Lipid nanoparticles and methods of delivering nucleic acids

Abstract

A lipid nanoparticle and a method of delivering a nucleic acid encapsulated in the lipid nanoparticle to a cell or a subject. The lipid nanoparticle may contain 55-65 mol% of a cationic lipid to the total lipid amount of the lipid nanoparticle; 30-40 mol% of a sterol to the total lipid amount of the lipid nanoparticle; 1-3 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to the total lipid amount of the lipid nanoparticle; and 1-5 mol% of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) to the total lipid amount of the lipid nanoparticle. The lipid nanoparticle may also contain 55-65 mol% of a cationic lipid to a total lipid amount of the lipid nanoparticle; 30-40 mol% of a sterol to the total lipid amount of the lipid nanoparticle; 4-6 mol% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to the total lipid amount of the lipid nanoparticle; and 1-5 mol% of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) to the total lipid amount of the lipid nanoparticle.

Description

NDJP.024WO PATENTLIPID NANOPARTICLES AND METHODS OF DELIVERING NUCLEIC ACIDSINCORPORATION BY REFERENCE TO PRIORITY APPLICATIONThis application claims priority to U. S. Provisional Application No. 63 / 739,079, filed December 26, 2024, which is hereby incorporated by reference in its entirety.FIELD

[0001] The present disclosure relates to a lipid nanoparticle and a method of delivering a nucleic acid encapsulated in the lipid nanoparticle to a cell or a subject.BACKGROUND

[0002] Lipid nanoparticles (LNPs) are used as carriers to encapsulate lipophilic drugs and nucleic acids such as siRNA (short interfering RNA) and mRNA to deliver to target cells and organs. For example, lipid nanoparticles comprising cationic lipids as constituent lipids are reported as lipid nanoparticles which serve as carriers to efficiently deliver nucleic acids such as siRNA into target cells (See, U. S. Pat. Nos. 8,058,069 and 8,158,601).SUMMARY

[0003] If the drugs in the LNPs could be delivered to desired organs, less off-target effects will be seen. Thus, there is a need to develop LNPs which show preferential delivery to specific organs.

[0004] The present disclosure relates to a lipid nanoparticle that has preferential delivery property to a liver in a subject.

