intended to include B cell polypeptide products within the immunoglobulin class of polypeptides that can bind to a specific molecular antigen and consist 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 each amino-terminal portion of each chain containing a variable region of approximately 100–130 amino acids or more, and each carboxy-terminal portion of each chain containing a constant region. See, for example, Antibody Engineering (Borrebaeck ed., 2d ed. 1995) and Kuby, Immunology (3d ed. 1997). In certain embodiments, a specific molecular antigen may be bound by an antibody provided herein, comprising a polypeptide, a fragment thereof, or an epitope. Antibodies include, but are not limited to, synthetic antibodies, recombinant antibodies, camelized antibodies, intrabodies, anti-idiotype (anti-Id) antibodies, and any of the functional fragments described above, where a functional fragment refers to a portion of the heavy or light chain polypeptide of an antibody that retains some or all of the binding activity of the antibody from which the fragment originated. Non-limiting examples of functional fragments include single-chain Fv(scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv(dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, the antibodies provided herein include immunoglobulin molecules and molecules containing immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or antigen-binding sites (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. 178:497-515, and Day, Advanced Immunochemistry (2nd ed. 1990). The antibodies provided herein may be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA), or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0068] The terms “administer” or “dosage” refer to the act of injecting or otherwise physically delivering an extracorporeal substance (e.g., the lipid nanoparticle composition described herein) to a patient by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. If a disease, disorder, illness, or its symptoms is being treated, the administration of the substance is typically performed after the onset of the disease, disorder, illness, or its symptoms. If a disease, disorder, illness, or its symptoms is being prevented, the administration of the substance is typically performed before the onset of the disease, disorder, illness, or its symptoms.

[0069] "Chronic" administration, in contrast to acute mode, refers to the administration of one or more drugs in a continuous mode (for example, over a period of several days, weeks, months, or years) to maintain the initial therapeutic effect (activity) for an extended period. "Intermittent" administration is a periodic treatment rather than a continuous one without interruption.

[0070] As used herein, the terms “targeted delivery” or the verb form “target” refer to a process that facilitates the arrival 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 intracellular component (referred to as a targeted site) rather than to any other organ, tissue, cell, or intracellular component (referred to as a non-targeted site). 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 a concentration at least twice as high at the targeted site compared to the non-targeted site.

[0071] An "effective dose" is generally defined as an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, prevent the onset of symptoms and / or their underlying causes, and / or improve or correct damage resulting from or associated with a disease, disorder, or illness, including, for example, infection and tumors. In some embodiments, the effective dose is a therapeutic effective dose or a preventive effective dose.

[0072] As used herein, the term “therapeutic dose” means an amount of an agent (e.g., a vaccine composition) sufficient to reduce and / or improve the severity and / or duration of a given disease, disorder, or illness and / or symptoms associated therewith (e.g., an infectious disease such as one caused by a viral infection, or a neoplastic disease such as cancer). The “therapeutic dose” of a substance / molecule / agent of this disclosure (e.g., a lipid nanoparticle composition described herein) may vary depending on factors such as the individual’s medical condition, age, sex, and weight, as well as the ability of the substance / molecule / agent to induce a desired response in the individual. The therapeutic dose encompasses the amount in which the therapeutically beneficial effects of the substance / molecule / agent outweigh the toxic or adverse effects. In certain embodiments, the term “therapeutic dose” means an amount of a lipid nanoparticle composition described herein or a therapeutic or prophylactic agent (e.g., therapeutic mRNA) contained therein that is effective in “treating” a disease, disorder, or illness in a subject or mammal.

[0073] The “preventive effective dose” is the amount of a pharmaceutical composition that, when administered to a subject, would prevent, delay, or reduce the likelihood of the onset (or recurrence) of a disease, disorder, illness, or associated symptoms (e.g., an infectious disease caused by a viral infection, or a neoplastic disease such as cancer). Typically, a preventive dose is used in a subject before or in the early stages of a disease, disorder, or illness, and therefore, the preventive effective dose may not necessarily be less than the therapeutic effective dose. A complete therapeutic or preventive effect may not necessarily occur with a single dose, but may only occur after a series of doses. Therefore, a therapeutic or preventive effective dose may be administered in one or more doses.

[0074] As used herein, unless otherwise specified, the terms “to treat,” “to treat,” and “treatment” mean the overall or partial relief of a disorder, disease, or illness, or one or more symptoms associated with such disorder, disease, or illness; or the delay or cessation of further progression or worsening of those symptoms; or the relief or eradication of the cause(s) of the disorder, disease, or illness itself.

[0075] As used herein, unless otherwise specified, the terms “prevent,” “prevent,” and “prevention” mean reducing the likelihood of the onset (or recurrence) of a disease, disorder, illness, or related condition (for example, an infectious disease such as one caused by a viral infection, or a neoplastic disease such as cancer).

[0076] As used herein, unless otherwise specified, the terms “to manage,” “to control,” and “to control” refer to the beneficial effects that a subject obtains from a therapy that does not result in a cure for a disease (e.g., a preventive or therapeutic agent). In certain embodiments, a subject is subjected to one or more therapies (e.g., a preventive or therapeutic agent such as the lipid nanoparticle composition described herein) to prevent the progression or worsening of a disease, thereby “controlling” an infectious or neoplastic disease, or one or more symptoms thereof.

[0077] The term "preventive agent" refers to any drug that can completely or partially inhibit the onset, recurrence, onset, or spread of a disease and / or related symptoms in a subject.

[0078] The term "therapeutic agent" refers to any drug that may be used to treat, prevent, or alleviate one or more symptoms of a disease, disorder, or illness, and / or symptoms associated therewith.

[0079] The term “therapy” refers to any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease, disorder, or illness. In certain embodiments, the terms “therapy(plural)” and “therapy(singular)” refer to biological therapies, supportive therapies, and / or other therapies known to those skilled in the art, such as healthcare professionals, that are useful for preventing, managing, treating, and / or improving a disease, disorder, or illness.

[0080] As used herein, “prophylactically effective serum titer” is the serum titer of an antibody in a subject (e.g., a human) that completely or partially inhibits the onset, recurrence, development, or progression of a disease, disorder, or illness and / or symptoms associated therewith in the subject.

[0081] In certain embodiments, “therapeutically effective serum titer” is the 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 illness in the subject.

[0082] 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 approximately 100, 1000, or more.

[0083] The term “adverse effects” encompasses undesirable and / or harmful effects of a therapy (e.g., a preventive or therapeutic agent). Undesirable effects are not necessarily harmful. Adverse effects from a therapy (e.g., a preventive or therapeutic agent) may be harmful, unpleasant, or dangerous. Examples of adverse 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 injection site, flu-like symptoms such as fever, chills, and fatigue, digestive problems, and allergic reactions. There are numerous further undesirable effects that patients may experience, and many are known in the art. Many are described in Physician's Desk Reference (68th ed. 2014).

[0084] The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, the subject is a mammal such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) with an infectious or neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious or neoplastic disease.

[0085] The term "detectable probe" refers to a composition that provides a detectable signal. This term includes, but is not limited to, any fluorophore, chromophore, radiolabel, enzyme, antibody, or antibody fragment that provides a detectable signal through its activity.

[0086] 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 may be a substance that can be visualized or otherwise determined and / or measured (e.g., by quantification).

[0087] "Substantially all" means 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%.

[0088] As used herein, unless otherwise indicated, the terms “about” or “approximately” mean a tolerance for a particular value measured by a person skilled in the art, depending in part to how that value is measured or determined. In certain embodiments, the terms “about” or “approximately” mean that the standard deviation is within 1, 2, 3, or 4. In certain embodiments, the terms “about” or “approximately” mean that the value is within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0089] As used herein, the singular terms “a,” “an,” and “the” include plural references unless the context explicitly indicates otherwise.

[0090] All publications, patent applications, accession numbers, and other references cited herein are incorporated in whole, as each individual publication or patent application is specifically and individually indicated as being invoked. Publications discussed herein are provided only for their disclosures prior to the filing date of this application. Nothing herein should be construed as acknowledging that the present invention has no prior rights to such publications by prior invention. Furthermore, the publication dates provided may differ from the actual publication dates and may need to be independently verified.

[0091] 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 the examples are intended to be illustrative and not to limit the scope of the invention as described in the claims.

[0092] 6.3 Lipid compounds Unless otherwise specified, the descriptions provided herein apply to all formulas provided herein (for example, formulas (I) and (II) including their subformulas) to the extent applicable.

[0093] In one embodiment, in this specification, formula (I): [ka] A compound of, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein, X 1 is a bond or O, X 2 is a bond or O, X 3 is a bond or O, G 1 and G 2 Each is independent, and the combination is C2~C 12 Alkylene, or C2~C 12 It is an alkenylene, however, G 1and G 2 One or more of the -CH2- in is independently 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 ), -(C6~C 10 arylene)-R 1 -(6~10 member heteroarylene)-R 1 or R 1 wherein, 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 ), -(C6~C 10 Arilen)-R 2 ,-(6-10 member heteroalylene)-R 2 , or R 2 And, R 1 and R 2 Each of these is independent of C6~C 32 Alkyl or C6-C 32 It is alkenyl, R a , R b , R d , and R e These are H, C1~C, and are independent of each other. 24 Alkyl, or C2-C 24 It is alkenyl, R c and R f Each of these is independent of C1~C 32 Alkyl or C2-C 32 It is alkenyl, G 3 is C2~C 12 Alkylene or C2~C 12 It is alkenylene, G 1 and G 2 If the -CH2- inside is not substituted by -O-, -C(=O)O-, or -OC(=O)-, then R 4 and R 5 teeth: (i)R 4 However, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 It is an aryl or 4- to 8-membered heterocycloalkyl, R 5 However, C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 It is either an aryl or a 4- to 8-membered heterocycloalkyl, or (ii)R 4 C1~C 12 Alkyl or C2-C 12 It is an alkenyl, R 5 C1-C is substituted with at least one hydroxyl group. 12 It is alkyl, G 1 and G 2 If one or more of the -CH2- groups are independently substituted by -O-, -C(=O)O-, or -OC(=O)-, then R 4 and R 5 teeth: R 4 However, C1~C 12 Alkyl, C2~C 12 Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 It is an aryl or 4- to 8-membered heterocycloalkyl group. R 5 However, C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 It is an aryl or 4- to 8-membered heterocycloalkyl group. a is either 0 or 1. n is 1, 2, or 3. m is 1, 2, or 3. i is either 0 or 1. j is either 0 or 1. Each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene are independently and optionally substituted. The above-mentioned compounds, or pharmaceutically acceptable salts, prodrugs, or stereoisomers thereof, are provided.

[0094] In one embodiment, in this specification, formula (I): [ka] A compound of, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein, X 1 is a bond or O, X 2 is a bond or O, X 3 is a bond or O, G 1 and G 2 Each is independent, and the combination is C2~C 12 Alkylene, or C2~C 12 It is alkenylene, Each L 1 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 ), -(C6~C 10 Arirene)-R 1,-(6-10 member heteroarylene)-R 1 , or R 1 And, Each L 2 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 ), -(C6~C 10 Arirene)-R 2 ,-(6-10 member heteroarylene)-R 2 , or R 2 And, R 1 and R 2 Each of these is independent of C6~C 32 Alkyl or C6-C 32 It is alkenyl, R a , R b , R d , and R e These are H, C1~C, and are independent of each other. 24 Alkyl, or C2-C 24 It is alkenyl, R c and R fEach of these is independent of C1~C 32 Alkyl or C2-C 32 It is alkenyl, G 3 is C2~C 12 Alkylene or C2~C 12 It is alkenylene, R 4 and R 5 teeth: (i)R 4 However, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 It is an aryl or 4- to 8-membered heterocycloalkyl, R 5 However, C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 It is either an aryl or a 4- to 8-membered heterocycloalkyl, or (ii)R 4 C1~C 12 Alkyl or C2-C 12 It is an alkenyl, R 5 C1-C is substituted with at least one hydroxyl group. 12 It is alkyl, a is either 0 or 1. n is 1, 2, or 3. m is 1, 2, or 3. i is either 0 or 1. j is either 0 or 1. Each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene are independently and optionally substituted. The above-mentioned compounds, or pharmaceutically acceptable salts, prodrugs, or stereoisomers thereof, are provided.

[0095] In one embodiment, in this specification, formula (II): [ka] A compound of, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein, X 1 is a bond or O, X 2 is a bond or O, X 3 is a bond or O, G 4 and G 5 Each of these is independently a C2-C6 alkylene or a C2-C6 alkenylene. G 1 and G 2 Each of these operates independently, C2~C 12 Alkylene, or C2~C 12 It is alkenylene, Each L 1 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 ), -(C6~C 10 Arirene)-R 1 ,-(6-10 member heteroarylene)-R 1 , or R 1And, Each L 2 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 ), -(C6~C 10 Arirene)-R 2 ,-(6-10 member heteroarylene)-R 2 , or R 2 And, R 1 and R 2 Each of these is independent of C6~C 32 Alkyl or C6-C 32 It is alkenyl, R a , R b , R d , and R e These are H, C1~C, and are independent of each other. 24 Alkyl, or C2-C 24 It is alkenyl, R c and R f Each of these is independent of C1~C 32 Alkyl or C2-C 32It is alkenyl, G 3 is C2~C 12 Alkylene or C2~C 12 It is alkenylene, R 4 C1~C 12 Alkyl, C2~C 12 Alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 It is an aryl or 4- to 8-membered heterocycloalkyl group. R 5 C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 It is an aryl or 4- to 8-membered heterocycloalkyl group. a is either 0 or 1. n is 1, 2, or 3. m is 1, 2, or 3. i is either 0 or 1. j is either 0 or 1. Each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene are independently and optionally substituted. The above-mentioned compounds, or pharmaceutically acceptable salts, prodrugs, or stereoisomers thereof, are provided.

[0096] In one embodiment, a is 0. In another embodiment, a is 1.

[0097] In one embodiment, the above compound is of formula (IA): [ka] It is a compound of, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer.

[0098] In one embodiment, the above compound is of formula (IB): [ka] It is a compound of, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer.

[0099] In one embodiment, the above compound is of formula (II-A): [ka] It is a compound of, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer.

[0100] In one embodiment, the above compound is of formula (II-B): [ka] It is a compound of, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer.

[0101] In one embodiment, i is 0. In one embodiment, i is 1. In one embodiment, j is 0. In one embodiment, j is 1. In one embodiment, i is 0 and j is 0. In one embodiment, i is 1 and j is 1.

[0102] In one embodiment, X 1 is a bond. In one embodiment, X 1 is O. In one embodiment, X 2 is a bond. In one embodiment, X 2 is O. In one embodiment, X 1 X is a bond. 2 is a bond. In one embodiment, X 1 is O and X 2 is a bond. In one embodiment, X 1 is O and X 2 It is O.

[0103] In one embodiment, X 3 is a bond. In one embodiment, X 3 It is O.

[0104] In one embodiment, the above compound is of formula (III): [ka] It is a compound of, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer.

[0105] In one embodiment, the above compound is of formula (IV): [ka] It is a compound of, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer.

[0106] In one embodiment, the above compound is of formula (V): [ka] It is a compound of, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer.

[0107] In one embodiment, the above compound is of formula (VI): [ka] It is a compound of, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer.

[0108] In one embodiment, the above compound is of formula (III-A), (III-B), (III-C), (III-D), (IV-A), (IV-B), (IV-C), (IV-D), (VA), (VB), (VC), or (VD): [ka] (In the formula, y and z are each independent integers from 0 to 9. (t is an integer between 2 and 12) It is a compound of, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0109] In one embodiment, the above compound is of formula (IV-E): [ka] (In the formula, u is an integer between 2 and 6. y is an integer from 0 to 9. (t is an integer between 2 and 12) It is a compound of, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer.

[0110] In one embodiment, the above compound is of formula (VI-A) or (VI-B): [ka] (In the formula, u and v are each independent integers between 2 and 6. y and z are each independent integers between 0 and 9. (t is an integer between 2 and 12) It is a compound of, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0111] In one embodiment, y is an integer from 0 to 9. In one embodiment, y is an integer from 0 to 3. In one embodiment, y is 0. In one embodiment, y is 1. 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. In one embodiment, y is 7. In one embodiment, y is 8. In one embodiment, y is 9.

[0112] In one embodiment, in formula (III-A), (IV-A), or (VA), y is 2. In one embodiment, in formula (III-B), (IV-B), or (VB), y is 3.

[0113] In one embodiment, z is an integer from 0 to 9. In one embodiment, z is an integer from 0 to 3. In one embodiment, z is 0. In one embodiment, z is 1. 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. In one embodiment, z is 7. In one embodiment, z is 8. In one embodiment, z is 9.

[0114] In one embodiment, y and z are different. In one embodiment, y and z are the same. In one embodiment, y and z are the same and selected from 0, 1, 2, and 3. In one embodiment, y is 0 and z is 0. In one embodiment, y is 2 and z is 2. In one embodiment, y is 3 and z is 3.

[0115] In one embodiment, in equations (III-C), (IV-C), or (VC), y is 2 and z is 2. In one embodiment, in equations (III-D), (IV-D), or (VD), y is 3 and z is 3. In one embodiment, in equation (VI-B), y and z are each independently integers between 4 and 7.

[0116] In one embodiment, u is an integer between 2 and 6. In one embodiment, u is 2. In one embodiment, u is 3. In one embodiment, u is 4. In one embodiment, u is 5. In one embodiment, u is 6.

[0117] In one embodiment, v is an integer between 2 and 6. In one embodiment, v is 2. In one embodiment, v is 3. In one embodiment, v is 4. In one embodiment, v is 5. In one embodiment, v is 6.

[0118] In one embodiment, u and v are different. In one embodiment, u and v are the same. In one embodiment, u is 2 and v is 2.

[0119] In one embodiment, t is an integer between 2 and 12. In one embodiment, t is an integer between 2 and 10. In one embodiment, t is an integer between 2 and 8. In one embodiment, t is an integer between 2 and 6. In one embodiment, t is an integer between 2 and 4. In one embodiment, t is 1. In one embodiment, t is 2. In one embodiment, t is 3. In one embodiment, t is 4. In one embodiment, t is 5. In one embodiment, t is 6. In one embodiment, t is 7.

[0120] In one embodiment, in equation (IV-E), u is 2 and y is 1.

[0121] In one embodiment, G 1 is a bond. In one embodiment, G 1 is C2~C 12 It is an alkylene. 1 These are C2-C6 alkylenes. In one embodiment, G 1 is a C2 alkylene. In one embodiment, G 1 is a C3 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 C6 alkylene. In one embodiment, G 1 is C2~C 12 It is an alkenylene. In one embodiment, G 1 These are C2-C6 alkenylenes.

[0122] In one embodiment, G 1 The -CH2- in the middle is not substituted by -O-, -C(=O)O-, or -OC(=O)-. In one embodiment, G 1 One or more of the -CH2- groups are independently substituted by -O-, -C(=O)O-, or -OC(=O)-. In one embodiment, G 1 is C2~C 12 It is alkylene, G 1 One of the -CH2- molecules is replaced by -O-. In one embodiment, G 1 is C2~C12 It is alkylene, G 1 One of the -CH2- molecules is replaced by -C(=O)O-. In one embodiment, G 1 is C2~C 12 It is alkylene, G 1 One of the -CH2- molecules is replaced by -OC(=O)-.

[0123] 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] This applies when described herein, unless otherwise specified. 1 The left-hand connection point (for example, G 1 The -C(=O)O-) inside is X 1 It is facing in the direction of G 1 The connection point (or multiple connection points) on the right side is L 1 It is facing in that direction.

[0124] In one embodiment, G 2 is a bond. In one embodiment, G 2 is C2~C 12 It is an alkylene. 2 These are C2-C6 alkylenes. In one embodiment, G 2is a C2 alkylene. In one embodiment, G 2 is a C3 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 C6 alkylene. In one embodiment, G 2 is C2~C 12 It is an alkenylene. In one embodiment, G 2 These are C2-C6 alkenylenes.

[0125] In one embodiment, G 2 The -CH2- in the middle is not substituted by -O-, -C(=O)O-, or -OC(=O)-. In one embodiment, G 2 One or more of the -CH2- groups are independently substituted by -O-, -C(=O)O-, or -OC(=O)-. In one embodiment, G 2 is C2~C 12 It is alkylene, G 2 One of the -CH2- molecules is replaced by -O-. In one embodiment, G 2 is C2~C 12 It is alkylene, G 2 One of the -CH2- molecules is replaced by -C(=O)O-. In one embodiment, G 2 is C2~C 12 It is alkylene, G 2 One of the -CH2- molecules is replaced by -OC(=O)-.

[0126] 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] This applies when described herein, unless otherwise specified. 2 The left-hand connection point (for example, G 2 The -C(=O)O-) inside is X 2 It is facing in the direction of G 2 The connection point (or multiple connection points) on the right side is L 2 It is facing in that direction.

[0127] In one embodiment, G 1 and G 2 Each of them is independent, C2~C 12 It is an alkylene. 1 and G 2 Each of these is independently a C2 alkylene. In one embodiment, G 1 and G 2 Each of these is independently a C3 alkylene. In one embodiment, G 1 and G 2 Each of these is independently a C6 alkylene.

[0128] In one embodiment, G 2 G is a bond. 1 is C2~C 12 It is an alkylene. 2 G is a bond. 1 is a C2 alkylene. In one embodiment, G 2 G is a bond. 1 is a C3 alkylene. In one embodiment, G 2 G is a bond. 1 It is a C6 alkylene.

[0129] In one embodiment, G 1is non-substitutable. In one embodiment, G 1 is replaced. In one embodiment, G 2 is non-substitutable. In one embodiment, G 2 It has been replaced.

[0130] In one embodiment, n is 1. In one embodiment, n is 2. In one embodiment, n is 3. In one embodiment, m is 1. In one embodiment, m is 2. In one embodiment, m is 3.

[0131] In one embodiment, L 1 is -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 , or -P(=O)(OR b )(OR c ) is. In one embodiment, L 1 ha-(C6~C 10 Arirene)-R 1 In one embodiment, L 1 ha-(6-10 member heteroarylene)-R 1 In one embodiment, L 1 is R 1 That is the case.

[0132] In one embodiment, L 1 is -C(=O)R 1 -OC(=O)R 1 , -C(=O)OR 1 -C(=O)SR 1 -SC(=O)R 1 , -NR a C(=O)R 1 , or -C(=O)NR b R c In one embodiment, L 1 is -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 1 is -OR 1 -OC(=O)R 1 , or -C(=O)OR 1 In one embodiment, L 1 は-OR 1 In one embodiment, L 1 is -C(=O)R 1 In one embodiment, L 1 -OC(=O)R 1 In one embodiment, L 1 is -C(=O)OR 1 In one embodiment, L 1 -NR a C(=O)R 1 In one embodiment, L 1 is -C(=O)NR b R c In one embodiment, L 1 -NR a C(=O)NR b R c In one embodiment, L 1 -OC(=O)NR b R c In one embodiment, L 1 -NR a C(=O)OR 1 That is the case.

