Compounds and Compositions for Delivery of Therapeutic Agents

Abstract

The present disclosure features novel lipids and compositions comprising the same. The lipid nanoparticles (e.g., empty or loaded LNPs) include novel cationic lipids and additional lipids, such as ionizable lipids, phospholipids, structural lipids, and PEG lipids. The lipid nanoparticles (e.g., empty or loaded LNPs) further comprising therapeutic and / or prophylactic agents, such as RNA, are useful in delivering therapeutic and / or prophylactic agents to mammalian cells or organs, for example, to control polypeptide, protein, or gene expression.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and benefit of U.S. Application No. 63 / 288,317, filed December 10, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure provides novel cationic lipids, compositions comprising such lipids, and methods involving lipid nanoparticle compositions for delivering one or more therapeutic and / or prophylactic agents to mammalian cells or organs and / or for producing polypeptides in mammalian cells or organs. In addition to the novel cationic lipids, the lipid nanoparticle compositions of the present disclosure may include, in specific proportions, one or more ionizable amino lipids, phospholipids including polyunsaturated lipids, PEG lipids, structured lipids, and / or therapeutic and / or prophylactic agents. [Background technology]

[0003] Effective targeted delivery of biologically active substances, such as small molecule drugs, proteins, and nucleic acids, represents a continuing medical challenge. In particular, delivery of nucleic acids into cells is made difficult by the relative instability and poor cell permeability of such species. Thus, there is a need to develop methods and compositions for facilitating the delivery of therapeutic and / or prophylactic agents, such as nucleic acids, into cells.

[0004] Lipid-containing nanoparticle compositions, liposomes, and lipoplexes have proven effective as delivery vehicles for biologically active substances, such as small molecule drugs, proteins, and nucleic acids, into cells and / or intracellular compartments. Such compositions typically contain one or more "cationic" and / or amino (ionizable) lipids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), and / or lipids containing polyethylene glycol (PEG lipids). Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated. Although a variety of such lipid-containing nanoparticle compositions have been demonstrated, improvements in safety, efficacy, and specificity remain lacking. Summary of the Invention

[0005] The present disclosure provides novel cationic lipids and compositions (e.g., lipid nanoparticles) and methods involving same. In some aspects, the present disclosure provides a cationic lipid of formula (I):

[0006] [ka]

[0007] or an isomer thereof (wherein R' x teeth,

[0008] [ka]

[0009] and R' y teeth,

[0010] [ka]

[0011] and R' z teeth,

[0012] [ka]

[0013] and;

[0014] [ka]

[0015] indicates the point of attachment; R xα , R xβ , R xγ , and R xδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R yα , R yβ , R yγ , and R yδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R zα , R zβ , R zγ , and R zδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R H is -(CH2) q OH, and q is selected from 1, 2, 3, 4, and 5; Each R T independently, C 1-12 Alkyl and C 2-12 alkenyl; a is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; b is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; c is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; A -is any pharmaceutically acceptable anion) Regarding.

[0016] In some embodiments, the cationic lipid of formula (I) has the following structure:

[0017] [ka]

[0018] (In the formula, A - is selected from chloride, bromide, iodide, hydroxide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate).

[0019] In some embodiments, the cationic lipid of formula (I) has the following structure:

[0020] [ka]

[0021] (In the formula, A - is bromide, chloride, hydroxide, or a combination thereof). In some embodiments, A - is bromide or hydroxide. In some embodiments, A - is chloride or hydroxide. In some embodiments, A - is bromide. In some embodiments, A - In some embodiments, A is chloride. - is hydroxide. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present disclosure provides novel cationic lipids comprising a central amine moiety and at least one biodegradable group. The cationic lipids described herein can be advantageously used in lipid nanoparticles (e.g., empty LNPs or loaded LNPs) for delivery of therapeutic and / or prophylactic agents to mammalian cells or organs. For example, the cationic lipids described herein can be advantageously used in lipid nanoparticles (e.g., empty LNPs or loaded LNPs) for delivery of therapeutic and / or prophylactic agents to specific mammalian cells or organs. In some embodiments, the cationic lipids described herein can be advantageously used in lipid nanoparticles (e.g., empty LNPs or loaded LNPs) for delivery of therapeutic and / or prophylactic agents to endothelial cells or lungs.

[0023] The present disclosure also provides lipid nanoparticles (LNPs) comprising novel cationic lipids for delivery of therapeutic and / or prophylactic agents to endothelial cells. For example, such LNPs can be used to deliver therapeutic and / or prophylactic agents, such as mRNA therapeutics, to endothelial cells. In some embodiments, such LNPs can be used to deliver nucleic acid molecules, small molecules, or other payloads for gene editing to improve endothelial cell dysfunction. In some embodiments, such LNPs can be used to deliver antigens to endothelial cells, which are key participants and regulators of inflammatory responses. Quiescent endothelial cells prevent clotting, control the passage of proteins from the bloodstream and blood into tissues, and inhibit inflammation. In some embodiments, the antigen is in the form of an mRNA construct present in the LNP that results in the expression of a polypeptide or peptide such that an immune response against the antigen is generated.

[0024] LNPs are an ideal platform for the safe and effective delivery of therapeutic and / or prophylactic agents, e.g., mRNA, to target cells. LNPs have the unique ability to deliver therapeutic and / or prophylactic agents (e.g., nucleic acids, e.g., mRNA) by mechanisms involving cellular uptake, intracellular trafficking, and endosomal release or endosomal escape. Some embodiments provided herein feature LNPs with improved properties. In some embodiments, LNPs provided herein have a lipid nanoparticle core, a therapeutic and / or prophylactic agent encapsulated within the core for delivery to cells, and a cationic agent primarily disposed on the outer surface of the nanoparticle. Without being bound by a particular theory, LNPs with a cationic agent primarily disposed on the outer surface of the core may improve the accumulation of LNPs in cells, e.g., human pulmonary endothelial cells, and may also improve the function of the therapeutic and / or prophylactic agent, as measured by mRNA expression in the cells, e.g., endothelial cells in the lung.

[0025] In some embodiments, provided herein is a loaded LNP, comprising: (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) located primarily on the outer surface of the core. wherein the loaded LNP has a zeta potential greater than neutral at physiological pH.

[0026] In some embodiments, provided herein is a loaded LNP, comprising: (a) an onboard LNP core; (i) ionizable lipids; (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid Including the onboard LNP core and (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) An on-board LNP is provided, including:

[0027] In some embodiments, provided herein is a loaded LNP, comprising: (a) a lipid nanoparticle core, (i) ionizable lipids; (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid a lipid nanoparticle core comprising: (b) a polynucleotide or polypeptide payload therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) located primarily on the outer surface of the core. An on-board LNP is provided, including:

[0028] In some embodiments, provided herein is a loaded LNP, comprising: (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) The loaded LNPs are provided, wherein the loaded LNPs exhibit cellular accumulation of at least about 20% of cells and exhibit expression in about 5% or more of cells in a population of cells to which the loaded LNPs are administered. In some embodiments, the loaded LNPs exhibit cellular accumulation of about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% of cells in a population of cells to which the loaded LNPs are administered.

[0029] In some embodiments, the loaded LNP exhibits expression in about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% of cells in which the loaded LNP accumulates.

[0030] In some aspects of embodiments, provided herein is an on-board LNP, comprising: (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) located primarily on the outer surface of the core. Provided are loaded LNPs comprising: a) a loaded LNP that exhibits cellular accumulation in at least about 20% of cells in a population of cells to which the loaded LNP is administered, and that exhibits expression in about 5% or more in the cells to which the loaded LNP accumulates; In some embodiments, the loaded LNP exhibits cellular accumulation in about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% of cells in a population of cells to which the loaded LNP is administered; In some embodiments, the loaded LNP exhibits expression in about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% in the cells to which the loaded LNP accumulates.

[0031] In some embodiments, provided herein is a loaded LNP, comprising: (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) located primarily on the outer surface of the core. The therapeutic and / or prophylactic agent expresses a protein, and the loaded LNP exhibits about 0.5% to about 50% protein expression in cells in a population of cells to which the loaded LNP is administered. In some embodiments, the loaded LNP exhibits about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% protein expression in cells to which the loaded LNP accumulates.

[0032] In some embodiments, provided herein is a loaded LNP, comprising: (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) The loaded LNP comprises: a) a protein expression level of about 0.5% to about 50% in cells in which the loaded LNP accumulates; b) a protein expression level of about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% in cells in which the loaded LNP accumulates;

[0033] In some embodiments, provided herein is a loaded LNP, comprising: (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) Provided are loaded LNPs comprising: a) a loaded LNP that exhibits at least about 20% cellular accumulation in endothelial cells and about 5% or greater expression in endothelial cells in which the loaded LNP has accumulated; b) a loaded LNP that exhibits about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% cellular accumulation in endothelial cells in a population of endothelial cells to which the loaded LNP has been administered; c) a loaded LNP that exhibits about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% expression in endothelial cells in which the loaded LNP has accumulated;

[0034] In some embodiments, provided herein is a loaded LNP, comprising: (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) The loaded LNPs are provided, wherein the loaded LNPs exhibit protein expression of about 0.5% to 50% of the endothelial cells in which the loaded LNPs have accumulated. In some embodiments, the loaded LNPs exhibit protein expression of about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of the endothelial cells in which the loaded LNPs have accumulated.

[0035] In some embodiments, provided herein is a loaded LNP, comprising: (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) The loaded LNPs are provided, wherein the loaded LNPs exhibit protein expression in about 0.5% to about 50% of lung cells in which the loaded LNPs have accumulated. In some embodiments, the loaded LNPs exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of lung endothelial cells in which the loaded LNPs have accumulated.

[0036] In some embodiments, provided herein is a loaded LNP, comprising: (a) Lipid-loaded LNP core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) Provided are loaded LNPs comprising: a) a loaded LNP that exhibits at least about 20% cellular accumulation in respiratory endothelial cells and about 5% or more expression in respiratory endothelial cells to which the loaded LNP has accumulated; b) a loaded LNP that exhibits about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% cellular accumulation in respiratory endothelial cells in a population of respiratory endothelial cells to which the loaded LNP has been administered; c) a loaded LNP that exhibits about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% expression in respiratory endothelial cells to which the loaded LNP has accumulated;

[0037] In some embodiments, provided herein is a loaded LNP, comprising: (a) Lipid-loaded LNP core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) The loaded LNPs are provided, wherein the loaded LNPs exhibit protein expression in about 0.5% to about 50% of respiratory endothelial cells in which the loaded LNPs accumulate. In some embodiments, the loaded LNPs exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of respiratory endothelial cells in which the loaded LNPs accumulate.

[0038] In some embodiments, provided herein is a loaded LNP, comprising: (a) Lipid-loaded LNP core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) The loaded LNPs are provided, wherein the loaded LNPs exhibit protein expression in about 0.5% to about 50% of HeLa cells in which the loaded LNPs have accumulated. In some embodiments, the loaded LNPs exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of HeLa cells in which the loaded LNPs have accumulated.

[0039] In some embodiments, provided herein is a loaded LNP, comprising: (a) Lipid-loaded LNP core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent (e.g., a cationic lipid of formula (I)) The loaded LNP comprises: a) a cellular accumulation of at least about 20% in bronchial endothelial cells in a population of bronchial endothelial cells to which the loaded LNP is administered; and b) an expression of about 5% or more in bronchial endothelial cells to which the loaded LNP has accumulated. In some embodiments, the loaded LNP exhibits a cellular accumulation of about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% in respiratory endothelial cells in a population of respiratory endothelial cells to which the loaded LNP has been administered. In some embodiments, the loaded LNP exhibits an expression of about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% in pulmonary endothelial cells to which the loaded LNP has accumulated.

[0040] In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 0.1:1 to about 20:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 1.5:1 to about 10:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 1.5:1 to about 9:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 1.5:1 to about 8:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 1.5:1 to about 7:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 1.5:1 to about 6:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 1.5:1 to about 5:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 1.5:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 2:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 3:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 4:1. In some embodiments, the molar ratio of cationic agent to therapeutic and / or prophylactic agent is about 5:1.

[0041] In some embodiments, nanoparticles of the present disclosure (e.g., empty LNPs or loaded LNPs) have a zeta potential of about 5 mV to about 20 mV. In some embodiments, nanoparticles have a zeta potential of about 5 mV to about 15 mV. In some embodiments, nanoparticles have a zeta potential of about 5 mV to about 12 mV. In some embodiments, nanoparticles have a zeta potential of about 5 mV to about 10 mV.

[0042] Cationic Agents Cationic agents can include any aqueous / organic soluble molecule or substance that has a net positive charge. Such agents can also be lipid-soluble, but can also be soluble in aqueous solutions. Cationic agents can be charged at physiological pH. Physiological pH is the pH level normally observed in the human body. Physiological pH can be about 7.30-7.45 or about 7.35-7.45. Physiological pH can be about 7.40. Generally speaking, cationic agents are characterized by a net positive charge at physiological pH because they contain one or more basic functional groups that are protonated at physiological pH in aqueous media. For example, cationic agents can contain one or more amine groups, e.g., primary, secondary, or tertiary amines, each with a pKa of 8.0 or greater. The pKa can be greater than about 9. In some embodiments, the cationic agent is a cationic lipid (i.e., a lipid that has a positive or partial positive charge at physiological pH). In some embodiments, the cationic agent is a cationic lipid of formula (I).

[0043] In some embodiments, the lipids of formula (I) include one or more of the following characteristics, where applicable: In some embodiments, R' x teeth,

[0044] [ka]

[0045] and; R' y teeth,

[0046] [ka]

[0047] and; R' z teeth,

[0048] [ka]

[0049] and;

[0050] [ka]

[0051] indicates the point of attachment; R xγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R yγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R zγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c are each independently 1-14 Alkyl and C 2-14 alkenyl.

[0052] In some embodiments, R' a teeth,

[0053] [ka]

[0054] and R' b teeth,

[0055] [ka]

[0056] and R' c teeth,

[0057] [ka]

[0058] and;

[0059] [ka]

[0060] indicates the point of attachment; R xγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R yγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R 2c and R 3c are each independently 1-14 Alkyl and C 2-14 alkenyl.

[0061] In some embodiments, R xγ and R yγ are H, respectively. In some embodiments, R xγ and R yγ are respectively, C 1-12 Alkyl or C 2-12 It is alkenyl.

[0062] In some embodiments, R xγ is C 1-12 Alkyl or C 2-12 alkenyl, and R yγ is H. In some embodiments, q is 2.

[0063] In some embodiments, the cationic lipids of formula (I) described herein are suitable for making nanoparticle compositions for intramuscular administration. In some embodiments, the cationic lipid of formula (I) is selected from the lipids in Table 1 or an isomer thereof.

[0064] [Table 1]

[0065] definition As used herein, the term "alkyl" or "alkyl group" means a linear or branched saturated hydrocarbon containing one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms), which is optionally substituted. 1-14 "Alkyl" means an optionally substituted, linear or branched, saturated hydrocarbon containing 1 to 14 carbon atoms. Unless otherwise specified, alkyl groups described herein refer to both unsubstituted and substituted alkyl groups. As used herein, "linear" alkyl means a straight-chain alkyl (methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, or n-dodecyl) (the point of attachment is at the C1 carbon).

[0066] As used herein, the term "alkenyl" or "alkenyl group" means an optionally substituted linear or branched hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one double bond. 2-14 "Alkenyl" means an optionally substituted linear or branched hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group can contain 1, 2, 3, 4, or more carbon-carbon double bonds. For example, C 18 Alkenyl may contain one or more double bonds. C containing two double bonds 18 The alkenyl group can be a linoleyl group. Unless otherwise specified, the alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups.

[0067] As used herein, the term "alkynyl" or "alkynyl group" means an optionally substituted linear or branched hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one carbon-carbon triple bond. 2-14 "Alkynyl" means an optionally substituted linear or branched hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups can contain 1, 2, 3, 4, or more carbon-carbon triple bonds. For example, C 18 Alkynyl can include one or more carbon-carbon triple bonds. Unless otherwise specified, alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.

[0068] As used herein, the term "carbocycle" or "carbocyclic group" means an optionally substituted mono- or polycyclic system containing one or more rings of carbon atoms. The rings can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-membered rings. 3-6 "Carbocycle" means a carbon ring, including a single ring, having 3 to 6 carbon atoms. A carbocycle may contain one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., a cycloalkyl or aryl group). A carbocycle may be a mono- or polycyclic (e.g., fused, bridged, or spirocyclic) system. Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. The term "cycloalkyl," as used herein, means a non-aromatic carbocycle, which may or may not contain any double or triple bonds. Unless otherwise specified, carbocycles described herein refer to both unsubstituted and substituted carbocyclic groups, i.e., optionally substituted carbocycles. In some embodiments, a carbocycle is a C 3-8 In some embodiments, the carbocycle is C 3-6 In some embodiments, the carbocycle is C 6-10 It is aryl.

[0069] "Aryl" includes groups having aromatic character, including "conjugated" or polycyclic systems having at least one aromatic ring, and does not contain any heteroatoms in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl, and the like. In some embodiments, "aryl" refers to C 6-10 A carbocyclic ring (e.g., "aryl" is C 6-10 (aryl).

[0070] As used herein, the term "heterocycle" or "heterocyclic group" refers to an optionally substituted mono- or polycyclic system containing one or more rings, at least one of which contains at least one heteroatom. The heteroatom can be, for example, a nitrogen, oxygen, or sulfur atom. The ring can be 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered. The heterocycle can contain one or more double or triple bonds and can be non-aromatic or aromatic (e.g., a heterocycloalkyl or heteroaryl group). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. The term "heterocycloalkyl" as used herein refers to a non-aromatic heterocycle, which may or may not contain any double or triple bonds. Unless otherwise specified, heterocycles described herein refer to both unsubstituted and substituted heterocyclic groups, i.e., optionally substituted heterocycles. In some embodiments, the heterocycle is a 4- to 12-membered heterocycloalkyl. In some embodiments, the heterocycle is a 5- or 6-membered heteroaryl.

[0071] A "heteroaryl" group is an aryl group as defined above, except having 1 to 4 heteroatoms in the ring structure, and may also be referred to as an "aryl heterocycle" or "heteroaromatic." As used herein, the term "heteroaryl" is intended to include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic heteroaromatic ring consisting of carbon atoms and one or more heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur, and boron, e.g., 1, 1-2, 1-3, 1-4, 1-5, or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms. The nitrogen atom can be substituted or unsubstituted (i.e., N or NR (where R is H or other defined substituent)). The nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., N→O and S(O)). p (p=1 or 2)).

[0072] Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0073] Furthermore, the terms "aryl" and "heteroaryl" include polycyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, such as naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, quinoline, isoquinoline, naphtholidine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

[0074] As used herein, a "biodegradable group" is a group that can facilitate more rapid metabolism of lipids in mammalian organisms. The biodegradable group may be selected from the group consisting of, but is not limited to, -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group. As used herein, an "aryl group" is an optionally substituted carbocyclic group containing one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, a "heteroaryl group" is an optionally substituted heterocyclic group containing one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups can be optionally substituted. For example, M and M' can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the formulas herein, M and M' can be independently selected from the list of biodegradable groups above. Unless otherwise specified, the aryl or heteroaryl groups described herein refer to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0075] Alkyl, alkenyl, and cyclyl (eg, carbocyclyl and heterocyclyl) groups may be optionally substituted, unless otherwise specified. Optional substituents include, but are not limited to, halogen atoms (e.g., chloride, bromide, fluoride, or iodide groups), carboxylic acids (e.g., —C(O)OH), alcohols (e.g., hydroxyl, —OH), esters (e.g., —C(O)OR or —OC(O)R), aldehydes (e.g., —C(O)H), carbonyls (e.g., —C(O)R, alternatively represented by C═O), acyl halides (e.g., —C(O)X, where X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (e.g., —OC(O)OR), alkoxy (e.g., —OR), acetals (e.g., —C(OR)R″″, where each OR is an alkoxy group, which can be the same or different, and R″″ is an alkyl or alkenyl group), phosphates (e.g., P(O) 3- ), thiol (e.g., -SH), sulfoxide (e.g., -S(O)R), sulfinic acid (e.g., -S(O)OH), sulfonic acid (e.g., -S(O)2OH), thial (e.g., -C(S)H), sulfate (e.g., S(O)4 2-), sulfonyl (e.g., -S(O)2-), amido (e.g., -C(O)NR2, or -N(R)C(O)R), azido (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR2, -NRH, or -NH2), carbamoyl (e.g., -OC(O)NR2 , -OC(O)NRH, or -OC(O)NH), sulfonamide (e.g., -S(O)NR, -S(O)NRH, -S(O)NH, -N(R)S(O)R, -N(H)S(O)R, -N(R)S(O)H, or -N(H)S(O)H), alkyl, alkenyl, and cyclyl (e.g., carbocyclyl or heterocyclyl) groups. In any of the foregoing, R is an alkyl or alkenyl group as defined herein. In some embodiments, the substituent itself can be further substituted, for example, with 1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, C 1-6 Alkyl groups can be further substituted with 1, 2, 3, 4, 5, or 6 substituents described herein.

[0076] The nitrogen-containing lipids of the present disclosure can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and / or hydrogen peroxide) to yield other lipids of the present disclosure. Thus, all of the nitrogen-containing lipids shown and claimed, when permitted by valency and structure, can be converted to N-oxide derivatives (N→O or N) of the shown lipids and their N-oxide derivatives. + -O -(which may be designated as "N-hydroxy or N-alkoxy"). Additionally, in other examples, nitrogen in the lipids of the present disclosure can be converted to N-hydroxy or N-alkoxy lipids. For example, N-hydroxy lipids can be prepared by oxidation of the parent amine with an oxidizing agent, such as m-CPBA. All depicted and claimed nitrogen-containing lipids are also considered to encompass both the depicted lipid and its N-hydroxy (i.e., N-OH) and N-alkoxy (i.e., N-OR, where R is substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3- to 14-membered carbocyclic or 3- to 14-membered heterocyclic) derivatives, where permitted by valency and structure.

[0077] About, Approximately: As used herein, the terms "approximately" and "about," when applied to one or more values ​​of interest, refer to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise stated or otherwise clear from the context (unless such number would exceed 100% of the possible values). For example, when used in the context of the amount of a given lipid in the lipid component of a nanoparticle composition, "about" can mean + / - 10% of the stated value. For example, a nanoparticle composition comprising a lipid component with about 40% of a given lipid may contain 30-50% lipid.

[0078] As used herein, the term "lipid" means to include all isomers and isotopes of the structure shown. "Isotope" refers to atoms that have the same atomic number but have different mass numbers due to the different number of neutrons in the nucleus.For example, the isotopes of hydrogen include tritium and deuterium.In addition, the lipid, salt or complex of the present disclosure can be prepared by conventional methods with solvent or water molecules to form solvate and hydrate.

[0079] As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle are caused to share a physical connection. Methods for contacting cells with external entities, both in vivo and ex vivo, are well known in the biological arts. For example, contacting a nanoparticle composition with a mammalian cell located within a mammal can be performed by various routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve various amounts of lipid nanoparticles (e.g., empty LNPs or loaded LNPs). Moreover, multiple mammalian cells can be contacted with the nanoparticle composition.

[0080] As used herein, the term "delivering" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject can involve administering to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes) a nanoparticle composition comprising the therapeutic and / or prophylactic agent. Administering a nanoparticle composition to a mammal or mammalian cells can involve contacting one or more cells with the nanoparticle composition.

[0081] As used herein, the term "enhanced delivery" refers to delivery of a therapeutic and / or prophylactic agent by a nanoparticle to a target tissue (e.g., the liver or lungs of a mammal) that is greater (e.g., at least 1.5-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, or at least 10-fold greater) than the level of delivery of a therapeutic and / or prophylactic agent by a control nanoparticle (e.g., MC3, KC2, or DLinDMA) to the target tissue. The level of nanoparticle delivery to a particular tissue can be measured by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of therapeutic and / or prophylactic agent in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the total amount of protein in the tissue, or comparing the amount of therapeutic and / or prophylactic agent in the tissue to the total amount of therapeutic and / or prophylactic agent in the tissue. It will be understood that the improved delivery of nanoparticles to target tissues need not be determined in the subject being treated; it can be determined in a surrogate, for example, an animal model (e.g., a rat model). In certain embodiments, nanoparticle compositions comprising the cationic lipids of the present disclosure have substantially the same level of improved delivery regardless of the route of administration. For example, certain lipids disclosed herein exhibit similar improved delivery when used to deliver therapeutic and / or prophylactic agents either intravenously or intramuscularly. In other embodiments, certain lipids disclosed herein exhibit higher levels of improved delivery when used to deliver therapeutic and / or prophylactic agents intramuscularly than intravenously.