[0005] Specifically, the present disclosure includes the following embodiments [1] to [9]:[1] A lipid nanoparticle, comprising:55-65 mol% of a cationic lipid to the total lipid amount of the lipid nanoparticle; 30-40 mol% of a sterol to the total lipid amount of the lipid nanoparticle;1-3 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to the total lipid amount of the lipid nanoparticle; and1-5 mol% of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG- PEG) to the total lipid amount of the lipid nanoparticle.[2] A lipid nanoparticle, comprising:55-65 mol% of a cationic lipid to the total lipid amount of the lipid nanoparticle; 30-40 mol% of a sterol to the total lipid amount of the lipid nanoparticle;4-6 mol% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to the total lipid amount of the lipid nanoparticle; and1-5 mol% of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG- PEG) to the total lipid amount of the lipid nanoparticle.[3] The lipid nanoparticle of [1] or [2], wherein the cationic lipid is a compound represented by Formula I or a pharmaceutically acceptable salt thereof:(R1)(R2)C(OH)–(CH2)a–(O–CO)b–XFormula Iwherein:a represents an integer of 3-5;b represents 0 or 1;R1and R2each independently represent a group represented by Formula A:(R11)(R12)–CH–(CO–O)c–(CH2)v–Formula Awherein:R11and R12each independently represent a linear or branched C5-15 alkyl group; each c represents 0 or 1;each v represents an integer of 4-12; andX represents a 5- to 7-membered non-aromatic heterocyclic group, wherein a carbon atom of said heterocyclic group is bound to (O-CO)b- and one or two hydrogen atoms of said heterocyclic group may optionally be replaced with a C-M alkyl group or C2-4 alkenyl group, orX represents a group represented by Formula B:–(CH2)d–N(R3)(R4)Formula Bwherein:d represents an integer of 0-3; andR3and R4each independently represent a Ci.?, alkyl group or C2-4 alkenyl group, orR3and R4are bound to each other to form a 5- to 7-membered non-aromatic heterocyclic group, wherein one or two hydrogen atoms of said heterocyclic group may optionally be replaced with a C1-4alkyl group or C2-4alkenyl group.[4] The lipid nanoparticle of [3], wherein the cationic lipid is a compound represented by Formula IA or a pharmaceutically acceptable salt thereof, or Formula IB or a pharmaceutically acceptable salt thereof:[5] The lipid nanoparticle of [1] or [2], wherein the cationic lipid is a compound represented by Formula II or a pharmaceutically acceptable salt thereof:Formula IIwherein:R1and R3are each independently a linear C5-Ci8alkyl group,R2and R4are each independently a hydrogen or a linear C5-C18alkyl group, provided that R2and R4are not hydrogen at the same time,n1 and n2 are each independently 2 or 3,A1 isthe arrow indicates a connection to the carbonyl group,R11 and R12 are the same or different and are a hydrogen or a hydroxy group, R13, R15, R16, and R17 are the same or different and are a linear C1 to C4 alkyl group,R14 is a linear C1 to C4 alkyl group or — (CH2)2 — OH, andX- is a pharmaceutically acceptable counter anion.[6] The lipid nanoparticle of [5], wherein R1 and R3 are n-octyl group, and R2 and R4 are n-hexyl group.[7] The lipid nanoparticle of [5], wherein n1 and n2 are 2.[8] The lipid nanoparticle of [5], wherein the cationic lipid is a compound represented by Formula HA or a pharmaceutically acceptable salt thereof:Formula IIA.[9] The lipid nanoparticle of [5], wherein the cationic lipid is a compound represented by Formula III or a pharmaceutically acceptable salt thereof:HO.,0^,'N9 oCompound III

[0010] The lipid nanoparticle of any one of [1] to [9], wherein a nucleic acid is encapsulated in the lipid nanoparticle.

[0011] A method of delivering the nucleic acid to a cell, comprising contacting the cell with the lipid nanoparticle of

[0010] ,

[0012] A method of delivering the nucleic acid to a subject in need thereof, comprising administering to the subject the lipid nanoparticle of

[0010] ,

[0013] A compound represented by Formula II or a pharmaceutically acceptable salt thereof:Formula Iwherein:R1 and R3 are each independently a linear C5-C18 alkyl group,R2 and R4 are each independently a hydrogen or a linear C5-C18 alkyl group, provided that R2 and R4 are not hydrogen at the same time,n1 and n2 are each independently 2 or 3,the arrow indicates a connection to the carbonyl group,R11 and R12 are the same or different and are a hydrogen or a hydroxy group, R13, R15, R16, and R17 are the same or different and are a linear C1 to C4 alkyl group,R14 is a linear C1 to C4 alkyl group or — (CH2)2 — OH, andX- is a pharmaceutically acceptable counter anion.

[0014] The compound of

[0013] or a pharmaceutically acceptable salt thereof, wherein R1and R3are n-octyl group, and R2and R4are n-hexyl group.

[0015] The compound of

[0013] or a pharmaceutically acceptable salt thereof, wherein n1 and n2 are 2.

[0016] The compound of

[0013] or a pharmaceutically acceptable salt thereof, wherein the cationic lipid is a compound represented by Formula IIA or a pharmaceutically acceptable salt thereof:Formula IIA.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Fig. 1 is a plot illustrating plasma hEPO protein levels of mice as measured after the lipid nanoparticles (LNPs) 1a, 1b, or R1 a were administered.

[0007] Fig. 2 is a plot illustrating average Flue mRNA levels in the lung, liver, spleen, kidney and heart of mice as measured after the LNPs (2a-1, 2b- 1, 2b-2, 2c-1, 2c-2, R2a, R2b, R2c) were administered.