[0133] In one embodiment, L 2 is -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 , or -P(=O)(OR e )(OR f ) is. In one embodiment, L 2 is, -(C6~C 10 Arirene)-R 2 In one embodiment, L 2 is -(6-10 member heteroarylene)-R 2 In one embodiment, L 2 is R 2 That is the case.

[0134] In one embodiment, L 2 is -C(=O)R 2 -OC(=O)R 2 , -C(=O)OR 2 -C(=O)SR 2 -SC(=O)R 2 , -NR d C(=O)R 2 , or -C(=O)NR e R fIn one embodiment, L 2 is -OC(=O)R 2 , -C(=O)OR 2 , -NR d C(=O)R 2 , or -C(=O)NR e R f In one embodiment, L 2 is -OR 2 -OC(=O)R 2 , or -C(=O)OR 2 In one embodiment, L 2 は-OR 2 In one embodiment, L 2 is -C(=O)R 2 In one embodiment, L 2 -OC(=O)R 2 In one embodiment, L 2 is -C(=O)OR 2 In one embodiment, L 2 -NR d C(=O)R 2 In one embodiment, L 2 is -C(=O)NR e R f In one embodiment, L 2 -NR d C(=O)NR e R f In one embodiment, L 2 -OC(=O)NR e R f In one embodiment, L 2 -NR d C(=O)OR 2 That is the case.

[0135] In one embodiment, G 1 -(L 1 ) n is R 1 In one embodiment, G 1 These are C2-C6 alkylenes, and each L 1 These are independent, -OR 1 -OC(=O)R 1 , or -C(=O)OR 1 In one embodiment, G1 is two L 1 These are C2-C6 alkylenes bonded to each L 1 These are independent, -OR 1 -OC(=O)R 1 , or -C(=O)OR 1 In one embodiment, G 1 is two -OR 1 It is a C2-C6 alkylene bonded to it. In one embodiment, G 1 This is two -OC(=O)R 1 It is a C2-C6 alkylene bonded to it. In one embodiment, G 1 This is two -C(=O)OR 1 It is a C2-C6 alkylene bonded to a nucleotide.

[0136] In one embodiment, G 1 is C2~C 12 It is alkylene, however, G 1 One of the -CH2- molecules in each L is substituted by -O-, -C(=O)O-, or -OC(=O)-. 1 These are independent, -OR 1 -OC(=O)R 1 , or -C(=O)OR 1 In one embodiment, G 1 is two L 1 C2~C bonded 12 It is alkylene, however, G 1 One of the -CH2- molecules in each L is substituted by -O-, -C(=O)O-, or -OC(=O)-. 1 These are independent, -OR 1 -OC(=O)R 1 , or -C(=O)OR 1 In one embodiment, G 1 is two L 1 C6~C bonded 10 It is alkylene, however, G 1 One of the -CH2- molecules in the middle is replaced by -O-, and each L 1 -OC(=O)R 1 In one embodiment, G 1 is two L 1C6~C bonded 10 It is alkylene, however, G 1 One of the -CH2- molecules is substituted by -C(=O)O-, and each L 1 -OC(=O)R is independent 1 That is the case.

[0137] In one embodiment, G 2 -(L 2 ) m is R 2 In one embodiment, G 2 These are C2-C6 alkylenes, and each L 2 These are independent, -OR 2 -OC(=O)R 2 , or -C(=O)OR 2 In one embodiment, G 2 is two L 2 These are C2-C6 alkylenes connected to each L 2 These are independent, -OR 2 -OC(=O)R 2 , or -C(=O)OR 2 In one embodiment, G 2 is two -OR 2 It is a C2-C6 alkylene bonded to it. In one embodiment, G 2 This is two -OC(=O)R 2 It is a C2-C6 alkylene bonded to it. In one embodiment, G 2 This is two -C(=O)OR 2 It is a C2-C6 alkylene bonded to a nucleotide.

[0138] In one embodiment, G 1 -(L 1 ) n and G 2 -(L 2 ) m Each is independent, C6~C 32 Alkyl, C6~C 32 Alkyl, [ka] That is the case.

[0139] In one embodiment, G 1 -(L 1 ) n teeth [ka] G 2 -(L 2 ) m is R 2 That is the case.

[0140] In one embodiment, G 1 -(L 1 ) n teeth [ka] G 2 -(L 2 ) m is R 2 That is the case.

[0141] In one embodiment, G 1 -(L 1 ) n teeth [ka] G 2 -(L 2 ) m is R 2 That is the case.

[0142] In one embodiment, G 1 -(L 1 ) n teeth [ka] G 2 -(L 2 ) m teeth [ka] That is the case.

[0143] In one embodiment, G 1 -(L1 ) n teeth [ka] G 2 -(L 2 ) m teeth [ka] That is the case.

[0144] In one embodiment, G 1 -(L 1 ) n teeth [ka] G 2 -(L 2 ) m teeth [ka] That is the case.

[0145] In one embodiment, G 3 is C2~C 12 It is an alkylene. 3 These are C2-C8 alkylenes. In one embodiment, G 3 These are C2-C6 alkylenes. In one embodiment, G 3 is a C2-C4 alkylene. In one embodiment, G 3 is a C2 alkylene. In one embodiment, G 3 It is a C4 alkylene.

[0146] In one embodiment, G 3 is C2~C 12 It is an alkenylene. In one embodiment, G 3 is a C2-C8 alkenylene. In one embodiment, G 3 is a C2-C6 alkenylene. In one embodiment, G 3 These are C2-C4 alkenylenes.

[0147] In one embodiment, G 3 is non-substitutable. In one embodiment, G 3 It has been replaced.

[0148] In one embodiment, G 4 These are C2-C6 alkylenes. In one embodiment, G 4 is a C2-C4 alkylene. In one embodiment, G 4 is a C2 alkylene. In one embodiment, G 4 is a C4 alkylene. In one embodiment, G 4 is a C2-C6 alkenylene. In one embodiment, G 4 is a C2-C4 alkenylene. In one embodiment, G 4 is a C2 alkenylene. In one embodiment, G 4 It is a C4 alkenylene.

[0149] In one embodiment, G 5 These are C2-C6 alkylenes. In one embodiment, G 5 is a C2-C4 alkylene. In one embodiment, G 5 is a C2 alkylene. In one embodiment, G 5 is a C4 alkylene. In one embodiment, G 5 is a C2-C6 alkenylene. In one embodiment, G 5 is a C2-C4 alkenylene. In one embodiment, G 5 is a C2 alkenylene. In one embodiment, G 5 It is a C4 alkenylene.

[0150] In one embodiment, G 4 and G 5 Both are C2 alkylenes.

[0151] In one embodiment, R 4 is C1~C 12 It is alkyl. In one embodiment, R 4 is a C1-C8 alkyl group. In one embodiment, R 4 is a C1-C6 alkyl group. In one embodiment, R 4is a C1-C4 alkyl group. In one embodiment, R 4 is methyl. In one embodiment, R 4 is ethyl. In one embodiment, R 4 is n-propyl. 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 It is n-nonyl.

[0152] In one embodiment, R 4 is C2~C 12 It is an alkenyl. In one embodiment, R 4 is a C2-C8 alkenyl. In one embodiment, R 4 is a C2-C6 alkenyl. In one embodiment, R 4 is a C2-C4 alkenyl. In one embodiment, the alkenyl is a linear alkenyl. In one embodiment, the alkenyl is a branched alkenyl. In one embodiment, R 4 is etenyl. In one embodiment, R 4 This is an allele.

[0153] In one embodiment, R 4 is a C3-C8 cycloalkyl group. 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. In one embodiment, R 4 It is cyclooctyl.

[0154] In one embodiment, R 4 is a C3-C8 cycloalkenyl. In one embodiment, R 4 is cyclopropenyl. In one embodiment, R 4is cyclobutenyl. In one embodiment, R 4 is cyclopentenyl. In one embodiment, R 4 is cyclohexenyl. In one embodiment, R 4 is cycloheptenyl. In one embodiment, R 4 It is cyclooctenyl.

[0155] In one embodiment, R 4 is C6~C 10 It is an arrow. In one embodiment, R 4 It is phenyl.

[0156] In one embodiment, R 4 R is a 4-8 member heterocyclyl. In one embodiment, R 4 is a 4-8 member heterocycloalkyl. In one embodiment, R 4 is oxetanil. In one embodiment, R 4 is tetrahydrofuranil. In one embodiment, R 4 is tetrahydropyranyl. In one embodiment, R 4 is tetrahydrothiopyranil. In one embodiment, R 4 It is N-methylpiperidinyl.

[0157] In one embodiment, R 4 This is a non-substitution.

[0158] 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 , and -OR i Substituted with one or more substituents selected from the group consisting of -OH, R g In each of these configurations, independently, it is either H or a C1-C6 alkyl group. R hIn each of these configurations, they are independently C1-C6 alkyl groups. R i These are C1-C6 alkylenes, independently of each other in their respective existence.

[0159] In one embodiment, R 4 R is substituted with one or more hydroxyls. In one embodiment, R 4 It is substituted with one hydroxyl group.

[0160] In one embodiment, R 4 R is substituted with one or more hydroxyls and one or more oxos. In one embodiment, R 4 It is substituted with one hydroxyl and one oxo.

[0161] In one embodiment, R 5 is C1~C 12 It is alkyl. In one embodiment, R 5 is C1~C 10 It is alkyl. In one embodiment, R 5 is a C1-C8 alkyl group. In one embodiment, R 5 is a C1-C6 alkyl group. In one embodiment, R 5 is a C1-C4 alkyl group. In one embodiment, R 5 is a C1-C2 alkyl group. In one embodiment, R 5 is methyl. In one embodiment, R 5 is ethyl. In one embodiment, R 5 is propyl. In one embodiment, R 5 is n-butyl. In one embodiment, R 5 is n-hexyl. In one embodiment, R 5 is n-octyl. In one embodiment, R 5 It is n-nonyl.

[0162] In one embodiment, R 5 is a C3-C8 cycloalkyl group. In one embodiment, R 5 is cyclopropyl. In one embodiment, R 5 is cyclobutyl. In one embodiment, R5 is cyclopentyl. In one embodiment, R 5 is cyclohexyl. In one embodiment, R 5 is cycloheptyl. In one embodiment, R 5 It is cyclooctyl.

[0163] In one embodiment, R 5 is a C3-C8 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 It is cyclooctenyl.

[0164] In one embodiment, R 5 is C6~C 10 It is an arrow. In one embodiment, R 5 It is phenyl.

[0165] In one embodiment, R 5 R is a 4-8 member heterocyclyl. In one embodiment, R 5 is a 4-8 member heterocycloalkyl. In one embodiment, R 5 is oxetanil. In one embodiment, R 5 is tetrahydrofuranil. In one embodiment, R 5 is tetrahydropyranyl. In one embodiment, R 5 It is tetrahydrothiopyranil.

[0166] In one embodiment, R 5 This is a non-substitution.

[0167] In one embodiment, R 5 is oxo, -OR g , -NR g C(=O)R h -C(=O)NRg R h 、 -C(=O)R h 、 -OC(=O)R h 、 -C(=O)OR h 、 and -O-R i substituted with one or more substituents selected from the group consisting of -OH, R g in each occurrence, is independently H or C1-C6 alkyl, R h in each occurrence, is independently C1-C6 alkyl, R i in each occurrence, is independently C1-C6 alkylene.

[0168] In one embodiment, R 5 is substituted with one or more hydroxyls. In one embodiment, R 5 is substituted with one hydroxyl. In one embodiment, R 5 is substituted with hydroxyl-substituted alkoxy. In one embodiment, R 5 is substituted with -OCH2CH2OH.

[0169] In one embodiment, R 5 is substituted with one or more hydroxyls and one or more oxos. In one embodiment, R 5 is substituted with one hydroxyl and one oxo. In one embodiment, R 5 is -CH2CH2OH.

[0170] In one embodiment, R 5 is -(CH2) p Q, -(CH2) p CHQR, -CHQR, or -CQ(R)2, wherein Q is C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4-8 member heterocyclyl, C6-C 10 aryl, 5-10 member heteroaryl, -OR, -O(CH2) pN(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)2C(O)OR, where each p is independently 1, 2, 3, 4, or 5. R 22 These include C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4-8 membered heterocyclyl, and C6-C 10 It is an aryl or a 5-10 membered heteroaryl. R 23 These are 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, 4-8 membered heterocyclyl, C6-C 10 It is an aryl or a 5-10 membered heteroaryl. Each R is independently H, C1-C3 alkyl, or C2-C3 alkenyl, or two Rs in the N(R)2 moiety, together with the nitrogen they are bonded to, form a cyclic moiety. Each X is independently F, Cl, Br, or I.

[0171] In one embodiment, R 1 is a straight chain C6~C 32It is alkyl. In one embodiment, R 1 is a straight chain C6~C 24 It is alkyl. In one embodiment, R 1 is a straight chain C7~C 15 It is alkyl. In one embodiment, R 1 is a linear C7 alkyl group. In one embodiment, R 1 R is a linear C8 alkyl group. In one embodiment, R 1 R is a linear C9 alkyl group. In one embodiment, R 1 is a straight chain C 10 It is alkyl. In one embodiment, R 1 is a straight chain C 11 It is alkyl. In one embodiment, R 1 is a straight chain C 12 It is alkyl. In one embodiment, R 1 is a straight chain C 13 It is alkyl. In one embodiment, R 1 is a straight chain C 14 It is alkyl. In one embodiment, R 1 is a straight chain C 15 It is alkyl.

[0172] In one embodiment, R 1 is a straight chain C6~C 32 It is an alkenyl. In one embodiment, R 1 is a straight chain C6~C 24 It is an alkenyl. In one embodiment, R 1 is a straight chain C7~C 17 It is an alkenyl. In one embodiment, R 1 R is a linear C7 alkenyl. In one embodiment, R 1 R is a linear C8 alkenyl. In one embodiment, R 1 R is a linear C9 alkenyl. In one embodiment, R 1 is a straight chain C 10 It is an alkenyl. In one embodiment, R 1 is a straight chain C 11 It is an alkenyl. In one embodiment, R 1 is a straight chain C 12 It is an alkenyl. In one embodiment, R 1 is a straight chain C 13 It is an alkenyl. In one embodiment, R1 is a linear C 14 alkenyl. In one embodiment, R 1 is a linear C 15 alkenyl. In one embodiment, R 1 is a linear C 16 alkenyl. In one embodiment, R 1 is a linear C 17 alkenyl.

[0173] In one embodiment, R 1 is a branched C6 - C 32 alkyl. In one embodiment, R 1 is a branched C6 - C 24 alkyl. In one embodiment, R 1 is -R 7 -CH(R 8 )(R 9 ), where R 7 is C0 - C5 alkylene and R 8 and R 9 are independently C2 - C 10 alkyl. In one embodiment, R 1 is -R 7 -CH(R 8 )(R 9 ), where R 7 is C0 - C1 alkylene and R 8 and R 9 are independently C4 - C8 alkyl.

[0174] In one embodiment, R 1 is a branched C6 - C 32 alkenyl. In one embodiment, R 1 is a branched C6 - C 24 alkenyl. In one embodiment, R 1 is -R 7 -CH(R 8 )(R<00​​​​​​​​​​​7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C1 alkylenes, and R 8 and R 9 It is independent of C6~C 10 It is Alkenil.

[0175] In one embodiment, R 2 is a straight chain C6~C 32 It is alkyl. In one embodiment, R 2 is a straight chain C6~C 24 It is alkyl. In one embodiment, R 2 is a straight chain C7~C 15 It is alkyl. In one embodiment, R 2 is a linear C7 alkyl group. In one embodiment, R 2 R is a linear C8 alkyl group. In one embodiment, R 2 R is a linear C9 alkyl group. In one embodiment, R 2 is a straight chain C 10 It is alkyl. In one embodiment, R 2 is a straight chain C 11 It is alkyl. In one embodiment, R 2 is a straight chain C 12 It is alkyl. In one embodiment, R 2 is a straight chain C 13 It is alkyl. In one embodiment, R 2 is a straight chain C 14 It is alkyl. In one embodiment, R 2 is a straight chain C 15 It is alkyl.

[0176] In one embodiment, R 2 is a linear C6~C 32 It is an alkenyl. In one embodiment, R 2 is a straight chain C6~C 24 It is an alkenyl. In one embodiment, R 2 is a straight chain C7~C 17 It is an alkenyl. In one embodiment, R 2 R is a linear C7 alkenyl. In one embodiment, R 2 R is a linear C8 alkenyl. In one embodiment, R2 R is a linear C9 alkenyl. In one embodiment, R 2 is a straight chain C 10 It is an alkenyl. In one embodiment, R 2 is a straight chain C 11 It is an alkenyl. In one embodiment, R 2 is a straight chain C 12 It is an alkenyl. In one embodiment, R 2 is a straight chain C 13 It is an alkenyl. In one embodiment, R 2 is a straight chain C 14 It is an alkenyl. In one embodiment, R 2 is a straight chain C 15 It is an alkenyl. In one embodiment, R 2 is a straight chain C 16 It is an alkenyl. In one embodiment, R 2 is a straight chain C 17 It is Alkenil.

[0177] In one embodiment, R 2 This is the branched chain C6~C 32 It is alkyl. In one embodiment, R 2 is branched chain C6~C 24 It is alkyl. In one embodiment, R 2 ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C5 alkylenes, and R 8 and R 9 These are independent of C2~C 10 It is alkyl. In one embodiment, R 2 ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C1 alkylenes, and R 8 and R 9 These are independently C4-C8 alkyl groups.

[0178] In one embodiment, R 2 This is the branched chain C6~C 32 It is an alkenyl. In one embodiment, R 2is branched chain C6~C 24 It is an alkenyl. In one embodiment, R 2 ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C5 alkylenes, and R 8 and R 9 These are independent of C2~C 10 It is an alkenyl. In one embodiment, R 2 ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C1 alkylenes, and R 8 and R 9 These are independent, C6~C 10 It is Alkenil.

[0179] In one embodiment, R c is a straight chain C5~C 32 It is alkyl. In one embodiment, R c is a straight chain C6~C 32 It is alkyl. In one embodiment, R c is a straight chain C6~C 24 It is alkyl. In one embodiment, R c is a straight chain C7~C 15 It is alkyl. In one embodiment, R c is a linear C7 alkyl group. In one embodiment, R c R is a linear C8 alkyl group. In one embodiment, R c R is a linear C9 alkyl group. In one embodiment, R c is a straight chain C 10 It is alkyl. In one embodiment, R c is a linear C 11 It is alkyl. In one embodiment, R c is a straight chain C 12 It is alkyl. In one embodiment, R c is a straight chain C 13 It is alkyl. In one embodiment, R c is a straight chain C 14 It is alkyl. In one embodiment, R c is a straight chain C 15 It is alkyl.

[0180] In one embodiment, R c is a straight chain C5~C 32 It is an alkenyl. In one embodiment, R c is a straight chain C6~C 32 It is an alkenyl. In one embodiment, R c is a straight chain C6~C 24 It is an alkenyl. In one embodiment, R c is a straight chain C7~C 17 It is an alkenyl. In one embodiment, R c R is a linear C7 alkenyl. In one embodiment, R c R is a linear C8 alkenyl. In one embodiment, R c R is a linear C9 alkenyl. In one embodiment, R c is a straight chain C 10 It is an alkenyl. In one embodiment, R c is a linear C 11 It is an alkenyl. In one embodiment, R c is a straight chain C 12 It is an alkenyl. In one embodiment, R c is a straight chain C 13 It is an alkenyl. In one embodiment, R c is a straight chain C 14 It is an alkenyl. In one embodiment, R c is a straight chain C 15 It is an alkenyl. In one embodiment, R c is a straight chain C 16 It is an alkenyl. In one embodiment, R c is a straight chain C 17 It is Alkenil.

[0181] In one embodiment, R c This is the branching chain C5~C 32 It is alkyl. In one embodiment, R c is branched chain C6~C 32 It is alkyl. In one embodiment, R c This is the branched chain C6~C 24 It is alkyl. In one embodiment, R c ha-R 7 -CH(R 8 )(R 9) and in the formula, R 7 These are C0-C5 alkylenes, and R 8 and R 9 These are, independently, C2~C 10 It is alkyl. In one embodiment, R c ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C1 alkylenes, and R 8 and R 9 These are independently C4-C8 alkyl groups.

[0182] In one embodiment, R c This is the branching chain C5~C 32 In one embodiment, R c is branched chain C6~C 32 It is an alkenyl. In one embodiment, R c is branched chain C6~C 24 It is an alkenyl. In one embodiment, R c ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C5 alkylenes, and R 8 and R 9 These are, independently, C2~C 10 It is an alkenyl. In one embodiment, R c ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C1 alkylenes, and R 8 and R 9 It is independent of C6~C 10 It is Alkenil.

[0183] In one embodiment, R f is a straight chain C5~C 32 It is alkyl. In one embodiment, R f is a straight chain C6~C 32 It is alkyl. In one embodiment, R f is a straight chain C6~C 24 It is alkyl. In one embodiment, Rf is a straight chain C7~C 15 It is alkyl. In one embodiment, R f is a linear C7 alkyl group. In one embodiment, R f R is a linear C8 alkyl group. In one embodiment, R f R is a linear C9 alkyl group. In one embodiment, R f is a straight chain C 10 It is alkyl. In one embodiment, R f is a straight chain C 11 It is alkyl. In one embodiment, R f is a linear C 12 It is alkyl. In one embodiment, R f is a straight chain C 13 It is alkyl. In one embodiment, R f is a straight chain C 14 It is alkyl. In one embodiment, R f is a straight chain C 15 It is alkyl.

[0184] In one embodiment, R f is a straight chain C5~C 32 It is an alkenyl. In one embodiment, R f is a straight chain C6~C 32 It is an alkenyl. In one embodiment, R f is a straight chain C6~C 24 It is an alkenyl. In one embodiment, R f is a straight chain C7~C 17 It is an alkenyl. In one embodiment, R f R is a linear C7 alkenyl. In one embodiment, R f R is a linear C8 alkenyl. In one embodiment, R f R is a linear C9 alkenyl. In one embodiment, R f is a straight chain C 10 It is an alkenyl. In one embodiment, R f is a straight chain C 11 It is an alkenyl. In one embodiment, R f is a straight chain C 12 It is an alkenyl. In one embodiment, R f is a straight chain C 13 It is an alkenyl. In one embodiment, R f is a straight chain C14 It is an alkenyl. In one embodiment, R f is a straight chain C 15 It is an alkenyl. In one embodiment, R f is a straight chain C 16 It is an alkenyl. In one embodiment, R f is a straight chain C 17 It is Alkenil.

[0185] In one embodiment, R f This is the branching chain C5~C 32 It is alkyl. In one embodiment, R f is branched chain C6~C 32 It is alkyl. In one embodiment, R f is branched chain C6~C 24 It is alkyl. In one embodiment, R f is, -R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C5 alkylenes, and R 8 and R 9 These are, independently, C2~C 10 It is alkyl. In one embodiment, R f ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C1 alkylenes, and R 8 and R 9 These are independently C4-C8 alkyl groups.