[0082] As used herein, the terms "specific delivery," "specifically deliver," or "specifically delivering" refer to delivery of a therapeutic and / or prophylactic agent by a nanoparticle to a target tissue of interest (e.g., the liver or lungs of a mammal) in greater amounts (e.g., at least 1.5-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, or at least 10-fold greater) than an off-target tissue (e.g., the spleen of a mammal). The level of nanoparticle delivery to a particular tissue can be measured by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of therapeutic and / or prophylactic agent in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the amount of total protein in the tissue, or comparing the amount of therapeutic and / or prophylactic agent in the tissue to the amount of total therapeutic and / or prophylactic agent in the tissue. For example, for renal vascular targeting, a therapeutic and / or prophylactic agent is specifically provided to the kidney of a mammal compared to the liver and spleen if 1.5, 2, 3, 5, 10, 15, or 20 times more therapeutic and / or prophylactic agent is delivered to the kidney per gram of tissue compared to that delivered to the liver or spleen following systemic administration of the therapeutic and / or prophylactic agent. It will be understood that the ability of a nanoparticle to be specifically delivered to a target tissue need not be determined in the subject being treated, but can be determined in a surrogate, for example, an animal model (e.g., a rat model).

[0083] As used herein, "encapsulation efficiency" refers to the amount of a therapeutic and / or prophylactic agent that becomes part of a nanoparticle composition relative to the initial total amount of the therapeutic and / or prophylactic agent used in preparing the nanoparticle composition. For example, if 97 mg of a therapeutic and / or prophylactic agent is encapsulated in a nanoparticle composition out of a total of 100 mg of the therapeutic and / or prophylactic agent initially provided in the composition, the encapsulation efficiency can be given as 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial surrounding, enclosing, surrounding, or packaging.

[0084] As used herein, "encapsulation," "encapsulated," "loaded," and "associated" can refer to complete, substantial, or partial surrounding, confinement, envelopment, or packaging. As used herein, "encapsulation" or "association" can refer to the process of confining an individual nucleic acid molecule within a nanoparticle and / or establishing a physiochemical association between an individual nucleic acid molecule and a nanoparticle. As used herein, an "empty nanoparticle" can refer to a nanoparticle that is substantially free of a therapeutic or prophylactic agent. As used herein, an "empty nanoparticle" or "empty lipid nanoparticle" can refer to a nanoparticle that is substantially free of nucleic acids. As used herein, an "empty nanoparticle" or "empty lipid nanoparticle" can refer to a nanoparticle that is substantially free of nucleotides or polypeptides. As used herein, an "empty nanoparticle" or "empty lipid nanoparticle" can refer to a nanoparticle that is substantially composed of only lipid components. As used herein, a "loaded LNP," "loaded nanoparticle," or "loaded lipid nanoparticle" (also referred to as a "full nanoparticle" or "full lipid nanoparticle") can refer to a nanoparticle that contains the components of an empty nanoparticle and a substantial amount of a therapeutic or prophylactic agent. In some embodiments, the loaded LNP comprises a therapeutic or prophylactic agent that is at least partially internal to the LNP. In some embodiments, the loaded LNP comprises a substantial amount of a therapeutic or prophylactic agent associated with the surface of the LNP or conjugated to the exterior of the LNP. As used herein, "loaded LNP," "loaded nanoparticle," or "loaded lipid nanoparticle" (also referred to as "full nanoparticle" or "full lipid nanoparticle") can refer to a nanoparticle that comprises the components of an empty nanoparticle and a substantial amount of a nucleotide or polypeptide. In some embodiments, the loaded LNP comprises a nucleotide or polypeptide that is at least partially internal to the LNP. In some embodiments, the loaded LNP comprises a nucleotide or polypeptide that is associated with the surface of the LNP or conjugated to the exterior of the LNP.As used herein, "loaded LNP," "loaded nanoparticle," or "loaded lipid nanoparticle" (also referred to as "full nanoparticle" or "full lipid nanoparticle") can refer to a nanoparticle that includes the components of an empty nanoparticle and a substantial amount of nucleic acid. In some embodiments, a loaded LNP includes nucleic acid (e.g., mRNA) that is at least partially within the interior of the LNP. In some embodiments, a loaded LNP includes nucleic acid (e.g., mRNA) associated with the surface of the LNP or conjugated to the exterior of the LNP.

[0085] As used herein, "expression" of a nucleic acid sequence refers to translation of mRNA into a polypeptide or protein and / or post-translational modification of a polypeptide or protein. As used herein, "expression of loaded LNPs" refers to expression by an agent contained in the loaded LNPs. For example, in some embodiments, the agent is a nucleic acid (e.g., mRNA). For example, in some embodiments, the agent expresses a protein or polypeptide. In some embodiments, the protein is a fluorescent protein. For example, in some embodiments, the term "loaded LNPs exhibit expression" means that loaded LNPs that accumulate in a cell deliver an agent to the cell, and that the agent (e.g., a nucleic acid, e.g., mRNA) expresses, for example, a protein or polypeptide in the cell.

[0086] As used herein, the "hydrophobicity" of a lipid refers to the tendency of the lipid to exclude water. In some embodiments, the hydrophobicity of the lipid nanoparticle surface affects the penetration of the lipid nanoparticle through the lipid bilayer of a cell. In some embodiments, hydrophobic nanoparticles exhibit increased cellular uptake compared to hydrophilic lipid nanoparticles.

[0087] As used herein, the term "in vitro" refers to events that occur not within a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc.

[0088] As used herein, the term "in vivo" refers to an event that takes place within an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof). As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof). An ex vivo event can occur in an environment that is minimally modified from the natural (e.g., in vivo) environment.

[0089] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound (e.g., a lipid of the present disclosure). A compound (e.g., a lipid of the present disclosure) may contain one or more chiral centers and / or double bonds and thus may exist as a stereoisomer, e.g., a double bond isomer (i.e., a geometric E / Z isomer) or diastereomer (e.g., an enantiomer (i.e., (+) or (-)) or cis / trans isomer). The present disclosure encompasses any and all isomers of the lipids described herein, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of lipids and means for resolving them into their component enantiomers or stereoisomers are well known.

[0090] A "tautomer" is one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom, achieved by the switch of adjacent conjugated double bonds. Tautomers exist as mixtures of tautomeric sets in solution. In solutions where tautomerization is possible, a chemical equilibrium of tautomers will be reached. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that are interconvertible by tautomerization is called tautomerism.

[0091] Of the various types of tautomerism possible, two are commonly observed: keto-enol tautomerism, in which a simultaneous shift of an electron and a hydrogen atom occurs; and ring-chain tautomerism, which occurs when an aldehyde group (-CHO) in a sugar molecule reacts with one of the hydroxyl groups (-OH) in the same molecule, giving it a cyclic (ring-shaped) form, such as that exhibited by glucose.

[0092] Common tautomeric pairs are ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomers in heterocyclic rings (e.g., in nucleobases such as guanine, thymine, and cytosine), imine-enamine, and enamine-enamine. An example of tautomerism in a disubstituted guanidine is shown below.

[0093] [ka]

[0094] It should be understood that the lipids of the present disclosure may be represented as different tautomeric forms. It should also be understood that where a lipid has tautomeric forms, all tautomeric forms are intended to be included within the scope of the present disclosure, and that the naming of the lipid does not exclude any tautomeric form.

[0095] As used herein, a "lipid component" refers to a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component can include one or more cationic lipids, ionizable lipids, PEGylated, structural, or other lipids, such as phospholipids. In some embodiments of empty or loaded LNPs of the present disclosure, the lipid component includes at least one cationic lipid and at least one ionizable lipid.

[0096] As used herein, a "linker" refers to a moiety that connects two moieties, such as the connection between two nucleosides of a capped species. The linker may include one or more groups, including, but not limited to, a phosphate group (e.g., phosphate, boranophosphate, thiophosphate, selenophosphate, and phosphonate), an alkyl group, an amidate, or glycerol. For example, the two nucleosides of a capped analog may be linked at their 5' positions by a triphosphate group or by a chain containing two phosphate moieties and a boranophosphate moiety.

[0097] As used herein, a "method of administration" can include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. The method of administration can be selected to target (e.g., specifically deliver) delivery to a particular area or system of the body.

[0098] As used herein, "modified" means non-natural. For example, RNA can be modified RNA. That is, RNA can contain one or more nucleobases, nucleosides, nucleotides, or linkers that do not occur in nature. "Modified" species can also be referred to herein as "altered" species. Species can be chemically, structurally, or functionally modified or altered. For example, modified nucleobase species can contain one or more substitutions that do not occur in nature.

[0099] As used herein, "N:P ratio" is the molar ratio of ionizable nitrogen atoms in lipids (in the physiological pH range) to phosphate groups in RNA, for example, in a nanoparticle composition comprising a lipid component and RNA.

[0100] As used herein, a "nanoparticle composition" refers to a composition containing one or more lipids. Nanoparticle compositions are typically sized on the order of micrometers or less and may contain a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0101] As used herein, "naturally occurring" means occurring in nature without artificial assistance. As used herein, "patient" refers to a subject who may seek or need treatment, who needs treatment, who is undergoing treatment, who will receive treatment, or who is under the care of a trained professional for a particular disease or condition.

[0102] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that includes a polyethylene glycol moiety. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, anions, cations, substances, compositions, carriers, and / or dosage forms that are, within the scope of reasonable medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0103] The phrase "pharmaceutically acceptable excipient," as used herein, refers to any ingredient (e.g., a vehicle capable of suspending, complexing, or dissolving an active lipid) other than the lipids described herein that has substantially non-toxic and non-inflammatory properties in a patient. Excipients can include, for example, antiadhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other species disclosed herein.

[0104] In this specification, the structural formula of lipid is sometimes used for convenience to represent a certain isomer, but the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, etc., and it is understood that not all isomers can have the same level of activity.In addition, crystalline polymorphism can exist for the lipid represented by formula.It should be noted that any crystalline form, crystalline mixture, or its anhydrous or hydrate are included in the scope of the present disclosure.

[0105] The term "crystalline polymorphism," "polymorphism," or "crystalline form" refers to a crystalline structure in which a compound (e.g., a lipid of the present disclosure; or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors may cause one crystalline form to predominate. Crystalline polymorphs of lipids can be prepared by crystallization under different conditions.

[0106] The compounds of the present disclosure include the compounds themselves, as well as their salts and solvates, if applicable. Salts can be formed, for example, between an anion and a positively charged group, such as a quaternary amino group. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, hydroxide, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. It is understood that in the salt form, the ratio of the compound to the cation or anion of the salt can be 1:1, or any ratio other than 1:1, for example, 3:1, 2:1, 1:2, or 1:3.

[0107] The composition can also include one or more lipid salts.Salt can be pharmaceutically acceptable salt.As used herein, " pharmaceutically acceptable salt " refers to the disclosed lipid derivatives, in which parent lipid is modified by converting existing acid or basic moiety into its salt form (for example, by reacting free base group with suitable organic acid).Examples of pharmaceutically acceptable salts include but are not limited to mineral acid or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids, etc. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxy-ethanesulfonate. , lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations (including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like). Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent lipid formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent lipid containing a basic or acidic moiety by conventional chemical methods.Typically, such salts can be prepared by reacting the free acid or base forms of these lipids with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; usually, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0108] It is understood that the compounds of the present disclosure include the compounds themselves and, if applicable, their ionic derivatives.As used herein, the term "ionic derivative" refers to the ionic form of the structure being referred to.The ionic form can be a free cation, a free anion, or a zwitterion.For example, when a structure is described herein as a salt, the present disclosure is intended to include the corresponding free cation of that structure or the corresponding free anion of that structure.For example, a compound of formula (I):

[0109] [ka]

[0110] is its corresponding free cation:

[0111] [ka]

[0112] is intended to include. As used herein, "phospholipid" refers to a lipid that contains a phosphate moiety and one or more carbon chains, for example, unsaturated fatty acid chains. Phospholipids can contain one or more multiple (for example, double or triple) bonds (for example, one or more unsaturated). Certain phospholipids can facilitate fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in membranes (for example, cell membranes or intracellular membranes). The fusion of phospholipids with membranes can allow one or more components of lipid-containing compositions to pass through the membrane (for example, delivery of one or more components to cells).

[0113] As used herein, "polydispersity index," or "PDI," is a ratio that describes the uniformity of the particle size distribution of a system. A low value, e.g., less than 0.3, indicates a narrow particle size distribution.

[0114] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues typically joined by peptide bonds, which can be naturally occurring (e.g., isolated or purified) or synthetically produced. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can contain modified amino acids. The term also encompasses amino acid polymers modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, e.g., homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides can be monomers or multimolecular complexes, such as dimers, trimers, or tetramers. They can also include single-chain or multi-chain polypeptides. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids. In some embodiments, a "peptide" can be 50 amino acids or less in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0115] As used herein, "RNA" refers to a ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleic acid bases, nucleosides, nucleotides, or linkers. RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence that encodes a polypeptide of interest.

[0116] As used herein, "DNA" refers to deoxyribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, DNA may be a synthetic molecule, e.g., a synthetic DNA molecule produced in vitro. In some embodiments, the DNA molecule is a recombinant molecule. As used herein, a "recombinant DNA molecule" refers to a DNA molecule that does not occur as a natural product but is produced using molecular biology techniques.

[0117] As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose / one time / single route / single point of contact, i.e., a single administration event. As used herein, a "divided dose" is a single unit dose or a division of the total daily dose into two or more doses.

[0118] As used herein, a "total daily dose" is the amount given or prescribed in a 24-hour period. It may be administered as a single unit dose. As used herein, "size" or "average size" in the context of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) refers to the average diameter of the nanoparticle composition.

[0119] As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0120] As used herein, "targeted cells" refers to any one or more cells of interest. Cells may be found in vitro, in vivo, in situ, or in the tissues or organs of an organism. The organism may be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0121] As used herein, "target tissue" refers to any one or more tissue types to which delivery of a therapeutic and / or prophylactic agent will result in a desired biological and / or pharmacological effect. Examples of targeted target tissues include specific tissues, organs, and systems or groups thereof. In particular applications, the target tissue may be the kidney, lung, spleen, vascular endothelium in blood vessels (e.g., in the coronary arteries or femoral arteries), or tumor tissue (e.g., via intratumoral injection). "Off-target tissue" refers to any one or more tissue types in which expression of the encoded protein does not result in a desired biological and / or pharmacological effect. In particular applications, off-target tissues may include the liver and spleen.

[0122] The term "therapeutic agent" or "prophylactic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or exerts a desired biological and / or pharmacological effect. A therapeutic agent may also be referred to as an "activator" or "active agent." Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0123] As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) delivered that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0124] As used herein, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0125] As used herein, the term "treating" refers to partially or completely alleviating, reversing, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the frequency of occurrence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting tumor survival, growth, and / or spread. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or to subjects who show only early signs of the disease, disorder, and / or condition for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0126] As used herein, "zeta potential" is the electrokinetic potential of lipids, for example, in a particle composition. Nanoparticle Composition The present disclosure also features lipid nanoparticles (eg, empty LNPs or loaded LNPs) that include cationic lipids according to Formula (I) described herein.

[0127] In some embodiments, the largest dimension of the nanoparticle composition is 1 μm or less (e.g., 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or less), as measured, for example, by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. Nanoparticle compositions include, for example, lipid nanoparticles (LNPs; e.g., empty or loaded LNPs), liposomes, lipid vesicles, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and / or crosslinked to one another. The lipid bilayers can include one or more ligands, proteins, or channels.

[0128] Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) of the present disclosure comprise at least one cationic lipid according to Formula (I). In some embodiments, empty or loaded LNPs of the present disclosure include one or more of the cationic lipids in Table 1. A variety of other components can be included in the nanoparticle composition. For example, in some embodiments, empty or loaded LNPs include one or more ionizable lipids in addition to a lipid according to Formula (I).

[0129] Ionizable lipids In some embodiments, the lipid nanoparticles of the present disclosure (eg, empty LNPs or loaded LNPs) further comprise one or more ionizable lipids in addition to the cationic lipid of formula (I).

[0130] In some embodiments, the ionizable lipid is a compound of formula (IL-A):

[0131] [ka]

[0132] or its N-oxide, or a salt or isomer thereof, wherein R 1 is C 5-30 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R 2 and R 3 are independently H, C 1-14 Alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 form, together with the atoms to which they are attached, a heterocyclic or carbocyclic ring; R 4 is hydrogen, C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 12 )2(CH2) n-o Q, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted C 1-6 alkyl, and Q is selected from the group consisting of carbocycle, heterocycle, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -OC(O)O-, -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 8 , -N(R)S(O)R 8 , -O(CH2) n OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR 9 )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 9 )N(R)2, -N(OR)C(=CHR 9 )N(R)2, -C(=NR 9)N(R)2, -C(=NR 9 )R, -C(O)N(R)OR, -(CH2) n N(R)2 and -C(R)N(R)2C(O)OR, NR A S(O)2R SX , and

[0133] [ka]

[0134] wherein A is a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O, and S; and a is 1, 2, 3, or 4;

[0135] [ka]

[0136] indicates the point of attachment; each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; R 8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 are H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; R 12 are H, OH, and C 1-3 Alkyl, and C 2-3 alkenyl; Each R is independently C 1-6 Alkyl, C 1-3 Alkyl-aryl, C 2-3 selected from the group consisting of alkenyl, and H; R A is H and C 1-3 alkyl; R SX is C3-8 Carbocycle, 3- to 14-membered heterocycle containing one or more heteroatoms selected from N, O, and S, C 1-6 Alkyl, C 2-6 Alkenyl, (C 1-3 Alkoxy)C 1-3 Alkyl, (CH2) p1 O(CH2) p2 R SX1 , and (CH2) p1 R SX1 and the carbocyclic and heterocyclic rings are selected from oxo, C 1-6 Alkyl, and (C 1-3 Alkoxy)C 1-3 optionally substituted with one or more groups selected from alkyl; R SX1 is C(O)NR 14 R 14 ', C 3-8 and a 3- to 14-membered carbocycle containing one or more heteroatoms selected from N, O, and S, wherein the carbocycle and heterocycle are each selected from oxo, halo, C 1-3 Alkyl, (C 1-3 Alkoxy)C 1-3 Alkyl, C 1-6 Alkylamino, di-(C 1-6 optionally substituted with one or more groups selected from alkyl)amino, and NH; Each R 13 OH, oxo, halo, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 1-6 Alkylamino, di-(C 1-6 selected from the group consisting of (alkyl)amino, NH, C(O)NH, CN, and NO; R 14 and R 14’ are each independently H and C 1-6 selected from the group consisting of alkyl; p1 is selected from 1, 2, 3, 4, and 5; p2 is selected from 1, 2, 3, 4, and 5; Each R 5 are independently OH, C 1-3Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R 7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)-M"-C(O)O-, -C(O)N(R M )-, -N(R M )C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR M )O—, —S(O)2—, —SS—, an aryl group, and a heteroaryl group; M′ is a bond, C 1-13 Alkyl or C 2-13 is alkenyl; Each R M are independently H, C 1-6 Alkyl and C 2-6 alkenyl; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R*YR", -YR", (CH2) q’ OR*, and H; each q' is independently selected from 1, 2, and 3; Each R" is independently C 3-15 Alkyl and C 3-15 alkenyl; Each R* is independently C 1-12 Alkyl and C 2-12 alkenyl; Each Y is independently C 3-6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13 is.

[0137] In some embodiments, the ionizable lipid is a compound of formula (IL-B):

[0138] [ka]

[0139] or its N-oxide, or its salt or isomer (In the formula, R' a is R' 分岐状 and; R' 分岐状 teeth,

[0140] [ka]

[0141] and;

[0142] [ka]

[0143] indicates the point of attachment; R aα , R aβ , R aγ , and R aδ are independently H, C 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and

[0144] [ka]

[0145] is selected from the group consisting of

[0146] [ka]

[0147] indicates the point of attachment; R 10 is N(R)2; each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 independently, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13. is.

[0148] In some embodiments, the ionizable lipid is a compound of formula (IL-C):

[0149] [ka]

[0150] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; M1 is M'; R4 is -(CH2) n Q, where Q is OH and n is selected from 1, 2, 3, 4, or 5; M and M' are independently selected from -C(O)O- and -OC(O)-; R2 and R3 are both C 1-14 Alkyl, or C 2-14 is alkenyl; R' is C1-C 12 (linear alkyl) is.

[0151] In some embodiments, the ionizable lipid is a compound of formula (IL-D):

[0152] [ka]

[0153] or its N-oxide, or its salt or isomer (In the formula, R' a is R' 分岐状 or R' 環状 and; R' 分岐状 teeth,

[0154] [ka]

[0155] and R' b teeth,

[0156] [ka]

[0157] and;

[0158] [ka]

[0159] indicates the point of attachment; R aγ is C 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, and n is selected from the group consisting of 1, 2, 3, 4, and 5; R' is C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9 is.

[0160] In some embodiments, the ionizable lipid is a compound of formula (IL-I):

[0161] [ka]

[0162] or an N-oxide thereof, or a salt or isomer thereof, wherein R 1 is C 5-30 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R 2 and R 3 are independently H, C 1-14 Alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3form, together with the atoms to which they are attached, a heterocyclic or carbocyclic ring; R 4 is hydrogen, C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o Q, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, and Q is selected from the group consisting of carbocycle, heterocycle, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R 8 , -N(R)S(O)R 8 , -O(CH2) n OR, -N(R)C(=NR 9 )N(R)2, -N(R)C(=CHR 9 )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 9 )N(R)2, -N(OR)C(=CHR 9 )N(R)2, -C(=NR 9 )N(R)2, -C(=NR 9 )R, —C(O)N(R)OR, and —C(R)N(R)C(O)OR, where each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; Each R 5 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; Each R 6 are independently OH, C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; M and M′ are independently selected from —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)—, —SS—, an aryl group, and a heteroaryl group; M″ is a bond, C 1-13 Alkyl or C 2-13 is alkenyl; R 7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R 8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 are H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; R 10 are H, OH, and C 1-3 Alkyl, and C 2-3 alkenyl; Each R is independently C 1-3 Alkyl, C 2-3 Alkenyl, (CH2) q OR*, and H; each q is independently selected from 1, 2, and 3; Each R' is independently C 1-18 Alkyl, C 2-18 selected from the group consisting of alkenyl, -R*YR", -YR", and H; Each R" is independently C 3-15 Alkyl and C 3-15 alkenyl; Each R* is independently C 1-12 Alkyl and C 2-12 alkenyl; Each Y is independently C 3-6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. is.

[0163] In some embodiments, the ionizable lipid is a compound of formula (IL-IA):

[0164] [ka]

[0165] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M 1 is a bond or M'; R4 is an unsubstituted C 1-3 Alkyl, or -(CH2) n Q, wherein Q is OH, -NHC(S)N(R), -NHC(O)N(R), -N(R)C(O)R, -N(R)S(O)R, -N(R)R, -NHC(=NR)N(R), -NHC(=CHR)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR)N(R), -N(OR)C(=CHR)N(R), or heteroaryl, and each n is selected from 1, 2, 3, 4, or 5; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R2 and R3 are both C 1-14 Alkyl or C 2-14 is alkenyl; R8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl, and H; R' is C 1-18 Alkyl or C 2-18 alkenyl) is.

[0166] In some embodiments, the ionizable lipid is a compound of formula (IL-IB):

[0167] [ka]

[0168] or its N-oxide, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; R ’ is C 1-14 Alkyl and C 2-14 alkenyl; R 2 and R 3 independently, C 1-14 Alkyl, and C 2-14 alkenyl; M and M' are independently selected from -C(O)O- and -OC(O)-; R N is H or C 1-3 is alkyl; X a and X b are each independently O or S; R 10is H, halo, -OH, R, -N(R)2, -CN, -N3, -C(O)OH, -C(O)OR, -OC(O)R, -OR, -SR, -S(O)R, -S(O)OR, -S(O)2OR, -NO2, -S(O)2N(R)2, -N(R)S(O)2R, -NH(CH2) t1 N(R)2, -NH(CH2) p1 O(CH2) q1 N(R)2, -NH(CH2) s1 OR, -N((CH2) s OR), -N(R)-carbocycle, -N(R)-heterocycle, -N(R)-aryl, -N(R)-heteroaryl, -N(R)(CH) t1 -carbocycle, -N(R)(CH2) t1 -heterocycle, -N(R)(CH2) t1 -aryl, -N(R)(CH2) t1 - selected from the group consisting of heteroaryl, carbocycle, heterocycle, aryl and heteroaryl; Each R is independently C 1-12 Alkyl, C 2-12 alkenyl, and H; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; r is 0 or 1; t 1 is selected from 1, 2, 3, 4, and 5; p 1 is selected from 1, 2, 3, 4, and 5; q 1 is selected from 1, 2, 3, 4, and 5; s 1 is selected from 1, 2, 3, and 4) is.

[0169] In some embodiments, the ionizable lipid is a compound of formula (IL-IC):

[0170] [ka]

[0171] or its N-oxide, or its salt or isomer (In the formula, R' a is R' 分岐状 or R' 環状 and; R' 分岐状 teeth,

[0172] [ka]

[0173] and R' 環状 teeth,

[0174] [ka]

[0175] and; R' b teeth,

[0176] [ka]

[0177] and;

[0178] [ka]

[0179] indicates the point of attachment; R aγ , R aγ , and R aγ are respectively, C 1-12 Alkyl or C 2-12 is alkenyl; R bγ is H, C 1-12 Alkyl or C 2-12 is alkenyl; R 2 and R 3are each independently 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH; or

[0180] [ka]

[0181] and

[0182] [ka]

[0183] indicates the point of attachment; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl; R 10 is N(R)2; each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H; n and n2 are each selected from the group consisting of 1, 2, 3, 4, and 5; Y a is C 3-6 It is a carbocyclic ring; R*” a is C 1-15 Alkyl and C 2-15 alkenyl; l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; s is 2 or 3) is.