[0008] Fig. 3 is a plot illustrating the ratio of Flue mRNA expression in the liver to mRNA expression in the spleen, and the ratio of Flue mRNA expression in the liver to total Flue mRNA expression after the LNPs (2a-1, 2b- 1, 2b-2, 2c-1, 2c-2, R2a, R2b, R2c) were administered.

[0009] Fig. 4 is a plot illustrating the body weight of mice as measured at various times after the LNPs (4a, R4a) were administered.

[0010] Fig. 5 is a plot illustrating the AST (aspartate aminotransferase) levels of mice as measured at various times after the LNPs (4a, R4a) were administered.

[0011] Fig. 6 is a plot illustrating the ALT (alanine aminotransferase) levels of mice as measured at various times after the LNPs (4a, R4a) were administered.

[0012] Fig. 7 is a plot illustrating the MCP-1 (monocyte chemotactic protein-1) levels of mice as measured at various times after the LNPs (4a, R4a) were administered.DETAILED DESCRIPTIONDefinition

[0013] In this application, " X1-X2” (where X1 and X2 are real numbers satisfying X1 < X2) means " X1 or more and X2 or less".

[0014] The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0015] As used herein, the term “cationic lipid” includes an ionizable cationic lipid and permanently cationic lipid. As used herein, the term “ionizable cationic lipid” refers to a lipid that comprises an ionizable moiety capable of bearing a positive charge under certain conditions (e.g., at a certain pH range, e.g., under physiological conditions). The ionizable moiety may comprise an amine. In addition to the ionizable moiety, the ionizable cationic lipid may contain an alkyl or alkenyl group. As used herein, the term “permanently cationic lipid” refers to a lipid that comprises a cationic moiety that is positively charged at any pH range. The permanently cationic moiety may comprise a quaternary amine. In addition to the cationic moiety, the permanently cationic lipid may contain an alkyl or alkenyl group.

[0016] As used herein, the term “alkyl” refers to a linear or a branched hydrocarbyl radical of a saturated aliphatic group, which can be of any length unless otherwise specified. The term “C1-C4 alkyl” in the present disclosure may include methyl, ethyl, n-propyl, n-butyl, isopropyl, tert-butyl, and isobutyl. The term “C5-15 alkyl” in the present disclosure may include pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, and branched isomers thereof. The term “C5-18 alkyl” in the present disclosure may include pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, and branched isomers thereof.

[0017] As used herein, the term “alkenyl” refers to a linear or a branched hydrocarbyl radical having at least one carbon-carbon double bond, which can be of anylength unless otherwise specified. The term “C2- alkenyl” in the present disclosure may include ethenyl, propenyl, 1-butenyl, and 2-butenyl.

[0018] The term “5- to 7-membered non-aromatic heterocyclic group” in the present disclosure may include tetra hydrofuran, pyrrolidine, tetrahydrothiophene, piperidine, morpholine, thiopyran, azepane, oxepane, and thiepane.

[0019] As used herein, the term “pharmaceutically acceptable” refers to being compatible with use in subjects, for example, mammals such as human.

[0020] The pharmaceutically acceptable counter anion of the present disclosure includes, but is not limited to, a chloride, bromide, fluoride, iodide, nitrate, sulfate, methyl sulfate, phosphate, acetate, benzoate, citrate, glutamate, and lactate.

[0021] The pharmaceutically acceptable salt of the present disclosure includes, but not limited to, salts containing a chloride, bromide, fluoride, iodide, nitrate, sulfate, methyl sulfate, phosphate, acetate, benzoate, citrate, glutamate, and / or lactate. The pharmaceutically acceptable salt of the compound of Formula I of the present disclosure can be synthesized by conventional chemical methods. For example, the salt of the compound is prepared either by ion exchange chromatography or by reacting the free base in the compound with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.