[0186] In one embodiment, R f This is the branching chain C5~C 32 It is an alkenyl. In one embodiment, R f is branched chain C6~C 32 It is an alkenyl. In one embodiment, R f is branched chain C6~C 24 It is an alkenyl. In one embodiment, R f ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C5 alkylenes, and R8 and R 9 These are, independently, C2~C 10 It is an alkenyl. In one embodiment, R f ha-R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C1 alkylenes, and R 8 and R 9 It is independent of C6~C 10 It is Alkenil.

[0187] In one embodiment, R 1 , R 2 , R c , and R f Each of them operates independently, as a linear C6~C 18 Alkyl, linear C6-C 18 Alkenyl, or -R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C5 alkylenes, and R 8 and R 9 These are, independently, C2~C 10 Alkyl or C2-C 10 It is Alkenil.

[0188] In one embodiment, R 1 , R 2 , R c , and R f Each of them operates independently, linear C7~C 15 Alkyl, linear C7-C 15 Alkenyl, or -R 7 -CH(R 8 )(R 9 ) and in the formula, R 7 These are C0-C1 alkylenes, and R 8 and R 9 These are independently C4-C8 alkyl or C6-C 10 It is Alkenil.

[0189] In one embodiment, R 1 , R 2 , R c , and Rf Each of these is independently one of the following structures: [ka]

[0190] In one embodiment, R 1 , R 2 , R c , and R f Each of these is replaced independently and at will.

[0191] In one embodiment, R a and R d Each of these is independently H. In one embodiment, R a , R b , R d , and R e Each of these is independently H. In one embodiment, R a and R d Each of these is independent of C1~C 24 It is alkyl. In one embodiment, R a and R d Each of these is independent of C1~C 18 It is alkyl. In one embodiment, R a and R d Each of these is independent of C1~C 12 It is alkyl. In one embodiment, R a and R d Each of these is independently a C1-C6 alkyl group.

[0192] In one embodiment, R b , R c , R e , and R f Each of these is independently either n-hexyl or n-octyl.

[0193] In one embodiment, R c and R f Each of these is independent of the branched chain C6~C 24 Alkyl or branched C6-C 24 It is an alkenyl. In one embodiment, R c and R fEach of these is independently -R 7 -CH(R 8 )(R 9 ) and, however, R 7 These are C1-C5 alkylenes, and R 8 and R 9 These are independent of C2~C 10 Alkyl or C2-C 10 It is Alkenil.

[0194] In one embodiment, R 1 and R 2 Each of these is independent of the branched chain C6~C 24 Alkyl or branched C6-C 24 It is an alkenyl. In one embodiment, R 1 and R 2 Each of these is independent of -R 7 -CH(R 8 )(R 9 ) and, however, R 7 These are C1-C5 alkylenes, and R 8 and R 9 These are independent of C2~C 10 Alkyl or C2-C 10 It is Alkenil.

[0195] In one embodiment, R 1 is a straight chain C6~C 24 It is alkyl, R 2 is a branched chain C6~C 24 It is alkyl. In one embodiment, R 1 is a straight chain C6~C 24 It is alkyl, R 2 ha-R 7 -CH(R 8 )(R 9 ) and, however, R 7 These are C1-C5 alkylenes, and R 8 and R 9 These are independent of C2~C 10 It is alkyl.

[0196] In one embodiment, the compound is one of the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer. [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]

[0197] In one embodiment, the compound is one of the compounds listed in Table 2, or a pharmaceutically acceptable salt thereof, prodrug, or stereoisomer. [Table 2-1] [Table 2-2]

[0198] It is understood that any embodiment of the compounds provided herein, and any particular substituents and / or variables in the compounds provided herein, may independently combine with substituents and / or variables of other embodiments and / or compounds to form embodiments not specifically described above. In addition, it is understood that if a list of substituents and / or variables is enumerated for any particular group or variable, each individual substituent and / or variable may be omitted from a particular embodiment and / or claim, and the remaining list of substituents and / or variables will be considered to be within the scope of the embodiments provided herein.

[0199] In this specification, it should be understood that combinations of substituents and / or variables in the given formulas are only permissible if such contributions result in a stable compound.

[0200] 6.4 Nanoparticle Compositions In one embodiment, what is described herein is a nanoparticle composition comprising a lipid compound as described herein. In a particular embodiment, the nanoparticle composition comprises a compound relating to formula (I) or formula (II) (and its subformula) as described herein.

[0201] In some embodiments, the maximum dimensions of the nanoparticle compositions provided herein are 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) when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or other methods. 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.

[0202] Examples of nanoparticle compositions that may be used in connection with this disclosure include 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 an aqueous compartment. The lipid bilayers may be functionalized and / or crosslinked with each other. The lipid bilayers may comprise one or more ligands, proteins, or channels.

[0203] The properties of a nanoparticle composition may depend on its constituent elements. For example, a nanoparticle composition containing cholesterol as a structural lipid may have different properties than 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 constituent elements. For example, a nanoparticle composition containing a higher mole fraction of phospholipids may have different properties than a nanoparticle composition containing a lower mole fraction of phospholipids. The properties may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0204] Nanoparticle compositions can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to investigate the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure the zeta potential. Particle size can also be measured using dynamic light scattering. Multiple properties of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential, can also be measured using instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, and Worcestershire, UK).

[0205] Dh (Size): The average size of the nanoparticle composition can range from tens of nanometers to hundreds of nanometers. For example, the average size can range from approximately 40 nm to approximately 150 nm, such as 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 may 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 may be about 70 nm to about 100 nm. In some embodiments, the average size may be about 80 nm. In other embodiments, the average size may be about 100 nm.

[0206] PDI: Nanoparticle compositions can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of a 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. Nanoparticle compositions may have polydispersity indices from 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 a nanoparticle composition may be from about 0.10 to about 0.20.

[0207] Encapsulation efficiency: The efficiency of encapsulation of therapeutic and / or prophylactic agents describes the amount of therapeutic and / or prophylactic agent encapsulated or otherwise associated with the nanoparticle composition after preparation, relative to the initial amount provided. High encapsulation efficiency is desirable (e.g., close to 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 decomposition of 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 the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of therapeutic and / or prophylactic agents may 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%.

[0208] The apparent pKa: The zeta potential of a nanoparticle composition can be used to indicate the interfacial dynamic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low positive or negative charges are generally preferable, as higher charged species can lead to undesirable interactions with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition may be approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0209] In another embodiment, self-replicating RNA may be formulated into liposomes. As a non-limiting example, self-replicating RNA may be formulated into liposomes as described in International Publication WO20120067378, which is incorporated herein by reference in whole. In one embodiment, the liposomes may contain lipids having a pKa value that may be advantageous for mRNA delivery. In another embodiment, the liposomes may have a surface charge that is essentially neutral at physiological pH and therefore may be effective for immunization (see, for example, the liposomes described in International Publication WO20120067378, which is incorporated herein by reference in whole).

[0210] In some embodiments, the nanoparticle compositions described herein include a lipid component comprising at least one lipid, such as a compound relating to one of the formulas (I) or (II) (and its subformulas) described herein. For example, in some embodiments, the nanoparticle compositions may include a lipid component comprising one of the compounds provided herein. The nanoparticle compositions may also include one or more other lipid or non-lipid components described below.

[0211] 6.4.1 Cationic / Ionizable Lipids As described herein, in some embodiments, the nanoparticle compositions provided herein include one or more charged or ionizable lipids in addition to lipids according to formula (I) or formula (II) (and its subformulas). Although not bound by theory, it is thought that certain charged or zwitterionic lipid components of the nanoparticle composition may resemble lipid components in cell membranes, thereby improving the cellular uptake of nanoparticles.Examples of charged or ionizable lipids that can form part of this nanoparticle composition include 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethaneamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), and 1,2-dilinoleyloxy-N,N-dimethyl Diylaminopropane (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-diene-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-diene-1-yloxy]propan-1-amine(octyl-CLinDMA( Examples include (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}heptadecane-8-amine.Further exemplary charged or ionizable lipids that can form part of the nanoparticle composition include the lipids 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), which is incorporated herein by reference in its entirety.

[0212] In some embodiments, preferred 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), and N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino [Butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamide-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 2Examples include (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), 1,2-dimyristreoyl-sn-glycero-3-ethylphosphocholine (MOEPC), (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-Cl), (R)-5-guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentane-1-aminium chloride (DOTAPen). Primary amines (e.g., DODAG) Cationic lipids having a head group charged at physiological pH, such as (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DOPen-G), are also preferred. Another preferred cationic lipid is (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DOPen-G)). The ionic lipid is an yl dioleate hydrochloride (DOTAP-Cl). In certain embodiments, the cationic lipid is a specific enantiomer or racemic form and includes various salt forms of the cationic lipid described above (e.g., chloride or sulfate). For example, in some embodiments, the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-Cl) or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulfate (DOTAP-sulfate).In some embodiments, the cationic lipids are ionizable cationic lipids such as dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[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 cationic lipids as described above).

[0213] In addition, in some embodiments, the charged or ionizable lipids that can form part of the nanoparticle composition are lipids containing cyclic amine groups. Further cationic lipids suitable for the formulations and methods disclosed herein are those described in WO2015199952, WO2016176330, and WO2015011633, the entirety of each of which is incorporated herein by reference.

[0214] 6.4.2 Polymer-complexed lipids In some embodiments, the lipid components of the nanoparticle composition may include one or more polymer-compounded lipids, such as PEGylated lipids (PEG lipids). While not bound by theory, polymer-compounded lipid components in nanoparticle compositions are thought to improve colloidal stability and / or reduce protein absorption of nanoparticles. Exemplary cationic lipids that may be used in connection with this 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, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, or Chol-PEG2000.

[0215] In one embodiment, the polymer-compounded lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerols (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamines (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), PEGylated ceramides (PEG-cer), or PEG dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl) carbamate or 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl) carbamate.

[0216] In one embodiment, the polymer-compounded lipid is present at a concentration in the range of 1.0 to 2.5 mole percent. In one embodiment, the polymer-compounded lipid is present at a concentration of about 1.7 mole percent. In one embodiment, the polymer-compounded lipid is present at a concentration of about 1.5 mole percent.

[0217] In one embodiment, the molar ratio of cationic lipids to polymer-compounded lipids is in the range of approximately 35:1 to approximately 25:1. In another embodiment, the molar ratio of cationic lipids to polymer-compounded lipids is in the range of approximately 100:1 to approximately 20:1.

[0218] In one embodiment, the pegylated lipid is given by the following formula: [ka] or having a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, in the formula, R 12 and R 13 Each of these is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds. w has an average value in the range of 30 to 60.

[0219] In one embodiment, R 12 and R 13 Each is independently a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, the average w is in the range of 42 to 55, for example, 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.

[0220] In one embodiment, the pegylated lipid has the following formula: [ka] (In the formula, the average value w is approximately 49.) It has.

[0221] 6.4.3 Structural lipids In some embodiments, the lipid components of the nanoparticle composition may include one or more structural lipids. While not theoretically bound, structural lipids are thought to be able to stabilize the amphiphilic structure of nanoparticles, such as, but not limited to, the lipid bilayer structure of the nanoparticles. Exemplary structural lipids that may be used in connection with this 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 includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

[0222] In one embodiment, the lipid nanoparticles provided herein include a steroid or a steroid analogue. In one embodiment, the steroid or steroid analogue is cholesterol. In one embodiment, the steroid is present at concentrations ranging from 39–49 mol percent, 40–46 mol percent, 40–44 mol percent, 40–42 mol percent, 42–44 mol percent, or 44–46 mol percent. In one embodiment, the steroid is present at concentrations of 40, 41, 42, 43, 44, 45, or 46 mol percent.

[0223] In one embodiment, the molar ratio of cationic lipids to steroids 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 molar ratio of cationic lipids to cholesterol is in the range of approximately 5:1 to 1:1. In one embodiment, the steroid is present at a concentration in the range of 32 to 40 mole percent of steroids.

[0224] 6.4.4 Phospholipids In some embodiments, the lipid components of the nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Although not bound by theory, phospholipids are thought to 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), and 1,2-dipalmitoyl-sn- 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-difytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine Examples include, but are not limited to, phosphoethanolamine, 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 includes both DSPC and DOPE.

[0225] Further exemplary neutral lipids include, for example, dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearyl-2-oleoylphosphatidineethanolamine (SOPE), and 1,2-dieryloyl-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.

[0226] In one embodiment, the neutral lipid is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylic acid (PA), or phosphatidylglycerol (PG).

[0227] In addition, the phospholipids that may form part of this nanoparticle composition include those described in WO2017 / 112865, the entirety of which is incorporated herein by reference.

[0228] 6.4.5 Therapeutic Payload According to this disclosure, the nanoparticle compositions described herein may further comprise one or more therapeutic and / or prophylactic agents. These therapeutic and / or prophylactic agents may be referred to herein as “therapeutic payloads” or “payloads.” In some embodiments, therapeutic payloads may be administered in vivo or in vitro using nanoparticles as a delivery vehicle.

[0229] In some embodiments, the nanoparticle composition is used as a therapeutic payload, containing antitumor agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (antitumor agents) (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methothotrexate and purines and pyrimidine analogs), antiinfective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensive agents (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., This includes small molecule compounds (e.g., small molecule drugs) such as phenytoin, antihistamines (diphenhydramine, chlorpheniramine, promethazine), antibiotics / antimicrobial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungal agents (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 agents.

[0230] In some embodiments, the therapeutic payload includes cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that induce immune responses, and / or other therapeutic and / or prophylactic agents. Cytotoxins or cytotoxic agents include any agents 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, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, mytansinoids, e.g., meitansinol, rakelmycin (CC-1065), and their analogues or homologues. 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.

[0231] In other embodiments, the therapeutic payload of the nanoparticle composition includes antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechloretamine, thiotepachlorambucil, racermycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, Therapeutic and / or prophylactic agents may include, but are not limited to, cis-dichlorodiamine platinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and meitansinoids).

[0232] In some embodiments, the nanoparticle composition includes biological molecules such as peptides and polypeptides as a therapeutic payload. The biomolecules forming part of the nanoparticle composition may be of natural origin or synthetic. For example, in some embodiments, the therapeutic payload of the nanoparticle composition may include, but is not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogues, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, cholera vaccine, and peptides and polypeptides.

[0233] 6.4.5.1 Nucleic acids In some embodiments, the nanoparticle composition comprises one or more nucleic acid molecules (e.g., DNA or RNA molecules) as a therapeutic payload. Exemplary forms of nucleic acid molecules that may be included in the nanoparticle composition as a therapeutic payload include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), their hybrids, RNAi inducers, 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 may be included in this nanoparticle composition as a therapeutic payload include, but are not limited to, short-mer RNAs, agomyl, antagomil, antisense RNAs, ribozymes, small interfering RNAs (siRNA), asymmetric interfering RNAs (aiRNA), microRNAs (miRNA), dicer substrate RNAs (dsRNA), small hairpin RNAs (shRNA), transfer RNAs (tRNA), messenger RNAs (mRNA), and other forms of RNA molecules known in the art. In certain embodiments, RNA is mRNA.

[0234] In other embodiments, the nanoparticle composition includes 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 specific disease, disorder, or illness when administered to a subject requiring the siRNA-containing nanoparticle composition. In some embodiments, the siRNA molecule includes a sequence complementary to the mRNA sequence encoding the protein product of interest. In some embodiments, the siRNA molecule is an immunomodulatory siRNA.

[0235] In some embodiments, the nanoparticle composition includes an shRNA molecule or a vector encoding an shRNA molecule as a therapeutic payload. In particular, in some embodiments, the therapeutic payload, when administered to target cells, produces shRNA within the target cells. Constructs and mechanisms related to shRNA are well known in the relevant art.

[0236] In some embodiments, the nanoparticle composition includes an mRNA molecule as a therapeutic payload. In particular, in some embodiments, the mRNA molecule encodes a polypeptide of interest, comprising any natural, non-natural, or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA payload may have a therapeutic effect when expressed in cells.

[0237] In some embodiments, the nucleic acid molecule of this disclosure comprises an mRNA molecule. In certain embodiments, the nucleic acid molecule comprises at least one coding region (e.g., an open reading frame (ORF)) encoding the peptide or polypeptide of interest. In some embodiments, the nucleic acid molecule further comprises at least one untranslated region (UTR). In certain embodiments, the untranslated region (UTR) is located upstream (to the 5' end) of the coding region and is referred to herein as the 5'-UTR. In certain embodiments, the untranslated region (UTR) is located downstream (to the 3' end) of the coding region and is referred to herein as the 3'-UTR. In certain embodiments, the nucleic acid molecule comprises both the 5'-UTR and the 3'-UTR. In some embodiments, the 5'-UTR comprises a 5' cap structure. In some embodiments, the nucleic acid molecule comprises a Kozak sequence (e.g., in the 5'-UTR). In some embodiments, the nucleic acid molecule comprises a poly-A region (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecule comprises a polyadenylation signal (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecule includes a stabilizing region (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecule includes a secondary structure. In some embodiments, the secondary structure is a stem-loop. In some embodiments, the nucleic acid molecule includes a stem-loop sequence (e.g., in the 5'-UTR and / or 3'-UTR). In some embodiments, the nucleic acid molecule includes one or more intron regions that can be excised during splicing. In certain embodiments, the nucleic acid molecule includes one or more regions selected from the 5'-UTR and the coding region. In certain embodiments, the nucleic acid molecule includes one or more regions selected from the coding region and the 3'-UTR. In certain embodiments, the nucleic acid molecule includes one or more regions selected from the 5'-UTR, the coding region, and the 3'-UTR.

[0238] Code region In some embodiments, the nucleic acid molecules of the present disclosure include at least one coding region. In some embodiments, the coding region is an open reading frame (ORF) encoding a single peptide or protein. In some embodiments, the coding region includes at least two ORFs, each encoding a peptide or protein. In those embodiments, where the coding region includes two or more ORFs, the encoded peptides and / or proteins may be the same as or different from each other. In some embodiments, multiple ORFs in the coding region are separated by a non-coding sequence. In certain embodiments, the non-coding sequence separating two ORFs includes an internal ribosome entry site (IRES).

[0239] While not constrained by theory, internal ribosome entry sites (IRESs) are thought to act as either a single ribosome binding site or as one of several ribosome binding sites on mRNA. mRNA molecules containing two or more functional ribosome binding sites can encode several peptides or polypeptides that are independently translated by ribosomes (e.g., multicistronic mRNA). Therefore, in some embodiments, the nucleic acid molecules (e.g., mRNA) of this disclosure contain one or more internal ribosome entry sites (IRESs). Examples of IRES sequences that may be used in connection with this disclosure include, but are not limited to, those derived from picomaviruses (e.g., FMDV), plague virus (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0240] In various embodiments, the nucleic acid molecules of this 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 may be the same or different. In some embodiments, the nucleic acid molecules of this disclosure encode dipeptides (e.g., camosine and anserine). In some embodiments, the nucleic acid molecules encode tripeptides. In some embodiments, the nucleic acid molecules encode tetrapeptides. In some embodiments, the nucleic acid molecules encode pentapeptides. In some embodiments, the nucleic acid molecules encode hexapeptides. In some embodiments, the nucleic acid molecules encode heptapeptides. In some embodiments, the nucleic acid molecules encode octapeptides. In some embodiments, the nucleic acid molecules encode nonapeptides. In some embodiments, the nucleic acid molecules encode decapeptides. In some embodiments, the nucleic acid molecules encode peptides or polypeptides having at least about 15 amino acids. In some embodiments, the nucleic acid molecules encode peptides or polypeptides having at least about 50 amino acids. In some embodiments, the 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.

[0241] In some embodiments, the nucleic acid molecule of the Disclosure is at least about 30 nucleotides (nt) long. In some embodiments, the nucleic acid molecule is at least about 35 nt long. In some embodiments, the nucleic acid molecule is at least about 40 nt long. In some embodiments, the nucleic acid molecule is at least about 45 nt long. In some embodiments, the nucleic acid molecule is at least about 50 nt long. In some embodiments, the nucleic acid molecule is at least about 55 nt long. In some embodiments, the nucleic acid molecule is at least about 60 nt long. In some embodiments, the nucleic acid molecule is at least about 65 nt long. In some embodiments, the nucleic acid molecule is at least about 70 nt long. In some embodiments, the nucleic acid molecule is at least about 75 nt long. In some embodiments, the nucleic acid molecule is at least about 80 nt long. In some embodiments, the nucleic acid molecule is at least about 85 nt long. In some embodiments, the nucleic acid molecule is at least about 90 nt long. In some embodiments, the nucleic acid molecule is at least about 95 nt long. In some embodiments, the nucleic acid molecule is at least about 100 nt long. 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 long. In some embodiments, the nucleic acid molecule is at least about 1200 nt long. In some embodiments, the nucleic acid molecule is at least about 1300 nt long. In some embodiments, the nucleic acid molecule is at least about 1400 nt long. In some embodiments, the nucleic acid molecule is at least about 1500 nt long. In some embodiments, the nucleic acid molecule is at least about 1600 nt long. In some embodiments, the nucleic acid molecule is at least about 1700 nt long. In some embodiments, the nucleic acid molecule is at least about 1800 nt long. In some embodiments, the nucleic acid molecule is at least about 1900 nt long. In some embodiments, the nucleic acid molecule is at least about 2000 nt long. In some embodiments, the nucleic acid molecule is at least about 2500 nt long. In some embodiments, the nucleic acid molecule is at least about 3000 nt long. In some embodiments, the nucleic acid molecule is at least about 3500 nt long. In some embodiments, the nucleic acid molecule is at least about 4000 nt long. 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.

[0242] In certain embodiments, the therapeutic payload comprises a vaccine composition described herein (e.g., a gene vaccine). In some embodiments, the therapeutic payload comprises a compound capable of inducing immunity to one or more target diseases or disorders. In some embodiments, the target diseases are associated with or caused by infection with pathogens such as coronaviruses (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 characteristic of the pathogen, or its antigenic fragment or epitope. When administered to a vaccinated subject, the vaccine enables the expression of the encoded pathogenic protein (or its antigenic fragment or epitope), thereby inducing immunity in the subject to the pathogen.

[0243] In some embodiments, the target disease is associated with or caused by the neoplastic growth of cells, such as cancer. In some embodiments, the therapeutic payload includes a nucleic acid sequence (e.g., mRNA) encoding a tumor-associated antigen (TAA) or its antigen fragment or epitope, which is characteristic of cancer. When administered to a vaccinated subject, the vaccine enables the expression of the encoded TAA (or its antigen fragment or epitope), thereby inducing immunity in the subject against tumor cells expressing the TAA.