[0184] In some embodiments, the ionizable lipid is a compound selected from Table IL-1.

[0185] [Table 2-1]

[0186] [Table 2-2]

[0187] [Table 2-3]

[0188] [Table 2-4]

[0189] [Table 2-5]

[0190] [Table 2-6]

[0191] [Table 2-7]

[0192] [Table 2-8]

[0193] [Table 2-9]

[0194] [Table 2-10]

[0195] In some embodiments, the ionizable lipid is a compound selected from Table IL-2.

[0196] [Table 3-1]

[0197] [Table 3-2]

[0198] [Table 3-3]

[0199] In some embodiments, the ionizable lipid is a compound of formula (IL-IIA):

[0200] [ka]

[0201] or an N-oxide thereof, or a salt or isomer thereof, wherein m is selected from 5, 6, 7, 8, and 9; R 2 and R 3 are independently H, C 1-14 Alkyl, and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from 1, 2, 3, 4, and 5, and

[0202] [ka]

[0203] wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R 10 is -N(R)2, and each R is independently C 1-6 Alkyl, C 2-3alkenyl, and H; M is -OC(O)O-, -C(O)O-, -OM"-O-, and -N(R M )C(O)—, and M″ is selected from -(CH2) z C(O)—, and z is 1, 2, 3, or 4; M' is -OC(O)O-, -C(O)O-, -OM''-O-, -N(R M )C(O)O-, and -ON=C(R M )-selected from M” is -(CH2) z C(O)-, C 1-13 alkyl, -B(R**)-, or -Si(R**)2-; z is 1, 2, 3, or 4; Each R M are independently H and C 1-6 alkyl; Each R** is independently H and C 1-12 alkyl; R' a is C 1-18 Alkyl, C 2-18 alkenyl, or -R*YR*" Each R*" is independently C 1-15 is alkyl; Each R* is independently C 1-12 is alkyl; Each Y is independently C 3-6 It is a carbocyclic ring; R" is a C3-C optionally substituted with OH 13 alkyl) is.

[0204] In some embodiments, the ionizable lipid is a compound of formula (IL-IIAX):

[0205] [ka]

[0206] or an N-oxide thereof, or a salt or isomer thereof, R 1 is -R"M'R', Each R' is independently C 1-18 is alkyl; M' is -C(O)O- and -ON=C(R M )-, and each R M are independently H and C 1-6 alkyl; Each R" is independently C 3-15 is alkyl; R 2 and R 3 are independently H, C 1-14 Alkyl, and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from 1, 2, 3, 4, and 5, and

[0207] [ka]

[0208] wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R 10 is -N(R)2, and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H; Each R 5 is H; Each R 6 is H; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. is.

[0209] In some embodiments, the ionizable lipid is a compound selected from Table IL-3.

[0210] [Table 4-1]

[0211] [Table 4-2]

[0212] [Table 4-3]

[0213] In some embodiments, the ionizable lipid is a compound of formula (IL-IIB):

[0214] [ka]

[0215] or its N-oxide, or its salt or isomer (In the formula, R' a teeth,

[0216] [ka]

[0217] and R' b teeth,

[0218] [ka]

[0219] and;

[0220] [ka]

[0221] indicates the point of attachment; R aβ , R aγ , and R aδare independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R bβ , R bγ , and R bδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R bβ , R bγ , and R bδ At least one of the 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n NRTQ, -(CH2) n NRS(O)2TQ, -(CH2) n NRC(O)H and -(CH2) n NRC(O)TQ, where n is selected from 1, 2, 3, 4, and 5; T is a bond or C 1-3 Alkyl linker, C 2-3 Alkenyl linker, or C 2-3 is an alkynyl linker; Q is a 3- to 14-membered heterocycle containing 1 to 5 heteroatoms selected from N, O, and S; C 3-10 carbocycle, C 1-6 Alkyl, and C 2-6 alkenyl, and each of alkyl, alkenyl, heterocycle, and carbocycle is selected from one or more R Q optionally substituted with; Each R Q are independently oxo, hydroxyl, cyano, amino, C 1-6 Alkylamino, Di-C 1-6 Alkylamino, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6Alkenyl, C 1-6 Alkanoyl, -C(O)C 1-6 Alkyl, and -NRC(O)C 1-6 selected from the group consisting of alkyl; Each R is independently H, C 1-6 Alkyl, and C 2-6 alkenyl; Each R' is independently C 1-12 Alkyl and C 2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9 is.

[0222] In some embodiments, the ionizable lipid is a compound selected from Table IL-4.

[0223] [Table 5-1]

[0224] [Table 5-2]

[0225] [Table 5-3]

[0226] In some embodiments, the ionizable lipid is a compound selected from Table IL-5.

[0227] [Table 6-1]

[0228] [Table 6-2]

[0229] [Table 6-3]

[0230] In some embodiments, the ionizable lipid is a compound of formula (IL-IIC):

[0231] [ka]

[0232] or an N-oxide thereof, or a salt or isomer thereof, R' 分岐状 teeth,

[0233] [ka]

[0234] and;

[0235] [ka]

[0236] indicates the point of attachment; R aα and R aβ are each independently H and C 1-2 alkyl; R aα and R aβ at least one of is C1 or C2 alkyl; R' is C 1-18 Alkyl and C 2-18 alkenyl; R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl; R 4is -(CH2) n Q, n is independently selected from 1, 2, 3, 4, and 5, and Q is NRS(O)R SX and

[0237] [ka]

[0238] wherein A is a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O, and S; and a is 1, 2, 3, or 4;

[0239] [ka]

[0240] indicates the point of attachment; R is H and C 1-3 alkyl; R SX is C 3-8 Carbocycle, 3- to 14-membered heterocycle containing one or more heteroatoms selected from N, O, and S, C 1-6 Alkyl, C 2-6 Alkenyl, (C 1-3 Alkoxy)C 1-3 Alkyl, (CH2) p1 O(CH2) p2 R SX1 , and (CH2) p1 R SX1 and the carbocyclic and heterocyclic rings are selected from oxo, C 1-6 Alkyl, and (C 1-3 Alkoxy)C 1-3 optionally substituted with one or more groups selected from alkyl; R SX1 is C(O)NR 14 R 14 ', C 3-8 and a 3- to 14-membered carbocycle containing one or more heteroatoms selected from N, O, and S, wherein the carbocycle and heterocycle are each selected from oxo, halo, C 1-3 Alkyl, (C 1-3Alkoxy)C 1-3 Alkyl, C 1-6 Alkylamino, di-(C 1-6 optionally substituted with one or more groups selected from alkyl)amino, and NH; Each R 13 OH, oxo, halo, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 1-6 Alkylamino, di-(C 1-6 selected from the group consisting of (alkyl)amino, NH, C(O)NH, CN, and NO; R 14 and R 14’ are each independently H and C 1-6 selected from the group consisting of alkyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; p1 is selected from 1, 2, 3, 4, and 5; p2 is selected from 1, 2, 3, 4, and 5 is.

[0241] In some embodiments, the ionizable lipid is a compound selected from Table IL-6.

[0242] [Table 7-1]

[0243] [Table 7-2]

[0244] [Table 7-3]

[0245] [Table 7-4]

[0246] In some embodiments, the ionizable lipid is a compound of formula (IL-III):

[0247] [ka]

[0248] or a salt or isomer thereof, wherein W is

[0249] [ka]

[0250] and; Ring A is

[0251] [ka]

[0252] and; t is 1 or 2; A1 and A2 are each independently selected from CH or N; Z is CH2 or absent, and when Z is CH2, the dashed lines (1) and (2) each represent a single bond; when Z is absent, both dashed lines (1) and (2) are absent; R1, R2, R3, R4, and R5 are independently C 5-20 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; R X1 and R X2 are each independently H or C alkyl; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group; M* is C1-C6 alkyl; W 1 and W 2 are each independently selected from the group consisting of -O- and -N(R6)-; Each R6 is independently H and C 1-5 selected from the group consisting of alkyl; X 1 , X 2 , and X 3 are independently a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, or -(CH2) n -C(O)-, -C(O)-(CH2) n -, -(CH2) n -C(O)O-, -OC(O)-(CH2) n -, -(CH2) n -OC(O)-, -C(O)O-(CH2) n selected from the group consisting of -, -CH(OH)-, -C(S)-, and -CH(SH)-; Each Y is independently C 3-6 It is a carbocyclic ring; Each R* is independently C 1-12 Alkyl and C 2-12 alkenyl; Each R is independently C 1-3 Alkyl and C 3-6 selected from the group consisting of carbocycles; Each R' is independently C 1-12 Alkyl, C 2-12 selected from the group consisting of alkenyl, and H; Each R" is independently C 3-12 Alkyl, C 3-12 selected from the group consisting of alkenyl and -R*MR'; n is an integer from 1 to 6. is.

[0253] In some embodiments, the ionizable lipid is a compound of formula (IL-IIIA):

[0254] [ka]

[0255] or a salt or isomer thereof, wherein R1, R2, R3, R4, and R5 are independently C 5-20 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group; X 1 , X 2 , and X 3 is independently selected from the group consisting of a bond, —CH—, —(CH)—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH—, —CH—C(O)—, —C(O)O—CH—, —OC(O)—CH—, —CH—C(O)O—, —CH—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—; Each Y is independently C 3-6 It is a carbocyclic ring; Each R* is independently C 1-12 Alkyl and C 2-12 alkenyl; Each R is independently C 1-3 Alkyl and C 3-6 selected from the group consisting of carbocycles; Each R' is independently C 1-12 Alkyl, C 2-12selected from the group consisting of alkenyl, and H; Each R" is independently C 3-12 Alkyl and C 3-12 alkenyl) is.

[0256] In some embodiments, the ionizable lipid is a compound selected from Table IL-7.

[0257] [Table 8-1]

[0258] [Table 8-2]

[0259] [Table 8-3]

[0260] [Table 8-4]

[0261] [Table 8-5]

[0262] [Table 8-6]

[0263] [Table 8-7]

[0264] [Table 8-8]

[0265] [Table 8-9]

[0266] [Table 8-10]

[0267] In some embodiments, the ionizable lipid is

[0268] [ka]

[0269] The compound is selected from In some embodiments, the ionizable lipid is a lipid disclosed in published International Patent Application Nos. WO / 2017 / 049245, WO / 2017 / 112865, WO / 2018 / 170306, WO / 2018 / 232120, WO / 2021 / 055835, WO / 2021 / 055833, and WO / 2021 / 055849, each of which is incorporated by reference in its entirety.

[0270] structural lipids Lipid nanoparticles (e.g., empty or loaded LNPs) can include one or more structured lipids. The structured lipids can be selected from the group consisting of, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. In some embodiments, the structured lipid is

[0271] [ka]

[0272] is. phospholipids Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can include one or more phospholipids, e.g., one or more (poly)unsaturated lipids. The phospholipids can be organized into one or more lipid bilayers. Typically, the phospholipids can include a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipids can be represented by the formula (PhL-IV):

[0273] [ka]

[0274] (In the formula, R p represents the phospholipid moiety, and R A and R Brepresents a fatty acid moiety with or without unsaturation, which may be the same or different. The phospholipid moiety may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-naturally occurring species, including naturally occurring species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also contemplated. For example, phospholipids can be functionalized with or crosslinked to one or more alkynes (e.g., alkenyl groups in which one or more double bonds have been replaced with triple bonds). Under appropriate reaction conditions, the alkyne groups can undergo copper-catalyzed cycloaddition upon exposure to azides. Such reactions can be useful in functionalizing the lipid bilayer of lipid nanoparticles (e.g., empty or loaded LNPs) to facilitate membrane permeation or cellular recognition, or in conjugating lipid nanoparticles (e.g., empty or loaded LNPs) to useful components, such as targeting or imaging moieties (e.g., dyes).

[0275] Phospholipids useful in the compositions and methods include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl- ... The lipid nanoparticles may be selected from the non-limiting group consisting of DSPC, dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof. In some embodiments, the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) include DSPC. In certain embodiments, the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) include DOPE. In some embodiments, the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) include both DSPC and DOPE.

[0276] PEG lipids Lipid nanoparticles (e.g., empty or loaded LNPs) can include one or more PEGs or PEG-modified lipids. Such species may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipids can be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DEG), PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0277] In certain embodiments, the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, and a PEG-modified dialkylglycerol.

[0278] In some embodiments, the PEG lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetol, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA). For example, in some embodiments, the PEG lipid is PEG-DMG.

[0279] In certain embodiments, the PEG lipid is a compound of formula (PL-I):

[0280] [ka]

[0281] or a salt thereof (wherein R 3PL1 -OR OPL1 and; R OPL1 is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r PL1 is an integer between 1 and 100 (inclusive); L 1 is an arbitrarily substituted C 1-10 alkylene and optionally substituted C 1-10 At least one methylene of the alkylene is independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), -C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ) has been replaced by; D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula:

[0282] [ka]

[0283] is of; L 2 Each occurrence of is independently a bond or an optionally substituted C 1-6 alkylene and optionally substituted C 1-6One methylene unit of alkylene is O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), C(O)O, OC(O), OC(O)O, -OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ) are optionally replaced by; R 2SL Each occurrence of may independently represent an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally R 2SL one or more methylene units of are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, N(R NPL1 ), O, S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), -NR NPL1 C(O)N(R NPL1 ), C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, C(O)S, -SC(O), C(=NR NPL1 ), C(=NR NPL1 )N(R NPL1 ), NR NPL1 C(=NR NPL1 ), NR NPL1 C(=NR NPL1 )N(R NPL1 ), C(S), C(S)N(R NPL1 ), NR NPL1 C(S), NR NPL1 C(S)N(R NPL1 ), S(O), OS(O), S(O)O, OS(O)O, -OS(O)2, S(O)2O, OS(O)2O, N(R NPL1 )S(O), S(O)N(R NPL1 ), N(R NPL1 )S(O)N(R NPL1 ), -OS(O)N(R NPL1 ), N(RNPL1 )S(O)O, S(O)2, N(R NPL1 )S(O)2, S(O)2N(R NPL1 ), -N(R NPL1 )S(O)2N(R NPL1 ), OS(O)2N(R NPL1 ), or N(R NPL1 )S(O)2O; R NPL1 each occurrence is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p SL is either 1 or 2) is.

[0284] In certain embodiments, the PEG lipid is a compound of formula (PL-I-OH):

[0285] [ka]

[0286] or a salt thereof. In certain embodiments, the PEG lipid is a compound of formula (PL-II-OH):

[0287] [ka]

[0288] or a salt or isomer thereof, wherein R 3PEG -OR O and; R O is hydrogen, C 1-6 is an alkyl or oxygen protecting group; r PEG is an integer between 1 and 100; R 5PEG is C 10-40 Alkyl, C 10-40Alkenyl, or C 10-40 alkynyl; optionally R 5PEG One or more methylene groups in 3-10 Carbocyclylene, 4-10 membered heterocyclylene, C 6-10 Arylene, 4- to 10-membered heteroarylene, -N(R NPEG )-, -O-, -S-, -C(O)-, -C(O)N(R NPEG )-, -NR NPEG C(O)-, -NR NPEG C(O)N(R NPEG )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R NPEG )-, -NR NPEG C(O)O-, -C(O)S-, -SC(O)-, -C(=NR NPEG )-, -C(=NR NPEG )N(R NPEG )-, -NR NPEG C(=NR NPEG )-, -NR NPEG C(=NR NPEG )N(R NPEG )-, -C(S)-, -C(S)N(R NPEG )-, -NR NPEG C(S)-, -NR NPEG C(S)N(R NPEG )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R NPEG )S(O)-, -S(O)N(R NPEG )-, -N(R NPEG )S(O)N(R NPEG )-, -OS(O)N(R NPEG )-, -N(R NPEG )S(O)O-, -S(O)2-, -N(R NPEG )S(O)2-, -S(O)2N(R NPEG )-, -N(R NPEG )S(O)2N(R NPEG )-, -OS(O)2N(R NPEG )-, or -N(R NPEG )S(O)2O- is replaced by; R NPEG Each instance of is independently hydrogen, C1-6 alkyl, or nitrogen protecting group) is.

[0289] In certain embodiments, in the PEG lipid of formula (PL-II-OH), r is an integer between 40 and 50. For example, r is selected from the group consisting of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. For example, r is 45.

[0290] In certain embodiments, in the PEG lipid of formula (PL-II-OH), R 5 is C 17 It is alkyl. In certain embodiments, the PEG lipid is a compound of formula (PL-II):

[0291] [ka]

[0292] (In the formula, r PEG is an integer from 1 to 100). In certain embodiments, the PEG lipid is a compound of formula (PEG-1):

[0293] [ka]

[0294] is. In certain embodiments, the PEG lipid is a compound of formula (PL-III):

[0295] [ka]

[0296] or a salt or isomer thereof (wherein, s PL1 is an integer from 1 to 100). In certain embodiments, the PEG lipid is a compound of the following formula:

[0297] [ka]

[0298] is. In certain embodiments, the nanoparticle formulations may comprise a compound of formula (PL-I), (PL-I-OH), (PL-II), (PL-II-OH), (PL-III), PEG 2k Incorporation of lipids of formula (PL-II-OH), (PL-IIa-OH), (PL-II), or PEG-1 can improve the pharmacokinetics and / or biodistribution of lipid nanoparticle formulations. For example, incorporation of lipids of formula (PL-II-OH), (PL-IIa-OH), (PL-II), or PEG-1 in nanoparticle formulations can reduce the accelerated blood clearance (ABC) effect.

[0299] Adjuvants In some embodiments, lipid nanoparticles comprising one or more lipids described herein (e.g., empty LNPs or loaded LNPs) may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxynucleotides (e.g., class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0300] therapeutic agent Lipid nanoparticles (e.g., empty or loaded LNPs) can include one or more therapeutic and / or prophylactic agents. The disclosure features methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs, methods for producing a polypeptide of interest in mammalian cells, and methods for treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal and / or contacting mammalian cells with lipid nanoparticles (e.g., empty or loaded LNPs) that include a therapeutic and / or prophylactic agent.

[0301] Therapeutic and / or prophylactic agents include biologically active substances and are alternatively referred to as "active agents." Therapeutic and / or prophylactic agents can be substances that, when delivered to a cell or organ, effect a desired change in the cell, organ, or other body tissue or system. Such species can be useful in the treatment of one or more diseases, disorders, or conditions. In some embodiments, therapeutic and / or prophylactic agents are small molecule drugs useful in the treatment of a particular disease, disorder, or condition.

[0302] In some embodiments, the therapeutic and / or prophylactic agent is a vaccine, a compound that elicits an immune response (e.g., a polynucleotide or nucleic acid molecule encoding a protein, polypeptide, or peptide, or a protein, polypeptide, or protein), and / or another therapeutic and / or prophylactic agent. Vaccines include compounds and preparations capable of providing immunity against one or more pathologies associated with infectious diseases and may include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines also include compounds and preparations that induce an immune response against cancer cells and may include mRNA encoding tumor cell-derived antigens, epitopes, and / or neoepitopes. In some embodiments, vaccines and / or compounds capable of eliciting an immune response are administered intramuscularly via compositions of the present disclosure.

[0303] In other embodiments, the therapeutic and / or prophylactic agent is a protein, e.g., a protein necessary to augment or replace a naturally occurring protein of interest. Such proteins or polypeptides can be naturally occurring or can be modified using methods known in the art, e.g., to increase half-life. Exemplary proteins are intracellular, transmembrane, or secreted.

[0304] Polynucleotides and Nucleic Acids In some embodiments, a therapeutic agent is an agent that enhances (i.e., increases, stimulates, upregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used to enhance protein expression include RNA, mRNA, dsRNA, CRISPR / Cas9 technology, ssDNA, and DNA (e.g., expression vectors). Agents that upregulate protein expression can upregulate the expression of naturally occurring or non-naturally occurring proteins (e.g., chimeric proteins modified to improve half-life or those containing desired amino acid changes). Exemplary proteins include intracellular, transmembrane, or secreted proteins, peptides, or polypeptides.

[0305] In some embodiments, the therapeutic agent is a DNA therapeutic agent. The DNA molecule can be double-stranded DNA, single-stranded DNA (ssDNA), or partially double-stranded DNA, i.e., a molecule having portions that are double-stranded and portions that are single-stranded. In some cases, the DNA molecule is triple-stranded or partially triple-stranded, i.e., having portions that are triple-stranded and portions that are double-stranded. The DNA molecule can be a circular DNA molecule or a linear DNA molecule.

[0306] A DNA therapeutic agent can be a DNA molecule capable of transferring a gene into a cell, e.g., encoding and expressing a transcription product. In other embodiments, the DNA molecule is a synthetic molecule, e.g., a synthetic DNA molecule generated in vitro. In some embodiments, the DNA molecule is a recombinant molecule. Non-limiting exemplary DNA therapeutic agents include plasmid expression vectors and viral expression vectors.

[0307] The DNA therapeutic agents described herein, for example, DNA vectors, may include a variety of different features. The DNA therapeutic agents described herein, for example, DNA vectors, may include non-coding DNA sequences. For example, the DNA sequence may include at least one regulatory element for a gene, such as a promoter, enhancer, termination element, polyadenylation signal element, splicing signal element, etc. In some embodiments, the non-coding DNA sequence is an intron. In some embodiments, the non-coding DNA sequence is a transposon. In some embodiments, the DNA sequence described herein may have a non-coding DNA sequence operably linked to a transcriptionally active gene. In other embodiments, the DNA sequence described herein may have a non-coding DNA sequence that is not linked to a gene. That is, the non-coding DNA does not control the gene on the DNA sequence.

[0308] In some embodiments, in the loaded LNPs of the present disclosure, the one or more therapeutic and / or prophylactic agents are nucleic acids, hi some embodiments, the one or more therapeutic and / or prophylactic agents are selected from the group consisting of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

[0309] For example, in some embodiments, when the therapeutic and / or prophylactic agent is DNA, the DNA is selected from the group consisting of double-stranded DNA, single-stranded DNA (ssDNA), partially double-stranded DNA, triple-stranded DNA, and partially triple-stranded DNA. In some embodiments, the DNA is selected from the group consisting of circular DNA, linear DNA, and mixtures thereof.

[0310] In some embodiments, in the loaded LNPs of the present disclosure, the one or more therapeutic and / or prophylactic agents are selected from the group consisting of a plasmid expression vector, a viral expression vector, and mixtures thereof.

[0311] For example, in some embodiments, when the therapeutic and / or prophylactic agent is RNA, the RNA is selected from the group consisting of single-stranded RNA, double-stranded RNA (dsRNA), partially double-stranded RNA, and mixtures thereof, hi some embodiments, the RNA is selected from the group consisting of circular RNA, linear RNA, and mixtures thereof.

[0312] For example, in some embodiments, when the therapeutic and / or prophylactic agent is RNA, the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecule, microRNA (miRNA), antagomir, antisense RNA, ribozyme, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), locked nucleic acid (LNA), and CRISPR / Cas9 technology, and mixtures thereof.

[0313] For example, in some embodiments, when the therapeutic and / or prophylactic agent is RNA, the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0314] In some embodiments, one or more therapeutic and / or prophylactic agents are mRNA. In some embodiments, one or more therapeutic and / or prophylactic agents are modified mRNA (mmRNA).

[0315] In some embodiments, the one or more therapeutic and / or prophylactic agents are mRNAs incorporating microRNA-binding sites (miR-binding sites). Further, in some embodiments, the mRNAs include one or more of a stem-loop, a chain-terminating nucleoside, a polyA sequence, a polyadenylation signal, and / or a 5' cap structure.

[0316] mRNA can be naturally occurring or non-naturally occurring mRNA. As described below, mRNA can contain one or more modified nucleic acid bases, nucleosides, or nucleotides, in which case it can be referred to as "modified mRNA" or "mmRNA". As described herein, "nucleoside" is defined as a compound that contains a sugar molecule (e.g., pentose or ribose) or its derivative, combined with an organic base (e.g., purine or pyrimidine) or its derivative (also referred to herein as "nucleobase"). As described herein, "nucleotide" is defined as a nucleoside that contains a phosphate group.

[0317] An mRNA can include a 5'-untranslated region (5'-UTR), a 3'-untranslated region (3'-UTR), and / or a coding region (e.g., an open reading frame). An mRNA can include any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900), or thousands (e.g., 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000) of base pairs. Any number (e.g., all, some, or none) of the nucleobases, nucleosides, or nucleotides can be substituted, modified, or otherwise analogs of non-naturally occurring standard species. In certain embodiments, all of a particular nucleobase type can be modified. In some embodiments, all uracils or uridines are modified. When all nucleic acid bases, nucleosides, or nucleotides are modified (e.g., all uracils or uridines), the mRNA can be referred to as "fully modified" for example, uracils or uridines.