[0022] In general, a compound may contain one or more chiral centers. Compounds containing one or more chiral centers may include those described as an “isomer”, a “stereoisomer”, a “diastereomer”, an “enantiomer”, an “optical isomer”, or a “racemic mixture”. Conventions for stereochemical nomenclature, for example the stereoisomer naming rules of Cahn, Ingold and Prelog, as well as methods for the determination of stereochemistry and the separation of stereoisomers are known in the art. See, for example, Michael B. Smith and Jerry March, March's Advanced Organic Chemistry, 5th edition, 2001. The compounds and structures of the present disclosure, including chemical drawings, are meant to encompass all possible isomers, chemically reasonable positional isomers, stereoisomers, diastereomers, enantiomers, and / or optical isomers that would be understood to exist for the specified compound or structure, including any mixture, racemic or otherwise, thereof.

[0023] As used herein, the “average particle size of the lipid nanoparticles” means Z-average particle size measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be carried out by usual method using commercially available DLS equipment, etc.

[0024] As used herein, the “N / P ratio” is the ratio of the number of cationic nitrogen atoms (N) of a cationic lipid in a lipid nanoparticle to the number of phosphate residues (P) of nucleic acids encapsulated in the lipid nanoparticle.Lipid nanoparticle

[0025] In one aspect, the present disclosure relates to a lipid nanoparticle comprising a cationic lipid, a sterol, a phospholipid and a polyalkylene glycol-modified lipid (hereinafter may be referred to as “the lipid nanoparticle according to the present disclosure”).

[0026] Examples of the cationic lipid in the lipid nanoparticle according to the present disclosure includes, but not limited to, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N–(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N–(1-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. CI), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. CI), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N, N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N, N-Dioleylamino)-1,2-propanedio (DOAP), bis(3-pentyloctyl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate, 1,2-Dilinoleyloxo-3-(2-N, N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N, N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS) — N, N-dimethyl-2,2-di((9Z,12Z)-octadeca-9, 12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN 100), (6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1, 1 '-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [(4-Hydroxybutyl)azanediyl]di(hexane-6, 1 - diyl) bis(2-hexyldecanoate) (ALC-0315), 2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N, N-dimethyl-2-oxoethan-aminium bromide (HEDC), ((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate (S104), ((2- (3,4-dihydroxypyrrolidin-1 -yl)acetyl)azanediyl)bis(ethane-2, 1 -diyl) (9Z,9'Z, 12Z, 12'Z)-bis(octadeca-9,12-dienoate), ((2-((3S,4R)-3,4-dihydroxypyrrolidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate), or a mixture thereof. Other lipids are described, e.g., in U. S. Pat. Nos. 9,011,903, 9,308,267, 10,167,253, and U. S. Pat. Pub. No. 2024 / 0024252, which are incorporated herein by references.

[0027] In some embodiments, the cationic lipid in the lipid nanoparticle according to the present disclosure is a compound of Formula I or a pharmaceutically acceptable salt thereof:(R1)(R2)C(OH)–(CH2)a–(O–CO)b–XFormula Iwherein:a represents an integer of 3-5;b represents 0 or 1;R1 and R2 each independently represent a group represented by Formula A:(R" XR’!)-CH-(CO-O)C-(CH!)V- Formula Awherein:R11 and R12 each independently represent a linear or branched C5-15 alkyl group; each c represents 0 or 1;each v represents an integer of 4-12; andX represents a 5- to 7-membered non-aromatic heterocyclic group, wherein a carbon atom of said heterocyclic group is bound to (O-CO)b- and one or two hydrogen atoms of said heterocyclic group may optionally be replaced with a C1-4 alkyl group or C2-4 alkenyl group, orX represents a group represented by Formula B:-CGH^d-NCR’XR4)Formula Bwherein:d represents an integer of 0-3; andR3 and R4 ach independently represent a C1-4 alkyl group or C2-4 alkenyl group, or R3 and R4 are bound to each other to form a 5- to 7-membered non-aromatic heterocyclic group, wherein one or two hydrogen atoms of said heterocyclic group may optionally be replaced with a C1-4 alkyl group or C2-4 alkenyl group.