[0244] 5' cap structure While not bound by theory, it is thought that the 5' cap structure of polynucleotides is involved in nuclear transport and increased polynucleotide stability, and that it binds to mRNA cap-binding proteins (CBPs) involved in polynucleotide stability and translational capacity in cells through association with CBPs with poly(A)-binding proteins to form mature circular mRNA species. The 5' cap structure further assists in the removal of 5' proximal introns during mRNA splicing. Therefore, in some embodiments, the nucleic acid molecules of this disclosure include a 5' cap structure.

[0245] Nucleic acid molecules can be capped at the 5' end by the cell's endogenous transcription mechanism, creating a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue of the polynucleotide and the 5'-terminal transcription sense nucleotide. This 5' guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugars of the 5' terminal and / or anteterminal transcription nucleotides of the polynucleotide can also be optionally 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.

[0246] In some embodiments, the nucleic acid molecules of this disclosure include one or more modifications to the native 5' cap structure produced by endogenous processes. While not bound by theory, modifications on the 5' cap may increase the stability of the polynucleotide, increase the half-life of the polynucleotide, and increase the translation efficiency of the polynucleotide.

[0247] Exemplary modifications to the natural 5' cap structure include the creation of a non-hydrolyzable cap structure that prevents detachment and thus increases the polynucleotide half-life. In some embodiments, hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester linkage; therefore, in some embodiments, modified nucleotides may be used during the capping reaction. For example, in some embodiments, a vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) may be used with α-thio-guanosine nucleoti, according to the manufacturer's instructions, to create the phosphorothioate linkage at the 5'-ppp-5' cap. Further modified guanosine nucleotides, such as α-methyl-phosphonate and seleno-phosphonate nucleotides, may be used.

[0248] Further exemplary modifications to the natural 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 the carbocyclic ring) with a methylene moiety (CH2), modifications at the triphosphate crosslinking portion of the cap structure, or modifications at the nucleic acid base (G) portion.

[0249] Further exemplary modifications to the natural 5' cap structure include, but are not limited to, 2'-O-methylation of the ribose sugar of the 5' terminus and / or 5' preterminus nucleotide of a polynucleotide (as described above) on the 2' hydroxyl 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. Further exemplary 5' cap structures that may be used in connection with this disclosure include, but are not limited to, those described in International Patent Publications WO2008127688, WO2008016473, and WO2011015347, the entirety of each of which is incorporated herein by reference.

[0250] In various embodiments, the 5' terminal cap may include cap analogues. Cap analogues, also referred to herein as synthetic cap analogues, chemical caps, chemical cap analogues, or structural or functional cap analogues, retain cap function while differing in their chemical structure from the natural (i.e., endogenous, wild-type, or physical) 5' cap. Cap analogues may be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or linked to polynucleotides.

[0251] For example, an anti-reverse cap analog (ARCA) cap contains two guanosines linked by a 5'-5'-triphosphate group, and one guanosine contains an N7-methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7G-3'mppp-G, which may be equivalently designated as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of other unmodified guanosine is ligated to the 5' terminal nucleotide (e.g., mRNA) of the capped polynucleotide. N7- and 3'-O-methylated guanosine provide the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary capping 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).

[0252] In some embodiments, the cap analog may be a dinucleotide cap analog. In non-limiting examples, the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group, such as the dinucleotide cap analog described in U.S. Patent No. 8,519,110, the entire scope of which is incorporated herein by reference.

[0253] In some embodiments, the cap analogues may be N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analogues known in the Art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analogues include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogues (see, for example, Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574, the various cap analogues and methods for synthesizing them, the entire contents of which are incorporated herein by reference). In other embodiments, a useful cap analogue in relation to the nucleic acid molecules of this disclosure is a 4-chloro / bromophenoxyethyl analogue.

[0254] In various embodiments, the cap analog may include guanosine analogs. 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.

[0255] While not constrained by theory, it is conceivable that cap analogs enable co-capping of polynucleotides in in vitro transcription reactions, but up to 20% of the transcript remains uncapped. This, like structural differences between cap analogs and the natural 5' cap structure of polynucleotides produced by the cell's endogenous transcription mechanism, could lead to reduced translational capacity and decreased cellular stability.

[0256] Accordingly, in some embodiments, nucleic acid molecules of the present disclosure may be post-transcriptionally capped using an enzyme to produce a more authentic 5' cap structure. As used herein, the term “more authentic” refers to a feature that more closely reflects or mimics an endogenous or wild-type feature, either structurally or functionally. That is, a “more authentic” feature better represents an endogenous wild-type, natural, or physiological cellular function and / or structure, or is superior in one or more respects to the corresponding endogenous wild-type, natural, or physiological feature, compared to a synthetic feature or analogue of the prior art. Non-limiting examples of more authentic 5' cap structures useful in relation to nucleic acid molecules of the present disclosure include, among other things, those having enhanced cap-binding protein binding, increased half-life, reduced sensitivity 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, recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5'-5'-triphosphate bond between the 5' terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, where the cap guanosine includes N7 methylation and the 5' terminal nucleotide of the polynucleotide includes 2'-O-methylation. Such a structure is referred to as a cap 1 structure. This cap results in higher translational capacity, cell stability, and reduced activation of pro-inflammatory cytokines compared, for example, with 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).

[0257] While not bound by theory, it is conceivable that the nucleic acid molecules of this disclosure can be capped after transcription, and since this process is more efficient, nearly 100% of the nucleic acid molecules can be capped.

[0258] Untranslated area (UTR) In some embodiments, the nucleic acid molecules of this disclosure include one or more untranslated regions (UTRs). In some embodiments, the UTRs are located upstream of the coding region in the nucleic acid molecule and are referred to as 5'-UTRs. In some embodiments, the UTRs are located downstream of the coding region in the nucleic acid molecule and are referred to as 3'-UTRs. The sequences of the UTRs may be homologous or heterologous to the sequences of the coding region found in the nucleic acid molecule. Multiple UTRs may be included in the nucleic acid molecule, and may have the same or different sequences and / or be of genetic origin. According to this disclosure, any portion (including none) of the UTRs in the nucleic acid molecule may be codon-optimized, and each may independently contain one or more different structural or chemical modifications before and / or after codon optimization.

[0259] In some embodiments, the nucleic acid molecule (e.g., mRNA) of the Disclosure comprises a UTR and coding region that are homologous with respect to each other. In other embodiments, the nucleic acid molecule (e.g., mRNA) of the Disclosure comprises a UTR and coding region that are heterogeneous with respect to each other. In some embodiments, a nucleic acid molecule comprising a UTR and a coding sequence of a detectable probe can be administered in vitro (e.g., in a cell or tissue culture) or in vivo (e.g., to a subject) to monitor the activity of the UTR sequence, and the effect of the UTR sequence (e.g., regulation 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.

[0260] In some embodiments, the UTR of a nucleic acid molecule (e.g., mRNA) of the Disclosure includes at least one translational enhancer element (TEE) that functions to increase the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the TEE is located at the 5'-UTR of the nucleic acid molecule. In other embodiments, the TEE is located at the 3'-UTR of the nucleic acid molecule. In yet another embodiment, at least two TEEs are located at the 5'-UTR and 3'-UTR of the nucleic acid molecule, respectively. In some embodiments, a nucleic acid molecule (e.g., mRNA) of the Disclosure may contain one or more copies of a TEE sequence, or two or more different TEE sequences. In some embodiments, the different TEE sequences present in the nucleic acid molecule of the Disclosure may be homologous or heterogeneous with respect to one another.

[0261] Various TEE sequences known in the art may be used in connection with this disclosure. For example, in some embodiments, the TEE may 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. Further internal ribosome entry sites (IRESs) that may be used in connection with this disclosure include, but are not limited to, those described in U.S. Patent No. 7,468,275, U.S. Patent Publication No. 2007 / 0048776, U.S. Patent Publication No. 2011 / 0124100, and International Patent Publication No. WO2007 / 025008 and International Patent Publication No. WO2001 / 055369, the entire contents of each of these publications are incorporated herein by reference. In some embodiments, the TEEs may be those described in Supplementary Tables 1 and 2 of Wellensiek et al. Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug;10(8):747-750, the entire contents of which are incorporated herein by reference.

[0262] Further exemplary TEEs that may be used in connection with this disclosure include U.S. Patent Nos. 6,310,197, 6,849,405, 7,456,273, 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, and Examples of TEE sequences disclosed in International Patent Publication WO2009 / 075886, International Patent Publication WO2012 / 009644, and International Patent Publication WO1999 / 024595, International Patent Publication WO2007 / 025008, International Patent Publication WO2001 / 055371, European Patent No. 2610341, and European Patent No. 2610340 include, but are not limited to, the contents of each of these, which are incorporated herein by reference in their entirety.

[0263] In various embodiments, the nucleic acid molecule (e.g., mRNA) of the Disclosure comprises at least one UTR containing at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, at least thirty, at least thirty-five, at least forty, at least forty-five, at least fifty, at least fifty, or more than sixty 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 within the UTR region of the nucleic acid molecule. For illustrative purposes only, the repeating patterns may be, for example, ABABAB, AABBAABBAABB, ABCABCABC, and in these exemplary patterns, each capital letter (A, B, or C) represents a different TEE sequence. In some embodiments, at least two TEE sequences are contiguous with each other in the UTR of the nucleic acid molecule (i.e., there are no spacer sequences in between). In other embodiments, at least two TEE sequences are separated by spacer sequences. In some embodiments, the UTR may include TEE sequence spacer sequence modules that are 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 within the UTR. In any of the embodiments described in this paragraph, the UTR may be the 5'-UTR, 3'-UTR, or both the 5'-UTR and 3'-UTR of the nucleic acid molecule.

[0264] In some embodiments, the UTR of a nucleic acid molecule (e.g., mRNA) of the Disclosure comprises at least one translational repressor element that functions to reduce the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the UTR of a 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 a 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 nucleic acid molecules are known in the art. In any of the embodiments described in this paragraph, the UTR may be the 5'-UTR, 3'-UTR, or both the 5'-UTR and 3'-UTR of the nucleic acid molecule.

[0265] Polyadenylated (Poly-A) region During processing of natural RNA, the long chain of adenosine nucleotides (poly-A region) is typically added to the messenger RNA (mRNA) molecule, increasing its stability. Immediately after transcription, the 3' end of the transcript is cleaved to release 3'-hydroxyl. Then, poly-A polymerase adds the adenosine nucleotide chain to the RNA. This process, called polyadenylation, adds a poly-A region of between 100 and 250 residues in length. While not bound by theory, it is thought that the poly-A region can confer various advantages to the nucleic acid molecules of this disclosure.

[0266] Accordingly, in some embodiments, the nucleic acid molecule (e.g., mRNA) of the Disclosure includes a polyadenylation signal. In some embodiments, the nucleic acid molecule (e.g., mRNA) of the Disclosure includes one or more polyadenylation (poly-A) regions. In some embodiments, the poly-A region consists entirely of an adenine nucleotide or a functional analog thereof. In some embodiments, the nucleic acid molecule includes at least one poly-A region at its 3' end. In some embodiments, the nucleic acid molecule includes at least one poly-A region at its 5' end. In some embodiments, the nucleic acid molecule includes at least one poly-A region at its 5' end and at least one poly-A region at its 3' end.

[0267] According to this disclosure, the length of the polyA region can vary in different embodiments. In particular, in some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 30 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 35 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 40 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 45 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 50 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 55 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 60 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 65 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 70 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of this disclosure is at least 75 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 80 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 85 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 90 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 95 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 100 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 110 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 120 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 130 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 140 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 150 nucleotides long.In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 160 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 170 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 180 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 190 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 200 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 225 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 250 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 275 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 300 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 350 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 400 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 450 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 500 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 600 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 700 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 800 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 900 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1000 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1100 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1200 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1300 nucleotides long.In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1400 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1500 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1600 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1700 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1800 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 1900 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 2000 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 2250 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 2500 nucleotides long. In some embodiments, the polyA region of the nucleic acid molecule of the disclosure is at least 2750 nucleotides long. In some embodiments, the poly(A) region of the nucleic acid molecule of the present disclosure is at least 3000 nucleotides long.

[0268] In some embodiments, the length of the polyA region in a nucleic acid molecule may 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). For example, in some embodiments, the polyA region occupies 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 containing the polyA region.

[0269] While not theoretically bound, certain RNA-binding proteins are thought to be able to bind to the poly(A) region located at the 3' end of the mRNA molecule. These poly(A)-binding proteins (PABPs) can regulate mRNA expression, such as by interacting with the translation initiation mechanism in cells and / or protecting the 3' poly(A) tail from degradation. Therefore, in some embodiments, the nucleic acid molecule (e.g., mRNA) of this disclosure contains at least one binding site for a poly(A)-binding protein (PABP). In other embodiments, the nucleic acid molecule is complexed or complexed with a PABP before being loaded onto a delivery vehicle (e.g., lipid nanoparticles).

[0270] In some embodiments, the nucleic acid molecule (e.g., mRNA) of this disclosure comprises a polyAG quartet. The G quartet is a cyclic hydrogen-bonded array of four guanosine nucleotides that can be formed by a G-rich sequence in both DNA and RNA. In this embodiment, the G quartet is incorporated at the end of a polyA region. The resulting polynucleotide (e.g., mRNA) can be assayed at various time points for other parameters, including stability, protein production, and half-life. It has been found that the polyAG quartet structure yields protein production equal to at least 75% of the protein production seen using only a 120-nucleotide polyA region.

[0271] In some embodiments, the nucleic acid molecule (e.g., mRNA) of the Disclosure may include a poly-A region and may be stabilized by the addition of a 3' stabilizing region. In some embodiments, the 3' stabilizing region that may be used to stabilize the nucleic acid molecule (e.g., mRNA) includes a poly-A or poly-AG quartet structure described in International Patent Publication WO2013 / 103659, the entirety of which is incorporated herein by reference.

[0272] In other embodiments, the 3' stabilizing region that may be used in relation to the nucleic acid molecules of this disclosure is 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. This includes any 2',3'-dideoxynucleoside, 2'-deoxynucleoside, or O-methylnucleoside, 3'-deoxynucleoside, 2',3'-dideoxynucleoside, 3'-O-methylnucleoside, 3'-O-ethylnucleoside, 3'-arabinoside, and other alternative nucleosides known in the art and / or described herein, but not limited to these.

[0273] secondary structure While not constrained by theory, stem-loop structures can potentially function as substrates for enzymatic reactions, oriented towards RNA folding, protecting the structural stability of nucleic acid molecules (e.g., mRNA), providing recognition sites for RNA-binding proteins, and facilitating enzymatic reactions. For example, the incorporation of miR and / or TEE sequences alters the shape of the stem-loop region, potentially increasing and / or decreasing 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 entire content of which is incorporated herein by reference).

[0274] Accordingly, in some embodiments, nucleic acid molecules (e.g., mRNA) or parts thereof described herein may take the form of 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 having a length of about 25 or about 26 nucleotides, such as but not limited to the one described in International Patent Publication WO2013 / 103659, the entire contents of which are incorporated herein by reference. Further examples of stem-loop sequences include those described in International Patent Publication WO2012 / 019780 and International Patent Publication WO201502667, the contents of which are incorporated herein by reference. In some embodiments, the stem-loop sequence includes a TEE described herein. In some embodiments, the stem-loop sequence includes a miR sequence described herein. In certain embodiments, the stem-loop sequence may include a miR-122 seed sequence. In certain embodiments, the nucleic acid molecule comprises two stem-loop sequences described in International Patent Publication WO2021204175 (which is incorporated herein by reference in its entirety).

[0275] In some embodiments, the nucleic acid molecule of the Disclosure (e.g., mRNA) includes a stem-loop sequence located upstream (towards the 5' end) of the 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, the nucleic acid molecule of the Disclosure (e.g., mRNA) includes a stem-loop sequence located downstream (towards the 3' end) of the 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, the nucleic acid molecule may contain two or more stem-loop sequences. In some embodiments, the nucleic acid molecule includes at least one stem-loop sequence in the 5'-UTR and at least one stem-loop sequence in the 3'-UTR.

[0276] In some embodiments, the nucleic acid molecule containing the stem-loop structure further includes a stabilizing region. In some embodiments, the stabilizing region includes at least one chain-terminating nucleoside that functions to slow degradation and thus increase the half-life of the nucleic acid molecule. Examples of chain-terminating nucleosides that may be used in connection with this 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 can be stabilized by modification of the 3' region of the polynucleotide, which can prevent and / or inhibit the addition of oligo(U) (the entirety of which is incorporated herein by reference, International Patent Publication WO2013 / 103659).

[0277] In some embodiments, the nucleic acid molecules of this disclosure include at least one stem-loop sequence and a poly-A region or polyadenylation signal. Non-limiting examples of polynucleotide sequences including at least one stem-loop sequence and a poly-A region or polyadenylation signal are described in International Patent Publication WO2013 / 120497, International Patent Publication WO2013 / 120629, International Patent Publication WO2013 / 120500, International Patent Publication WO2013 / 120627, International Patent Publication WO2013 / 120498, International Patent Publication WO2013 / 120626, International Patent Publication WO2013 / 120499, and International Patent Publication WO2013 / 120628, the contents of each of these, in whole, are incorporated herein by reference.

[0278] In some embodiments, nucleic acid molecules comprising a stem-loop sequence and a poly-A region or polyadenylation signal can encode pathogenic antigens or fragments thereof, such as polynucleotide sequences described in International Patent Publication WO2013 / 120499 and International Patent Publication WO2013 / 120628, the contents of which are incorporated herein by reference in whole.

[0279] In some embodiments, nucleic acid molecules comprising a stem-loop sequence and a poly-A region or polyadenylation signal can encode therapeutic proteins such as polynucleotide sequences described in International Patent Publication WO2013 / 120497 and International Patent Publication WO2013 / 120629, the contents of which are incorporated herein by reference in whole.

[0280] In some embodiments, nucleic acid molecules comprising a stem-loop sequence and a poly-A region or polyadenylation signal can encode tumor antigens or fragments thereof, such as polynucleotide sequences described in International Patent Publication WO2013 / 120500 and International Patent Publication WO2013 / 120627, the contents of which are incorporated herein by reference in whole.

[0281] In some embodiments, nucleic acid molecules comprising a stem-loop sequence and a poly-A region or polyadenylation signal can encode allergic antigens or autoimmune autoantigens, such as polynucleotide sequences described in International Patent Publication WO2013 / 120498 and International Patent Publication WO2013 / 120626, the entire contents of which are incorporated herein by reference.

[0282] 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). While not bound by theory, certain functional nucleotide analogs are thought to be able to confer useful properties to nucleic acid molecules. Examples of useful properties in the context of this disclosure include, but are not limited to, increased stability of nucleic acid molecules, reduced immunogenicity of nucleic acid molecules in innate immune responses, enhanced production of proteins encoded by nucleic acid molecules, increased intracellular delivery and / or retention of nucleic acid molecules, and / or reduced cytotoxicity of nucleic acid molecules.

[0283] Therefore, in some embodiments, the 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 the nucleic acid base, sugar group, and / or phosphate group. Therefore, a payload nucleic acid molecule comprising at least one functional nucleotide analog contains at least one chemical modification to the nucleic acid base, sugar group, and / or nucleoside linkage. Exemplary chemical modifications to the nucleic acid base, sugar group, or nucleoside linkage of a nucleic acid molecule are provided herein.

[0284] As described herein, the range of 0% to 100% of all nucleotides in a payload nucleic acid molecule may be functional nucleotide analogs 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%, and about 20% to about 70% of all nucleotides in a nucleic acid molecule. Approximately 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% are functional nucleotide analogs as described herein. In any of these embodiments, the functional nucleotide analog may be located at any position(s) of the nucleic acid molecule, including the 5' end, the 3' end, and / or one or more internal positions. In some embodiments, a single nucleic acid molecule may contain different sugar modifications, different nucleic acid base modifications, and / or different types of internucleoside linkages (e.g., skeletal structures).

[0285] As described herein, a range of 0% to 100% of certain nucleotides in a payload nucleic acid molecule (e.g., all purine-containing nucleotides as a species, or all pyrimidine-containing nucleotides as a species, or all A, G, C, T, or U as a species) may be functional nucleotide analogs as described herein. For example, in various embodiments, approximately 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, and 20% to 70% of certain nucleotides in a nucleic acid molecule. Approximately 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% are functional nucleotide analogs as described herein. In any of these embodiments, the functional nucleotide analog may be located at any position(s) of the nucleic acid molecule, including the 5' end, the 3' end, and / or one or more internal positions. In some embodiments, a single nucleic acid molecule may contain different sugar modifications, different nucleic acid base modifications, and / or different types of internucleoside linkages (e.g., skeletal structures).

[0286] Modification of nucleic acid bases In some embodiments, the functional nucleotide analogs contain non-standard nucleic acid bases. In some embodiments, standard nucleic acid bases in nucleotides (e.g., adenine, guanine, uracil, thymine, and cytosine) may be modified or substituted to provide one or more functional analogs of nucleotides. Exemplary modifications to nucleic acid bases include, but are not limited to, one or more substitutions or modifications, including alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions, one or more condensations or ring openings, oxidation, and / or reductions.

[0287] In some embodiments, the non-standard nucleic acid base is modified uracil. Exemplary nucleic acid bases and nucleosides having modified uracil include pseudouridine (ψ), pyridine-4-onribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, and 2-thiouracil (s 2 U), 4-thiouracil(s 4 U), 4-thiopsoiduridine, 2-thiopsoiduridine, 5-hydroxyuracil (ho 5 U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyluracil (m 3 U), 5-methoxyuracil (mo 5 U), uracil 5-oxyacetic acid (cmo 5 U), uracil 5-oxyacetate methyl ester (mcmo 5 U), 5-carboxymethyl-uracil (cm 5 U), 1-carboxymethyl-psoidouridine, 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-thiouracil (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-pseudolacil, 5-taurinomethyl-uracil (τm 5 U), 1-taurinomethyl-psoidouridine, 5-taurinomethyl-2-thiouracil (τm 5 5s 2 U), 1-taurinomethyl-4-thiopsoidouridine, 5-methyluracil (m 5 U, i.e., having the nucleic acid base deoxythymine), 1-methyl-psoiduridine (m 1 ψ), 1-ethyl-psoidouridine (Et 1 ψ), 5-methyl-2-thiouracil (m 5 s 2 U), 1-methyl-4-thio-psoidouridine (m 1 s 4 ψ), 4-thio-1-methyl-psoidouridine, 3-methyl-psoidouridine (m 3 ψ), 2-thio-1-methyl-psoidouridine, 1-methyl-1-deaz-psoidouridine, 2-thio-1-methyl-1-deaz-psoidouridine, dihydrouracil(D), dihydropsoidouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil(m 5D) 2-thio-dihydrouracil, 2-thio-dihydropsoidouridine, 2-methoxy-uracil, 2-methoxy-4-thiouracil, 4-methoxy-psoidouridine, 4-methoxy-2-thiopsoidouridine, N1-methylpsoidouridine, 3-(3-amino-3-carboxypropyl)uracil (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)psoidouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uracil (m 5 U), 5-(isopentenylaminomethyl)-2-thiouracil(m 5 s 2 U), 5,2'-O-dimethyluridine (m 5 Um), 2-thio-2'-O-methyluridine(s) 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-Carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), and 5-(isopentenylaminomethyl)-2'-O-methyluridine(inm 5 Examples include Um), 1-thiouracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyluracil, 5-methoxy-2-thiouracil, and 5-[3-(1-E-propenylamino)]uracil.