[0318] In some embodiments, the mRNAs described herein may include a 5' cap structure, chain-terminating nucleotides, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem-loop, a polyA sequence, and / or a polyadenylation signal.

[0319] A 5' cap structure or cap species is a compound containing two nucleoside moieties connected by a linker and can be selected from naturally occurring caps, non-naturally occurring caps or cap analogs, or anti-reverse cap analogs (ARCAs). A cap species can contain one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap can contain a guanine nucleotide and a guanine (G) nucleotide methylated at the 7th position connected by a triphosphate linkage at their 5' positions, such as m7G(5')ppp(5')G (commonly represented as m7GpppG). A cap species can also be an anti-reverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, m27,O2'GppppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, and m27,O2'GppppG.

[0320] Alternatively or additionally, mRNA may include chain-terminating nucleosides. For example, chain-terminating nucleosides may include those nucleosides deoxygenated at the 2' and / or 3' positions of their sugar groups. Such species may include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, and 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymine. In some embodiments, incorporation of chain-terminating nucleotides into mRNA, for example, at the 3'-terminus, may result in stabilization of the mRNA.

[0321] Alternatively or additionally, the mRNA may include a stem loop, e.g., a histone stem loop. The stem loop may include 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, the stem loop may include 4, 5, 6, 7, or 8 nucleotide base pairs. The stem loop may be located in any region of the mRNA. For example, the stem loop may be located in an untranslated region (5' untranslated region or 3' untranslated region), a coding region, or before or after a polyA sequence or tail. In some embodiments, the stem loop may affect one or more functions of the mRNA, such as translation initiation, translation efficiency, and / or transcription termination.

[0322] Alternatively or additionally, the mRNA may include a polyA sequence and / or a polyadenylation signal. The polyA sequence may be composed entirely or mostly of adenine nucleotides or their analogs or derivatives. The polyA sequence may also include stabilizing nucleotides or analogs. For example, the polyA sequence may include deoxythymidine, e.g., inverted (or reverse-linked) deoxythymidine (dT), as the stabilizing nucleotide or analog. Details regarding the use of inverted dT and other stabilizing polyA sequence modifications can be found, for example, in WO2017 / 049275A2 (the contents of which are incorporated herein by reference). The polyA sequence may be a tail located adjacent to the 3' untranslated region of the mRNA. In some embodiments, the polyA sequence may affect the nuclear export, translation, and / or stability of the mRNA.

[0323] Alternatively or additionally, mRNA may contain a microRNA binding site. MicroRNA binding sites (or miR binding sites) can be used to control mRNA expression in various tissues or cell types. In an exemplary embodiment, a miR binding site is engineered into the 3'UTR sequence of an mRNA to control, e.g., enhance, mRNA degradation in cells or tissues that express the cognate miR. Such control is useful for controlling or regulating "off-target" expression of mRNA, i.e., expression in undesirable cells or tissues in vivo. Details regarding the use of miR binding sites can be found, for example, in WO2017 / 062513A2 (the contents of which are incorporated herein by reference).

[0324] In some embodiments, the mRNA is a bicistronic mRNA containing a first coding region and a second coding region, with an intervening sequence containing an internal ribosome entry site (IRES) sequence between the first and second coding regions that allows internal translation initiation, or an intervening sequence encoding a self-cleaving peptide, e.g., a 2A peptide. IRES sequences and 2A peptides are typically used to improve expression of multiple proteins from the same vector. A variety of IRES sequences, including, for example, the encephalomyocarditis virus IRES, are known and available in the art and can be used.

[0325] In some embodiments, mRNA of the present disclosure comprises one or more modified nucleic acid bases, nucleosides, or nucleotides (referred to as "modified mRNA" or "mmRNA"). In some embodiments, modified mRNA may have useful properties, including improved stability, intracellular retention, improved translation, and / or lack of substantial induction of an innate immune response in cells into which the mRNA is introduced, when compared to a reference unmodified mRNA. Thus, the use of modified mRNA may improve the efficiency of protein production, intracellular retention of nucleic acids, and possess reduced immunogenicity.

[0326] In some embodiments, the mRNA includes one or more (e.g., 1, 2, 3, or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified mRNA may have reduced degradation in cells into which the mRNA is introduced, compared to the corresponding unmodified mRNA.

[0327] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine. (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5 -taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy Pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α -thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl These include thymidine-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0328] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-his(2-methyl-2-cytidine), 5-amino-2-cytidine (5-amino ... Hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine cytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-di These include methyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0329] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include α-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8- ... -amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxyno norvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,These include 2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0330] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include a-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OhyW), undermodified hydroxywyobutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), Galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl -guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl These include thio-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0331] In some embodiments, the mRNA of the present disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0332] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the mRNA of the present disclosure includes a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases). In some embodiments, the modified nucleobase is N1-methylpseudouridine (m1ψ), and the mRNA of the present disclosure is fully modified with N1-methylpseudouridine (m1ψ). In some embodiments, N1-methylpseudouridine (m1ψ) represents 75-100% of the uracils in the mRNA. In some embodiments, N1-methylpseudouridine (m1ψ) represents 100% of the uracils in the mRNA.

[0333] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine. In some embodiments, the mRNA of the present disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0334] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A). In some embodiments, the mRNA of the present disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0335] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine. In some embodiments, the mRNA of the present disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0336] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, the mRNA of the disclosure includes a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases).

[0337] In some embodiments, the mRNA comprises pseudouridine (ψ). In some embodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2'-O-methyluridine. In some embodiments, the mRNA comprises 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0338] In some embodiments, the mRNA of the present disclosure is uniformly modified for a particular modification (i.e., completely modified, modified throughout the entire sequence). For example, the mRNA can be uniformly modified with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C), which means that all uridine or all cytosine nucleosides in the mRNA sequence are replaced with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C). Similarly, the mRNA of the present disclosure can be uniformly modified for any type of nucleoside residue present in the sequence by replacing it with a modified residue, such as those listed above.

[0339] In some embodiments, mRNAs of the present disclosure may be modified in the coding region (e.g., the open reading frame encoding a polypeptide). In other embodiments, mRNAs may be modified in regions other than the coding region. For example, in some embodiments, a 5'-UTR and / or a 3'-UTR are provided, either or both of which may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region.

[0340] The mmRNAs of this disclosure can include combinations of modifications to sugars, nucleobases, and / or internucleoside linkages, which can include any one or more of the modifications described herein.

[0341] When a single modification is listed, the listed nucleoside or nucleotide represents 100% of the A, U, G, or C nucleotides or nucleosides that are modified. When percentages are listed, they represent the percentage of that particular A, U, G, or C nucleobase triphosphate out of the total amount of A, U, G, or C triphosphates present. For example, the combination: 25% 5-aminoallyl-CTP + 75% CTP / 25% 5-methoxy-UTP + 75% UTP refers to a polynucleotide in which 25% of the cytosine triphosphates are 5-aminoallyl-CTP, while 75% of the cytosines are CTP; while 25% of the uracils are 5-methoxy-UTP, while 75% of the uracils are UTP. If no modified UTP is listed, then naturally occurring ATP, UTP, GTP, and / or CTP are used at 100% of the nucleotide positions found in the polynucleotide. In this example, all of the GTP and ATP nucleotides remain unmodified.

[0342] The mRNA of the present disclosure, or a region thereof, can be codon-optimized. Codon optimization methods are known in the art and can be useful for a variety of purposes, namely, to match codon frequencies in a host organism and ensure proper folding, to bias GC content to increase mRNA stability or reduce secondary structure, to minimize tandem repeat codons or runs of bases that may impair gene organization or expression, to customize transcriptional and translational regulatory regions, to insert or remove protein transport sequences, to remove / add post-translational modification sites (e.g., glycosylation sites) of the encoded protein, to add, remove, or swap protein domains, to insert or delete restriction enzyme recognition sites, to modify ribosome binding sites and mRNA degradation sites, to adjust the translation rate and correctly fold various domains of the protein, or to reduce or remove problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art; non-limiting examples include services from GeneArt (Life technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. In some embodiments, mRNA sequences are optimized using optimization algorithms, e.g., to optimize expression in mammalian cells or to improve mRNA stability.

[0343] In certain embodiments, the present disclosure includes polynucleotides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the polynucleotide sequences described herein.

[0344] The mRNA of the present disclosure can be produced by means available in the art, including, but not limited to, in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combined synthesis, small-area synthesis, and ligation methods can be used. In some embodiments, the mRNA is produced using an IVT enzymatic synthesis method. Thus, the present disclosure also includes polynucleotides, such as DNA, constructs, and vectors, that can be used to in vitro transcribe the mRNA described herein.

[0345] Unnatural modified nucleobases can be introduced into polynucleotides, such as mRNA, during or after synthesis.In some embodiments, the modification can be in internucleoside linkages, purine or pyrimidine bases, or sugars.In certain embodiments, the modification can be introduced at the end of polynucleotide chain or at other locations in polynucleotide chains; by chemical synthesis or polymerase enzyme.

[0346] Either enzymatic or chemical ligation methods can be used to conjugate polynucleotides or regions thereof with different functional moieties, e.g., targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Therapeutic Agents for Reducing Protein Expression In some embodiments, the therapeutic agent is one that reduces (i.e., decreases, inhibits, downregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used to reduce protein expression include mRNA incorporating microRNA binding site(s) (miR binding sites), microRNA (miRNA), antagomir, small (short) interfering RNA (siRNA) (including shortmers and Dicer-substrate RNA), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acid (LNA), and CRISPR / Cas9 technology.

[0347] Peptide / Polypeptide Therapeutics In some embodiments, the therapeutic agent is a peptide therapeutic agent. In some embodiments, the therapeutic agent is a polypeptide therapeutic agent.

[0348] In some embodiments, the peptide or polypeptide is naturally occurring, e.g., isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, e.g., a synthetic peptide or polypeptide produced in vitro. In some embodiments, the peptide or polypeptide is a recombinant molecule. In some embodiments, the peptide or polypeptide is a chimeric molecule. In some embodiments, the peptide or polypeptide is a fusion molecule. In some embodiments, the peptide or polypeptide therapeutic of the composition is a naturally occurring peptide or polypeptide. In some embodiments, the peptide or polypeptide therapeutic of the composition is a modified version of a naturally occurring peptide or polypeptide (e.g., containing fewer than 3, fewer than 5, fewer than 10, fewer than 15, fewer than 20, or fewer than 25 amino substitutions, deletions, or additions compared to its wild-type, naturally occurring peptide or polypeptide counterpart).

[0349] In some embodiments, in the loaded LNPs of the present disclosure, the one or more therapeutic and / or prophylactic agents are polynucleotides or polypeptides. Other ingredients Lipid nanoparticles (e.g., empty or loaded LNPs) can include one or more components in addition to those described in the preceding section. For example, lipid nanoparticles (e.g., empty or loaded LNPs) can include one or more small hydrophobic molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0350] Lipid nanoparticles (e.g., empty or loaded LNPs) can also include one or more permeability-enhancing molecules, carbohydrates, polymers, surface-modifying agents, or other moieties. Carbohydrates can include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0351] A polymer can be included in the nanoparticle composition and / or used to encapsulate or partially encapsulate the nanoparticle composition. The polymer can be biodegradable and / or biocompatible. The polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), and poly(D,L-lactide-co-PPO-co-D,L-lactide).L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl ... Dimethylcellulose, polymers of acrylic acid, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), Poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[0352] Surface modifications can include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants, e.g., dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letostein, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexin, and erdosteine), and DNase (e.g., rhDNase). Surface-altering agents can be disposed within the nanoparticle and / or on the surface of the lipid nanoparticle (e.g., empty or loaded LNP) (e.g., by coating, adsorption, covalent attachment, or other process).

[0353] Lipid nanoparticles (e.g., empty or loaded LNPs) may also contain one or more functionalized lipids. For example, lipids may be functionalized with alkyne groups that can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. In particular, lipid bilayers may be functionalized in this manner with one or more groups useful for facilitating membrane permeabilization, cell recognition, or imaging. The surface of lipid nanoparticles (e.g., empty or loaded LNPs) may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane permeabilization are well known in the art.

[0354] In addition to these components, lipid nanoparticles (e.g., empty or loaded LNPs) can include any substance useful in pharmaceutical compositions. For example, lipid nanoparticles (e.g., empty or loaded LNPs) can include one or more pharmaceutically acceptable excipients or accompanying ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, tonicity agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, and other species. Excipients, such as waxes, butters, colorants, coating agents, flavors, and fragrances, can also be included.

[0355] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof. The granulating and dispersing agent may be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0356] Surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]) , polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinylpyrrolidone), diethylene glycol monolaurate,Triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof may be included.

[0357] The binder may be starch (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabinogalactan); alginate; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylate; wax; water; alcohol; and combinations thereof, or any other suitable binder.

[0358] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, edetate dipotassium, edetate, fumaric acid, malic acid, phosphoric acid, edetate sodium, tartaric acid, and / or edetate trisodium. Examples of antibacterial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™, KATHON™, and / or EUXYL®.

[0359] Examples of buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. The lubricant may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0360] Examples of oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grapeseed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, lily of the valley, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange Gillaffe, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oils, as well as butyl stearate, glyceryl tricaprylate, glyceryl tricaprate, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0361] Generation of nanoparticle compositions In some embodiments, lipid-containing nanoparticles of the present disclosure can be prepared using, for example, ethanol droplet nanoprecipitation followed by solvent exchange into a suitable aqueous buffer using dialysis to prepare a cationic lipid according to Formula (I), an ionizable lipid according to Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid (e.g., DSPC), a PEG lipid (e.g., 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG, e.g., PEG 2kIt is prepared by combining a structural lipid (e.g., cholesterol) with a PEG-1 (also known as PEG-DMG or PEG-1) and a structural lipid (e.g., cholesterol).

[0362] Characterization of nanoparticle compositions Zeta potential measures the electrokinetic potential in a colloidal dispersion. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between similarly charged adjacent particles in the dispersion. Zeta potential can be measured with a Wyatt Technologies Mobius Zeta Potential instrument. This instrument characterizes mobility and zeta potential through the principles of "massively parallel phase analysis light scattering," or MP-PALS. Without wishing to be bound by theory, this measurement is more sensitive and induces less stress than ISO Method 13099-1:2012, which uses only one detection angle and requires higher voltages for operation. In some embodiments, the zeta potential of empty or loaded LNPs of the present disclosure is measured using an instrument that employs the principles of MP-PALS.

[0363] UV-visible spectroscopy can be used to determine the concentration of a therapeutic and / or prophylactic agent (e.g., RNA) in the loaded LNP. 100 μL of a diluted formulation in 1× PBS is added to 900 μL of a 4:1 (v / v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded, for example, at 230 nm to 330 nm, on a DU800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of the therapeutic and / or prophylactic agent in the loaded LNP can be calculated based on the extinction coefficient of the therapeutic and / or prophylactic agent used in the composition and the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm.

[0364] For loaded LNPs containing RNA, the QUANT-IT™ RIBOGREEN® RNA Assay (Invitrogen Corporation, Carlsbad, CA) can be used to assess RNA encapsulation by LNPs. Samples are diluted to a concentration of approximately 5 μg / mL in TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of the diluted sample is transferred to a polystyrene 96-well plate, and either 50 μL of TE buffer or 50 μL of 2% Triton X-100 solution is added to each well. The plate is incubated at 37°C for 15 minutes. RIBOGREEN® reagent is diluted 1:100 in TE buffer, and 100 μL of this solution is added to each well. Fluorescence intensity can be measured using a fluorescence plate reader (Wallac Victor 1420 Multilabel Counter; Perkin Elmer, Waltham, MA) at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence value of the reagent blank is subtracted from the fluorescence value of each sample, and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (no Triton X-100 added) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).

[0365] In vivo formulation studies To monitor how effectively various nanoparticle compositions deliver therapeutic and / or prophylactic agents to targeted cells, different nanoparticle compositions containing a particular therapeutic and / or prophylactic agent (e.g., modified or naturally occurring RNA, e.g., mRNA) are prepared and administered to a population of animals. Animals (e.g., mice, rats, or non-human primates) are intravenously, intramuscularly, intraarterially, or intratumorally administered a single dose containing a nanoparticle composition containing a lipid and a protein of the present disclosure, e.g., mRNA expressing OX40L or tdTomato. A control composition containing PBS can also be used.

[0366] By administering nanoparticle compositions to animals, the dose delivery profile, dose response, and toxicity of a particular formulation and dose can be measured by enzyme-linked immunosorbent assay (ELISA), bioluminescence imaging, or other methods. For nanoparticle compositions containing mRNA, the time course of protein expression can also be assessed. Samples collected from animals for evaluation can include blood, serum, and tissues (e.g., muscle tissue and internal tissues from the site of intramuscular injection); sample collection can involve sacrificing the animal.

[0367] Nanoparticle compositions (e.g., loaded LNPs) containing mRNA are useful in evaluating the efficacy and usefulness of various formulations for the delivery of therapeutic and / or prophylactic agents. Higher levels of protein expression induced by administration of a composition containing mRNA would indicate higher mRNA translation and / or mRNA delivery efficiency of the nanoparticle composition. Because non-RNA components are not believed to affect the translation machinery itself, higher levels of protein expression are likely to indicate higher efficiency of therapeutic and / or prophylactic agent delivery by a given nanoparticle composition compared to other nanoparticle compositions or their absence.

[0368] formulation Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can include a lipid component and one or more additional components, such as therapeutic and / or prophylactic agents. Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be designed for one or more specific applications or targets. The components of a lipid nanoparticle (e.g., empty LNPs or loaded LNPs) can be selected based on the specific application or target and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, a particular formulation of a nanoparticle composition can be selected for a particular application or target according to, for example, the efficacy and toxicity of a particular combination of components.

[0369] In some embodiments, the lipid component of a lipid nanoparticle composition (e.g., empty or loaded LNP) includes, for example, a cationic lipid according to Formula (I), an ionizable lipid according to Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid (e.g., an unsaturated lipid, e.g., DOPE or DSPC), a PEG-lipid, and a structural lipid. The elements of the lipid component may be provided in specific ratios.

[0370] In some embodiments, the lipid components of an empty or loaded LNP include a cationic lipid according to formula (I), an ionizable lipid according to formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a PEG-lipid, and a structural lipid. In certain embodiments, the lipid component of the nanoparticle composition includes about 20 mol% to about 40 mol% of a cationic lipid according to Formula (I), about 15 mol% to about 40 mol% of an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), about 0 mol% to about 30 mol% of a phospholipid, about 18.5 mol% to about 48.5 mol% of a structural lipid, and about 0 mol% to about 10 mol% of a PEG-lipid, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 20 mol% to about 40 mol% cationic lipid according to Formula (I), about 20 mol% to about 25 mol% ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), about 5 mol% to about 25 mol% phospholipid, about 30 mol% to about 40 mol% structural lipid, and about 0 mol% to about 10 mol% PEG lipid.

[0371] In some embodiments, empty lipid nanoparticles (empty LNPs) comprise a cationic lipid of formula (I), an ionizable lipid according to formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structural lipid, and a PEG-lipid.

[0372] In some embodiments, the loaded lipid nanoparticles (loaded LNPs) comprise a cationic lipid of formula (I), an ionizable lipid according to formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structural lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents.

[0373] In some embodiments, empty or loaded LNPs comprise cationic lipids of formula (I) in an amount of about 20 mol% to about 40 mol%. In some embodiments, empty or loaded LNPs comprise an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA) in an amount of about 15 mol% to about 40 mol%.

[0374] In some embodiments, empty or loaded LNPs comprise phospholipids in an amount of about 0 mol% to about 20 mol%. For example, in some embodiments, empty or loaded LNPs comprise DSPC in an amount of about 0 mol% to about 20 mol%.

[0375] In some embodiments, empty or loaded LNPs comprise structural lipids in an amount of about 30 mol% to about 50 mol%. For example, in some embodiments, empty or loaded LNPs comprise cholesterol in an amount of about 30 mol% to about 50 mol%.

[0376] In some embodiments, empty or loaded LNPs comprise PEG lipids in an amount of about 0 mol% to about 5 mol%. For example, in some embodiments, empty or loaded LNPs comprise PEG-1 or PEG lipids. 2k-Contains DMG in an amount of about 0 mol % to about 5 mol %.

[0377] In some embodiments, empty or loaded LNPs comprise about 20 mol% to about 40 mol% cationic lipid of formula (I), about 15 mol% to about 40 mol% ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), about 0 mol% to about 20 mol% phospholipid, about 30 mol% to about 50 mol% structural lipid, and about 0 mol% to about 5 mol% PEG-lipid.

[0378] In some embodiments, empty or loaded LNPs comprise about 20 mol% to about 40 mol% cationic lipid of formula (I), about 15 mol% to about 40 mol% ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG. 2k In some embodiments, empty or loaded LNPs comprise about 20 mol% to about 40 mol% of a lipid from Table 1, about 15 mol% to about 40 mol% of a lipid from Tables IL-1 to IL-7, about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG. 2k -Includes DMG.

[0379] In some embodiments, empty or loaded LNPs comprise about 20 mol% to about 40 mol% cationic lipid of formula (I), about 15 mol% to about 40 mol% ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG-1. In some embodiments, empty or loaded LNPs comprise about 20 mol% to about 40 mol% lipids from Table 1, about 15 mol% to about 40 mol% lipids from Tables IL-1 to IL-7, about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG-1.

[0380] In some embodiments, empty or loaded LNPs comprise a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structured lipid, and a PEG-lipid, where the phospholipid is DSPC and the structured lipid is cholesterol. In some embodiments, empty or loaded LNPs comprise a cationic lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, a phospholipid, a structured lipid, and a PEG-lipid, where the phospholipid is DSPC and the structured lipid is cholesterol.

[0381] In some embodiments, the empty or loaded LNPs comprise a cationic lipid of formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structural lipid, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG. 2k In some embodiments, the empty or loaded LNPs comprise a lipid from Table 1, an ionizable lipid from Tables IL-1 through IL-7, a phospholipid, a structural lipid, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG. 2k -DMG.

[0382] In some embodiments, empty or loaded LNPs comprise a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structural lipid, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG-1. In some embodiments, empty or loaded LNPs comprise a cationic lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, a phospholipid, a structural lipid, and a PEG lipid, wherein the structural lipid is cholesterol and the PEG lipid is PEG-1.

[0383] In some embodiments, the empty or loaded LNPs comprise a cationic lipid of formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG. 2kIn some embodiments, the empty or loaded LNPs comprise a lipid from Table 1, an ionizable lipid from Tables IL-1 through IL-7, a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG. 2k -DMG.

[0384] In some embodiments, empty or loaded LNPs comprise a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC and the PEG lipid is PEG-1. In some embodiments, empty or loaded LNPs comprise a lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, a phospholipid, a structural lipid, and a PEG lipid, where the phospholipid is DSPC and the PEG lipid is PEG-1.

[0385] In some embodiments, the empty or loaded LNPs comprise a lipid of formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structural lipid, and a PEG-lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG-lipid is PEG. 2k In some embodiments, the empty or loaded LNP comprises a cationic lipid from Table 1, an ionizable lipid from Tables IL-1 through IL-7, a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG. 2kIn some embodiments, the empty or loaded LNP comprises a cationic lipid from Table 1, an ionizable lipid from Tables IL-1 through IL-7, a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG. 2k -DMG.

[0386] In some embodiments, empty or loaded LNPs comprise a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG-1. In some embodiments, empty or loaded LNPs comprise a cationic lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, a phospholipid, a structural lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is PEG-1.

[0387] Lipid nanoparticles (e.g., empty or loaded LNPs) can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof in the mammalian body. The physiochemical properties of lipid nanoparticles (e.g., empty or loaded LNPs) can be modified to increase selectivity for specific bodily targets. For example, particle size can be adjusted based on the aperture size of different organs. The therapeutic and / or prophylactic agents included in the nanoparticle composition can also be selected based on the desired delivery target(s). For example, therapeutic and / or prophylactic agents can be selected for a particular indication, condition, disease, or disorder and / or for delivery (e.g., localized or specific delivery) to specific cells, tissues, organs, or systems or groups thereof. In certain embodiments, the nanoparticle composition can include mRNA encoding a polypeptide of interest that can be translated intracellularly to produce the polypeptide of interest. Such compositions can be designed for specific delivery to a specific organ. In some embodiments, the composition can be designed for specific delivery to the mammalian liver. In some embodiments, the composition may be designed to be delivered specifically to the lungs of a mammal.

[0388] The amount of therapeutic and / or prophylactic agent in a nanoparticle composition can depend on the size, composition, desired target and / or use, or other characteristics of the nanoparticle composition, as well as the properties of the therapeutic and / or prophylactic agent. For example, the amount of RNA useful in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agent and other components (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the wt / wt ratio of lipid component to therapeutic and / or prophylactic agent in the nanoparticle composition can be from about 5:1 to about 60:1, e.g., 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 wt / wt ratio of lipid component to therapeutic and / or prophylactic agent can be from about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20: 1.