[0028] The compound of Formula I is disclosed in U. S. Pat. Pub. No.2024 / 0024252, which is hereby expressly incorporated by reference in its entirety.

[0029] In some embodiments, the cationic lipid in the lipid nanoparticle according to the present disclosure is a compound of Formula IA or a pharmaceutically acceptable salt thereof, or Formula IB or a pharmaceutically acceptable salt thereof.. OHNFormula IA. OHNFormula IB

[0030] In some embodiments, the cationic lipid in the lipid nanoparticle according to the present disclosure is a compound of Formula II or a pharmaceutically acceptable salt thereof.Formula IIwherein:R1and R3are each independently a linear C5-C18alkyl group,R2and R4are each independently a hydrogen or a linear C5-C18alkyl group, provided that R2and R4are not hydrogen at the same time,n1 and n2 are each independently 2 or 3,the arrow indicates a connection to the carbonyl group,R11and R12are the same or different and are a hydrogen or a hydroxy group, R13, R15, R16, and R17are the same or different and are a linear C1to C4alkyl group, R14is a linear C1to C4alkyl group or —(CH2)2—OH, andX‘ is a pharmaceutically acceptable counteranion.

[0031] In some embodiments, R1and R3in Formula II are n-octyl group, and R2and R4in Formula II are n-hexyl group.

[0032] in some embodiments, n1 and n2 in Formula II are 2.

[0033] In some embodiments, a compound of Formula II is a compound of Formula HA:.0 ooFormula IIA.

[0034] The compound of Formula II can be prepared in view of the synthesis of the ionizable lipid compound of formula I in PCT Publication No. WO2013 / 185116, which is hereby expressly incorporated by reference in its entirety, and the following synthesis of Compound HA.

[0035] The phospholipid in the lipid nanoparticle according to the present disclosure can include a glycerophospholipid such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphorylcholine, cardiolipin, plasmalogen, ceramide phosphorylglycerol phosphate, phosphatidic acid; sphingophospholipids such as sphingomyelin, ceramide phosphorylglycerol, ceramide phosphoryl ethanolamine; etc. In addition, phospholipids derived from natural products such as egg yolk lecithin and soy lecithin can also be used. Fatty acid residues in glycerophospholipids and sphingophospholipids are not particularly limited, but can include, for example, saturated or unsaturated fatty acid residues having carbon number of 12-24, saturated or unsaturated fatty acid residues having carbon number of 14-20 are preferable. Specifically, acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, and lignoceric acid can be included. When these glycerolipids or sphingolipids have two or more fatty acid residues, all fatty acid residues may be the same group or may be different group from each other. Examples of the phospholipids includes diphytanoyl phosphatidyl ethanolamine (DPhPE) and 1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine (DPhPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0036] The sterol in the lipid nanoparticle according to the present disclosure include, for example, animal-derived sterols such as cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, p-sitosterol, campesterol, brassicasterol; microorganism-derived sterols such as zymosterol and ergosterol, etc.

[0037] The polyalkylene glycol in the polyalkylene glycol-modified lipid in the lipid nanoparticle according to the present disclosure includes, for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, etc. The average molecular weight of polyalkylene glycol is, for example, approximately between 200 and 10,000, preferably approximately between 500 and 10,000, further preferably approximately between 1,000 and 5,000. In some embodiments, the molecular weight of polyalkylene glycol is about 200, 300, 350, 400, 500, 550, 750, 1000, 1500, 2000, 3000, 3500, 4000, 5000 or 10,000 Da.