[0288] In some embodiments, the non-standard nucleic acid base is modified cytosine. Exemplary nucleic acid bases and nucleosides having modified cytosine include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methylcytosine (m4C), 5-methylcytosine (m5C), 5-halocytosine (e.g., 5-iodocytosine), 5- Hydroxymethyl-cytosine (hm5C), 1-methyl-psoidisocytidine, pyrrol-cytosine, pyrrol-psoidisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytidine, 4-thio-psoidisocytidine, 4-thio-1-methyl-psoidisocytidine, 4-thio-1-methyl-1-deazapse-psoidisocytidine, 1-methyl-1-deazapse Doisocytidine, Zebralin, 5-Aza-Zebralin, 5-Methyl-Zebralin, 5-Aza-2-Thio-Zebralin, 2-Thio-Zebralin, 2-Methoxycytosine, 2-Methoxy-5-Methylcytosine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, Lysidine (k2C), 5,2'-O-Dimethylcytidine (m5Cm), N4-A Examples include cetyl-2'-O-methylcytidine (ac4Cm), N4,2'-O-dimethylcytidine (m4Cm), 5-formyl-2'-O-methylcytidine (fSCm), N4,N4,2'-O-trimethylcytidine (m42Cm), 1-thiocytosine, 5-hydroxycytosine, 5-(3-azidopropyl)cytosine, and 5-(2-azidoethyl)cytosine.

[0289] In some embodiments, the non-standard nucleic acid base is a modified adenine. Exemplary nucleic acid bases and nucleosides having alternative adenines include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8- Aza-2,6-diaminopurine, 1-methyl-adenine (m1A), 2-methyl-adenine (m2A), N6-methiadenine (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-glycinyl Bamoyl-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 Examples include 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.

[0290] In some embodiments, the non-standard nucleic acid base is modified guanine. Exemplary nucleic acid bases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), waiosine (imG), methylwaiosine (mimG), 4-demethylwaiosine (imG-14), isowyosine (imG2), waibutosine (yW), peroxywaibutosine (o2yW), hydroxywaibutosine (OHyW), and low-modified hydroxywaibutosine (OHyW) * ), 7-deaza-guanine, quosin (Q), epoxyquosin (oQ), galactosylquosin (galQ), mannosylquosin (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQ1), alkaeosin (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, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7- Examples include 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-6-thio-guanine, N2-methyl-2'-O-methyl-guanine (m2Gm), N2,N2-dimutyl-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.

[0291] In some embodiments, the non-standard nucleic acid bases of the functional nucleotide analog may independently be purines, pyrimidines, or purine or pyrimidine analogs. For example, in some embodiments, the non-standard nucleic acid bases may be modified adenine, cytosine, guanine, uracil, or hypoxanthine. In other embodiments, non-standard nucleic acid bases 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-thioalkyl, 8-hydroxy and other 8- This may also include naturally occurring and synthetic derivatives of bases containing substituted adenines and guanines, 5-halos, particularly 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-triazinon, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazine-2-one, 1,2,4-triazine, pyridazine, or 1,3,5-triazine.

[0292] Modification of sugars In some embodiments, the functional nucleotide analog contains a non-standard sugar group. In various embodiments, the non-standard sugar group may be a five-carbon or six-carbon sugar having one or more substitutions such as a halo group, hydroxyl group, thiol group, alkyl group, alkoxy group, alkenyloxy group, alkynyloxy group, cycloalkyl group, aminoalkoxy group, alkoxyalkoxy group, hydroxyalkoxy group, amino group, azide group, aryl group, aminoalkyl group, aminoalkenyl group, aminoalkynyl group, etc. (e.g., pentose, ribose, arabinose, xylose, glucose, galactose, or their deoxy derivatives).

[0293] Generally, RNA molecules contain a ribose sugar group, which is a five-membered ring containing oxygen. Exemplary and non-limiting alternative nucleotides include substitution of oxygen in ribose (e.g., S, Se, or alkylene such as methylene or ethylene), addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl), ring contraction of ribose (e.g., to form a four-membered ring of cyclobutane or oxetane), and additional carbon (e.g., anhydrohexitol, althritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino (which also has a phosphoramide backbone)). This includes ring expansions of ribose (to form a 6 or 7-membered ring having an element or heteroatom), polycyclic forms (e.g., “unlocked” forms such as tricyclo and glycol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units bonded to a phosphodiester linkage)), threose nucleic acids (TNA, where ribose is replaced by α-L-treophranosyl-(3'→2')), and peptide nucleic acids (PNA, where a 2-amino-ethyl-glycine linkage is replaced by ribose and a phosphodiester backbone).

[0294] In some embodiments, the sugar group contains one or more carbons having the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, the nucleic acid molecule may include a nucleotide containing, for example, arabinose or L-ribose as the sugar. In some embodiments, the nucleic acid molecule contains at least one nucleoside, and the sugar is L-ribose, 2'-O-methylribose, 2'-fluororibose, arabinose, hexitol, LNA, or PNA.

[0295] Modification of internucleoside linkages In some embodiments, the payload nucleic acid molecule of the present disclosure may include one or more modified internucleoside links (e.g., a phosphate skeleton). The phosphate group of the skeleton may be modified by replacing one or more oxygen atoms with different substituents.

[0296] In some embodiments, functional nucleotide analogs may include substitution of the unmodified phosphate moiety in another nucleoside linkage 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 phosphotryesters. Phosphorodithioates replace both unbound oxygen atoms with sulfur. The phosphate linker may also be modified by replacing the linked oxygen atoms with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate).

[0297] Alternative nucleosides and nucleotides may involve replacing one or more of the non-crosslinked oxygen atoms with borane moieties (BH3), sulfur (thio), methyl, ethyl, and / or methoxy. As a non-limiting example, two non-crosslinked oxygen atoms at the same position (e.g., alpha (α), beta (β), or gamma (γ) positions) may be replaced with sulfur (thio) and methoxy. Replacement of one or more oxygen atoms at the phosphate moiety position (e.g., α-thiophosphate) is provided to confer stability (e.g., against exonucleases and endonucleases) to RNA and DNA via unnatural phosphorothioate skeletal linkage. Phosphothioate DNA and RNA exhibit increased nuclease resistance and subsequently longer half-lives in the cellular environment.

[0298] Other internucleoside links that may be used in accordance with this disclosure, including internucleoside links that do not contain phosphorus atoms, are described herein.

[0299] Further examples of nucleic acid molecules (e.g., mRNA), compositions, formulations and / or related methods that may be used in connection with this 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 This includes the contents described in 15 / 024669, WO2015 / 024668, WO2015 / 024667, WO2015 / 024665, WO2015 / 024666, WO2015 / 024664, WO2015101415, WO2015101414, WO2015024667, WO2015062738, and WO2015101416, the contents of each of these in whole being incorporated herein.

[0300] 6.5 Formulations According to this disclosure, the nanoparticle compositions described herein may comprise at least one lipid component and one or more additional components, such as therapeutic and / or prophylactic agents. The nanoparticle compositions may be designed for one or more specific uses or targets. The elements of the nanoparticle compositions may be selected based on a specific use or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more elements. Similarly, a particular formulation of a nanoparticle composition may be selected for a specific use or target, for example, depending on the efficacy and toxicity of a particular combination of elements.

[0301] The lipid components of the nanoparticle composition may include, for example, lipids of one of the formulas (I) or (II) (and its subformulas) described herein, phospholipids (unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. The elements of the lipid components may be provided in specific fractions.

[0302] In one embodiment, the herein provides a nanoparticle composition comprising a cationic or ionizable lipid compound, a therapeutic agent, and one or more excipients provided herein. In one embodiment, the cationic or ionizable lipid compound comprises a compound according to one of formulas (I) or (II) (and its subformulas) 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-compounded lipids. In one embodiment, the therapeutic agent is encapsulated within lipid nanoparticles or associated with lipid nanoparticles.

[0303] In one embodiment, provided herein is: i) 40-50 mole percent of cationic lipids, ii) Neutral lipids and, iii) Steroids and, iv) Polymer-complexed lipids, v) A nanoparticle composition (lipid nanoparticles) comprising a therapeutic agent.

[0304] As used herein, “mol percent” refers to the ratio of moles of a component to the total moles of all lipid components in an LNP (i.e., the total moles of cationic lipids, neutral lipids, steroids, and polymer-complexed lipids).

[0305] In one embodiment, the lipid nanoparticles contain 41-49 mol percent, 41-48 mol percent, 42-48 mol percent, 43-48 mol percent, 44-48 mol percent, 45-48 mol percent, 46-48 mol percent, or 47.2-47.8 mol percent of cationic lipids. In one embodiment, the lipid nanoparticles contain approximately 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol percent of cationic lipids.

[0306] In one embodiment, neutral lipids are present at concentrations ranging from 5 to 15 mole percent, 7 to 13 mole percent, or 9 to 11 mole percent. In one embodiment, neutral lipids are present at concentrations of about 9.5, 10, or 10.5 mole percent. In one embodiment, the molar ratio of cationic lipids to neutral lipids 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.

[0307] In one embodiment, the steroid is present at concentrations ranging from 39–49 mol%, 40–46 mol%, 40–44 mol%, 40–42 mol%, 42–44 mol%, or 44–46 mol%. In one embodiment, the steroid is present at concentrations of 40, 41, 42, 43, 44, 45, or 46 mol%. In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9–1.0:1.2, or 1.0:1.0–1.0:1.2. In one embodiment, the steroid is cholesterol.

[0308] In one embodiment, the therapeutic agent-to-lipid ratio in the LNP (i.e., N / P, where N represents the moles of cationic lipids and P represents the moles of phosphate present as part of the nucleic acid backbone) is in the range of 2:1 to 30:1, for example, 3:1 to 22:1. In one embodiment, N / P is in the range of 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.

[0309] In one embodiment, provided herein is: i) Cationic lipids having an effective pKa greater than 6.0, ii) Neutral lipids in a 5-15 mole percent range, iii) 1 to 15 mole percent of anionic lipids, iv) 30-45 mole percent of steroids, v) Polymerized lipids, vi) Lipid nanoparticles comprising a therapeutic agent, or a pharmaceutically acceptable salt or prodrug thereof, The mole percentage is determined based on the total moles of lipids present within the lipid nanoparticles.

[0310] In one embodiment, the cationic lipid may 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 below herein. 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.

[0311] In one embodiment, the lipid nanoparticles contain 40 to 45 mole percent of cationic lipids. In another embodiment, the lipid nanoparticles contain 45 to 50 mole percent of cationic lipids.

[0312] In one embodiment, the molar ratio of cationic lipids to neutral lipids is in the range of approximately 2:1 to approximately 8:1. In one embodiment, the lipid nanoparticles contain 5 to 10 mole percent of neutral lipids.

[0313] Examples of 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).

[0314] In one embodiment, the lipid nanoparticles contain 1 to 10 mole percent of anionic lipids. In one embodiment, the lipid nanoparticles contain 1 to 5 mole percent of anionic lipids. In one embodiment, the lipid nanoparticles contain 1 to 9 mole percent, 1 to 8 mole percent, 1 to 7 mole percent, or 1 to 6 mole percent of anionic lipids. In one embodiment, the molar ratio of anionic lipids to neutral lipids is in the range of 1:1 to 1:10.

[0315] In one embodiment, the steroid is cholesterol. In one embodiment, the molar ratio of cationic lipids to cholesterol is in the range of approximately 5:1 to 1:1. In one embodiment, the lipid nanoparticles contain 32 to 40 mole percent of the steroid.

[0316] In one embodiment, the sum of the mole percent of neutral lipids and the mole percent of anionic lipids is in the range of 5 to 15 mole percent. In another embodiment, the sum of the mole percent of neutral lipids and the mole percent of anionic lipids is in the range of 7 to 12 mole percent.

[0317] In one embodiment, the molar ratio of anionic lipids to neutral lipids is in the range of 1:1 to 1:10. In one embodiment, the sum of the molar percentages of neutral lipids and steroids is in the range of 35 to 45 molar percentages.

[0318] In one embodiment, the lipid nanoparticles include the following: i) 45-55 mole percent of cationic lipids, ii) 5-10 mole percent of neutral lipids, iii) 1 to 5 mole percent of anionic lipids, and iv) 32-40 mole percent of steroids.

[0319] In one embodiment, the lipid nanoparticles contain 1.0 to 2.5 mole percent of composite lipids. In another embodiment, the polymer-composite lipids are present at a concentration of about 1.5 mole percent.

[0320] In one embodiment, neutral lipids are present at concentrations ranging from 5 to 15 mole percent, 7 to 13 mole percent, or 9 to 11 mole percent. In one embodiment, neutral lipids are present at concentrations of about 9.5, 10, or 10.5 mole percent. In one embodiment, the molar ratio of cationic lipids to neutral lipids 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.

[0321] In one embodiment, the steroid is cholesterol. In some embodiments, the steroid is present at concentrations ranging from 39–49 mol%, 40–46 mol%, 40–44 mol%, 40–42 mol%, 42–44 mol%, or 44–46 mol%. In one embodiment, the steroid is present at concentrations of 40, 41, 42, 43, 44, 45, or 46 mol%. In some embodiments, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9–1.0:1.2, or 1.0:1.0–1.0:1.2.

[0322] In one embodiment, the molar ratio of cationic lipids to steroids is in the range of 5:1 to 1:1.

[0323] In one embodiment, the lipid nanoparticles contain 1.0 to 2.5 mole percent of composite lipids. In another embodiment, the polymer-composite lipids are present at a concentration of about 1.5 mole percent.

[0324] In one embodiment, the molar ratio of cationic lipids to polymer-compounded lipids is in the range of approximately 100:1 to approximately 20:1. In another embodiment, the molar ratio of cationic lipids to polymer-compounded lipids is in the range of approximately 35:1 to approximately 25:1.

[0325] 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.

[0326] In one embodiment, the composition comprises cationic lipids, DSPCs, cholesterol, and PEG lipids provided herein, as well as mRNA. In one embodiment, the cationic lipids, DSPCs, cholesterol, and PEG lipids provided herein are in a molar ratio of approximately 50:10:38.5:1.5.

[0327] Nanoparticle compositions may be designed for one or more specific applications or targets. For example, a nanoparticle composition may be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof within the body of a mammal. The physiological and chemical properties of the nanoparticle composition may be modified to increase selectivity for specific bodily targets. For example, particle size may be adjusted based on the window sizes of various organs. The therapeutic and / or prophylactic agents contained in the nanoparticle composition may be selected based on the desired delivery target(s). For example, the therapeutic and / or prophylactic agents may be selected for specific indications, diseases, disorders, or impairments, and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery). In certain embodiments, the nanoparticle composition may contain mRNA encoding a target polypeptide that can be translated within a cell to produce the target polypeptide. Such a composition may be designed to be specifically delivered to a specific organ. In certain embodiments, the composition may be designed to be specifically delivered to the mammalian liver.

[0328] The amount of therapeutic and / or prophylactic agents in a nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition, as well as the properties of the therapeutic and / or prophylactic agents. For example, the amount of RNA useful in a nanoparticle composition may depend on the size, sequence, and other properties of the RNA. The relative amounts of therapeutic and / or prophylactic agents and other elements (e.g., lipids) in the nanoparticle composition may also vary. In some embodiments, the weight / weight ratio of lipid components to therapeutic and / or prophylactic agents in the nanoparticle composition may be approximately 5:1 to approximately 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 components to therapeutic and / or prophylactic agents may range 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 agents in the nanoparticle composition may be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0329] In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and their amounts may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in RNA. In some embodiments, a lower N:P ratio is selected. The one or more RNAs, lipids, and their amounts may 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 may 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 could be approximately 5.0:1, 5.5:1, 5.67:1, 6.0:1, 6.5:1, or 7.0:1.

[0330] The physical properties of a nanoparticle composition may depend on its constituent elements. For example, a nanoparticle composition containing cholesterol as a structural lipid may have different properties compared to a nanoparticle composition containing different structural lipids. Similarly, the properties of a nanoparticle composition may depend on the absolute or relative amounts of its constituent elements. For example, a nanoparticle composition containing a higher mole fraction of phospholipids may have different properties than a nanoparticle composition containing a lower mole fraction of phospholipids. The properties may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0331] Nanoparticle compositions can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to investigate the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure the zeta potential. Particle size can also be measured using dynamic light scattering. Multiple properties of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential, can also be measured using instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK).

[0332] In various embodiments, the average size of the nanoparticle composition can range from tens of nanometers to hundreds of nanometers. For example, the average size may range from approximately 40 nm to approximately 150 nm, such as approximately 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 may 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 may be about 70 nm to about 100 nm. In some embodiments, the average size may be about 80 nm. In other embodiments, the average size may be about 100 nm.

[0333] Nanoparticle compositions can be relatively uniform. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. Small polydispersity indices (e.g., less than 0.3) generally indicate a narrow particle size distribution. Nanoparticle compositions may have polydispersity indices ranging from 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 a nanoparticle composition may be about 0.10 to about 0.20.

[0334] The zeta potential of a nanoparticle composition can be used to indicate its interfacial dynamic potential. For example, the zeta potential can explain the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low positive or negative charges are generally preferable, as higher charged species can lead to undesirable interactions with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition may be approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0335] The encapsulation efficiency of therapeutic and / or prophylactic agents describes the amount of therapeutic and / or prophylactic agent encapsulated or otherwise associated with the nanoparticle composition after preparation, relative to the initial amount provided. High encapsulation efficiency is desirable (e.g., close to 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 decomposition of 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 the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of therapeutic and / or prophylactic agents may 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%.

[0336] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated into capsules, films, or tablets having the coatings. Capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0337] 6.6 Pharmaceutical Compositions In accordance with this disclosure, nanoparticle compositions may be formulated whole or in part as pharmaceutical compositions. A pharmaceutical composition may comprise one or more nanoparticle compositions. For example, a pharmaceutical composition may comprise one or more nanoparticle compositions comprising one or more different therapeutic and / or prophylactic agents. A pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients or auxiliary components, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and drugs can be found, for example, in Remington's The Science and Practice of Pharmacy, 21 st Available in Edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and auxiliary components may be used in any pharmaceutical composition unless any conventional excipient or auxiliary component may be incompatible with one or more components of the nanoparticle composition. An excipient or auxiliary component may be incompatible with the components of the nanoparticle composition if its combination with the components may result in any undesirable biological or other adverse effect.

[0338] In some embodiments, one or more excipients or auxiliary components may constitute more than 50% of the total mass or volume of the pharmaceutical composition, including the nanoparticle composition. For example, one or more excipients or auxiliary components may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, pharmaceutically acceptable excipients are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipients are approved for human and veterinary use. In some embodiments, the excipients are approved by the United States Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and / or International Pharmacopoeia.

[0339] The relative amounts of one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical composition according to the present invention may vary depending on the identity, size, and / or disease of the target being treated, and / or further depending on the route through which the composition is administered. For example, the pharmaceutical composition may contain one or more nanoparticle compositions in amounts from 0.1% to 100% (by weight / weight).

[0340] In certain embodiments, the nanoparticle compositions and / or pharmaceutical compositions of the Disclosure are refrigerated or frozen for storage and / or shipment (for example, stored at temperatures of 4°C or less, such as about -150°C to about 0°C, or about -80°C to about -20°C (for example, 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 either compound of formula (I) or formula (II) (and its subformulas) is a solution refrigerated for storage and / or shipment at about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C). In certain embodiments, the disclosure also relates to a method for increasing the stability of nanoparticle compositions and / or pharmaceutical compositions containing a compound of formula (I) or formula (II) (and its subformulas) by storing the nanoparticle compositions and / or pharmaceutical compositions at temperatures of 4°C or less, such as about -150°C to about 0°C, or about -80°C to about -20°C, for example, 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 at temperatures below 4°C (e.g., about 4°C to -20°C) for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 14 months, 16 months, 18 months, 20 months, 22 months, or 24 months. In one embodiment, the formulation is stabilized at about 4°C for at least 4 weeks. In a particular embodiment, the pharmaceutical composition of the Disclosure comprises the nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, acetates (e.g., sodium acetate), citrates (e.g., sodium citrate), saline, PBS, and sucrose.In certain embodiments, the pharmaceutical compositions of the Disclosure have a pH value of about 7 to 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 to 8, or 7 to 7.8). For example, the pharmaceutical compositions of the Disclosure comprise the nanoparticle compositions disclosed herein, Tris, physiological saline, and sucrose, and have a pH of about 7.5 to 8, suitable for storage and / or shipment at about -20°C. For example, the pharmaceutical compositions of the Disclosure comprise the nanoparticle compositions disclosed herein and PBS, and have a pH of about 7 to 7.8, suitable for storage and / or shipment at about 4°C or below. In the context of this disclosure, “stability,” “stabilized,” and “stable” refer to the resistance of the nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., decomposition, particle size changes, aggregation, encapsulation changes, etc.) when subjected to stresses such as shear force or freeze / thaw stress under given conditions during manufacturing, preparation, transport, storage, and / or use.

[0341] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions may be administered to any patient or subject, including patients or subjects who can benefit from the therapeutic effect provided by the delivery of therapeutic and / or prophylactic agents to one or more specific cells, tissues, organs, or systems or groups thereof, such as the renal system. The descriptions provided herein of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions are primarily directed toward compositions suitable for administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammals. Modifications of compositions suitable for administration to humans are well understood in order to provide compositions suitable for administration to various animals, and such modifications can be designed and / or carried out, if any, by ordinary experiments. Subjects to which the administration of compositions is intended include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0342] Pharmaceutical compositions comprising one or more nanoparticle compositions may be prepared by any method known or to be developed in the field of pharmacology. Generally, such preparation methods involve associating the active ingredient with excipients and / or one or more other auxiliary components, and then, if desired or as needed, dividing, shaping and / or packaging the product into desired single or multi-dose units.

[0343] The pharmaceutical compositions according to this disclosure may be prepared, packaged, and / or sold in bulk as single unit doses and / or as multiple single unit doses. As used herein, “unit dose” refers to an individual amount of a pharmaceutical composition containing a predetermined amount of the active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject and / or a convenient fraction of such dose, such as half or one-third of such dose.