[0389] The amount of therapeutic and / or prophylactic agent in the nanoparticle composition can be measured using, for example, absorbance spectroscopy (eg, UV-visible spectroscopy). In some embodiments, nanoparticle compositions include one or more RNAs, and the one or more RNAs, lipids, and their amounts can be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. Lower N:P ratios are generally preferred. The one or more RNAs, lipids, and their amounts can be selected to provide an N:P ratio of about 2:1 to about 30:1, e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio can be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio can be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. In some embodiments, the N:P ratio is about 5.67:1. In some embodiments, the N:P ratio is about 4.9:1.

[0390] physical properties The characteristics of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may depend on their components. For example, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) containing cholesterol as a structural lipid may have different characteristics from lipid nanoparticles (e.g., empty LNPs or loaded LNPs) containing different structural lipids. Similarly, the characteristics of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) may depend on the absolute or relative amounts of their components. For example, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) containing a higher molar fraction of phospholipids may have different characteristics from lipid nanoparticles (e.g., empty LNPs or loaded LNPs) containing a lower molar fraction of phospholipids. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0391] Lipid nanoparticles (e.g., empty or loaded LNPs) can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be utilized to determine particle size. Zeta potential can be measured with a Wyatt Technologies Mobius Zeta Potential instrument. This instrument characterizes mobility and zeta potential using the principle of "massively parallel phase analysis light scattering," or MP-PALS. Without wishing to be bound by theory, this measurement is more sensitive and induces less stress than ISO Method 13099-1:2012, which uses only one detection angle and requires higher voltages for operation. In some embodiments, the zeta potential of the lipids in the empty LNP compositions described herein is measured using an instrument employing the principle of MP-PALS. Zeta potential can be measured with a Malvern Zetasizer (Nano ZS).

[0392] In some embodiments, the mean diameter of lipid nanoparticles (e.g., empty or loaded LNPs) of the present disclosure is tens to hundreds of nanometers, as measured by dynamic light scattering (DLS). For example, in some embodiments, the mean diameter of lipid nanoparticles of the present disclosure is about 40 nm to about 150 nm. In some embodiments, the mean diameter of lipid nanoparticles of the present disclosure is about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean diameter of the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) is 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 150 nm, about 70 nm to about 130 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 150 nm, about 80 nm to about 130 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, about 90 nm to about 150 nm, about 90 nm to about 130 nm, or about 90 nm to about 100 nm. In certain embodiments, the mean diameter of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) of the present disclosure is about 70 nm to about 130 nm or about 70 nm to about 100 nm. In some embodiments, the mean diameter of nanoparticles of the present disclosure is about 80 nm. In some embodiments, the mean diameter of nanoparticles of the present disclosure is about 100 nm. In some embodiments, the mean diameter of nanoparticles of the present disclosure is about 110 nm. In some embodiments, the mean diameter of nanoparticles of the present disclosure is about 120 nm.

[0393] In some embodiments, the polydispersity index ("PDI") of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) formulated with lipids of the present disclosure is less than 0.3. In some embodiments, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) formulated with lipids of the present disclosure have a polydispersity index of about 0 to about 0.25. In some embodiments, lipid nanoparticles (e.g., empty LNPs or loaded LNPs) formulated with lipids of the present disclosure have a polydispersity index of about 0.10 to about 0.20.

[0394] The surface hydrophobicity of nanoparticles of the present disclosure can be measured by Laurdan generalized polarization (GPL). In this method, Laurdan, a fluorescent aminonaphthalene ketone lipid, is post-inserted onto the nanoparticle surface, and the Laurdan fluorescence spectrum is collected to determine the normalized generalized polarization (N-GP). In some embodiments, nanoparticles formulated with lipids of the present disclosure have a surface hydrophobicity, expressed as N-GP, of about 0.5 to about 1.5. For example, in some embodiments, nanoparticles formulated with lipids of the present disclosure have a surface hydrophobicity, expressed as N-GP, of about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, or about 1.5. In some embodiments, nanoparticles formulated with lipids of the present disclosure have a surface hydrophobicity, expressed as N-GP, of about 1.0 or about 1.1.

[0395] The zeta potential of lipid nanoparticles (e.g., empty or loaded LNPs) can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can represent the surface charge of a colloidal dispersion, e.g., a nanoparticle composition. Lipid nanoparticles (e.g., empty or loaded LNPs) with a relatively low charge (positive or negative) are usually desired, as more highly charged species may undesirably interact with cells, tissues, and other elements in the body. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between similarly charged adjacent particles in the dispersion. In some embodiments, the zeta potential of the lipid nanoparticles (e.g., empty or loaded LNPs) is about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about - It can be 5mV to about +15mV, about -5mV to about +10mV, about -5mV to about +5mV, about -5mV to about 0mV, about 0mV to about +20mV, about 0mV to about +15mV, about 0mV to about +10mV, about 0mV to about +5mV, about +5mV to about +20mV, about +5mV to about +15mV, or about +5mV to about +10mV.

[0396] The efficiency of encapsulation of a therapeutic and / or prophylactic agent represents the amount of therapeutic and / or prophylactic agent encapsulated or otherwise associated with the lipid nanoparticles (e.g., empty LNPs or loaded LNPs) after preparation, compared to the initial amount provided. A high encapsulation efficiency is desirable (e.g., approaching 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing loaded LNPs before and after disrupting the loaded LNPs 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 lipid-formulated loaded LNPs of the present disclosure, the encapsulation efficiency of the therapeutic and / or prophylactic agent is 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 is at least 80%. In some embodiments, the encapsulation efficiency is at least 90%. In some embodiments, the encapsulation efficiency of the therapeutic and / or prophylactic agent is between 80% and 100%.

[0397] Pharmaceutical Compositions Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can be formulated, in whole or in part, as pharmaceutical compositions. A pharmaceutical composition can include one or more lipid nanoparticles (e.g., empty LNPs or loaded LNPs). In one embodiment, a pharmaceutical composition includes a population of lipid nanoparticles (e.g., empty LNPs or loaded LNPs). For example, a pharmaceutical composition can include one or more lipid nanoparticles (e.g., empty LNPs or loaded LNPs) that include one or more different therapeutic and / or prophylactic agents. A pharmaceutical composition can further include one or more pharmaceutically acceptable excipients or accompanying ingredients, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and medicaments can be found in Remington's The Science and Practice of Pharmacy, 21 stEdition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and associated components can be used in any pharmaceutical composition, except insofar as any conventional excipient or associated component may be incompatible with one or more components of the nanoparticle composition. An excipient or associated component may be incompatible with a component of a lipid nanoparticle (e.g., empty or loaded LNP) if its combination with the component could result in any undesired biological or otherwise harmful effect.

[0398] In some embodiments, one or more excipients or accessory ingredients may comprise more than 50% of the total mass or volume of a pharmaceutical composition, including a nanoparticle composition. For example, one or more excipients or accessory ingredients may comprise 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeias.

[0399] The relative amounts of one or more lipid nanoparticles (e.g., empty or loaded LNPs), one or more pharmaceutically acceptable excipients, and / or any additional components in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated and also depending on the route by which the composition is administered. By way of example, a pharmaceutical composition may contain 0.1% to 100% (wt / wt) of one or more lipid nanoparticles (e.g., empty or loaded LNPs).

[0400] In certain embodiments, lipid nanoparticles (e.g., empty or loaded LNPs) and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at temperatures below 4°C, e.g., about -150°C to about 0°C or about -80°C to about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C)). For example, a pharmaceutical composition comprising a cationic lipid of Formula (I) is a solution that is refrigerated for storage and / or transport at, e.g., about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. In certain embodiments, the present disclosure also relates to methods of increasing the stability of lipid nanoparticles (e.g., empty or loaded LNPs) and / or pharmaceutical compositions comprising a cationic lipid of any of Formula (I) by storing the lipid nanoparticles (e.g., empty or loaded LNPs) and / or pharmaceutical compositions at a temperature of 4°C or below, e.g., from about -150°C to about 0°C or from about -80°C to about -20°C, e.g., from 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 lipid nanoparticles (e.g., empty or loaded LNPs) and / or pharmaceutical compositions disclosed herein are stable, e.g., at temperatures below 4°C (e.g., about 4°C to -20°C), for approximately at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In some embodiments, the formulations are stabilized at about 4°C for at least 4 weeks.In certain embodiments, a pharmaceutical composition of the present disclosure comprises a lipid nanoparticle (e.g., empty or loaded LNP) disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, a pharmaceutical composition of the present disclosure has a pH value of about 7-8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or 7.5-8, or 7-7.8). For example, a pharmaceutical composition of the present disclosure comprises a lipid nanoparticle (e.g., empty or loaded LNP) disclosed herein, Tris, saline, and sucrose and has a pH of about 7.5-8, which is suitable for storage and / or transportation, for example, at about -20°C. For example, a pharmaceutical composition of the present disclosure comprises a lipid nanoparticle (e.g., empty or loaded LNP) disclosed herein and PBS, e.g., having a pH of about 7 to 7.8, suitable for storage and / or transport at about 4° C. or below. In the context of the present disclosure, "stability," "stabilized," and "stable" refer to the resistance of the lipid nanoparticle (e.g., empty or loaded LNP) disclosed herein and / or pharmaceutical composition to chemical or physical changes (e.g., degradation, particle size change, aggregation, changes in encapsulation, etc.) under given manufacturing, preparation, transport, storage, and / or use conditions, e.g., when subjected to stresses such as shear forces, freeze / thaw stress, etc.

[0401] In some embodiments, the pharmaceutical compositions of the present disclosure comprise empty or loaded LNPs, a cryoprotectant, a buffer, or a combination thereof. In some embodiments, the cryoprotectant comprises one or more cryoprotectants, each of which is independently selected from the group consisting of a polyol (e.g., a diol or triol, such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a non-detergent sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG200), PEG400, PEG600, PEG1000, PEG), a PEG-400 lysine 1000, a PEG-6 ... 2k-DMG, PEG3350, PEG4000, PEG8000, PEG10000, PEG20000, polyethylene glycol monomethyl ether 550 (mPEG550), mPEG600, mPEG2000, mPEG3350, mPEG4000, mPEG5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K15), pentaerythritol propoxylate, or polypropylene glycol P400), organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugar (e.g., D-(+)-sucrose, D- In some embodiments, the cryoprotectant is a cryoprotectant selected from the group consisting of sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, and D-(+)-glucose monohydrate, or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or a hydrate of any of these), or any combination thereof. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant and / or excipient is sucrose. In some embodiments, the cryoprotectant comprises sodium acetate. In some embodiments, the cryoprotectant and / or excipient is sodium acetate. In some embodiments, the cryoprotectant comprises sucrose and sodium acetate.

[0402] In some embodiments, the buffer is selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, Tris buffer, and combinations thereof. Lipid nanoparticles (e.g., empty or loaded LNPs) and / or pharmaceutical compositions comprising one or more lipid nanoparticles (e.g., empty or loaded LNPs) can be administered to any patient or subject, including those who may benefit from a therapeutic effect provided by delivery of a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof. While the description provided herein of lipid nanoparticles (e.g., empty or loaded LNPs) and pharmaceutical compositions comprising lipid nanoparticles (e.g., empty or loaded LNPs) is primarily directed to compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Modifications of compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or perform such modifications using no more than routine, if any, experimentation. Subjects to which the compositions are intended to be administered include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals, such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats. The lipid nanoparticles can also be used for in vitro and ex vivo applications.

[0403] Pharmaceutical compositions containing one or more lipid nanoparticles (e.g., empty or loaded LNPs) can be prepared by any method known or hereafter developed in the art of pharmacology. Typically, such preparative methods include bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, as desired or necessary, dividing, shaping, and / or packaging the product into desired single or multiple dose units.

[0404] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is typically equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0405] Pharmaceutical compositions can be prepared in a variety of forms suitable for various routes and methods of administration. For example, pharmaceutical compositions can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0406] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may contain additional therapeutic and / or prophylactic agents, additional agents, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and / or fragrances. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent, such as Cremophor®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof.

[0407] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any sterile fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid, can be used in the preparation of injectables.

[0408] Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0409] To prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of drug absorption then depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the characteristics of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0410] Compositions for rectal or vaginal administration are typically suppositories which may be prepared by mixing the composition with a suitable non-irritating excipient such as cocoa butter, polyethylene glycol, or a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active ingredient.

[0411] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharmaceutically acceptable excipient, such as sodium citrate or dicalcium phosphate, and / or fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g., glycerol), disintegrants (e.g., agar, calcium carbonate, potato, etc.). The formulation may be mixed with an aqueous dispersion medium such as corn or tapioca starch, alginic acid, certain silicates, and sodium carbonate), dissolution inhibitors (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite clay, silicates), and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may contain buffering agents.

[0412] Solid compositions of a similar type may be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. Solid compositions of a similar type may be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0413] Dosage forms for topical and / or transdermal administration of the composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Typically, the active ingredient is mixed under sterile conditions with pharmaceutically acceptable excipients and / or any necessary preservatives and / or buffers that may be required. The present disclosure also contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispersing the compound in a suitable medium. Alternatively or additionally, the rate can be controlled by providing a rate-controlling membrane and / or dispersing the compound in a polymer matrix and / or gel.

[0414] Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered using devices that limit the effective penetration length of the needle into the skin. Jet injection devices that deliver liquid compositions to the dermis via a liquid jet injector and / or via a needle that penetrates the stratum corneum and creates a jet that reaches the dermis are suitable. Ballistic powder / particle delivery devices that use compressed gas to accelerate a powdered vaccine through the outer layer of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes can be used in the classic Mantoux method of intradermal administration.

[0415] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid preparations, such as liniments, lotions, oil-in-water and / or water-in-oil emulsions, such as creams, ointments and / or pastes, and / or solutions and / or suspensions. Topically administrable formulations may contain, for example, about 1% to about 10% (wt / wt) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further include one or more of the additional ingredients described herein.

[0416] Pharmaceutical compositions can be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the oral cavity. Such formulations can include dry particles containing the active ingredient. Such compositions are conveniently in the form of a dry powder for administration using a device containing a dry powder reservoir into which a stream of propellant can be induced to disperse the powder, and / or using a self-propelling solvent / powder dispensing container, such as a device containing the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Dry powder compositions can include a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dose form.

[0417] Low-boiling propellants typically include liquid propellants having a boiling point below 65°F at atmospheric pressure. Typically, the propellant may comprise 50% to 99.9% (wt / wt) of the composition, and the active ingredient may comprise 0.1% to 20% (wt / wt) of the composition. The propellant may further comprise additional components, such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size on the same order as the particles containing the active ingredient).

[0418] Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as optionally sterile aqueous and / or dilute alcoholic solutions and / or suspensions containing the active ingredient, and may conveniently be administered using any atomization and / or nebulization device. Such formulations may further contain one or more additional ingredients, including, but not limited to, flavoring agents, e.g., saccharin sodium, volatile oils, buffers, surfactants, and / or preservatives, e.g., methylhydroxybenzoate. The droplets provided by this administration route may have an average diameter ranging from about 1 nm to about 200 nm.

[0419] The formulations described herein that are useful for pulmonary delivery are also useful for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations are administered in the same manner as nasal aspiration, i.e., by rapid inhalation through the nasal passages from a container of powder held close to the nose.

[0420] Formulations suitable for nasal administration may contain, for example, from about as little as 0.1% (wt / wt) to as much as 100% (wt / wt) of the active ingredient, and may include one or more of the additional ingredients described herein. Pharmaceutical compositions may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may be, for example, in the form of tablets and / or lozenges made using conventional methods and may contain, for example, 0.1 to 20% (wt / wt) of the active ingredient, with the remainder comprising an orally dissolvable and / or disintegrable composition, and, optionally, one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder and / or aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range of about 0.1 nm to about 200 nm and may further include one or more of any of the additional ingredients described herein.

[0421] The pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for ocular administration. Such a formulation may be in the form of, for example, eye drops comprising a 0.1 / 1.0% (wt / wt) solution and / or suspension of the active ingredient in an aqueous or oily liquid vehicle. Such eye drops may further comprise a buffer, salts, and / or one or more other of any of the additional ingredients described herein. Other useful ophthalmically administrable formulations include those comprising the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or eye drops are contemplated as being within the scope of the present disclosure.

[0422] mRNA therapy mRNA as a drug modality has the potential to deliver secreted proteins as well as intracellular and transmembrane proteins. mRNA as a drug modality has the potential to deliver transmembrane and intracellular proteins, i.e., targets that are inaccessible when standard biologics are delivered in protein form due to their inability to cross the cell membrane. One major challenge to realizing mRNA-based therapy is identifying an optimal delivery vehicle. Due to its large size, chemical instability, and potential immunogenicity, mRNA requires a delivery vehicle that can provide protection from endo- and exonucleases and shield the cargo from immune surveillance. Lipid nanoparticles (LNPs) have been identified as an excellent option in this regard.

[0423] The key performance criteria for a lipid nanoparticle delivery system are maximizing cellular uptake and enabling efficient release of mRNA from endosomes. In one embodiment, the LNPs containing the novel lipids disclosed herein demonstrate improvement in at least one of cellular uptake and endosomal release. At the same time, the LNPs must provide a stable drug product and be able to be safely administered at therapeutically relevant levels. LNPs are typically multi-component systems consisting of amino lipids, phospholipids, cholesterol, and PEG-lipids. Each component is required for efficient delivery of nucleic acid cargo and particle stability. The amino lipid is the key component believed to induce cellular uptake, endosomal escape, and tolerability. While cholesterol and PEG-lipids contribute to the stability of the drug product both in vivo and during storage, the phospholipid provides additional fusogenicity to the LNPs, thereby helping to induce endosomal escape and making the nucleic acid bioavailable in the cytosol of cells.

[0424] Over the past few decades, several aminolipid series, including the aminolipid MC3 (DLin-MC3-DMA), have been developed for oligonucleotide delivery. MC3-based LNPs have been shown to be effective in delivering mRNA. This class of LNPs is rapidly opsonized by apolipoprotein E (ApoE) when delivered intravenously, allowing cellular uptake by the low-density lipoprotein receptor (LDLr). However, concerns remain that the long tissue half-life of MC3 may contribute to unwanted side effects that prevent its use for chronic therapy. Furthermore, extensive literature evidence suggests that chronic administration of lipid nanoparticles can result in several toxic side effects, including complement activation-associated pseudoallergy (CARPA) and liver damage. This calls for a class of LNPs with increased delivery efficiency along with metabolic and toxicity profiles that would enable chronic administration in humans to unlock the potential of mRNA and other nucleic acid, nucleotide, or peptide-based therapies for humans.

[0425] The ability to treat a wide range of diseases requires flexibility for safe chronic dosing at various dose levels. Through systematic optimization of the amino lipid structure, the lipids disclosed herein were identified as lipids with a balanced combination of chemical stability, improved delivery efficiency due to improved endosomal escape, rapid in vivo metabolism, and a clean toxicity profile. The combination of these features provides a drug candidate that can be administered chronically without activating the immune system. Initial rodent screening led to the identification of lead lipids with good delivery efficiency and pharmacokinetics. The lead LNPs were further profiled in non-human primates for delivery efficiency after single and repeated dosing. Finally, the optimized LNPs were evaluated in a 1-month repeated-dose toxicity study in rats and non-human primates. Without wishing to be bound by theory, the novel ionizable lipids of the present disclosure have improved cellular delivery, improved protein expression, and improved biodegradability properties, which may result in a 2-fold, 5-fold, 10-fold, 15-fold, or greater than 20-fold increase in mRNA expression in cells when compared to LNPs lacking the lipids of the present invention. In another embodiment, LNPs comprising the lipids of the present invention may provide specific (e.g., preferential) delivery to a given cell type(s) when compared to other cell types, thereby resulting in a 2-fold, 5-fold, 10-fold, 15-fold, or greater than 20-fold increase in mRNA expression in a given cell or tissue when compared to LNPs lacking the lipids of the present invention. These improvements to the technology enable the safe and effective use of mRNA-based therapies in acute and chronic diseases.

[0426] How to use In some aspects, the present disclosure provides methods for delivering a therapeutic and / or prophylactic agent to a cell (e.g., a mammalian cell). The method includes contacting the cell with a loaded LNP or pharmaceutical composition of the present disclosure, thereby delivering the therapeutic and / or prophylactic agent to the cell. In some embodiments, the cell is in a subject, and the contacting includes administering the cell to the subject. In some embodiments, the method includes administering to the subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structured lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agent to the cell.

[0427] In some embodiments, the disclosure provides a method of delivering a therapeutic and / or prophylactic agent to cells in a subject, the method comprising: a cationic lipid of formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG. 2k -DMG and lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA) are administered to a subject.

[0428] In some embodiments, the present disclosure provides a method of delivering a therapeutic and / or prophylactic agent to cells in a subject, the method comprising administering to the subject lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents selected from a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG-1, and a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA).

[0429] In some aspects, the present disclosure provides methods of delivering (e.g., specifically delivering) a therapeutic and / or prophylactic agent to a mammalian organ or tissue (e.g., liver, kidney, spleen, or lung). The methods include contacting a cell with a loaded LNP or pharmaceutical composition of the present disclosure, thereby delivering the therapeutic and / or prophylactic agent to the target organ or tissue. In some embodiments, the methods include administering to a subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structured lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agent to the target organ or tissue. In some embodiments, the target organ is the lung or the target tissue is the pulmonary endothelium.

[0430] In some embodiments, the disclosure provides a method for specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, the method comprising: a cationic lipid of formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG. 2k -DMG and lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA) are administered to a subject.

[0431] In some embodiments, the present disclosure provides a method for specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, the method comprising administering to a subject lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents selected from a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG-1, and a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA).

[0432] In some aspects, the disclosure features methods for enhanced delivery of a therapeutic and / or prophylactic agent (e.g., mRNA) to a target tissue (e.g., liver, spleen, or lung). The method includes contacting a cell with a loaded LNP or pharmaceutical composition of the disclosure, thereby delivering the therapeutic and / or prophylactic agent to the target tissue (e.g., liver, kidney, spleen, or lung). In some embodiments, the target tissue is pulmonary endothelium. In some embodiments, the method comprises administering to a subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structured lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agents to a target tissue (e.g., liver, kidney, spleen, or lung). In some embodiments, the target tissue is pulmonary endothelium.

[0433] In some embodiments, the present disclosure provides a method for enhanced delivery of a therapeutic and / or prophylactic agent to a target tissue, the method comprising a cationic lipid of formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG. 2k -DMG and lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA) are administered to a subject.

[0434] In some embodiments, the present disclosure provides a method for enhanced delivery of a therapeutic and / or prophylactic agent to a target tissue, the method comprising administering to a subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). In some embodiments, the target tissue is pulmonary endothelium.

[0435] In some aspects, the present disclosure provides methods for producing a polypeptide of interest in a cell (e.g., a mammalian cell). The method includes contacting the cell with a loaded LNP or pharmaceutical composition of the present disclosure, wherein the loaded LNP or pharmaceutical composition comprises mRNA, whereby the mRNA can be translated in the cell to produce the polypeptide. In some embodiments, the cell is in a subject, and the contacting includes administering the cell to the subject. In some embodiments, the method includes administering to the subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structured lipid, a PEG-lipid, and mRNA, whereby the mRNA can be translated in the cell to produce the polypeptide.

[0436] In some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell, the method comprising: combining a cationic lipid of formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG. 2k In some embodiments, the present disclosure provides a method for producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a cationic lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, DSPC, cholesterol, and PEG. 2k The present invention provides a method for administering to a subject lipid nanoparticles comprising -DMG and mRNA.

[0437] In some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG-1, and mRNA. For example, in some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell, the method comprising administering to a subject lipid nanoparticles comprising a cationic lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, DSPC, cholesterol, and PEG-1, and mRNA. In some aspects, the present disclosure provides methods of treating a disease or disorder in a mammal (e.g., a human) in need thereof. The method includes administering to the mammal a therapeutically effective amount of a loaded LNP or pharmaceutical composition of the present disclosure. In some embodiments, the method includes administering to the subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structured lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents, thereby delivering the therapeutic and / or prophylactic agents to cells. In some embodiments, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity. For example, the disease or disorder is selected from the group consisting of rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0438] In some embodiments, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising combining a cationic lipid of formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG. 2k In some embodiments, the present disclosure provides methods for treating a disease or disorder in a subject, the methods comprising administering to the subject lipid nanoparticles comprising -DMG and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the present disclosure provides methods for treating a disease or disorder in a subject, the methods comprising administering to the subject lipid nanoparticles comprising a cationic lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, DSPC, cholesterol, and PEG. 2k-DMG and lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA) are administered to a subject.

[0439] In some embodiments, the present disclosure provides methods of treating a disease or disorder in a subject, the method comprising administering to the subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents selected from a cationic lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, DSPC, cholesterol, and PEG-1, and a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA).

[0440] In yet another aspect, the disclosure features a method of reducing immunogenicity, comprising introducing a loaded LNP or pharmaceutical composition of the disclosure into a cell, wherein the loaded LNP or pharmaceutical composition reduces induction of a cellular immune response in the cell to the loaded LNP or pharmaceutical composition compared to induction of a cellular immune response in the cell induced by a reference composition. In some embodiments, the cell is in a subject, and the contacting comprises administering the cell to the subject. In some embodiments, the method includes administering to a subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid, a structured lipid, a PEG-lipid, and one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA), wherein the cationic lipid of Formula (I) and one or more therapeutic and / or prophylactic agents are selected from the group consisting of (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-I), (IL-I Lipid nanoparticles comprising an ionizable lipid of formula (IL-A), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA) reduce the induction of a cellular immune response in cells to a cationic lipid of formula (I) and lipid nanoparticles comprising an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), when compared to the induction of a cellular immune response in cells induced by a reference composition. For example, the cellular immune response can be an innate immune response, an adaptive immune response, or both.

[0441] In some embodiments, the disclosure provides a method of reducing immunogenicity in a subject, the method comprising: a cationic lipid of formula (I), an ionizable lipid of formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG. 2k In some embodiments, the present disclosure provides a method for reducing immunogenicity in a subject, the method comprising administering to a subject lipid nanoparticles comprising α-DMG and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the present disclosure provides a method for reducing immunogenicity in a subject, the method comprising administering to a subject lipid nanoparticles comprising a cationic lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, DSPC, cholesterol, and PEG. 2k -DMG and lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (e.g., RNA) are administered to a subject.

[0442] In some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, the method comprising administering to the subject lipid nanoparticles comprising a cationic lipid of Formula (I), an ionizable lipid of Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), DSPC, cholesterol, and PEG-1, and one or more therapeutic and / or prophylactic agents selected from a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA). For example, in some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, the method comprising administering to the subject lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents selected from a cationic lipid of Table 1, an ionizable lipid of Tables IL-1 through IL-7, DSPC, cholesterol, and PEG-1, and a nucleotide, a polypeptide, and a nucleic acid (e.g., RNA).

[0443] The present disclosure also includes methods for synthesizing cationic lipids of formula (I) and methods for making lipid nanoparticles (e.g., empty LNPs or loaded LNPs) that include lipid components that include cationic lipids of formula (I).

[0444] Method for producing polypeptides in cells The present disclosure provides a method for producing a polypeptide of interest in mammalian cells. The method for producing the polypeptide involves contacting the cells with lipid nanoparticles (e.g., empty LNPs or loaded LNPs) containing mRNA encoding the polypeptide of interest. By contacting the cells with the nanoparticle composition, the mRNA can be taken up by the cells and translated to produce the polypeptide of interest.

[0445] Typically, contacting mammalian cells with lipid nanoparticles (e.g., empty or loaded LNPs) containing mRNA encoding a polypeptide of interest can be performed in vivo, ex vivo, in culture, or in vitro. The amount of lipid nanoparticles (e.g., empty or loaded LNPs) contacted with cells and / or the amount of mRNA therein can depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics (e.g., size, charge, and chemical composition) of the lipid nanoparticles (e.g., empty or loaded LNPs) and the mRNA therein, and other factors. Typically, an effective amount of lipid nanoparticles (e.g., empty or loaded LNPs) will enable efficient polypeptide production in cells. Metrics for efficiency can include polypeptide translation (indicated by polypeptide expression), the level of mRNA degradation, and indicators of immune response.

[0446] Contacting cells with lipid nanoparticles (e.g., empty or loaded LNPs) containing mRNA can involve or result in transfection. Phospholipids contained in the lipid component of the lipid nanoparticles (e.g., empty or loaded LNPs) can facilitate transfection and / or increase transfection efficiency, for example, by interacting with and / or fusing with cell membranes or intracellular membranes. Transfection can allow translation of the mRNA within the cell.

[0447] In some embodiments, the lipid nanoparticles described herein (e.g., empty or loaded LNPs) can be used therapeutically. For example, the mRNA contained in the lipid nanoparticles (e.g., empty or loaded LNPs) can encode a therapeutic polypeptide (e.g., in a translatable region) and produce the therapeutic polypeptide upon contact with and / or entry into cells (e.g., transfection). In other embodiments, the mRNA contained in the lipid nanoparticles (e.g., empty or loaded LNPs) can encode a polypeptide that can improve or increase a subject's immunity. For example, the mRNA can encode granulocyte colony-stimulating factor or trastuzumab.

[0448] In certain embodiments, the mRNA contained in the lipid nanoparticles (e.g., empty or loaded LNPs) may encode a recombinant polypeptide that can replace one or more polypeptides that may be substantially absent in cells contacted within the nanoparticle composition. The one or more substantially absent polypeptides may be absent due to genetic mutations in the encoding gene or its regulatory pathway. Alternatively, the recombinant polypeptide produced by translation of the mRNA may antagonize the activity of an endogenous protein present in the cell, present on the surface of the cell, or secreted from the cell. Antagonizing recombinant polypeptides may be desirable to counteract deleterious effects caused by the activity of the endogenous protein, such as altered activity or localization caused by mutations. In another alternative, the recombinant polypeptide produced by translation of the mRNA may indirectly or directly antagonize the activity of a biological moiety present in the cell, present on the surface of the cell, or secreted from the cell. Antagonized biological moieties may include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins. The recombinant polypeptide produced by translation of the mRNA can be engineered for subcellular localization, such as within a specific compartment such as the nucleus, or can be engineered for secretion from the cell or translocation to the plasma membrane of the cell.

[0449] In some embodiments, contacting cells with lipid nanoparticles (e.g., empty or loaded LNPs) containing mRNA can reduce the cellular innate immune response to exogenous nucleic acids. Cells can be contacted with first lipid nanoparticles (e.g., empty or loaded LNPs) containing a first amount of a first exogenous mRNA that includes a translatable region, and the level of the cellular innate immune response to the first exogenous mRNA can be determined. The cells can then be contacted with a second composition containing a second amount of the first exogenous mRNA, the second amount being a lower amount of the first exogenous mRNA compared to the first amount. Alternatively, the second composition can contain a first amount of a second exogenous mRNA that is different from the first exogenous mRNA. The step of contacting cells with the first and second compositions can be repeated one or more times. Also, the efficiency of polypeptide production (e.g., translation) in the cells can optionally be determined, and the cells can be repeatedly re-contacted with the first and / or second compositions until a target protein production efficiency is achieved.

[0450] Methods for delivering therapeutic agents to cells and organs The present disclosure provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs. Delivery of the therapeutic and / or prophylactic agent to a cell involves administering lipid nanoparticles (e.g., empty or loaded LNPs) containing the therapeutic and / or prophylactic agent to a subject, and administration of the composition involves contacting the cell with the composition. For example, proteins, cytotoxic agents, radioactive ions, chemotherapeutic agents, or nucleic acids (e.g., RNA, e.g., mRNA) can be delivered to the cell or organ. In examples where the therapeutic and / or prophylactic agent is mRNA, contacting the cell with the nanoparticle composition allows translatable mRNA to be translated in the cell to produce a polypeptide of interest. However, substantially non-translatable mRNA can also be delivered to the cell. Substantially non-translatable mRNA can be useful as a vaccine and / or to sequester cellular translational components to reduce expression of other species in the cell.

[0451] In some embodiments, lipid nanoparticles (e.g., empty or loaded LNPs) can be targeted to a specific type or class of cells (e.g., cells of a specific organ or system thereof). For example, lipid nanoparticles (e.g., empty or loaded LNPs) containing a therapeutic and / or prophylactic agent of interest can be specifically delivered to the liver, kidney, spleen, or lung of a mammal. Specific delivery to a specific class of cells, organ, or system or group thereof means that a higher percentage of lipid nanoparticles (e.g., loaded LNPs) containing a therapeutic and / or prophylactic agent are delivered to the destination of interest (e.g., tissue) compared to other destinations. In some embodiments, specific delivery of loaded LNPs containing mRNA can result in a 2-fold, 5-fold, 10-fold, 15-fold, or greater than 20-fold increase in mRNA expression in cells of the targeted destination (e.g., tissue of interest, e.g., liver) compared to cells of another destination (e.g., spleen). In some embodiments, the tissue of interest is selected from the group consisting of liver, kidney, lung, spleen, and tumor tissue (e.g., via intratumoral injection).

[0452] As another example of targeted or specific delivery, nanoparticle compositions can include mRNA encoding protein binding partners (e.g., antibodies or functional fragments thereof, scaffolding proteins, or peptides) or cell surface receptors. Additionally or alternatively, mRNA can be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties. Alternatively, other therapeutic and / or prophylactic agents or components (e.g., lipids or ligands) of lipid nanoparticles (e.g., empty or loaded LNPs) can be selected based on their affinity for specific receptors (e.g., low-density lipoprotein receptors) so that the lipid nanoparticles (e.g., empty or loaded LNPs) can more readily interact with receptor-containing target cell populations. For example, ligands can include, but are not limited to, members of specific binding pairs, antibodies, monoclonal antibodies, Fv fragments, single-chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelidized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof; multivalent binding reagents including mono- or bispecific antibodies, e.g., disulfide-stabilized Fv fragments, scFv tandems, diabodies, tribodies, or tetrabodies; and aptamers, receptors, and fusion proteins.

[0453] In some embodiments, the ligand may be a surface-bound antibody, which may allow for tuning of cell targeting specificity. This may be particularly useful because highly specific antibodies may be established against epitopes of interest for a desired targeting site. In some embodiments, multiple antibodies may be expressed on the surface of a cell, each with a different specificity for a desired target. Such an approach may increase the avidity and specificity of the targeting interaction.

[0454] The ligand can be selected by one skilled in the art of biology based on, for example, the desired localization or function of a cell. Targeted cells may include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0455] Methods of Treating Diseases and Disorders Lipid nanoparticles (e.g., empty or loaded LNPs) can be useful for treating diseases, disorders, or conditions. In particular, such compositions can be useful in treating diseases, disorders, or conditions characterized by missing or aberrant protein or polypeptide activity. For example, lipid nanoparticles (e.g., empty or loaded LNPs) containing mRNA encoding a missing or aberrant polypeptide can be administered or delivered to cells. Subsequent translation of the mRNA can produce the polypeptide, thereby reducing or eliminating problems caused by the absence or aberrant activity caused by the polypeptide. Because translation can occur rapidly, the methods and compositions can be useful in treating acute diseases, disorders, or conditions, such as sepsis, stroke, and myocardial infarction. Therapeutic and / or prophylactic agents contained in lipid nanoparticles (e.g., empty or loaded LNPs) can also alter the rate of transcription of a given species, thereby affecting gene expression. Diseases, disorders, and / or conditions characterized by dysfunctional or abnormal protein or polypeptide activity for which the compositions can be administered include, but are not limited to, rare diseases, infectious diseases (both as vaccines and therapeutics), cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiac and renal vascular diseases, and metabolic diseases. Several diseases, disorders, and / or conditions can be characterized by protein activity that is absent (or substantially diminished such that proper protein function does not occur). Such proteins may be absent, or they may be essentially non-functional. The present disclosure provides methods for treating such diseases, disorders, and / or conditions in a subject by administering lipid nanoparticles (e.g., empty or loaded LNPs) comprising RNA and a cationic lipid component comprising a lipid according to Formula (I), an ionizable lipid component comprising a lipid according to Formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a phospholipid (optionally unsaturated), a PEG-lipid, and a structured lipid, wherein the RNA can be mRNA encoding a polypeptide that antagonizes or otherwise overcomes aberrant protein activity present in the subject's cells.

[0456] The present disclosure provides methods involving administering lipid nanoparticles (e.g., empty or loaded LNPs) containing one or more therapeutic and / or prophylactic agents and pharmaceutical compositions comprising the same. The terms therapeutic agent and prophylactic agent may be used interchangeably herein with respect to features and embodiments of the present disclosure. Therapeutic compositions, or imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any reasonable amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and / or condition, and / or for any other purpose. The specific amount administered to a given subject may vary depending on the subject's species, age, and general condition; the purpose of administration; the particular composition; the mode of administration; etc. Compositions according to the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. However, it will be understood that the overall daily usage of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment. The particular therapeutically effective, prophylactically effective, or otherwise appropriate dose level (e.g., for imaging) for any particular patient will depend upon a variety of factors, including the severity and identity (if any) of the disorder being treated; the one or more therapeutic and / or prophylactic agents used; the particular composition used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion of the particular pharmaceutical composition used; the duration of treatment; drugs used in combination with or concurrently with the particular pharmaceutical composition used; and similar factors well known in the medical arts.

[0457] The loaded LNPs can be administered by any route. In some embodiments, compositions, including prophylactic, diagnostic, or imaging compositions comprising one or more loaded LNPs described herein, are administered by one or more of a variety of routes, including orally, intravenously, intramuscularly, intraarterially, subcutaneously, transdermally or intradermally, intercutaneously, intraperitoneally, mucosally, nasally, intratumorally, intranasally; by inhalation; as an oral spray and / or powder, a nasal spray, and / or aerosol, and / or via a portal vein catheter. In some embodiments, the compositions can be administered intravenously, intramuscularly, intradermally, intraarterially, intratumorally, subcutaneously, or by any other parenteral administration route or by inhalation. However, the present disclosure encompasses delivery or administration of the compositions described herein by any appropriate route, taking into account potential advances in the science of drug delivery. Generally, the most appropriate route of administration will depend on a variety of factors, including the characteristics of the loaded LNP containing one or more therapeutic and / or prophylactic agents (e.g., its stability in various bodily environments, e.g., the bloodstream and gastrointestinal tract), the condition of the patient (e.g., whether the patient can tolerate a particular route of administration), and the like.

[0458] In certain embodiments, compositions according to the present disclosure may comprise a dose of about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 0.0001 mg / kg to about 5 mg / kg, Approximately 0.001mg / kg to approximately 5mg / kg, approximately 0.005mg / kg to approximately 5mg / kg, approximately 0.01mg / kg to approximately 5mg / kg, approximately 0.05mg / kg to approximately 5mg / kg, approximately 0.1mg / kg to approximately 5mg / kg, approximately 1mg / kg to approximately 5mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 2.5 mg / kg, about 0.001 mg / kg to about 2.5 mg / kg, about 0.005 mg / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, Approximately 0.05mg / kg to approximately 2.5mg / kg, approximately 0.1mg / kg to approximately 2.5mg / kg, approximately 1mg / kg to approximately 2.5mg / kg, approximately 2mg / kg to approximately 2.5mg / kg, approximately 0.0001mg / kg to approximately 1mg / kg, approximately 0.001mg / k g~about 1mg / kg, about 0.005mg / kg~about 1mg / kg, about 0.01mg / kg~about 1mg / kg, about 0.05mg / kg~about 1mg / kg, about 0.1mg / kg~about 1mg / kg, about 0.0001mg / kg~about 0.25mg / k The loaded LNPs may be administered at dosage levels sufficient to deliver about 0.001 mg / kg to about 0.25 mg / kg, about 0.005 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, or about 0.1 mg / kg to about 0.25 mg / kg of a therapeutic and / or prophylactic agent (e.g., mRNA) at a given dose, with a 1 mg / kg (mpk) dose providing 1 mg of therapeutic and / or prophylactic agent per kg of subject body weight. In some embodiments, a dose of about 0.001 mg / kg to about 10 mg / kg of the therapeutic and / or prophylactic agent loaded in the LNP may be administered. In other embodiments, a dose of about 0.005 mg / kg to about 2.5 mg / kg of the therapeutic and / or prophylactic agent may be administered.In certain embodiments, a dose of about 0.1 mg / kg to about 1 mg / kg may be administered. In other embodiments, a dose of about 0.05 mg / kg to about 0.25 mg / kg may be administered. Doses may be administered one or more times per day, in the same or different amounts, to achieve the desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered, for example, three times per day, twice per day, once per day, every other day, once every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). In some embodiments, a single dose may be administered, for example, before or after a surgical procedure or in the case of an acute disease, disorder, or condition.

[0459] Lipid nanoparticles (e.g., empty or loaded LNPs) containing one or more therapeutic and / or prophylactic agents can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. "In combination with" is not intended to imply that the agents must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of the present disclosure. For example, one or more lipid nanoparticles (e.g., empty or loaded LNPs) containing one or more different therapeutic and / or prophylactic agents can be administered in combination. The composition can be administered simultaneously with, before, or after one or more other desired therapeutic agents or medical treatments. Typically, each agent will be administered at a dose and / or time schedule determined for that agent. In some embodiments, the present disclosure encompasses delivery of a composition, or imaging, diagnostic, or prophylactic composition, in combination with agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution within the body.

[0460] It will be understood that therapeutic, prophylactic, diagnostic, or imaging agents utilized in combination can be administered together in a single composition or administered separately in different compositions. Typically, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination may be lower than those utilized individually.

[0461] The particular combination of therapies (therapeutic agents or procedures) to use in a combination regimen will take into account compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. It will also be understood that the therapies used may achieve a desired effect for the same disorder (e.g., a composition useful for treating cancer may be administered simultaneously with a chemotherapeutic agent), or they may achieve a different effect (e.g., control of any adverse effects, such as infusion-related reactions).

[0462] Lipid nanoparticles (e.g., empty or loaded LNPs) can be used in combination with drugs to increase the efficacy and / or therapeutic window of the composition. Such drugs can be, for example, anti-inflammatory compounds, steroids (e.g., corticosteroids), statins, estradiol, BTK inhibitors, S1P1 agonists, glucocorticoid receptor modulators (GRMs), or antihistamines. In some embodiments, lipid nanoparticles (e.g., empty or loaded LNPs) can be used in combination with dexamethasone, methotrexate, acetaminophen, H1 receptor blockers, or H2 receptor blockers. In some embodiments, methods of treating a subject in need of treatment or methods of delivering therapeutic and / or prophylactic agents to a subject (e.g., a mammal) can involve pre-treating the subject with one or more drugs prior to administering the nanoparticle composition. For example, a subject can be pretreated with a useful amount (e.g., 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or any other useful amount) of dexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, or an H2 receptor blocker. Pretreatment can occur within 24 hours (e.g., 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes) prior to administration of lipid nanoparticles (e.g., empty or loaded LNPs), and can occur once, twice, or more times, e.g., in increasing dosages.

[0463] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0464] In the claims, articles such as "a," "an," and "the" can mean one or more unless indicated to the contrary or otherwise clear from the context. A claim or description including "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. As used herein, the expressions "one or more of A, B, or C," "one or more of A, B, or C," "one or more of A, B, and C," "one or more of A, B, and C," "one or more of A, B, and C," "selected from A, B, and C," "selected from the group consisting of A, B, and C," etc. are used interchangeably and, unless otherwise specified, all refer to selection from the group consisting of A, B, and / or C, i.e., one or more A, one or more B, one or more C, or any combination thereof.

[0465] It is also noted that the term "comprising" is intended to be open, permitting but not requiring the inclusion of additional elements or steps. When the term "comprising" is used herein, the terms "consisting essentially of" and "consisting of" are therefore also encompassed and disclosed. Throughout the specification, when a composition is described as having, including, or comprising certain components, it is contemplated that the composition also consists essentially of or consists of the described components. Similarly, when a method or process is described as having, including, or comprising certain process steps, the process also consists essentially of or consists of the described process steps. Furthermore, it should be understood that the order of steps or order for performing certain operations is immaterial so long as the invention remains operable. Moreover, two or more steps or operations may be performed simultaneously.

[0466] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, it is to be understood that values ​​expressed as ranges can assume any specific value or subrange within the ranges described in different embodiments of this disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0467] The synthetic processes of the present disclosure can tolerate a variety of functional groups, and therefore various substituted starting materials can be used. The processes usually provide the desired final lipid at or near the end of the overall process, although in certain instances it may be desirable to further convert the lipid to its pharmaceutically acceptable salt.

[0468] The lipids of the present disclosure can be prepared in a variety of ways by using commercially available starting materials, compounds known in the literature, or from easily prepared intermediates, using standard synthetic methods and procedures known to those skilled in the art or that will become apparent to those skilled in the art in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from relevant scientific literature or from standard textbooks in the field. Examples include, but are not limited to, any one or several sources, such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th edition,John Wiley & Sons:New York,2001;Greene,TW,Wuts,PGM,Protective Groups in Organic Synthesis,3 rd Classic texts such as "The Encyclopedia of Reagents for Organic Synthesis" (John Wiley & Sons, 1995) (incorporated herein by reference) are useful and recognized reference texts for organic synthesis known to those skilled in the art. The following synthetic methodology description is designed to illustrate the general procedures for preparing the lipids of the present disclosure, but is not intended to be limiting.

[0469] The cationic lipids of the present disclosure having the formulas described herein can be prepared according to the procedures shown in the following schemes from commercially available starting materials or starting materials that can be prepared using literature procedures. Those skilled in the art will note that the order of certain steps can be changed in the reaction sequences and synthetic schemes described herein.

[0470] Those skilled in the art will recognize that certain groups may require protection from the reaction conditions through the use of protecting groups. Protecting groups to differentiate similar functional groups in a molecule may also be used. A list of protecting groups and how to install and remove these groups can be found in Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 5 th edition, John Wiley & Sons: New York, 2014.

[0471] Preferred protecting groups include, but are not limited to, the following: For hydroxyl sites: TBS, benzyl, THP, Ac. For carboxylic acids: benzyl ester, methyl ester, ethyl ester, allyl ester.

[0472] For amines: Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl, phthalimide, benzylideneamine. For diols: Ac(x2) TBS(x2), or acetonide if combined.

[0473] For thiols: Ac. For benzimidazoles: SEM, benzyl, PMB, DMB. For aldehydes: di-alkyl acetals, for example dimethoxy acetal or diethylacetyl.

[0474] In the reaction scheme described herein, multiple stereoisomers can be produced.When no specific stereoisomer is indicated, it is understood that all possible stereoisomers that can be produced from the reaction are meant.Those skilled in the art will recognize that the reaction can be optimized to preferentially obtain one isomer, or a new scheme can be devised to produce a single isomer.When a mixture is produced, techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC can be used to separate isomers.

[0475] General Scheme 1

[0476] [ka]

[0477] As shown in General Scheme 1 above, an ionizable lipid is reacted with the desired tail in a base (e.g., K2CO3) in the presence of potassium iodide and heated to reflux to provide the cationic lipid of the present disclosure. - is any pharmaceutically acceptable anion.

[0478] Those skilled in the art will recognize that in the above schemes, the order of certain steps may be interchangeable. In certain embodiments, the present disclosure also includes methods for synthesizing cationic lipids of formula (I) and intermediate(s) for synthesizing the lipids. Without wishing to be bound by theory, it is believed that the anion (i.e., "A") associated with the cationic lipids of formula (I) may be a cation. - It is understood that the cationic lipids of formula (I) can be provided by the starting materials involved in the synthesis of the cationic lipids of formula (I). For example, Br -The anion can be provided by undecyl 6-bromohexanoate, nonyl 8-bromooctanoate, or 3-butylheptyl 8-bromooctanoate. Without wishing to be bound by theory, the identification of the anion may require additional analysis by known methods (e.g., elemental analysis). However, it is understood that the identification of the anion is not a necessary step in the synthesis of the cationic lipid of formula (I). Without wishing to be bound by theory, during any of the syntheses described herein, the cationic lipid of formula (I) may be formed with an anion replaced by another anion during the purification process. For example, in some embodiments, the cationic lipid of formula (I) is a Br - ions or Cl - ions, but Br - ions or Cl - ions are converted to OH during the purification process. - For example, when NH4OH is included in the eluent for silica gel column purification, the initial counter ion (e.g., Br - or Cl - ) may be replaced with an OH- anion. In some embodiments, when multiple molecules of a compound of formula (I) are present, it is understood that a mixture of anions may be present. For example, without wishing to be bound by theory, when two molecules of a compound of formula (I) are present, each molecule may have a different counter ion. For example, a counter ion may be a Br in one molecule of a compound of formula (I). - and OH for other molecules - It could be.

[0479] The ionizable lipids described herein may be prepared according to the procedures disclosed in published International Patent Application Nos. WO / 2017 / 049245, WO / 2017 / 112865, WO / 2018 / 170306, WO / 2018 / 232120, WO / 2021 / 055835, WO / 2021 / 055833, and WO / 2021 / 055849, each of which is incorporated herein by reference in its entirety.

[0480] It should also be understood that any particular embodiment of the present disclosure that falls within the prior art may be expressly excluded from any one or more of the claims. Because such embodiments are deemed to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not expressly set forth herein.