[0344] Pharmaceutical compositions can be prepared in various 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), topical and / or transdermal dosage forms (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0345] 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, liquid dosage forms may include, for example, inert diluents and solubilizers commonly used in the art, such as water or other solvents, 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, as well as mixtures thereof. In addition to inert diluents, oral compositions may contain additional agents such as additional therapeutic and / or prophylactic agents, wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or fragrances. In specific embodiments for parenteral administration, the composition is mixed with solubilizers such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0346] Preparations for injection, such as sterile aqueous or oily suspensions for injection, can be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspensions. Sterile preparations for injection may also be sterile solutions, suspensions, and / or emulsions for injection, as solutions in non-toxic, parenterally acceptable diluents and / or solvents, such as 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile fixatives are conventionally used as solvents or suspensions. For this purpose, any non-irritating fixative, including synthetic mono- or diglycerides, may be used. Fatty acids, such as oleic acid, may be used in the preparation of injections.

[0347] Injectable formulations can be sterilized, for example, by filtering through a bacterial-retaining filter and / or by incorporating a bactericide in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile media for injection before use.

[0348] This disclosure provides a method for delivering a therapeutic and / or prophylactic agent to mammalian cells or organs to produce a polypeptide of interest in the mammalian cells and to treat a disease or disorder in a mammal that requires the same, the method comprising administering a nanoparticle composition comprising the therapeutic and / or prophylactic agent to a mammal and / or contacting mammalian cells with the nanoparticle composition. In one embodiment, the organ is the liver. In one embodiment, the organ is the heart. In one embodiment, the organ is the spleen. In one embodiment, the organ is the lungs. In one embodiment, the organ is the kidneys. In one embodiment, the organ is the brain. In one embodiment, the organ is the bone marrow. [Examples]

[0349] 7. Examples The examples in this section are provided for illustrative purposes only and are not intended to be limiting.

[0350] General method General preparative HPLC method: HPLC purification is generally performed on an Inertsil Pre-C8 OBD column in a Waters 2767 equipped with a diode array detector (DAD), using water containing 0.1% TFA as solvent A and acetonitrile as solvent B.

[0351] General LC-MS method: LC-MS analysis is performed using a Shimadzu (LC-MS2020) system. Chromatography is generally 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.

[0352] 7.1 Example 1: Preparation of starting materials and intermediates Preparation of compound A [ka] Compound V (5g, 40 mmol, 1.0 equivalent) was added to a mixture of compound A-1 (5.7g, 100 mmol, 2.5 equivalents) in EtOH (50 mL). The reaction mixture was stirred at 50°C for 16 hours. LC-MS indicated that the reaction was complete. After removing the solvent, compound A (6.6g, crude) was obtained as a yellow oily substance.

[0353] Preparation of compound B: [ka] A mixture of compound B-1 (8.0 g, 114 mol, 1.0 equivalent) and compound S (20.9 g, 342 mol, 3.0 equivalent) in methanol (100 mL) was stirred under argon at room temperature for 16 hours. Then, sodium borohydride (4.3 g, 114 mmol, 1.0 equivalent) was added at 0°C, and the resulting mixture was stirred for a further 16 hours. Next, the reaction mixture was concentrated under reduced pressure, water (200 mL) was added, and the mixture was extracted with DCM. The combined organic layer was dehydrated over Na2SO4, removed by distillation under reduced pressure, and purified by column chromatography (silica gel, 2% in DCM → 10% MeOH) to obtain compound B (3.9 g, yield 30%) as a pale yellow oil.

[0354] Preparation of compound C: [ka] A mixture of compound C-1 (16.8 g, 200 mmol, 1.0 equivalent) and compound S (13.4 g, 220 mmol, 1.1 equivalent) in MeOH (300 ml) containing 3 drops of AcOH was stirred overnight at room temperature. Then, NaBH4 (8.4 g, 220 mmol, 1.1 equivalent) was added to this mixture at 0°C. This mixture was stirred at room temperature for 2 hours. This mixture was quenched with water (100 mL), extracted with EA (3 × 100 mL), and dehydrated. After concentration, the residue was purified by silica gel column chromatography (MeOH:DCM = 0% → 10%) to obtain compound C (17.8 g, yield 49.0%) as a yellow oil.

[0355] Preparation of compound D: [ka] A mixture of compound D-1 (2.0 g, 20.0 mmol, 1.0 equivalent), titanium(IV) isopropoxide (7.4 g, 26 mmol, 1.3 equivalents), and compound S (3.66 g, 60.0 mmol, 3.0 equivalents) in methanol (10.0 mL) was stirred under argon at room temperature for 5 hours. Then, sodium borohydride (760.0 mg, 20.0 mmol, 1.0 equivalent) was added at 0°C, and the resulting mixture was stirred for a further 2 hours. The reaction was then quenched by adding water (10.0 mL). Stirring was continued at room temperature for 20 minutes, and then the reaction mixture was acidified with hydrochloric acid (1 M, 5 mL). After filtering on a Celite pad, the mixture was washed with water and EA. The organic layer was separated, dehydrated over Na2SO4, and the mixture was removed by distillation under reduced pressure. The mixture was then purified by FCC (PE / EA = 5 / 1 → 0 / 1) to obtain compound D (1.5 g, yield 52%) as a yellow oil.

[0356] Preparation of compound E: [ka] A mixture of compound E-1 (15 g, 134 mmol, 1 equivalent) and compound S (9 g, 147 mmol, 1.1 equivalents) in MeOH (250 ml) containing 3 drops of AcOH was stirred overnight at room temperature. Then, NaBH4 (5.6 g, 147 mmol, 1.1 equivalents) was added to this mixture at 0°C. This mixture was stirred at room temperature for 2 hours. This mixture was quenched with water (100 mL), extracted with EA (3 × 100 mL), and dehydrated. After concentration, the residue was purified by silica gel column chromatography (MeOH:DCM = 0% → 10%) to obtain compound E (10.3 g, yield 69.2%) as a yellow oil.

[0357] Preparation of compound F: [ka] A mixture of compound F-1 (2.0 g, 15.85 mmol, 1 equivalent) and compound S (1.07 g, 17.43 mmol, 1.1 equivalent) in MeOH (30 ml) containing 3 drops of AcOH was stirred overnight at room temperature. Then, NaBH4 (660 mg, 17.43 mmol, 1.1 equivalent) was added to this mixture at 0°C. This mixture was stirred at room temperature for 2 hours. This mixture was quenched with water (100 mL), extracted with EA (3 × 100 mL), and dehydrated. After concentration, the residue was purified by silica gel column chromatography (MeOH:DCM = 0% → 10%) to obtain compound F (960 mg, yield 35%) as a yellow oil.

[0358] Preparation of compound G: [ka] Step 1: Preparation of Compound G-2 Compound G-1 (2.18 g, 20.1 mmol, 1.0 equivalent), 1,4-dibromobutane (10.0 g, 46.3 mmol, 2.3 equivalents), and tetrabutylammonium bisulfate (171 mg, 0.50 mmol, 0.025 equivalents) were added to a 40 mL aqueous solution of NaOH (2.0 g, 50.3 mmol, 2.5 equivalents). The mixture was stirred at 80°C for 16 hours. The reaction mixture was extracted with EA. The combined organic layer was washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 50 / 1) to obtain compound G-2 (3.3 g, yield 67%) as a colorless oil.

[0359] Step 2: Preparation of Compound G-3 To a suspension of NaH (653 mg, 16.3 mmol, 1.2 equivalents) in THF (60 mL), dimethyl malonate (3.6 g, 27.2 mmol, 2.0 equivalents) was added dropwise. Then, a solution of compound G-2 (3.3 g, 13.6 mmol, 1.0 equivalent) in THF (10 mL) was added dropwise, and the resulting mixture was stirred under reflux for 16 hours. After cooling to room temperature, the mixture was poured into water and extracted with EA. The combined organic layer was washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 20 / 1 → 5 / 1) to obtain compound G-3 (3.2 g, yield 80%) as a colorless oil.

[0360] Step 3: Preparation of Compound G-4 A solution of compound G-3 (3.2 g, 10.9 mmol, 1.0 equivalent) in THF (20 mL) was added dropwise to a solution of LiAlH4 (828 mg, 21.8 mmol, 2.0 equivalent) in THF (40 mL). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was carefully combined with EA and water. 6 mL of 2N NaOH aqueous solution was added. The mixture was filtered through a Celite pad and washed with EA. The filtrate was dehydrated over Na2SO4 and purified by column chromatography on silica gel (DCM / MeOH = 30 / 1 → 20 / 1) to obtain compound G-4 (1.6 g, yield 62%) as a colorless oil.

[0361] Step 4: Preparation of Compound G-5 Compound G-4 (1.0 g, 4.2 mmol, 1.0 equivalent) and octanoic acid (1.8 g, 12.6 mmol, 3.0 equivalents) were dissolved in toluene (40 mL), to which TsOH·H2O (36 mg) was added. This mixture was stirred under reflux for 4 hours through a Dean-Stark trap. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 30 / 1) to obtain compound G-5 (926 mg, yield 46%) as a colorless oil.

[0362] Step 5: Preparation of Compound G Pd / C (82 mg) was added to a solution of compound G-5 (820 mg, 1.67 mmol, 1.0 equivalent) in MeOH (20 mL). This mixture was stirred under H2 at 35°C for 36 hours. The mixture was filtered through a Celite pad and washed with MeOH. The filtrate was concentrated to obtain the labeled compound (630 mg, yield 941%) as a colorless oil.

[0363] Preparation of compound H: [ka] To a solution of compound G (16.0 g, 40.0 mmol, 1.0 equivalent) in acetone (200 mL), Jones' reagent (24 mL, 80 mmol, 2.0 equivalents) was added at 0°C. The mixture was stirred at room temperature for 1 hour. The reaction mixture was poured into water and extracted with EA. The combined organic layer was washed with saturated brine, dehydrated over Na₂SO₄, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 4 / 1) to obtain compound H (14.3 g, yield 86%) as a colorless oil.

[0364] Preparation of compound L: [ka] Compound I (7.55 g, 50 mmol, 1.0 equivalent) and Compound L-1 (6.48 g, 60 mmol, 1.2 equivalents) were dissolved in toluene (100 mL), to which TsOH·H2O (950 mg, 5.0 mmol, 0.1 equivalent) was added. This mixture was stirred at 150 °C for 3 hours. The reaction mixture was concentrated and purified by FCC (PE / EA = 1 / 0 → 10 / 1) to obtain Compound L (13 g, crude) as a colorless oil.

[0365] Preparation of compound N: [ka] To a solution of compound N-1 (3.15 g, 30 mmol, 1.0 equivalent) and compound B-1 (2.1 g, 30 mmol, 1.0 equivalent) in MeOH (30 mL), AcOH (180 mg, 3.0 mmol, 0.1 equivalent) was added. This mixture was stirred at 50°C for 2 hours. Then NaBH4 (1.7 g, 45 mmol, 1.5 equivalents) was added and the mixture was stirred at 50°C for 16 hours. This reaction mixture was concentrated and purified by preparative HPLC to obtain compound N (2.0 g, crude) as a colorless oil.

[0366] Preparation of compound O: [ka] To a solution of compound N-1 (3.0 g, 28.5 mmol, 1.0 equivalent) and compound C-1 (4.8 g, 57.0 mmol, 1.0 equivalent) in MeOH (40 mL), AcOH (5 drops) and Pd / C (300 mg) were added. This reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered and concentrated to obtain compound O (4.8 g, crude) as a yellow oily substance.

[0367] Preparation of compound P: [ka] To a solution of compound N-1 (3.15 g, 30 mmol, 1.0 equivalent) and compound D-1 (5.88 g, 60 mmol, 1.0 equivalent) in MeOH (40 mL), AcOH (5 drops) and Pd / C (300 mg) were added. This reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered and concentrated to obtain compound P (3.5 g, crude) as a yellow oily substance.

[0368] Preparation of compound U: [ka] Step 1: Preparation of compound U-2 Compound U-1 (10g, 77.40 mmol, 1 equivalent) and C8H 17 A mixture of OH (30.2 g, 232.2 mmol, 3.0 equivalents) was stirred with PPTS (1 g, 3.870 mmol, 0.05 equivalents) at 110°C for 160 hours. This mixture was purified by FCC to obtain compound U-2 (10.5 g, yield 41.68%) as a yellow oily substance. 1 H NMR (400 MHz, CDCl3) δ: 0.87-0.90 (m, 6 H), 1.28-1.36 (m, 20 H), 1.54-1.61 (m, 4 H), 1.92-1.97 (m, 2 H), 2.40-2.44 (m, 2 H), 3.40-3.46 (m, 2H), 3.57-3.63 (m, 2H), 4.54-4.57 (m, 1H).

[0369] Step 2: Preparation of Compound U A mixture of compound U-2 (8 g, 24.58 mmol, 1.0 equivalent) in H2O (70 mL) and EtOH (70 mL) was mixed with KOH (4.1 g, 73.73 mmol, 3.0 equivalent). This mixture was stirred overnight at 110°C. The mixture was concentrated, the pH was adjusted to 7 with 1 M HCl, extracted with DCM, and the organic layer was concentrated to obtain compound U (6 g, yield 70.86%) as a yellow oil.

[0370] Preparation of compound SM4: [ka] Step 1: Preparation of compound SM4-2 Compound SM4-1 (12g, 90.84 mmol, 1.0 equivalent) was added to a mixture of NaH (12g, 227.1 mmol, 2.5 equivalents) in DMF (100mL) under N2 conditions at 0°C. This reaction mixture was stirred at 0°C for 1 hour. C8H 17 A solution of Br (44 g, 227.1 mmol, 2.5 equivalents) in DMF (100 mL) was added. The reaction mixture was stirred at room temperature for 16 hours. TLC indicated that the reaction was complete. The mixture was poured into water and washed with EA. The organic components were separated and dehydrated over Na₂SO₄. After removing the solvent, the mixture was purified by FCC to obtain compound SM4-2 (17.8 g, 54.96%) as a colorless oil. 1 H NMR (400 MHz, CCl3D) δ: 3.71 (s, 6H), 1.88-1.84 (m, 4H), 1.59 (s, 1H), 1.25 (s, 19H), 1.14-1.10 (m, 4H), 0.89-0.86 (m, 6H).

[0371] Step 2: Preparation of compound SM4-3 LiCl (21.17 g, 499.3 mmol, 10.0 equivalent) was added to a solution of SM4-2 (17.8 g, 49.93 mmol, 1.0 equivalent) in DMF (260 mL). The reaction mixture was stirred at 120 °C for 12 hours. TLC showed that the reaction was complete. The mixture was poured into water and washed with EA. The organic components were separated and dehydrated over Na2SO4. After removing the solvent, the mixture was purified by FCC to obtain compound SM4-3 (10 g, 67.10%) as a colorless oil. 1 H NMR (400 MHz, CCl3D) δ: 0.89-0.86 (m, 6H), 1.25 (s, 22H), 1.45-1.40 (m, 2H), 1.59 (s, 4H), 2.36-2.30 (m, 1H), 3.67 (s, 3H).

[0372] Step 3: Preparation of compound SM4 To a solution of compound SM4-3 (10 g, 33.50 mmol, 1.0 equivalent) in THF (100 mL), LiAlH4 (2.546 g, 67.00 mmol, 2.0 equivalents) was slowly added at 0°C. The reaction mixture was stirred under reflux for 1 hour. TLC indicated that the reaction was complete. After cooling to 0°C, the mixture was quenched by sequentially adding water (3.4 mL), 15% NaOH aqueous solution (3.4 mL), and water (10 mL). The resulting mixture was diluted with EA, and the precipitate was removed by filtration. The filtrate was removed under reduced pressure and purified by FCC to obtain compound SM4 (8.5 g, 93.80%) as a yellow oily substance. 1 H NMR (400 MHz, CCl3D) δ: 0.90-0.86 (m, 6H), 1.27 (s, 27H), 1.43 (s, 3H), 3.54 (d, J = 5.2 Hz, 2H).

[0373] Preparation of compound SM7: [ka] Compound W (6.8g, 34.8mmol, 1.2 equivalents) and TsOH (0.1g, 0.58mmol, 0.02 equivalents) were added to a toluene (50mL) solution of compound SM7-1 (5.0g, 29.0mmol, 1.0 equivalent). This mixture was stirred under reflux for 2 hours through a Dean-Stark trap. The reaction mixture was diluted with EA and washed with saturated NaHCO3 aqueous solution, water, and saturated brine. The combined organic layer was dehydrated over NaSO4 and concentrated. The residue was purified by silica gel column chromatography (PE / EA=50 / 1) to obtain compound SM7 (9.8g, yield 96.08%) as a colorless oil.

[0374] 7.2 Example 2: Preparation of Compound 1 [ka] Step 1: Preparation of Compounds 1-2 Compound 1-1 (1.06 g, 10.0 mmol, 1.0 equivalent) and linoleic acid (2.08 g, 10.0 mmol, 1.0 equivalent) were dissolved in DCM (20 mL). EDCI (2.88 g, 15.0 mmol, 1.5 equivalents), DIEA (1.93 g, 15.0 mmol, 1.5 equivalents), and DMAP (240 mg, 2.0 mmol, 0.2 equivalents) were added to this solution. The mixture was stirred under reflux for 16 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 1 / 1) to obtain compound 1-2 (0.7 g, crude) as a colorless oil.

[0375] Step 2: Preparation of Compounds 1-3 To a 20 mL solution of compound 1-2 (0.533 g, 1.45 mmol, 1.0 equivalent) and compound H (0.6 g, 1.45 mmol, 1.0 equivalent) in DCM, EDCI (0.556 g, 2.9 mmol, 2.0 equivalent), DIEA (0.561 g, 4.35 mmol, 3.0 equivalent), and DMAP (35 mg, 0.29 mmol, 0.2 equivalent) were added. This mixture was stirred under reflux for 16 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 4 / 1) to obtain compound 1-3 (0.4 g, yield 36%) as a colorless oil.

[0376] Step 3: Preparation of Compounds 1-4 To a solution of Compound 1-3 (0.2 g, 0.26 mmol, 1.0 equivalent) and Compound I (61 mg, 1.45 mmol, 1.0 equivalent) in DCM (10.0 mL), EDCI (0.1 g, 0.52 mmol, 2.0 equivalents), DIEA (0.1 g, 0.78 mmol, 3.0 equivalents), and DMAP (3 mg, 0.026 mmol, 0.2 equivalents) were added. This mixture was stirred under reflux for 16 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 4 / 1) to obtain Compound 1-4 (0.12 g, crude) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.27-1.37 (m, 38H), 1.59-1.63 (m, 8H), 2.01-2.06 (m, 6H), 2.28-2.33 (m, 8H), 4.04-4.24 (m, 10H), 5.30-5.35 (m, 4H).

[0377] Step 4: Preparation of Compound 1 To a solution of compounds 1-4 (100 mg, 0.11 mmol, 1.0 equivalent) and compound B (38 mg, 0.33 mmol, 3.0 equivalents) in THF (10 mL), DIPEA (71 mg, 0.55 mmol, 5.0 equivalents) and NaI (1.4 mg, 0.011 mmol, 0.1 equivalent) were added. This mixture was stirred at 70°C for 16 hours. LC-MS indicated that the reaction was complete. This mixture was concentrated and purified by preparative HPLC to obtain compound 1 (21 mg, yield 20%) as a yellow oil.

[0378] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.25-1.42 (m, 30H), 1.57-1.63 (m, 20H), 2.04-2.06 (m, 7H), 2.28-2.32 (m, 8H), 2.47-2.60 (m, 4H), 3.03 (s, 1H), 3.45 (s, 2H), 4.02-4.16 (m, 10H), 5.24-5.49 (m, 4H). LCMS: Rt (retention time): 1.440 min; MS m / z (ESI): 934.5 [M+H] + .

[0379] The following compounds were prepared using the corresponding starting materials in the same manner as compound 1. [Table 3-1] [Table 3-2]

[0380] 7.3 Example 3: Preparation of Compound 3 [ka] Step 1: Preparation of Compound 3-2 To a solution of compound 3-1 (1.3 g, 10.0 mmol, 1.0 equivalent) in THF (15 mL), NaH (0.52 g, 13.0 mmol, 1.3 equivalents) was added. This mixture was stirred at room temperature for 1 hour, and then compound L (3.0 g, 12.0 mmol, 1.2 equivalents) was added. This mixture was stirred at room temperature for 16 hours. This mixture was poured into water and extracted with DCM. The combined organic layer was washed with saturated brine, dehydrated on Na₂SO₄, and purified by column chromatography (PE / EA=4 / 1) on silica gel to obtain compound 3-2 (0.6 g, crude) as a colorless oil.

[0381] Step 2: Preparation of Compound 3-3 Compound 3-2 (0.6 g, 2.04 mmol, 1.0 equivalent) was dissolved in THF (10 mL) and HCl (2 mL, 6 M) was added. The mixture was stirred at room temperature for 2 hours. TLC indicated that the reaction was complete. The mixture was poured into water and extracted by DCM. The combined organic layer was washed with saturated brine, dehydrated on Na2SO4, and purified by column chromatography (PE / EA=0 / 1) on silica gel to obtain compound 3-3 (0.4 g, crude) as a colorless oil.

[0382] Step 3: Preparation of Compounds 3-4 Compound 3-3 (0.4 g, 1.57 mmol, 1.0 equivalent) and octanoic acid (0.56 g, 3.94 mmol, 2.5 equivalents) were dissolved in DCM (10 mL), to which EDCI (0.9 g, 4.7 mmol, 3.0 equivalents), DIEA (1.0 g, 7.85 mmol, 5.0 equivalents), and DMAP (38 mg, 0.31 mmol, 0.2 equivalents) were added. This mixture was stirred under reflux for 16 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 5 / 1) to obtain compound 3-4 (0.5 g, yield 63%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 6H), 1.27 (s, 10H), 1.60-1.62 (m, 4H), 2.28-2.32 (m, 4H), 2.61-2.65 (m, 2H), 3.72-3.75 (m, 1H), 3.77-3.89 (m, 1H), 4.07-4.18 (m, 4H), 4.53 (s, 1H), 7.33-7.36 (m, 5H).

[0383] Step 4: Preparation of Compounds 3-5 Pd / C (50 mg) was added to a solution of compound 3-4 (0.5 g, 0.97 mmol, 1.0 equivalent) in EA (10 mL). This mixture was stirred under H2 at room temperature for 16 hours. LC-MS indicated that the reaction was complete. The mixture was filtered and concentrated to obtain compound 3-5 (0.42 g, crude) as a colorless oil. LC-MS: Rt: 1.147 min; MS m / z (ESI): 415.2 [MH] - .

[0384] Step 5: Preparation of Compounds 3-6 To a solution of compound 1-2 (0.37 g, 1.0 mmol, 1.0 equivalent) and compound 3-5 (416 mg, 1.0 mmol, 1.0 equivalent) in DCM (10.0 mL), EDCI (0.38 g, 2.0 mmol, 2.0 equivalent), DIEA (0.38 g, 3.0 mmol, 3.0 equivalent), and DMAP (24 mg, 0.2 mmol, 0.2 equivalent) were added. This mixture was stirred under reflux for 16 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 4 / 1) to obtain compound 3-6 (0.3 g, crude) as a colorless oil.