[0481] All sources cited herein, e.g., references, publications, databases, database entries, and techniques, are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a conflict between the statements of the cited sources and this application, the statements in this application shall control. [Example]

[0482] Example 1: Synthesis of cationic lipids in Table 1 A. General Considerations All solvents and reagents used were commercially obtained and used as received unless otherwise stated. 1 H NMR spectra were recorded in CDCl3 at 300 K using a Bruker Ultrashield 300 MHz instrument. Chemical shifts are 1The values ​​are reported as parts per million (ppm) relative to TMS (0.00) for H. Silica gel chromatography was performed on an ISCO CombiFlash Rf+ Lumen Instrument using ISCO RediSep Rf Gold Flash Cartridges (particle size: 20-40 microns). Reverse-phase chromatography was performed on an ISCO CombiFlash Rf+ Lumen Instrument using a RediSep Rf Gold C18 High Performance column. All final lipids were confirmed to be >85% pure via analysis by reverse-phase UPLC-MS (retention time, RT (min)) using a gradient of 65 to 100% acetonitrile in water containing 0.1% TFA at 1.2 mL / min over 5 min using a Waters Acquity UPLC system equipped with a DAD and ELSD and a ZORBAX Rapid Resolution High Definition (RRHD) SB-C18 LC column (2.1 mm, 50 mm, 1.8 μm). The injection volume was 5 μL, and the column temperature was 80 °C. Detection was based on electrospray ionization (ESI) and evaporative light scattering detection in positive mode using a Waters SQD mass spectrometer (Milford, MA, USA). LCMS method: Instrument information: HPLC / MS-Agilent 1100 Column: Agela Technologies Durashell C18 3.5 μm, 100 Å, 4.6 x 50 mm Mobile phase A: Water / 0.1% trifluoroacetic acid Mobile phase B: acetonitrile / 0.1% trifluoroacetic acid Flow rate: 1mL / min Gradient: 70% B to 100% B in 5 min, hold at 100% B for 10 min, 100% B to 70% B in min, then stop. Column temperature: ambient Detector: ELSD The procedures described below are useful for the synthesis of the lipids in Table 1.

[0483] The following abbreviations are used herein: THF: tetrahydrofuran TLC: Thin Layer Chromatography MeCN: acetonitrile LAH: Lithium aluminum hydride DCM: dichloromethane DMAP: 4-dimethylaminopyridine LDA: lithium diisopropylamide rt: room temperature DME: 1,2-dimethoxyethane n-BuLi: n-butyllithium CPME: Cyclopentyl methyl ether i-Pr2EtN: N,N-diisopropylethylamine Preparation of 8-(heptadecan-9-yloxy)-N-(2-hydroxyethyl)-8-oxo-N,N-bis(6-oxo-6-(undecyloxy)hexyl)octan-1-aminium (Compound 1)

[0484] [ka]

[0485] Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)-octanoate (40 g, 51 mmol) and undecyl 6-bromohexanoate (90 g, 258 mmol) were dissolved in acetonitrile (220 mL). The flask was equipped with an air-cooled reflux condenser and stirred at 90 °C, and the solution was stirred under reflux under N2(g). After 5 days, the reaction was cooled to room temperature and the acetonitrile was evaporated. The residual oil was dissolved in heptane (600 mL), and the heptane solution was extracted with acetonitrile (3 × 250 mL). The heptane layer was collected and dried. A portion of the crude oil (4.84 g) was purified by two silica gel column chromatography runs. The first column was run with 100% isopropyl acetate for several column volumes, followed by 20% MeOH in dichloromethane. The second column was run using a gradient method [0 to 100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane)] in dichloromethane to give 998.4 mg of 8-(heptadecan-9-yloxy)-N-(2-hydroxyethyl)-8-oxo-N,N-bis(6-oxo-6-(undecyloxy)hexyl)octan-1-aminium. UPLC / ELSD: RT=3.13 min. HRMS (ESI): C 61 H 120 No. 7 + Calculated m / z for (M+H), 978.91; found, 979.40. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.85 (m, 1H);4.49-4.40 (br, m, 1H);4.19-4.09 (br, 2H);4.05 (t, 4H, J = 6.0 Hz);3.64-3.57 (br, m, 2H);3.45-3.31 (br, m, 6H);2.45-2.23 (m, 6H);1.83-1.55 (m, 19H);1.54-1.14 (m, 64H);0.98-0.82 (m, 15H).

[0486] The compounds heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate and undecyl 6-bromohexanoate were prepared as described in Sabnis, S.; Kumarasinghe, E.S.; Salerno, T.; Cosmin, M.; Ketoba, T.; Senn, J.J.; Lynn, A.; Bulychev, A.; Mcfadyen, I.; Chan, J.; Almarsson, O.; Stanton, M.G.A. Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates, Molecular Therapy, 2018, 1509-1519 (incorporated herein by reference in its entirety).

[0487] Preparation of 8-(heptadecan-9-yloxy)-N-(2-hydroxyethyl)-N,N-bis(8-(nonyloxy)-8-oxooctyl)-8-oxooctane-1-aminium (Compound 2)

[0488] [ka]

[0489] A 12 L three-neck round-bottom flask equipped with an overhead stirrer, heating mantle, temperature probe, air-cooled reflux condenser, and N2 inlet was charged with potassium carbonate (225 g, 1.63 mol), potassium iodide (74 g, 0.45 mol), and nonyl 8-((2-hydroxyethyl)amino)octanoate (215 g, 0.40 mol). The solid was suspended in acetonitrile (2.2 L). Nonyl 8-bromooctanoate (167.3 g, 0.47 mol) was added to the stirred suspension, followed by cyclopentyl methyl ether (2200 mL). The resulting mixture was heated to 81.0 °C. After 48 h, the reaction was cooled to room temperature and the white inorganic salts were filtered. The filtrate was concentrated to an oil, and the crude oil was dissolved in heptane (3 L) and washed with acetonitrile (5 × 1000 mL). The acetonitrile layer was collected and concentrated. A portion of the crude product (2 g) was purified by two silica gel column chromatography runs. The first silica gel column used a gradient method [0 to 80% (1% NH4OH, 20% MeOH mixture in dichloromethane)] in dichloromethane, and the second column was run with 100% isopropyl acetate for several column volumes, followed by a mixture of 1% NH4OH, 20% MeOH in dichloromethane. The desired product, 8-(heptadecan-9-yloxy)-N-(2-hydroxyethyl)-N,N-bis(8-(nonyloxy)-8-oxooctyl)-8-oxooctane-1-aminium, was obtained as a pale yellow oil (988.4 mg). UPLC / ELSD: RT = 3.16 min. HRMS (ESI): C 61 H 120 No. 7 + m / z calculated for (M+H), 978.91; observed, 979.64. 1H NMR (300 MHz, CDCl3) δ: ppm 4.85 (m, 1H);4.57-4.36 (br, m, 1H);4.18-4.09 (br, 2H);4.05 (t, 4H, J = 6.0 Hz);3.65-3.55 (br, m, 2H);3.45-3.30 (br, m, 6H);2.37-2.22 (m, 6H);1.75-1.55 (m, 19H);1.54-1.15 (m, 64H);0.97-0.82 (m, 15H).

[0490] The compounds nonyl 8-((2-hydroxyethyl)amino)octanoate and nonyl 8-bromooctanoate were prepared as described in Sabnis, S.; Kumarasinghe, E.S.; Salerno, T.; Cosmin, M.; Ketoba, T.; Senn, J.J.; Lynn, A.; Bulychev, A.; Mcfadyen, I.; Chan, J.; Almarsson, O.; Stanton, M.G.A. Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates, Molecular Therapy, 2018, 1509-1519 (incorporated herein by reference in its entirety).

[0491] Preparation of 8-((3-butylheptyl)oxy)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-N-(2-hydroxyethyl)-N-(8-(nonyloxy)-8-oxooctyl)-8-oxooctane-1-aminium (compound 3)

[0492] [ka]

[0493] Heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (2 g, 2.82 mmol) and 3-butylheptyl 8-bromooctanoate (5.31 g, 14.08 mmol) were dissolved in acetonitrile (10 mL). The reaction was stirred at 80 °C for 6 days. The reaction turned pale yellow-orange after 1 day and eventually pale yellow. The reaction was cooled to room temperature. Heptane (10 mL) was added to the solution, and the heptane layer was washed with acetonitrile (6 × 10 mL). The acetonitrile layer was concentrated and purified by two silica gel column chromatography runs. The first silica gel column used 100% isopropyl acetate for several column volumes, followed by a mixture of 1% NH4OH, 20% MeOH in dichloromethane. A second silica gel chromatography using a gradient method [0 to 80% (1% NH4OH, 20% MeOH mixture in dichloromethane) in dichloromethane] gave the product (1.06 g), 8-((3-butylheptyl)oxy)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-N-(2-hydroxyethyl)-N-(8-(nonyloxy)-8-oxooctyl)-8-oxooctane-1-aminium. UPLC / ELSD: RT = 3.07 min. HRMS (ESI): C 63 H 124 No. 7 + m / z calculated for (M+H), 1006.94; observed, 1007.40. 1 H NMR (300 MHz, CDCl3) δ: ppm 4.85 (m, 1H);4.17-4.00 (m, 7H);3.59-3.50 (br, m, 2H);3.45-3.33 (br, m, 6H);2.35-2.22 (m, 6H);1.76-1.45 (m, 23H);1.44-1.17 (m, 64H);0.96-0.82 (m, 15H).

[0494] The compound heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate was reported by Sabnis, S.; Kumarasinghe, E.S.; Salerno, T.; Cosmin, M.; Ketoba, T.; Senn, J.J.; Lynn, A.; Bulychev, A.; Mcfadyen, I.; Chan, J.; Almarsson, O.; Stanton, M.G.A. Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates, Molecular Therapy, 2018, 1509-1519 (incorporated herein by reference in its entirety), and the compound 3-butylheptyl 8-bromooctanoate was prepared as described in Benenato, K. E.; Cornebise, M.; Hennessy, E.; Kumarasinghe, E. S. Branched Tail Lipid Compounds and Compositions for Intracellular Delivery of Therapeutic Agents, US11066355B2 (incorporated herein by reference in its entirety).

[0495] Preparation of 8-((3-butylheptyl)oxy)-N-(8-((3-butylheptyl)oxy)-8-oxooctyl)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-N-(2-hydroxyethyl)-8-oxooctane-1-aminium (compound 4)

[0496] [ka]

[0497] In a round-bottom flask, heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (2 g, 2.709 mmol) and 3-butylheptyl 8-bromooctanoate (5.112 g, 13.55 mmol) were dissolved in acetonitrile (10 mL). The reaction was stirred at 80 °C for 6 days, at which point the reaction color changed from clear to pale yellow. The reaction was cooled to room temperature, and 10 mL of heptane was added to the solution. The heptane layer was washed with acetonitrile (6 × 10 mL). The acetonitrile fractions were collected, concentrated, and purified by silica gel chromatography. The first silica gel column was run with 100% isopropyl acetate for several column volumes, followed by a mixture of 1% NH4OH, 20% MeOH in dichloromethane. After the first column, a second silica gel chromatography was performed using a gradient method [0 to 80% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to collect the target product, 8-((3-butylheptyl)oxy)-N-(8-((3-butylheptyl)oxy)-8-oxooctyl)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-N-(2-hydroxyethyl)-8-oxooctane-1-aminium (1.274 g). UPLC / ELSD: RT=3.08 min. HRMS (ESI): C 65 H 128 No. 7 + m / z calculated for (M+H), 1034.97; observed, 1035.03. 1 NMR (300 MHz, CDCl3) δ: ppm 4.85 (m, 1H);4.20-4.01 (m, 6H);3.56-3.50 (br, m, 2H);3.45-3.32 (br, m, 6H);2.35-2.21 (m, 6H);1.75-1.46 (m, 25H);1.45-1.17 (m, 64H);0.98-0.81 (m, 18H).

[0498] The compounds heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)-octanoate and 3-butylheptyl 8-bromooctanoate were prepared as described in Benenato, K. E.; Cornebise, M.; Hennessy, E.; Kumarasinghe, E. S. Branched Tail Lipid Compounds and Compositions for Intracellular Delivery of Therapeutic Agents, US11066355B2 (incorporated herein by reference in its entirety).

[0499] Example 2: LNP formulation Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) containing therapeutic and / or prophylactic agents can be optimized according to the selection of cationic lipids according to Formula (I), the selection of additional lipids, the amount of each lipid in the lipid component, and the wt:wt ratio of lipid component to therapeutic and / or prophylactic agent.

[0500] Lipid nanoparticles (e.g., empty or loaded LNPs) were prepared containing DSPC and DOPE as phospholipids, cholesterol as structural lipid, PEG-1 as PEG lipid, an ionizable lipid according to formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), and a cationic lipid according to formula (I).

[0501] An exemplary lipid nanoparticle composition was prepared by dissolving lipids (e.g., an ionizable lipid according to formula (IL-A), (IL-B), (IL-C), (IL-D), (IL-I), (IL-IA), (IL-IB), (IL-IC), (IL-IIA), (IL-IIAX), (IL-IIB), (IL-IIC), (IL-III), or (IL-IIIA), a cationic lipid according to formula (I), DSPC, cholesterol, and PEG1) in ethanol to a concentration of 12.5 mM and at the molar ratios summarized in Table 2. The lipid to mRNA ratio was maintained at an N / P ratio of 4.9. The mRNA was then diluted with 25 mM sodium acetate (pH 5.0) and combined with the lipid mixture at a volume ratio of 3:1 (aqueous:ethanol). The resulting formulation was dialyzed against 20 mM Tris / 8% sucrose, 10 mM sodium chloride (pH 7.4) at a volume 300 times the volume of the major product for at least 18 hours using a Slide-A-Lyzer dialysis cassette (Thermo Scientific, Rockford, IL, USA) with a molecular cutoff of 10 kDa. The first dialysis was performed at 85 rpm on a digital orbital shaker at room temperature for 3 hours, followed by overnight dialysis at 4°C. The formulation was concentrated using a centrifugal filter, passed through a 0.22 μm filter, and stored at 4°C until use. The lipid nanoparticle solution was typically adjusted to a specific mRNA concentration of 0.1 mg / mL to 1 mg / mL.

[0502] Table 2 summarizes the ingredients and compositions of exemplary LNPs. Table 3 summarizes the characteristics of the LNP formulations, all of which were determined at room temperature on the day of preparation. As shown in Table 3, the majority of LNPs containing cationic lipids of Formula (I) exhibited small sizes (60-80 nm).

[0503] [Table 9]

[0504] [Table 10]

[0505] Example 3: Isolation of mouse endothelial lung cells for flow cytometry Reagents: Digestion medium: 3 mg / mL collagenase I, 0.1 mg / mL DNase I, Dulbecco's modified Eagle's medium (DMEM); Washing solution: Dulbecco's phosphate-buffered saline (DPBS) + 0.5% bovine serum albumin (BSA); FACS buffer: flow cytometry staining buffer (eBioscience™, ThermoFisher Scientific).

[0506] Minimum Marker Panel: · CD31: common endothelial marker; · CD45: common leukocyte marker; · mOX40L: transmembrane reporter; · tdTomato: a fluorescent protein reporter from Cre-mediated recombination in Ai14 mice.

[0507] Six-week-old female mice (Ai14) were aged in-house to approximately 7-8 weeks of age. N = 5 per group received Cre mRNA (i.e., mRNA that induces tdTomato expression)-formulated LNPs intravenously via the lateral tail vein 8 days before harvest. One week after the first administration, mice received the presently disclosed LNPs containing mRNA expressing OX40L intravenously (via the lateral tail vein). Twenty-four hours after the second intravenous administration, mice were euthanized under CO2 asphyxiation, and the right atrium was cut to allow perfusion of blood. The lungs were perfused with 5 mL of PBS via the right ventricle of the heart and then removed. The left lobe of the lung was stored in 3 mL of PBS and placed on ice. The left lobe was cut into <1 mm sections and placed in 8 mL of digestion medium. The tissue suspension was incubated in digestion medium for 45 minutes at 37°C, with inversion every 15 minutes. The tissue suspension was passed through a 3 mL syringe with a 20 G cannula until the mixture was triturated into a single-cell suspension. The cell mixture was filtered through a 70 μm strainer into ice-cold washing solution. Additional washing solution was then added to the top of the strainer. The cell mixture was centrifuged at 300 x g for 5 minutes at 4 °C. The supernatant was removed, and the cell pellet was resuspended in washing buffer. The cell mixture was centrifuged at 300 x g for 5 minutes at 4 °C. The supernatant was removed, and the cell pellet was resuspended in ammonium chloride potassium (ACK) lysis buffer for 1 minute, and buffer was added (2x volume). The cell mixture was centrifuged at 300 x g for 5 minutes at 4 °C, after which the supernatant was removed. The cell pellet was resuspended in flow cytometry staining (FACS) buffer and filtered through a 70 μm mesh. Lung cells were stained with viability dyes and antibodies against a marker panel according to the manufacturer's recommendations. Cells were resuspended in anti-mouse CD16 / 32 antibody (TruStain FcX™ BioLegend) 10 minutes before staining with the antibody cocktail. Compensation controls were run on the flow cytometer. Lung samples were run on an acoustic focusing flow cytometer (Attune™ NxT, ThermoFisher Scientific). Collected data were analyzed using flow cytometry analysis software (FlowJo™).

[0508] Example 4: Endothelial delivery by LNPs of the present disclosure To evaluate delivery of therapeutic agents contained in the loaded LNPs of the present disclosure to endothelial cells, expression of Cre and mOX40L mRNA encapsulated in the loaded LNPs of the present disclosure was measured in endothelial cells after administration of the loaded LNPs to Ai14 mice as described in Example 3. The results are summarized in Tables 4 and 5 below, showing the percentage of endothelial cells showing expression after administration of the LNPs of the present disclosure in a sample endothelial cell population positive for a given reporter as detected by flow cytometry. Expression was evaluated in LNPs containing 30 mol% of a cationic lipid of Formula (I) and in loaded LNPs that did not contain a cationic lipid of Formula (I) (i.e., LNPs containing no cationic lipid or LNPs containing dioleoyl-3-trimethylammoniumpropane (DOTAP)). The loaded LNPs also contained the ionizable lipid and PEG-lipid indicated in the tables.

[0509] [Table 11]

[0510] [Table 12]

[0511] Enumerated Embodiments Embodiment 1. A cationic lipid of formula (I):

[0512] [ka]

[0513] or its isomer (In the formula, R' x teeth,

[0514] [ka]

[0515] and R' y teeth,

[0516] [ka]

[0517] and R' z teeth,

[0518] [ka]

[0519] and;

[0520] [ka]

[0521] indicates the point of attachment; R xα , R xβ , R xγ , and R xδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R yα , R yβ , R yγ , and R yδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R zα , R zβ , R zγ , and R zδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R H is -(CH2) q OH, and q is selected from 1, 2, 3, 4, and 5; Each R T independently, C1-12 Alkyl and C 2-12 alkenyl; a is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; b is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; c is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; A - is any pharmaceutically acceptable anion).

[0522] Embodiment 2. A cationic lipid of formula (I-cat):

[0523] [ka]

[0524] or its isomer (In the formula, R' x teeth,

[0525] [ka]

[0526] and R' y teeth,

[0527] [ka]

[0528] and R' z teeth,

[0529] [ka]

[0530] and;

[0531] [ka]

[0532] indicates the point of attachment; R xα , R xβ , R xγ , and R xδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R yα , R yβ , R yγ , and R yδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R zα , R zβ , R zγ , and R zδ are independently H, C 1-12 Alkyl, and C 2-12 alkenyl; R H is -(CH2) q OH, and q is selected from 1, 2, 3, 4, and 5; Each R T independently, C 1-12 Alkyl and C 2-12 alkenyl; a is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; b is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; c is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9).

[0533] Embodiment 3. R' x teeth,

[0534] [ka]

[0535] and; R' y teeth,

[0536] [ka]

[0537] and; R' z teeth,

[0538] [ka]

[0539] and;

[0540] [ka]

[0541] indicates the point of attachment; R xγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R yγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R zγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c are each independently 1-14 Alkyl and C 2-14 alkenyl, 3. The cationic lipid of embodiment 1 or 2.

[0542] Embodiment 4. R' a teeth,

[0543] [ka]

[0544] and R' b teeth,

[0545] [ka]

[0546] and R' c teeth,

[0547] [ka]

[0548] and;

[0549] [ka]

[0550] indicates the point of attachment; R xγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R yγ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R 2c and R 3c are each independently 1-14 Alkyl and C 2-14 alkenyl, 1. The cationic lipid of any of the preceding embodiments.

[0551] Embodiment 5.R xγ and R yγ

[0023] The compound of any preceding embodiment, wherein each is H. Embodiment 6.R xγ and Ryγ are respectively, C 1-12 Alkyl or C 2-12 The compound of any of the preceding embodiments, wherein the compound is alkenyl.

[0552] Embodiment 7.R xγ is C 1-12 Alkyl or C 2-12 alkenyl, and R yγ is H. Embodiment 8.q is a compound of any of the preceding embodiments, wherein q is 2.

[0553] Embodiment 9. The compound is

[0554] [ka]

[0555] The compound of any preceding embodiment, selected from: Embodiment 10.

[0556] [ka]

[0557] A compound selected from: Embodiment 11.A - is selected from chloride, bromide, iodide, hydroxide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate.

[0558] Embodiment 12.A -The compound of any of the preceding embodiments, wherein is selected from bromide, chloride, and hydroxide. Embodiment 13.A -

[0023] The compound of any of the preceding embodiments, wherein is bromide.

[0559] Embodiment 14.A -

[0023] The compound of any of the preceding embodiments, wherein is chloride. Embodiment 15.A -

[0023] The compound of any of the preceding embodiments, wherein is bromide or hydroxide.

[0560] Embodiment 16.A -

[0023] The compound of any of the preceding embodiments, wherein is chloride or hydroxide. Embodiment 17.A -

[0023] The compound of any of the preceding embodiments, wherein is hydroxide.

[0561] Embodiment 18. An empty lipid nanoparticle (empty LNP) comprising a cationic lipid according to any of the preceding embodiments. Embodiment 19 The empty LNP of any one of the preceding embodiments, further comprising an ionizable lipid.

[0562] Embodiment 20 The empty LNP of any one of the preceding embodiments, further comprising a phospholipid. Embodiment 21 The empty LNP of any one of the preceding embodiments, further comprising a structured lipid.

[0563] Embodiment 22 The empty LNP of any one of the preceding embodiments, further comprising a PEG lipid. Embodiment 23. An empty LNP comprising a lipid component comprising about 20 mol% to about 40 mol% of the cationic lipid of any of the preceding embodiments; about 15 mol% to about 40 mol% of an ionizable lipid, about 0 mol% to about 30 mol% of a phospholipid, about 15 mol% to about 50 mol% of a structural lipid, and about 0 mol% to about 1 mol% of a PEG-lipid.

[0564] Embodiment 24. A loaded lipid nanoparticle (loaded LNP) comprising an empty LNP according to any one of the preceding embodiments and a therapeutic and / or prophylactic agent. Embodiment 25. An on-board LNP, (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent disposed primarily on the outer surface of the core wherein the loaded LNP has a zeta potential greater than neutral at physiological pH.

[0565] Embodiment 26. An on-board LNP, (a) an onboard LNP core; (i) ionizable lipids; (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid the mounted LNP core, (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent The on-board LNP includes:

[0566] Embodiment 27. An on-board LNP, (a) a lipid nanoparticle core, (i) ionizable lipids; (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid the lipid nanoparticle core comprising: (b) a polynucleotide or polypeptide payload therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell; and (c) a cationic agent disposed primarily on the outer surface of the core The on-board LNP includes:

[0567] Embodiment 28. An on-board LNP, (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent wherein the loaded LNP exhibits cellular accumulation of at least about 20% of cells and expression in about 5% or more of cells in a population of cells to which the loaded LNP was administered. In some embodiments, the loaded LNP exhibits cellular accumulation of about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% of cells in a population of cells to which the loaded LNP was administered.

[0568] Embodiment 29. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits expression in about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% of cells in which the loaded LNP accumulates.

[0569] Embodiment 30. In some embodiments, the present specification provides a loaded LNP, comprising: Embodiment 31. (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent disposed primarily on the outer surface of the core wherein the loaded LNP exhibits cellular accumulation in at least about 20% of cells in a population of cells to which the loaded LNP is administered, and exhibits expression in about 5% or more of the cells to which the loaded LNP accumulates.

[0570] Embodiment 32. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits cellular accumulation in about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% of cells in a population of cells to which the loaded LNP is administered.

[0571] Embodiment 33. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits expression of about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% in cells in which the loaded LNP accumulates.

[0572] Embodiment 34. An on-board LNP, (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) a cationic agent disposed primarily on the outer surface of the core wherein the therapeutic and / or prophylactic agent expresses a protein, and the loaded LNP exhibits protein expression of about 0.5% to 50% in cells in a population of cells to which the loaded LNP is administered.

[0573] Embodiment 35. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits protein expression of about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% in cells in which the loaded LNP accumulates.

[0574] Embodiment 36. An on-board LNP, (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent wherein the loaded LNP exhibits about 0.5% to 50% protein expression in cells in which the loaded LNP accumulates.

[0575] Embodiment 37. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits protein expression of about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% in cells in which the loaded LNP accumulates.

[0576] Embodiment 38. An on-board LNP, (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent wherein the loaded LNP exhibits at least about 20% cellular accumulation in endothelial cells and about 5% or more expression in endothelial cells in which the loaded LNP accumulates.

[0577] Embodiment 39. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits a cellular accumulation of endothelial cells of about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% in a population of endothelial cells to which the loaded LNP is administered.

[0578] Embodiment 40. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits expression of about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% in endothelial cells in which the loaded LNP accumulates.

[0579] Embodiment 41. An on-board LNP, (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent wherein the loaded LNP exhibits protein expression in about 0.5% to 50% of endothelial cells in which the loaded LNP accumulates.

[0580] Embodiment 42. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits protein expression of about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of endothelial cells to which the loaded LNP has accumulated.

[0581] Embodiment 43. An on-board LNP, (a) lipid nanoparticle core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent wherein the loaded LNP exhibits protein expression in about 0.5% to about 50% of lung cells in which the loaded LNP accumulates.

[0582] Embodiment 44. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of pulmonary endothelial cells to which the loaded LNP has accumulated.

[0583] Embodiment 45. An on-board LNP, (a) Lipid-loaded LNP core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent wherein the loaded LNP exhibits at least about 20% cellular accumulation in respiratory endothelial cells and about 5% or more expression in respiratory endothelial cells in which the loaded LNP accumulates.

[0584] Embodiment 46. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits a cellular accumulation of respiratory endothelial cells of about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% in a population of respiratory endothelial cells to which the loaded LNP is administered.

[0585] Embodiment 47. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits expression of about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% in respiratory endothelial cells in which the loaded LNP accumulates.

[0586] Embodiment 48. An on-board LNP, (a) Lipid-loaded LNP core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent wherein the loaded LNP exhibits protein expression in about 0.5% to about 50% of respiratory endothelial cells in which the loaded LNP accumulates.

[0587] Embodiment 49. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of respiratory endothelial cells to which the loaded LNP has accumulated.

[0588] Embodiment 50. An on-board LNP, (a) Lipid-loaded LNP core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent wherein the loaded LNPs exhibit protein expression in about 0.5% to about 50% of HeLa cells in which the loaded LNPs have accumulated.

[0589] Embodiment 51. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits protein expression of about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of the HeLa cells to which the loaded LNP accumulated.

[0590] Embodiment 52. An on-board LNP, (a) Lipid-loaded LNP core; (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell, and (c) Cationic Agent wherein the loaded LNP exhibits at least about 20% cellular accumulation in bronchial endothelial cells in a population of bronchial endothelial cells to which the loaded LNP is administered, and exhibits about 5% or more expression in bronchial endothelial cells to which the loaded LNP accumulates.

[0591] Embodiment 53 The loaded LNP of any one of the preceding embodiments, wherein the cationic agent is a cationic lipid. Embodiment 54. The loaded LNP of any one of the preceding embodiments, wherein the cationic agent is a cationic lipid of formula (I).

[0592] Embodiment 55. The loaded LNP of any one of the preceding embodiments, wherein the cationic agent is a cationic lipid of any one of embodiments 1-17. Embodiment 56. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits a cellular accumulation of respiratory endothelial cells of about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% in a population of respiratory endothelial cells to which the loaded LNP is administered.

[0593] Embodiment 57. The loaded LNP of any one of the preceding embodiments, wherein the loaded LNP exhibits expression of about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% in pulmonary endothelial cells in which the loaded LNP accumulates.

[0594] Embodiment 58. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 0.1:1 to about 15:1. Embodiment 59. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 0.2:1 to about 10:1.

[0595] Embodiment 60. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1:1 to about 10:1. Embodiment 61. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1:1 to about 8:1.

[0596] Embodiment 62. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1:1 to about 7:1. Embodiment 63. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1:1 to about 6:1.

[0597] Embodiment 64. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1:1 to about 5:1. Embodiment 65. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1:1 to about 4:1.

[0598] Embodiment 66. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1.25:1 to about 3.75:1. Embodiment 67. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is about 1.25:1.

[0599] Embodiment 68. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is about 2.5:1. Embodiment 69. The loaded LNP of any one of the preceding embodiments, wherein the weight ratio of the cationic agent to the therapeutic and / or prophylactic agent is about 3.75:1.

[0600] Embodiment 70. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 0.1:1 to about 20:1. Embodiment 71. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1.5:1 to about 10:1.

[0601] Embodiment 72. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1.5:1 to about 9:1. Embodiment 73. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1.5:1 to about 8:1.

[0602] Embodiment 74. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1.5:1 to about 7:1. Embodiment 75. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1.5:1 to about 6:1.

[0603] Embodiment 76. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is from about 1.5:1 to about 5:1. Embodiment 77. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is about 1.5:1.

[0604] Embodiment 78. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is about 2:1. Embodiment 79. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is about 3:1.

[0605] Embodiment 80. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is about 4:1. Embodiment 81. The loaded LNP of any one of the preceding embodiments, wherein the molar ratio of the cationic agent to the therapeutic and / or prophylactic agent is about 5:1.

[0606] Embodiment 82. The ionizable lipid is a compound of formula (IL-A):

[0607] [ka]

[0608] or its N-oxide, or a salt or isomer thereof, wherein R 1 is C 5-30 Alkyl, C 5-20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R 2 and R 3 are independently H, C 1-14 Alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 form, together with the atoms to which they are attached, a heterocyclic or carbocyclic ring; R 4 is hydrogen, C 3-6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 12 )2(CH2) n-o Q, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted C 1-6 alkyl, and Q is selected from the group consisting of carbocycle, heterocycle, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -OC(O)O-, -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 8 , -N(R)S(O)R 8 , -O(CH2) n OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR 9 )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 9)N(R)2, -N(OR)C(=CHR 9 )N(R)2, -C(=NR 9 )N(R)2, -C(=NR 9 )R, -C(O)N(R)OR, -(CH2) n N(R)2, -C(R)N(R)2C(O)OR, NR A S(O)2R SX , and

[0609] [ka]

[0610] wherein A is a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O, and S; and a is 1, 2, 3, or 4;

[0611] [ka]

[0612] indicates the point of attachment; each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; R 8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 are H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; R 12 are H, OH, and C 1-3 Alkyl, and C 2-3 alkenyl; Each R is independently C 1-6 Alkyl, C 1-3 Alkyl-aryl, C 2-3 selected from the group consisting of alkenyl, and H; R Ais H and C 1-3 alkyl; R SX is C 3-8 Carbocycle, 3- to 14-membered heterocycle containing one or more heteroatoms selected from N, O, and S, C 1-6 Alkyl, C 2-6 Alkenyl, (C 1-3 Alkoxy)C 1-3 Alkyl, (CH2) p1 O(CH2) p2 R SX1 , and (CH2) p1 R SX1 wherein the carbocyclic and heterocyclic...

Claims

1. Cationic lipids of formula (I): 【Chemistry 1】 or an isomer thereof, R' ｘ teeth, 【Chemistry 2】 and R' ｙ teeth, 【Chemistry 3】 and R' ｚ teeth, 【Chemistry 4】 and 【Chemistry 5】 indicates the point of attachment; R ｘα , R ｘβ , R ｘγ , and R ｘδ are each independently H, C １－１２ Alkyl, and C ２－１２ alkenyl; R ｙα , R ｙβ , R ｙγ , and R ｙδ are each independently H, C １－１２ Alkyl, and C ２－１２ alkenyl; R ｚα , R ｚβ , R ｚγ , and R ｚδ are each independently H, C １－１２ Alkyl, and C ２－１２ alkenyl; R Ｈ is -(CH ２ ) ｑ OH, and q is selected from 1, 2, 3, 4, and 5; Each R Ｔ is independently １－１２ Alkyl and C ２－１２ alkenyl; a is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; b is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; c is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; A － is any pharma- ceutically acceptable anion).

2. R' ｘ teeth, 【Chemistry 6】 and R' ｙ teeth, 【Chemistry 7】 and R' ｚ teeth, 【Chemistry 8】 and 【Chemistry 9】 indicates the point of attachment; R ｘγ is H, C １－１２ Alkyl, and C ２－１２ alkenyl; R ｙγ is H, C １－１２ Alkyl, and C ２－１２ alkenyl; R ｚγ is H, C １－１２ Alkyl, and C ２－１２ alkenyl; R ２ａ , R ２ｂ , R ２ｃ , R ３ａ , R ３ｂ , and R ３ｃ are each independently １－１４ Alkyl and C ２－１４ alkenyl, The cationic lipid according to claim 1.

3. R' ａ teeth, 【Chemistry 10】 and R' ｂ teeth, 【Chemistry 11】 and R' ｃ teeth, 【Chemistry 12】 and 【Chemistry 13】 indicates the point of attachment; R ｘγ is H, C １－１２ Alkyl, and C ２－１２ alkenyl; R ｙγ is H, C １－１２ Alkyl, and C ２－１２ alkenyl; R ２ｃ and R ３ｃ are each independently １－１４ Alkyl and C ２－１４ alkenyl, The cationic lipid according to claim 1.

4. R ｘγ and R ｙγ The compound according to any one of claims 1 to 3, wherein each is H.

5. R ｘγ and R ｙγ are respectively C １－１２ Alkyl or C ２－１２ The compound according to any one of claims 1 to 3, which is alkenyl.

6. R ｘγ is C １－１２ Alkyl or C ２－１２ alkenyl, R ｙγ The compound according to any one of claims 1 to 3, wherein is H.

7. 2. A compound according to any one of the preceding claims, wherein q is 2.

8. The compound is 【Chemistry 14】 Selected from: The compound according to any one of claims 1 to 3.

9. A － is selected from chloride, bromide, iodide, hydroxide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate.

10. A － 2. A compound according to any one of the preceding claims, wherein is selected from bromide, chloride and hydroxide.

11. 10. An empty lipid nanoparticle (empty LNP) comprising a cationic lipid according to any one of the preceding claims.

12. The empty LNP of claim 11 , further comprising an ionizable lipid.

13. The empty LNP of claim 11 or 12, further comprising a phospholipid.

14. The empty LNP of any one of claims 11 to 13, further comprising a structured lipid.

15. The empty LNP of any one of claims 11 to 14, further comprising PEG lipids.

16. Empty LNPs comprising a lipid component comprising about 20 mol% to about 40 mol% of a compound according to any one of claims 1 to 10; about 15 mol% to about 40 mol% of an ionizable lipid, about 0 mol% to about 30 mol% of a phospholipid, about 15 mol% to about 50 mol% of a structured lipid, and about 0 mol% to about 1 mol% of a PEG lipid.

17. A loaded lipid nanoparticle (loaded LNP) comprising the empty LNP of any one of claims 11 to 16 and a therapeutic and / or prophylactic agent.

18. An on-board LNP, (a) an on-board LNP core, (i) an ionizable lipid; (ii) phospholipids, (iii) structured lipids, and (iv) PEG-lipids The mounted LNP core, (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell; and (c) A cationic agent, the cationic agent being a cationic lipid according to any one of claims 1 to 10. The on-board LNP.

19. An on-board LNP, (a) an on-board LNP core, (i) an ionizable lipid; (ii) phospholipids, (iii) structured lipids, and (iv) PEG-lipids The mounted LNP core, (b) a therapeutic and / or prophylactic agent encapsulated within the core for delivery to a cell; and (c) Cationic Agent The on-board LNP.

20. 20. The loaded LNP of claim 19, wherein the cationic agent is a cationic lipid.

21. The loaded LNP of any one of claims 17 to 20, wherein the therapeutic and / or prophylactic agent is a nucleic acid.

22. The loaded LNP of claim 21 , wherein the therapeutic and / or prophylactic agent is ribonucleic acid (RNA).

23. 23. The loaded LNP of claim 22, wherein the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecules, microRNA (miRNA), antagomir, antisense RNA, ribozyme, dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

24. The loaded LNP of claim 23, wherein the RNA is mRNA.

25. The loaded LNP of claim 24, wherein the mRNA is a modified mRNA (mmRNA).

26. The loaded LNP of claim 24 or 25, wherein the mRNA incorporates a microRNA binding site (miR binding site).

27. The loaded LNP of any one of claims 24 to 26, wherein the mRNA comprises one or more of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and / or a 5' cap structure.

28. The ionizable lipid is a compound of formula (IL-A): 【Chemistry 15】 or an N-oxide thereof, or a salt or isomer thereof, R １ is C ５－３０ Alkyl, C ５－２０ selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R ２ and R ３ are independently H, C １－１４ Alkyl, C ２－１４ alkenyl, -R*YR", -YR", and -R*OR", or R ２ and R ３ together with the atom to which they are attached form a heterocyclic or carbocyclic ring; R ４ is hydrogen, C ３－６ Carbocycle, -(CH ２ ) ｎ Q, -(CH ２ ) ｎ CHQR, -(CH ２ ) ｏ C(R １２ ) ２ (CH ２ ) ｎ－ｏ Q, -CHQR, -CQ(R) ２ , -C(O)NQR and unsubstituted C １－６ alkyl; Q is selected from the group consisting of carbocycle, heterocycle, -OR, -O(CH ２ ) ｎ N (R) ２ , -C(O)OR, -OC(O)R, -OC(O)O-, -CX ３ , -CX ２ H, -CXH ２ , -CN, -N(R) ２ , -C(O)N(R) ２ , -N(R)C(O)R, -N(R)S(O) ２ R, -N(R)C(O)N(R) ２ , -N(R)C(S)N(R) ２ , -N(R)R ８ , -N(R)S(O) ２ R ８ , -O(CH ２ ) ｎ OR, -N(R)C(=NR ９ ) N (R) ２ , -N(R)C(=CHR ９ ) N (R) ２ , -OC(O)N(R) ２ , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) ２ R, -N(OR)C(O)OR, -N(OR)C(O)N(R) ２ , -N(OR)C(S)N(R) ２ , -N(OR)C(=NR ９ ) N (R) ２ , -N(OR)C(=CHR ９ ) N (R) ２ , -C(=NR ９ ) N (R) ２ , -C(=NR ９ )R, -C(O)N(R)OR, -(CH ２ ) ｎ N (R) ２ and -C(R)N(R) ２ C(O)OR,NR Ａ S (O) ２ R ＳＸ , and 【Chemistry 16】 where A is a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S; a is 1, 2, 3, or 4; 【Chemistry 17】 indicates the point of attachment; each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; R ８ is C ３－６ selected from the group consisting of carbocycle and heterocycle; R ９ H, CN, NO ２ , C １－６ Alkyl, -OR, -S(O) ２ R, -S(O) ２ N (R) ２ , C ２－６ Alkenyl, C ３－６ selected from the group consisting of carbocycle and heterocycle; R １２ is H, OH, C １－３ Alkyl, and C ２－３ alkenyl; Each R is independently C １－６ Alkyl, C １－３ Alkyl-Aryl, C ２－３ alkenyl, and H; R Ａ is H and C １－３ alkyl; R ＳＸ is C ３－８ Carbocycle, 3- to 14-membered heterocycle containing one or more heteroatoms selected from N, O and S, C １－６ Alkyl, C ２－６ Alkenyl, (C １－３ Alkoxy)C １－３ Alkyl, (CH ２ ) ｐ１ O (CH ２ ) ｐ２ R ＳＸ１ , and (CH ２ ) ｐ１ R ＳＸ１ wherein the carbocycle and heterocycle are selected from oxo, C １－６ Alkyl, and (C １－３ Alkoxy)C １－３ optionally substituted with one or more groups selected from alkyl; R ＳＸ１ is C(O)NR １４ R １４ ', C ３－８ and a 3- to 14-membered heterocycle containing one or more heteroatoms selected from N, O, and S, wherein the carbocycle and heterocycle are each selected from oxo, halo, C １－３ Alkyl, (C １－３ Alkoxy)C １－３ Alkyl, C １－６ Alkylamino, di-(C １－６ alkyl)amino, and NH ２ and optionally substituted with one or more groups selected from Each R １３ OH, oxo, halo, C １－６ Alkyl, C １－６ Alkoxy, C ２－６ Alkenyl, C １－６ Alkylamino, di-(C １－６ alkyl)amino, NH ２ , C(O)NH ２ , CN, and NO ２ selected from the group consisting of: R １４ and R １４’ are each independently H and C １－６ selected from the group consisting of alkyl; p １ is selected from 1, 2, 3, 4, and 5; p ２ is selected from 1, 2, 3, 4, and 5; Each R ５ are independently OH, C １－３ Alkyl, C ２－３ alkenyl, and H; Each R ６ are independently OH, C １－３ Alkyl, C ２－３ alkenyl, and H; R ７ is C １－３ Alkyl, C ２－３ alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)-M"-C(O)O-, -C(O)N(R Ｍ ) -, -N(R Ｍ )C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR Ｍ ) O-, -S(O) ２ M' is selected from -, -S-S-, an aryl group, and a heteroaryl group; M' is a bond, C １－１３ Alkyl or C ２－１３ alkenyl; Each R Ｍ are independently H, C １－６ Alkyl and C ２－６ alkenyl; Each R' is independently C １－１８ Alkyl, C ２－１８ Alkenyl, -R*YR", -YR", (CH ２ ) ｑ’ is selected from the group consisting of: OR*, and H; each q' is independently selected from 1, 2, and 3; Each R" is independently C ３－１５ Alkyl and C ３－１５ alkenyl; Each R* is independently C １－１２ Alkyl and C ２－１２ alkenyl; Each Y is independently C ３－６ is a carbocyclic ring; Each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; An empty or loaded LNP according to any one of claims 12 to 27.

29. The ionizable lipid is a compound of formula (IL-B): 【Chemistry 18】 or an N-oxide thereof, or a salt or isomer thereof, R' ａ is R' 分岐状 and R' 分岐状 teeth, 【Chemistry 19】 and 【Chemistry 20】 indicates the point of attachment; R ａα , R ａβ , R ａγ , and R ａδ are each independently H, C ２－１２ Alkyl, and C ２－１２ alkenyl; R ２ and R ３ are each independently １－１４ Alkyl and C ２－１４ alkenyl; R ４ is -(CH ２ ) ｎ OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; and 【Chemistry 21】 is selected from the group consisting of 【Chemical 22】 indicates the point of attachment; R １０ is N(R) ２ each R is independently C １－６ Alkyl, C ２－３ alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R ５ is independently １－３ Alkyl, C ２－３ alkenyl, and H; Each R ６ is independently １－３ Alkyl, C ２－３ alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C １－１２ Alkyl or C ２－１２ alkenyl; l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

29. The empty or loaded LNP of claim 28,

30. The ionizable lipid has the compound of formula (IL-C): 【Chemistry 23】 or a salt or isomer thereof, l is selected from 1, 2, 3, 4, and 5; M １ is M'; R ４ is -(CH ２ ) ｎ Q, Q is OH and n is selected from 1, 2, 3, 4, or 5; M and M' are independently selected from -C(O)O-, and -OC(O)-; R ２ and R ３ In either case, C １－１４ Alkyl, or C ２－１４ alkenyl; R' is C １ -C １２ is a linear alkyl 29. The empty or loaded LNP of claim 28,

31. The ionizable lipid has the compound of formula (IL-D): 【Chemistry 24】 or an N-oxide thereof, or a salt or isomer thereof, R' ａ is R' 分岐状 or R' 環状 and R' 分岐状 teeth, 【Chemistry 25】 and R' ｂ teeth, 【Chemistry 26】 and 【Chemistry 27】 indicates the point of attachment; R ａγ is C １－１２ Alkyl and C ２－１２ alkenyl; R ２ and R ３ are each independently １－１４ Alkyl and C ２－１４ alkenyl; R ４ is -(CH ２ ) ｎ OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; R' is C １－１２ Alkyl or C ２－１２ alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

29. The empty or loaded LNP of claim 28,

32. The ionizable lipid is a compound of formula (IL-III): 【Chemistry 28】 or a salt or isomer thereof, W is 【Chemical 29】 and Ring A is 【Chemistry 30】 and t is 1 or 2; A １ and A ２ are each independently selected from CH or N; Z is CH ２ or absent, Z is CH ２ when Z is absent, then dashed lines (1) and (2) each represent a single bond; when Z is absent, then dashed lines (1) and (2) are both absent; R １ , R ２ , R ３ , R ４ , and R ５ is independently ５－２０ Alkyl, C ５－２０ alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; R Ｘ１ and R Ｘ２ are each independently H or C １ - ３ is alkyl; Each M is independently -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O), ２ selected from the group consisting of -, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group; M* is C １ -C ６ is alkyl, W １ and W ２ are each independently —O— and —N(R ６ )- selected from the group consisting of; Each R ６ are independently H and C １－５ selected from the group consisting of alkyl; X １ , X ２ , and X ３ are independently a bond, -CH ２ -, -(CH ２ ) ２ -, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -(CH ２ ) ｎ -C(O)-, -C(O)-(CH ２ ) ｎ -, -(CH ２ ) ｎ -C(O)O-, -OC(O)-(CH ２ ) ｎ -, -(CH ２ ) ｎ -OC(O)-, -C(O)O-(CH ２ ) ｎ selected from the group consisting of -, -CH(OH)-, -C(S)-, and -CH(SH)-; Each Y is independently C ３－６ is a carbocyclic ring; Each R* is independently C １－１２ Alkyl and C ２－１２ alkenyl; Each R is independently C １－３ Alkyl and C ３－６ carbocycle; Each R' is independently C １－１２ Alkyl, C ２－１２ alkenyl, and H; Each R" is independently C ３－１２ Alkyl, C ３－１２ selected from the group consisting of alkenyl and -R*MR'; n is an integer from 1 to 6.

28. The empty or loaded LNP of any one of claims 12 to 27,

33. The ionizable lipid is a compound of formula (IL-IIIA): 【Chemistry 31】 or a salt or isomer thereof, R １ , R ２ , R ３ , R ４ , and R ５ is independently ５－２０ Alkyl, C ５－２０ alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; Each M is independently -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O), ２ -, an aryl group, and a heteroaryl group; X １ , X ２ , and X ３ are independently a bond, -CH ２ -, -(CH ２ ) ２ -, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH ２ --, --CH ２ -C(O)-, -C(O)O-CH ２ -, -OC(O)-CH ２ --, --CH ２ -C(O)O-, -CH ２ selected from the group consisting of -OC(O)-, -CH(OH)-, -C(S)-, and -CH(SH)-; Each Y is independently C ３－６ is a carbocyclic ring; Each R* is independently C １－１２ Alkyl and C ２－１２ alkenyl; Each R is independently C １－３ Alkyl and C ３－６ carbocycle; Each R' is independently C １－１２ Alkyl, C ２－１２ alkenyl, and H; Each R" is independently C ３－１２ Alkyl and C ３－１２ alkenyl) 33. The empty or loaded LNP of claim 32,

34. R １ , R ２ , R ３ , R ４ , and R ５ are respectively C ５－２０ is alkyl; １ is -CH ２ - and X ２ and X ３ The empty or loaded LNP of claim 32 or 33, wherein each of is -C(O)-.

35. The ionizable lipid is 【Chemistry 32】 The empty or loaded LNP of any one of claims 12 to 27, wherein the compound is selected from the group consisting of:

36. 28. The empty or loaded LNP of any one of claims 12 to 27, wherein the ionizable lipid is a compound selected from the compounds of tables IL-1 to IL-7.

37. The phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diureto ... 1-Oleyl-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-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE). The empty or loaded LNP of any one of claims 13 to 36, wherein the LNP is selected from the group consisting of sphingomyelin, sphingomyelin, and mixtures thereof.

38. 38. The empty or loaded LNP of claim 37, wherein the phospholipid is DSPC.

39. 39. The empty or loaded LNP of any one of claims 14 to 38, wherein the structured lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

40. The structured lipid is 【Chemical 33】 40. The empty or loaded LNP of claim 39, which is:

41. 41. The empty or loaded LNP of any one of claims 15-40, wherein the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

42. The PEG lipid has the following structure: 【Chemical 34】 or a salt thereof, wherein r is an integer from 1 to 100; and s is an integer from 1 to 100.

43. The PEG lipid may be selected from the group consisting of the following compounds: 【Chemistry 35】 or a salt thereof.

44. 44. A pharmaceutical composition comprising the loaded LNP of any one of claims 17 to 43 and a pharma- ceutically acceptable carrier.

45. A method for delivering a therapeutic and / or prophylactic agent to cells in a subject, the method comprising administering to the subject a loaded LNP described in any one of claims 17 to 43, or a pharmaceutical composition described in claim 44.

46. A method for producing a polypeptide of interest in a cell in a subject, the method comprising administering to the subject a loaded LNP described in any one of claims 17 to 43, or a pharmaceutical composition described in claim 44.

47. 47. The method of claim 45 or 46, wherein the cell is an endothelial cell.

48. 48. The method of claim 47, wherein the endothelial cell is a pulmonary endothelial cell, a respiratory endothelial cell, or a bronchial endothelial cell.

49. A method for specifically delivering a therapeutic and / or prophylactic agent to an organ of a subject, the method comprising administering to the subject a loaded LNP described in any one of claims 17 to 43, or a pharmaceutical composition described in claim 44.

50. 50. The method of claim 49, wherein the organ is a lung.

51. A method for specifically delivering a therapeutic and / or prophylactic agent to a tissue of a subject, the method comprising administering to the subject a loaded LNP described in any one of claims 17 to 43, or a pharmaceutical composition described in claim 44.

52. 52. The method of claim 51, wherein the tissue is endothelium.

53. 52. The method of claim 51, wherein the tissue is pulmonary endothelium.

54. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the loaded LNP of any one of claims 17 to 43, or the pharmaceutical composition of claim 44.

55. 55. The method of any one of claims 45 to 54, wherein said administering is performed parenterally, intramuscularly, intradermally, subcutaneously, and / or intravenously.

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