[0385] Step 6: Preparation of Compound 3 To a solution of compound 3-6 (300.0 mg, 0.4 mmol, 1.0 equivalent) in DCM (10.0 mL), pyridine (63.0 mg, 0.8 mmol, 2.0 equivalents), DMAP (9.0 mg, 0.08 mmol, 0.2 equivalents), and compound J (120.0 mg, 0.6 mmol, 2.0 equivalents) were added at 0°C. This mixture was stirred at room temperature for 2 hours, and then compound K (310.0 mg, 2.4 mmol, 6.0 equivalents) was added. This mixture was stirred at room temperature for 16 hours. LC-MS indicated that the reaction was complete. This mixture was poured into water and washed with DCM (20.0 mL x 2). This mixture was concentrated and purified by preparative HPLC to obtain compound 3 (30 mg, yield 8%) as a yellow oil.

[0386] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.04 (s, 6H),1.27-1.35 (m, 33H), 1.55-1.66 (m, 7H), 2.04-2.06 (m, 4H), 2.31-2.38 (m, 6H), 2.47-2.58 (m, 7H), 2.72-2.83 (m, 2H), 3.70-3.90 (m, 3H), 4.11-4.20(m, 12H), 5.24-5.49 (m, 4H). LCMS: Rt: 1.330 min; MS m / z (ESI): 925.5 [M+H] + .

[0387] 7.4 Example 4: Preparation of Compound 5 [ka] Step 1: Preparation of Compound 5-2 Compound 1-1 (1.06 g, 10.0 mmol, 1.2 equivalents) and C6H 13 To a 10 mL solution of COOH (1.08 g, 8.33 mmol, 1.0 equivalent) in DCM, EDCI (2.4 g, 12.5 mmol, 1.5 equivalent), DIPEA (2.15 g, 16.66 mmol, 2.0 equivalent), and DMAP (203 mg, 1.66 mmol, 0.2 equivalent) were added. This mixture was stirred overnight at 35°C. This mixture was concentrated and purified by FCC (PE / EA = 1 / 0 → 1 / 1) to obtain compound 5-2 (700 mg, yield 39%) as a yellow oily substance. 1 H NMR (400 MHz, CDCl3) δ: 0.88 (t, J = 6.8 Hz, 3H), 1.26-1.33 (m, 6H), 1.60-1.64 (m, 2H), 2.02-2.05 (m, 1H), 2.33 (t, J = 7.6 Hz, 3H), 2.63 (s, 2H), 3.74-3.80 (m, 4H), 4.23 (d, J = 6.4 Hz, 2H).

[0388] Step 2: Preparation of Compound 5-3 To a 16 mL solution of compound 5-2 (502 mg, 2.30 mmol, 1.0 equivalent) and compound H (955 mg, 2.30 mmol, 1.0 equivalent) in DCM, EDCI (661 mg, 3.45 mmol, 1.5 equivalents), DIPEA (595 mg, 4.60 mmol, 2.0 equivalents), and DMAP (56 mg, 0.46 mmol, 0.2 equivalents) were added. This mixture was stirred at 35°C for 16 hours. This mixture was poured into water and extracted with DCM. The combined organic layer was washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 5 / 1 → 3 / 1) to obtain compound 5-3 (516 mg, yield 36%) as a colorless oil.

[0389] Step 3: Preparation of Compounds 5-4 To a 10 mL solution of compound 5-3 (516 mg, 0.84 mmol, 1.0 equivalent) and compound I (257 mg, 1.68 mmol, 2.0 equivalents) in DCM, EDCI (403 mg, 2.10 mmol, 2.5 equivalents), DIPEA (326 mg, 2.52 mmol, 3.0 equivalents), and DMAP (21 mg, 0.17 mmol, 0.2 equivalents) were added. This mixture was stirred at 35°C for 16 hours. This mixture was poured into water and extracted with DCM. The combined organic layer was washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 6 / 1) to obtain compound 5-4 (167 mg, yield 27%) as a colorless oil.

[0390] Step 4: Preparation of Compound 5 To a solution of compound 5-4 (150 mg, 0.20 mmol, 1.0 equivalent) and compound B (46 mg, 0.40 mmol, 2.0 equivalents) in THF (6 mL), DIPEA (78 mg, 0.60 mmol, 3.0 equivalents) and NaI (9 mg, 0.06 mmol, 0.3 equivalents) were added. This mixture was stirred at 70°C for 16 hours. LC-MS indicated that the reaction was complete. This mixture was concentrated and purified by preparative HPLC to obtain compound 5 (37 mg, yield 27%) as a yellow oil.

[0391] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.26-1.30 (m, 24H), 1.36-1.40 (m, 2H), 1.59-1.71 (m, 9H), 1.82-1.89 (m, 2H), 1.97-2.06 (m, 3H), 2.33-2.33 (m, 8H), 2.37-2.54 (m, 5H), 2.77-2.80 (m, 2H), 3.09-3.16 (m, 1H), 3.52-3.55 (m, 2H), 4.01-4.16 (m, 10H). LCMS: Rt: 0.890 min; MS m / z (ESI): 784.4[M+H] + .

[0392] The following compounds were prepared using the corresponding starting materials in the same manner as compound 5. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10]

[0393] 7.5 Example 5: Preparation of Compound 12 [ka] Step 1: Preparation of Compound 12-1 To a solution of compound 1-3 (250.0 mg, 0.33 mmol, 1.0 equivalent) in DCM (10.0 mL), Py (78.0 mg, 0.99 mmol, 3.0 equivalents) and triphosgene (46.0 mg, 0.16 mmol, 0.5 equivalents) were added at 0°C. This mixture was stirred at 0°C for 0.5 hours, and then compound V (123.0 mg, 0.99 mmol, 3.0 equivalents) was added. This mixture was stirred at room temperature for 16 hours. TLC showed that the reaction was complete. This mixture was poured into water and washed with DCM (20.0 mL x 2). This mixture was concentrated and purified by FCC (PE / EA=5 / 1) to obtain compound 12-1 (230 mg, crude) as a yellow oily substance.

[0394] Step 2: Preparation of Compound 12 Compound 12-1 (200.0 mg, 0.21 mmol, 1.0 equivalent) and Compound B (72.0 mg, 0.63 mmol, 3.0 equivalents) were dissolved in THF (5.0 mL) and DIEA (135.0 mg, 1.05 mmol, 5.0 equivalents) and NaI (3.0 mg, 0.021 mmol, 0.1 equivalent) were added at 0°C. This mixture was stirred at 70°C for 16 hours. LC-MS indicated that the reaction was complete. The mixture was removed under reduced pressure and purified by preparative HPLC to obtain Compound 12 (25.0 mg, yield 12%) as a yellow oil.

[0395] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.28-1.40 (m, 33H), 1.59-1.68 (m, 12H), 2.02-2.06 (m, 8H), 2.28-2.35 (m, 9H), 2.50-2.55 (m, 1H), 2.75-2.78 (m, 4H), 3.20-3.23 (m, 1H), 3.53-3.56 (m, 2H), 4.04-4.20 (m, 12H), 5.34-5.37 (m, 4H). LCMS: Rt: 1.340 min; MS m / z (ESI): 950.4 [M+H] + .

[0396] The following compounds were prepared using the corresponding starting materials in the same manner as compound 12. [Table 5-1] [Table 5-2] [Table 5-3]

[0397] 7.6 Example 6: Preparation of Compound 13 [ka] Step 1: Preparation of Compound 13-2 To a solution of compound 13-1 (250 mg, 0.38 mmol, 1.0 equivalent) in DCM (10.0 mL), Py (91 mg, 1.14 mmol, 3.0 equivalents) and triphosgene (56 mg, 0.19 mmol, 0.5 equivalents) were added at 0°C. This mixture was stirred at 0°C for 0.5 hours, and then compound V (143 mg, 1.14 mmol, 3.0 equivalents) was added. This mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into water and extracted with DCM. The combined organic layer was washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA=5 / 1) to obtain compound 15-2 (220 mg, yield 72%) as a colorless oil.

[0398] Step 2: Preparation of Compound 13 To a solution of compound 13-2 (200 mg, 0.25 mmol, 1.0 equivalent) and compound B (57 mg, 0.50 mmol, 2.0 equivalents) in THF (6 mL), DIPEA (97 mg, 0.75 mmol, 3.0 equivalents) and NaI (11 mg, 0.075 mmol, 0.3 equivalents) were added. This mixture was stirred at 70°C for 16 hours. LC-MS indicated that the reaction was complete. This mixture was concentrated and purified by preparative HPLC to obtain compound 13 (30 mg, yield 14%) as a yellow oil.

[0399] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.26-1.30 (m, 29H), 1.36-1.42 (m, 2H), 1.59-1.73 (m, 13H), 1.99-2.02 (m, 1H), 2.09-2.19 (m, 2H), 2.28-2.33 (m, 8H), 2.40-2.45 (m, 1H), 2.53-2.87 (m, 4H), 3.50-3.82 (m, 2H), 4.01-4.22 (m, 12H). LCMS: Rt: 1.150 min; MS m / z (ESI): 842.4[M+H] + .

[0400] The following compounds were prepared using the corresponding starting materials in the same manner as compound 13. [Table 6-1] [Table 6-2]

[0401] 7.7 Example 7: Preparation of Compound 14 [ka] Compound j (185 mg, 0.92 mmol, 2.0 equivalents) was added to a 10 mL DCM solution of compound 13-1 (300 mg, 0.46 mmol, 1.0 equivalent) and pyridine (184 mg, 2.30 mmol, 5.0 equivalents). This mixture was stirred at room temperature for 2 hours. Then, compound P (258 mg, 1.38 mmol, 3.0 equivalents) was added. This mixture was stirred at room temperature for 16 hours. LC-MS indicated that the reaction was complete. This mixture was concentrated and purified by preparative HPLC to obtain compound 14 (69 mg, yield 17%) as a yellow oil.

[0402] 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.02-1.42 (m, 36H), 1.59-1.87 (m, 13H), 1.97-2.03 (m, 1H), 2.28-2.33 (m, 8H), 2.35-2.50 (m, 2H), 2.63-2.89 (m, 4H), 3.45-3.58 (m, 2H), 4.01-4.21 (m, 12H). LCMS: Rt: 1.010 min; MS m / z (ESI): 870.4[M+H] + .

[0403] The following compounds were prepared using the corresponding starting materials in the same manner as compound 14. [Table 7-1] [Table 7-2]

[0404] 7.8 Example 8: Preparation of Compound 38 [ka] Step 1: Preparation of Compound 38-1 Compound H (1.5 g, 3.618 mmol, 2.0 equivalents) and Compound 1-1 (192 mg, 1.809 mmol, 1.0 equivalent) were dissolved in DCM (30 mL), to which EDCI (1.0 g, 5.427 mmol, 3.0 equivalents), DMAP (111 mg, 0.9045 mmol, 0.5 equivalents), and DIEA (1.2 g, 9.045 mmol, 5.0 equivalents) were added. This mixture was stirred at room temperature for 16 hours. TLC indicated that the reaction was complete. The reaction mixture was concentrated and purified by column chromatography on silica gel to obtain Compound 38-1 (400 mg, yield 24.59%) as a yellow oil.

[0405] Step 2: Preparation of Compound 38 To a 10 mL solution of compound 38-1 (200 mg, 0.2224 mmol, 1.0 equivalent) in DCM, Py (35 mg, 0.4448 mmol, 2.0 equivalents), DMAP (5 mg, 0.04448 mmol, 0.2 equivalents), and compound j (90 mg, 0.4448 mmol, 2.0 equivalents) were added at room temperature. This mixture was stirred at room temperature for 2 hours, and then compound P (167 mg, 0.8896 mmol, 4.0 equivalents) was added. This mixture was stirred at room temperature for 16 hours. LC-MS indicated that the reaction was complete. This mixture was poured into water and washed with DCM. This mixture was concentrated and purified by preparative HPLC to obtain compound 38 (24 mg, yield 9.70%) as a yellow oil.

[0406] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H), 1.22-1.30 (m, 32H), 1.36-1.42 (m, 6H), 1.59-1.78 (m, 19H), 1.96-2.03 (m, 2H), 2.28-2.35 (m, 13H), 2.40-2.47 (m, 2H), 2.66-2.68 (m, 2H), 2.76-2.79 (m, 2H), 3.47-3.50 (m, 2H), 4.01-4.21 (m, 17H). LCMS: Rt: 1.190 min; MS m / z (ESI): 1111.9[M+H] + .

[0407] The following compounds were prepared using the corresponding starting materials in the same manner as compound 38. [Table 8]

[0408] 7.9 Example 9: Preparation of Compound 61 [ka] Step 1: Preparation of Compound 61-2 Compound 61-1 (2.0 g, 8.4 mmol, 1.0 equivalent) and octanoic acid (1.09 g, 7.6 mmol, 0.9 equivalents) were dissolved in DCM (50 mL), to which DIEA (3.26 g, 2.52 mmol, 0.3 equivalents), EDCI (2.1 g, 10.9 mmol, 1.3 equivalents), and DMAP (0.31 g, 2.52 mmol, 0.3 equivalents) were added. This mixture was stirred at 40°C for 10 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 10 / 1) to obtain compound 61-2 (1.318 g, yield 43%) as a colorless oil.

[0409] Step 2: Preparation of Compound 61-3 To a 50 mL solution of compound 61-2 (1.32 g, 3.7 mmol, 1.0 equivalent) in DCM, Et3N (0.76 g, 7.4 mmol, 2.0 equivalents) and hexanoyl chloride (0.609 g, 4.4 mmol, 1.2 equivalents) were added. This mixture was stirred at 25°C for 10 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 10 / 1) to obtain compound 61-3 (1.4 g, yield 78%) as a colorless oil.

[0410] Step 3: Preparation of Compound 61-4 To a solution of compound 61-3 (1.4 g, 4.5 mmol, 1.0 equivalent) in EA (50 mL), Pd / C (0.229 g) and concentrated HCl (0.1 mL) were added. This mixture was stirred under H2 at room temperature for 10 hours. The mixture was filtered through a Celite pad and washed with MeOH. The filtrate was concentrated to obtain compound 61-4 (0.9 g, yield 57%) as a yellow oily substance.

[0411] Step 4: Preparation of Compounds 61-5 A solution of compound 61-4 (2.6 g, 6.9 mmol, 1.0 equivalent) in acetone (50 mL) was mixed with ice-cooked Jones reagent (5 mL). The mixture was stirred at 0°C for 2 hours. The mixture was poured into water and extracted by DCM. The combined organic layer was washed with saturated brine and dehydrated over Na₂SO₄. The residue was purified by column chromatography on silica gel (PE / EA=5 / 1) to obtain compound 61-5 (1.6 g, yield 84.9%) as a colorless oil.

[0412] Step 5: Preparation of Compounds 61-6 To a 30 mL solution of compound 61-5 (0.5 g, 1.3 mmol, 1.0 equivalent) and compound 54-1 (480 mg, 1.2 mmol, 0.9 equivalents) in DCM, EDCI (322 mg, 1.6 mmol, 1.2 equivalents), DIEA (502 mg, 3.8 mmol, 2.9 equivalents), and DMAP (47 mg, 0.4 mmol, 0.3 equivalents) were added. This mixture was stirred at 40°C for 16 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 5 / 1) to obtain compound 61-6 (500 mg, yield 52%) as a yellow oil.

[0413] Step 6: Preparation of Compounds 61-7 Compound M (160 mg, 0.81 mmol, 1.35 equivalents), EDCI (200 mg, 1.04 mmol, 1.7 equivalents), DIEA (150 mg, 1.16 mmol, 1.9 equivalents), and DMAP (14 mg, 0.11 mmol, 0.2 equivalents) were added to a 10 mL solution of compound 61-6 (300 mg, 0.6 mmol, 1.0 equivalents). This mixture was stirred at 35°C for 10 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 5 / 1) to obtain compound 61-7 (288 mg, yield 77.5%) as a colorless oil.

[0414] Step 7: Preparation of Compound 61 Compound B (100 mg, 0.86 mmol, 2.8 equivalents), DIEA (300 mg, 1.56 mmol, 5.0 equivalents), and NaI (23 mg, 0.15 mmol, 0.5 equivalents) were added to a solution of compound 61-7 (288 mg, 0.31 mmol, 1.0 equivalent) in THF (30 mL). This mixture was stirred at 80°C for 10 hours. LC-MS indicated that the reaction was complete. This mixture was concentrated and purified by preparative HPLC to obtain compound 61 (39 mg, yield 13%) as a yellow oil.

[0415] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.91 (m, 9H), 1.29-1.46 (m, 48H), 1.63-1.80 (m, 11H), 1.89-1.99 (m, 2H), 2.01-2.28 (m, 3H), 2.31-2.33 (m, 13H), 2.43-2.56 (m, 2H), 3.14-3.18 (m, 1H), 3.51-3.52 (m, 2H), 4.06-4.13 (m, 10H). LCMS: Rt: 1.650 min; MS m / z (ESI): 952.0 [M+H] + .

[0416] 7.10 Example 10: Preparation of Compound 69 [ka] Step 1: Preparation of Compound 69-2 Compound 69-1 (2 g, 15.4 mmol, 1.0 equivalent) and nonanedioic acid (5.8 g, 30.8 mmol, 2.0 equivalents) were dissolved in DCM (40 mL), to which EDCI (3.3 g, 16.9 mmol, 1.1 equivalents), DIPEA (6.0 g, 42.6 mmol, 3.0 equivalents), and DMAP (0.4 g, 3.1 mmol, 0.2 equivalents) were added. This mixture was stirred overnight at 35°C. This mixture was concentrated and purified by FCC (PE / EA = 1 / 0 → 3 / 1) to obtain compound 69-2 (976 g, yield 21.1%) as a colorless oil.

[0417] Step 2: Preparation of Compound 69-3 To a 30 mL solution of compound 69-2 (976 mg, 3.25 mmol, 1.0 equivalent) and compound 1-1 (448 mg, 4.23 mmol, 1.3 equivalents) in DCM, EDCI (749 mg, 3.9 mmol, 1.2 equivalents), DIPEA (1.3 g, 10.1 mmol, 3.1 equivalents), and DMAP (79 mg, 0.7 mmol, 0.2 equivalents) were added. This mixture was stirred at 35°C for 16 hours. This mixture was poured into water and extracted with DCM. The combined organic layer was washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 1 / 0 → 0 / 1) to obtain compound 69-3 (605 mg, yield 47.9%) as a colorless oil.

[0418] Step 3: Preparation of Compound 69-4 To a 12 mL solution of compound 69-3 (595 mg, 1.53 mmol, 1.0 equivalent) and compound H (636 mg, 1.53 mmol, 1.0 equivalent) in DCM, EDCI (353 mg, 1.84 mmol, 1.2 equivalents), DIPEA (593 mg, 4.60 mmol, 3.0 equivalents), and DMAP (38 mg, 0.31 mmol, 0.2 equivalents) were added. This mixture was stirred at 35°C for 16 hours. This mixture was poured into water and extracted with DCM. The combined organic layer was washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 5 / 1 → 3 / 1) to obtain compound 69-4 (570 mg, yield 47.3%) as a pale yellow oil.

[0419] Step 4: Preparation of Compound 69-5 To a 6 mL solution of compound 69-4 (285 mg, 0.36 mmol, 1.0 equivalent) and compound M (79 mg, 0.44 mmol, 1.2 equivalents) in DCM, EDCI (84 mg, 0.44 mmol, 1.2 equivalents), DIPEA (117 mg, 0.91 mmol, 2.5 equivalents), and DMAP (9 mg, 0.07 mmol, 0.2 equivalents) were added. This mixture was stirred at 35°C for 16 hours. This mixture was poured into water and extracted with DCM. The combined organic layer was washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (PE / EA = 10 / 1 → 5 / 1) to obtain compound 69-5 (147 mg, yield 42.7%) as a pale yellow oil.

[0420] Step 5: Preparation of Compound 69 Compound Z (14 mg, 0.19 mmol, 1.2 equivalents), DIEA (60 mg, 0.47 mmol, 3.0 equivalents), and NaI (12 mg, 0.08 mmol, 0.5 equivalents) were added to a 3 ml THF solution of compound 69-5 (147 mg, 0.16 mmol, 1.0 equivalent) at room temperature. This mixture was stirred overnight at 70°C. The mixture was washed with water and saturated brine, the organic layer was concentrated, and purified by preparative HPLC to obtain compound 69 (15 mg, yield 10.3%) as a colorless oil.

[0421] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H), 1.25-1.43 (m, 33H), 1.57-1.73 (m, 29H), 2.01-2.06 (m, 2H), 2.27-2.44 (m, 9H), 2.85-2.88 (m, 1H), 3.04-3.30 (m, 2H), 4.02-4.14 (m, 10H). LCMS: Rt: 0.960 min; MS m / z (ESI): 942.0 [M+H] + .

[0422] The following compounds were prepared using the corresponding starting materials in the same manner as compound 69. [Table 9]

[0423] 7.11 Example 11: Preparation of Compound 100 [ka] Step 1: Preparation of Compound 100-2 Compound SM3 (10.2 g, 102 mmol, 10.0 equivalent) and Cs2CO3 (16.6 g, 51.0 mmol, 5.0 equivalent) were added to a solution of compound 100-1 (2.0 g, 10.2 mmol, 1.0 equivalent) in ACN (100 mL). This mixture was stirred at 50°C for 36 hours. The mixture was concentrated and purified by column chromatography on silica gel (PE / EA=6 / 1) to obtain compound 100-2 (900 mg, yield 23%) as a colorless oil.

[0424] Step 2: Preparation of Compound 100-3 To a solution of compound 100-2 (870 mg, 2.0 mol, 1.0 equivalent) in THF / H2O (8 mL / 8 mL), lithium hydroxide monohydrate (922 mg, 22.0 mmol, 10.0 equivalents) was added. The reaction mixture was stirred overnight at room temperature. LC-MS confirmed the completion of the reaction. The reaction mixture was concentrated under reduced pressure to remove the organic solvent. The aqueous layer was acidified to pH=4 with 2N HCl and extracted with EA. The combined organic layers were washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography (DCM / MeOH=20 / 1) on silica gel to obtain compound 100-3 (526 mg, 70% yield) as a yellow oil.

[0425] Step 3: Preparation of Compound 100-4 Compound 100-3 (520 mg, 1.53 mmol, 1.0 equivalent) and Compound SM4 (1.0 g, 3.83 mmol, 2.5 equivalents) were dissolved in DCM (20 mL) and EDCI (880 mg, 4.59 mmol, 3.0 equivalents), DMAP (187 mg, 1.53 mmol, 2.0 equivalents), and DIPEA (989 mg, 7.65 mmol, 5.0 equivalents) were added. This mixture was stirred under reflux for 16 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 20 / 1) to obtain Compound 100-4 (542 mg, yield 42%) as a yellow oil.

[0426] Step 4: Preparation of Compound 100-5 To a solution of compound 100-4 (542 mg, 0.64 mmol, 1.0 equivalent) in EA (10 mL), Pd / C (60 mg) and concentrated HCl (3 drops) were added. This mixture was stirred under H2 at room temperature for 2 hours. The mixture was filtered through a Celite pad and washed with EA. The filtrate was concentrated and purified by column chromatography on silica gel (PE / EA = 20 / 1 → 3 / 1) to obtain compound 100-5 (310 mg, yield 64%) as a colorless oil.

[0427] Step 5: Preparation of Compound 100-6 To a 10 mL solution of compound 100-5 (310 mg, 0.41 mmol, 1.0 equivalent) and compound M (148 mg, 0.82 mmol, 2.0 equivalents) in DCM, EDCI (236 mg, 1.23 mmol, 3.0 equivalents), DIPEA (265 mg, 2.05 mmol, 5.0 equivalents), and DMAP (25 mg, 0.21 mmol, 0.5 equivalents) were added. This mixture was stirred at 35°C for 16 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 10 / 1) to obtain compound 100-6 (205 mg, yield 54%) as a yellow oil.

[0428] Step 6: Preparation of Compound 100 To a solution of compound 100-6 (180 mg, 0.20 mmol, 1.0 equivalent) and compound B (46 mg, 0.40 mmol, 2.0 equivalents) in THF (10 mL), DIPEA (78 mg, 0.60 mmol, 3.0 equivalents) and NaI (9 mg, 0.06 mmol, 0.3 equivalents) were added. This mixture was stirred at 70°C for 16 hours. LC-MS indicated that the reaction was complete. This mixture was concentrated and purified by preparative HPLC to obtain compound 100 (29 mg, yield 16%) as a yellow oil.

[0429] 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H), 1.26-1.32 (m, 63H), 1.52-1.72 (m, 9H), 2.14-2.22 (m, 2H), 2.34-2.38 (m, 2H), 2.54-2.59 (m, 5H), 3.41-3.58 (m, 5H), 3.65-3.71 (m, 5H), 3.98-4.10 (m, 6H). LCMS: Rt: 2.340 min; MS m / z (ESI): 953.0 [M+H] + .

[0430] 7.12 Example 12: Preparation of Compound 116 [ka] Step 1: Preparation of Compound 116-1 To a 100 mL solution of Compound 1-2 (3.0 g, 8.139 mmol, 1.0 equivalent) and Compound U (2.8 g, 8.139 mmol, 1.0 equivalent) in DCM, EDCI (2.3 g, 12.21 mmol, 1.5 equivalents), DIPEA (3.2 g, 24.42 mmol, 3.0 equivalents), and DMAP (0.3 g, 2.442 mmol, 0.3 equivalents) were added. This mixture was stirred at 35°C for 16 hours. This mixture was poured into water and extracted with DCM. The combined organic layer was washed with saturated brine, dehydrated over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel to obtain Compound 161-1 (2.57 g, yield 45.45%) as a yellow oil.

[0431] Step 2: Preparation of Compound 116-2 Compound 116-1 (500 mg, 0.72 mmol, 1.0 equivalent) and Compound M (390 mg, 2.16 mmol, 3.0 equivalents) were dissolved in DCM (20 mL), to which EDCI (207 mg, 1.08 mmol, 1.5 equivalents), DMAP (27 mg, 0.22 mmol, 0.3 equivalents), and DIEA (280 mg, 2.16 mmol, 3.0 equivalents) were added. This mixture was stirred at 55°C for 16 hours. TLC indicated that the reaction was complete. This reaction mixture was concentrated and purified by column chromatography on silica gel to obtain Compound 116-2 (220 mg, yield 35.6%) as a yellow oil.

[0432] Step 3: Preparation of Compound 116 To a 10 mL ACN (ACN) solution of compound 116-2 (220 mg, 0.26 mmol, 1.0 equivalent) and compound B (88 mg, 0.76 mmol, 3.0 equivalents), K2CO3 (106 mg, 0.76 mmol, 3.0 equivalents), Cs2CO3 (26 mg, 0.08 mmol, 0.3 equivalents), and NaI (12 mg, 0.08 mmol, 0.3 equivalents) were added. This mixture was stirred at 80°C for 16 hours. LC-MS indicated that the reaction was complete. The reaction mixture was concentrated and purified by preparative HPLC to obtain compound 116 (23 mg, 9.9%) as a yellow oily substance.

[0433] 1 H NMR (400 MHz, CDCl3) δ: 0.79-0.84 (m, 9H), 1.19-1.23 (m, 28H), 1.45-1.61 (m, 19H), 1.77-2.01 (m, 10H), 2.22-2.48 (m, 11H), 2.69-2.72 (m, 2H), 3.05-3.10 (m, 1H), 3.30-3.52 (m, 6H), 4.06 (d, J = 6 Hz, 6H), 4.40-4.43 (m, 1H), 5.23-5.36 (m, 4H). LCMS: Rt: 1.265 min; MS m / z (ESI): 892.7 [M+H] + .

[0434] The following compounds were prepared using the corresponding starting materials in the same manner as compound 116. [Table 10]

[0435] 7.13 Example 13: Preparation of compound 124. [ka] Step 1: Preparation of Compound 124-2 To a 100 mL solution of compound 1-1 (1.0 g, 9.42 mmol, 1.0 equivalent) and compound 124-1 (5.29 g, 18.85 mmol, 2.0 equivalents) in DCM, DIEA (6.09 g, 47.12 mmol, 5.0 equivalents), EDCI (5.42 g, 28.27 mmol, 3.0 equivalents), and DMAP (2.3 g, 18.85 mmol, 2.0 equivalents) were added. This mixture was stirred at 45°C for 10 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 3 / 1) to obtain compound 124-2 (2.0 g, yield 33%) as a colorless oil.

[0436] Step 2: Preparation of Compound 124-3 Compound 124-2 (2.0 g, 3.17 mmol, 1.0 equivalent) and Compound X (1.41 g, 6.34 mmol, 2.0 equivalents) were dissolved in DCM (100 mL), to which DIEA (2.05 g, 15.85 mmol, 5.0 equivalents), EDCI (1.82 g, 9.51 mmol, 3.0 equivalents), and DMAP (0.78 g, 6.34 mmol, 2.0 equivalents) were added. This mixture was stirred at 45°C for 10 hours. The reaction mixture was concentrated and purified by column chromatography on silica gel (PE / EA = 10 / 1) to obtain Compound 124-3 (1.2 g, yield 45%) as a colorless oil.

[0437] Step 3: Preparation of Compound 124 To a solution of compound 124-3 (300 mg, 0.36 mmol, 1.0 equivalent) and compound B (85 mg, 0.72 mmol, 2.0 equivalents) in THF (10 mL), DIPEA (140 mg, 1.08 mmol, 3.0 equivalents) and NaI (55 mg, 0.36 mmol, 1.0 equivalent) were added. This mixture was stirred at 70°C for 10 hours. LC-MS indicated that the reaction was complete. This mixture was concentrated and purified by preparative HPLC to obtain the labeled compound (60 mg, yield 19%) as a yellow oil.

[0438] 1 H NMR (400 MHz, CDCl3) δ: 0.80-0.84 (m, 6H), 1.19-1.30 (m, 37H), 1.53-1.56 (m, 8H), 1.78-2.00 (m, 12H), 2.22-2.49 (m, 11H), 2.68-2.72 (m, 4H), 3.02-3.11 (m, 1H), 3.44-3.46 (m, 2H), 4.05-4.07 (m, 6H), 5.27-5.30 (m, 8H). LCMS: Rt: 1.437 min; MS m / z (ESI): 870.7 [M+H] + .

[0439] 7.14 Example 14: Preparation of Compound 68 [ka] Step 1: Preparation of Compound 68-2 To a 20 mL solution of 68-1 (1.4 g, 7.13 mmol, 1.0 equivalent) and octanoic acid (2.6 g, 17.83 mmol, 2.5 equivalents) in DCM, EDCI (5.5 g, 28.52 mmol, 4.0 equivalents), DMAP (174 mg, 1.43 mmol, 0.2 equivalents), and DIPEA (4.6 g, 35.65 mmol, 5.0 equivalents) were added. This mixture was stirred at 50°C for 16 hours. After TLC showed that the starting material 68-1 had completely disappeared, the reaction mixture was extracted with DCM, washed with saturated brine, and dehydrated over Na2SO4. The crude product was purified by column chromatography on silica gel (PE / EA = 15 / 1) to obtain compound 68-2 (2.85 g, yield 89.06%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 6H), 1.19-1.33 (m, 16H), 1.51-1.69 (m, 4H), 2.18 -2.43(m, 5H), 3.44-3.56 (m, 2H), 4.12-4.20 (m, 4H), 4.44-4.55 (m, 2H), 7.26-7.35(m, 5H).

[0440] Step 2: Preparation of Compound 68-3 To a 30 mL solution of 68-2 (2.85 g, 6.35 mmol, 1.0 equivalent) in EA (30 mL), Pd / C (285 mg, 2.407 mmol, 10 wt% of 68-2) and HCl (3 drops) were added. This mixture was stirred in an H2 environment for 16 hours. After TLC showed that the starting material 68-2 had completely disappeared, the reaction mixture was filtered through a Celite pad and washed with EA. The filtrate was extracted with EA, washed with saturated brine, and dehydrated over Na2SO4. The organic layer was purified by column chromatography on silica gel (PE / EA = 5 / 1) to obtain compound 68-3 (1.91 g, yield 83.77%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 6H), 1.28-1.40 (m, 16H), 1.58-1.64 (m, 4H), 2.16-2.23 (m, 1H), 2.24-2.70 (m, 5H), 3.61-2.71 (m, 2H), 4.04-4.30 (m, 4H).

[0441] Step 3: Preparation of Compound 68-4 To a 30 mL solution of 68-3 (1.7 g, 4.74 mmol, 1.0 equivalent) and succinic acid (1.68 g, 14.22 mmol, 3.0 equivalents) in DCM, EDCI (1.82 g, 9.48 mmol, 2.0 equivalents), DMAP (116 mg, 0.95 mmol, 0.2 equivalents), and DIPEA (4.9 g, 37.92 mmol, 8.0 equivalents) were added. This mixture was stirred at 50°C for 16 hours. After TLC showed that the starting material 68-3 had completely disappeared, the reaction mixture was extracted with DCM, washed with saturated brine, and dehydrated over Na2SO4. The crude product was purified by column chromatography on silica gel (PE / EA = 15 / 1) to obtain compound 68-4 (1.4 g, yield 64.52%) as a yellow solid.

[0442] Step 4: Preparation of Compound 68-5 To a 20 mL solution of compound 68-4 (1.4 g, 3.05 mmol, 1.0 equivalent) and compound 68-7 (1.25 g, 3.355 mmol, 1.1 equivalents) in DCM, EDCI (1.2 g, 6.1 mmol, 2.0 equivalents), DMAP (75 mg, 0.61 mmol, 0.2 equivalents), and DIPEA (1.97 g, 15.25 mmol, 5.0 equivalents) were added. This mixture was stirred at 50°C for 16 hours. After TLC showed that the starting material 68-4 had completely disappeared, the reaction mixture was extracted with DCM, washed with saturated brine, and dehydrated over Na2SO4. The crude product was purified by column chromatography on silica gel (PE / EA = 15 / 1) to obtain compound 68-5 (665 mg, yield 26.81%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ: 0.79-0.94 (m, 9H), 1.26-1.44 (m, 44H), 1.53-1.63 (m, 6H), 1.99-2.46 (m, 9H), 2.61-2.70 (m, 4H), 3.51-3.78 (m, 2H), 3.95-4.34 (m, 10H).

[0443] Step 5: Preparation of Compound 68-6 To a 10 mL solution of 68-5 (650 mg, 0.80 mmol, 1.0 equivalent) and 5-bromopentanoic acid (435 mg, 2.4 mmol, 3.0 equivalents) in DCM, EDCI (460 mg, 2.4 mmol, 3.0 equivalents), DMAP (49 mg, 0.4 mmol, 0.5 equivalents), and DIPEA (517 mg, 4.0 mmol, 5.0 equivalents) were added. This mixture was stirred at 50°C for 16 hours. After TLC showed that the starting material 68-5 had completely disappeared, the reaction mixture was extracted with DCM, washed with saturated brine, and dehydrated over Na2SO4. The crude product was purified by column chromatography on silica gel (PE / EA = 10 / 1) to obtain compound 68-6 (309 mg, yield 39.56%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ: 0.84-0.88 (m, 9H), 0.99-1.53 ​​(m, 46H), 1.60-1.63 (m, 8H), 1.74-2.05 (m, 3H), 2.23-2.44 (m, 9H), 2.58-2.68 (m, 4H), 3.51-3.58 (m, 1H), 4.12-4.17(m, 11H).

[0444] Step 6: Preparation of Compound 68 To a 10 mL solution of 68-6 (190 mg, 0.19 mmol, 1.0 equivalent) in THF, DIPEA (74 mg, 0.57 mmol, 3.0 equivalents), 2-(methylamino)ethane-1-ol (28 mg, 0.38 mmol, 2.0 equivalents), and NaI (9 mg, 0.06 mmol, 0.3 equivalents) were added. This reaction mixture was stirred at 70°C for 16 hours. LC-MS indicated that the reaction was complete. The mixture was concentrated and purified by preparative HPLC to obtain compound 68 (42 mg, yield 22.83%) as a colorless oil.

[0445] 1 H NMR (400 MHz, CDCl3) δ: 0.83-0.88 (m, 9H), 1.28-1.37(m, 46H), 1.53-1.82 (m, 16H), 1.95-1.98 (m, 1H), 2.26 -2.29(m, 6H), 2.33-2.48 (m, 4H), 2.64-2.70(m, 4H), 2.83-3.06 (m, 3H), 4.12-4.17(m, 10H). LCMS: Rt: 2.450 min; MS m / z (ESI): 970.9 [M+H] + .

[0446] 7.15 Example 15: Preparation and Characterization of Lipid Nanoparticles Briefly, the cationic lipids, DSPCs, cholesterol, and PEG lipids provided herein were solubilized in ethanol in a molar ratio of 50:10:38.5:1.5, and mRNA was diluted in 10–50 mM citrate buffer (pH=4). LNPs were prepared at a total lipid to mRNA weight ratio of approximately 10:1–30:1 by mixing the ethanol-soluble lipid solution with the mRNA aqueous solution in a 1:3 volume ratio using a microfluidic apparatus with a total flow rate ranging from 9–30 mL / min. The ethanol was then removed using dialysis and replaced with DPBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter.

[0447] Lipid nanoparticle size was measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) with a 173° backscatter detection mode. The encapsulation efficiency of lipid nanoparticles was measured using the Quant-it Ribogreen RNA Quantification Assay Kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions.

[0448] As reported in the literature, the apparent pKa of LNP formulations correlates with the delivery efficiency of LNPs to nucleic acids in vivo. The apparent pKa of each formulation was measured using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). LNP formulations consisting of cationic lipids / 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 formulations were diluted to 0.1 mg / ml of total lipids 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 in the range of 3 to 9. Aliquots of TNS solution were added to obtain a final concentration of 0.1 mg / ml, followed by vortex mixing. The fluorescence intensity was then measured at room temperature in a Molecular Devices Spectramax iD3 spectrometer using excitation and emission wavelengths of 325 nm and 435 nm. Sigmoid optimal fit analysis was applied to the fluorescence data, and the pKa value was determined as the pH that produced half of the maximum fluorescence intensity.

[0449] The properties of the specific lipid nanoparticles tested are listed in the table below. [Table 11]

[0450] 7.16 Example 16: Animal Testing Lipid nanoparticles containing the compounds listed in the table below, which encapsulate human erythropoietin (hEPO) mRNA, were administered systemically to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) at a dose of 0.5 mg / kg via tail vein injection. The mice's blood was sampled at a specific time point after administration (e.g., 6 hours). In addition to the above test group, lipid nanoparticles containing dilinoleyl methyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated as MC3), which encapsulates hEPO mRNA, were administered similarly to age and sex comparison groups of mice as a positive control at the same dose.

[0451] After the last sample collection, the mice were euthanized by CO2 overdose. Serum was separated from whole blood by centrifugation at 5000g at 4°C for 10 minutes, rapidly frozen, and stored at -80°C for analysis. An ELISA assay was performed using a commercially available kit (DEP00, R&D systems) according to the manufacturer's instructions.

[0452] The characteristics of the tested lipid nanoparticles, including the MC3 expression levels measured in the test group, are listed in the table below. [Table 12-1] [Table 12-2]

[0453] 7.17 Animal testing Lipid nanoparticles containing specific compounds provided herein, encapsulating luciferase-encoding (luciferase) mRNA, were administered systemically to 6-8 week old female Balb / c mice (Charles River Lab, ZheJiang) at a dose of 0.25 mg / kg via tail vein injection. Blood samples were collected from the mice at a specific time point after administration (e.g., 6 hours). Optical imaging was performed using an IVIS Spectrum CT scanner (PerkinElmer Inc., Paris, France). Emission levels were evaluated by applying an ROI to the injection site zone (Living Image software, PerkinElmer Inc., Paris, France). The results, expressed as total flux (photons / second), are shown in Figure 1.

[0454] Six hours later, the radiance 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 IVIS Spectrum CT (PerkinElmer, Hopkinton, MA). To quantify the bioluminescence signal, the same region of interest (ROI) was set up surrounding each organ region, and the imaging signal was quantified as mean radiance (photons / sec / cm² / steradians). The organ data are summarized in the table below. Significant transduction and luciferase expression were observed in the liver for all LNPs tested. These LNPs showed high absolute expression in the heart, spleen, lungs, kidneys, and brain, while compounds 78, 114, 124, 127, and 129 showed mild expression in the bone marrow. [Table 13]

Claims

1. Equation (I): 【Chemistry 1】 A compound thereof, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, During the ceremony, X １ is a bond or O, X ２ is a bond or O, X ３ is a bond or O, G １ and G ２ are each independently a linking group, C ２ to C １２ alkylene, or C ２ to C １２ alkenylene, provided that one or more of the -CH １ in G ２ and G ２ are independently optionally replaced by -O-, -C(=O)O-, or -OC(=O)-, Each L １ Independently, -OC(=O)R １ , -C (=O) OR １ , -OC(=O)OR １ , -C(=O)R １ , -OR １ , or R １ And, Each L ２ Independently, -OC(=O)R ２ , -C (=O) OR ２ , -OC(=O)OR ２ , -C(=O)R ２ , -OR ２ , or R ２ And, R １ and R ２ Each of them is independent of C ６ ~C ３２ Alkyl or C ６ ~C ３２ It is alkenyl, G ３ is C ２ ~C １２ Alkylene or C ２ ~C １２ It is alkenylene, R ４ However, C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, C ６ ~C １０ It is an aryl or 4- to 8-membered heterocycloalkyl group. R ５ However, C １ ~C １２ Alkyl, C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, C ６ ~C １０ It is an aryl or 4- to 8-membered heterocycloalkyl group. a is either 0 or 1, n is 1, 2, or 3. m is 1, 2, or 3. i is either 0 or 1, j is either 0 or 1. Each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene independently contains a halogen atom 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 It is optionally substituted with any substituent selected from the group consisting of NR'R', where R' is independently H, C in each presence. １ ~C １５ It is an alkyl or cycloalkyl group, where x is 0, 1, or 2. The aforementioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

2. G １ and G ２ CH inside ２ The compound according to claim 1, wherein the - is not substituted with -O-, -C(=O)O-, or -OC(=O)-.

3. Equation (I-A): 【Chemistry 2】 The compound described in claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

4. Formula (IB): 【Transformation 3】 The compound described in claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

5. Formulas (III), (IV), or (V): 【Chemistry 4】 The compound described in claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

6. Formula (III-A), (III-B), (III-C), (III-D), (IV-A), (IV-B), (IV-C), (IV-D), (VA), (V-B), (VC), or (V-D): 【Transformation 5】 (In the formula, y and z are each independent integers from 0 to 9. (t is an integer between 2 and 12) The compound described in claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

7. Equation (IV-E): 【Transformation 6】 (In the formula, u is an integer between 2 and 6. y is an integer from 0 to 9. (t is an integer between 2 and 12) The compound described in claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

8. Formula (II): 【Transformation 7】 A compound thereof, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, During the ceremony, X １ is a bond or O, X ２ is a bond or O, X ３ is a bond or O, G ４ and G ５ Each of them is independent of C ２ ~C ６ Alkylene, or C ２ ~C ６ It is alkenylene, G １ and G ２ Each of them is independent of C ２ ~C １２ Alkylene, or C ２ ~C １２ It is alkenylene, Each L １ Independently, -OC(=O)R １ , -C (=O) OR １ , -OC(=O)OR １ , -C(=O)R １ , -OR １ , or R １ And, Each L ２ is independently -OC(=O)R ２ , -C(=O)OR ２ , -OC(=O)OR ２ , -C(=O)R ２ , -OR ２ , or R ２ and is R １ and R ２ each independently is C ６ to C ３２ alkyl or C ６ to C ３２ alkenyl, G ３ is C ２ ~C １２ Alkylene or C ２ ~C １２ It is alkenylene, R ４ C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, C ６ ~C １０ It is an aryl or 4- to 8-membered heterocycloalkyl group. R ５ C １ ~C １２ Alkyl, C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, C ６ ~C １０ It is an aryl or 4- to 8-membered heterocycloalkyl group. a is either 0 or 1, n is 1, 2, or 3. m is 1, 2, or 3. i is either 0 or 1, j is either 0 or 1. Each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene independently contains a halogen atom 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 It is optionally substituted with any substituent selected from the group consisting of NR'R', where R' is independently H, C in each presence. １ ~C １５ The compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, which is alkyl or cycloalkyl, where x is 0, 1, or 2.

9. Equation (II-A): 【Transformation 8】 The compound described in claim 8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

10. Formula (II-B): 【Chemistry 9】 The compound described in claim 8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

11. Equation (VI): 【Chemistry 10】 The compound described in claim 8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

12. Equation (VI-A) or (VI-B): 【Chemistry 11】 (In the formula, u and v are each independent integers between 2 and 6. y and z are each independent integers between 0 and 9. (t is an integer between 2 and 12) The compound according to claim 8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof. 【Request Item 13】 【Chemistry 12-1】 【Chemistry 12-2】 【Chemistry 12-3】 【Chemistry 12-4】 【Chemistry 12-5】 【Chemistry 12-6】 【Chemistry 12-7】 【Chemistry 12-8】 【Chemistry 12-9】 【Chemistry 12-10】 【Chemistry 12-11】 【Chemistry 12-12】 [Chemistry 12-13] Compounds thereof, or pharmaceutically acceptable salts thereof, or stereoisomers thereof.

14. A composition comprising a compound according to any one of claims 1 to 13 and a therapeutic or prophylactic agent.

15. Lipid nanoparticles comprising the compound described in claim 1, or the composition described in claim 14.

16. A pharmaceutical composition comprising the compound described in claim 1, the composition described in claim 14, or the lipid nanoparticles described in claim 15, and a pharmaceutically acceptable excipient or diluent.

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