[0038] The polyalkylene glycol-modified lipid in the lipid nanoparticle according to the present disclosure includes, for example, stearylated polyethylene glycol (e.g., PEG-45 stearate (STR-PEG45), etc.), N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), N-[carbonyl-methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), N-[carbonyl-methoxypolyethylene glycol]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG). For example, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG2000), n-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG750), N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000), N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine(DSPE-PEG5000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-750 (DMG-PEG750), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DMG-PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG750), N- [carbonyl-methoxypolyethylene glycol2000]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2000), N-[carbonyl-methoxypolyethylene glycol5000]- 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG5000) etc. can be used.

[0039] In some embodiments, the lipid nanoparticle according to the present disclosure comprises:• 55-65 mol% of a cationic lipid to the total lipid amount of the lipid nanoparticle • 30-50 mol% of a sterol to the total lipid amount of the lipid nanoparticle;• 1-3 mol% of DOPE to the total lipid amount of the lipid nanoparticle; and• 1-5 mol% of DMG-PEG to the total lipid amount of the lipid nanoparticle.

[0040] This lipid nanoparticle may be referred to as “lipid nanoparticle A” hereinbelow.

[0041] In some embodiments, the lipid nanoparticle A comprises 55.00-65.00, 55.25-65.00, 55.50-65.00, 55.75-65.00, 56.00-65.00, 56.25-65.00, 56.50-65.00, 56.75-65.00, 57.00-65.00, 57.25-65.00, 57.50-65.00, 57.75-65.00, 58.00-65.00, 58.25-65.00, 58.50-65.00, 58.75-65.00, 59.00-65.00, 59.25-65.00, 59.50-65.00, 59.75-65.00, 60.00-65.00, 60.25-65.00, 60.50-65.00, 60.75-65.00, 61.00-65.00, 61.25-65.00, 61.50-65.00, 61.75-65.00, 62.00-65.00, 62.25-65.00, 62.50-65.00, 62.75-65.00, 63.00-65.00, 63.25-65.00, 63.50-65.00, 63.75-65.00, 64.00-65.00, 64.25-65.00, 64.50-65.00 or 64.75-65.00 mol% of the cationic lipid to the total lipid amount of the lipid nanoparticle. In some embodiments, the lipid nanoparticle A comprises 30.00-50.00, 30.25-50.00, 30.50-50.00, 30.75-50.00, 31.00-50.00, 31.25-50.00, 31.50-50.00, 31.75-50.00, 32.00-50.00, 32.25-50.00, 32.50-50.00, 32.75-50.00, 33.00-50.00, 33.25-50.00, 33.50-50.00, 33.75-50.00, 34.00-50.00, 34.25-50.00, 34.50-50.00, 34.75-50.00, 35.00-50.00, 35.25-50.00, 35.50-50.00, 35.75-50.00, 36.00-50.00, 36.25-50.00, 36.50-50.00, 36.75-50.00, 37.00-50.00, 37.25-50.00, 37.50-50.00, 37.75-50.00, 38.00-50.00, 38.25-50.00, 38.50-50.00, 38.75-50.00, 39.00-50.00, 39.25-50.00, 39.50-50.00, 39.75-50.00, 40.00-50.00, 40.25-50.00, 40.50-50.00, 40.75-50.00, 41.00-50.00, 41.25-50.00, 41.50-50.00, 41.75-50.00, 42.00-50.00, 42.25-50.00, 42.50-50.00, 42.75-50.00, 43.00-50.00, 43.25-50.00, 43.50-50.00, 43.75-50.00, 44.00-50.00, 44.25-50.00, 44.50-50.00, 44.75-50.00, 45.00-50.00, 45.25-50.00, 45.50-50.00, 45.75-50.00, 46.00-50.00, 46.25-50.00, 46.50-50.00, 46.75-50.00, 47.00-50.00, 47.25-50.00, 47.50-50.00, 47.75-50.00, 48.00-50.00, 48.25-50.00, 48.50-50.00, 48.75-50.00, 49.00-50.00, 49.25-50.00, 49.50-50.00, 49.75-50.00, 30.00-40.00, 30.25-40.00, 30.50-40.00, 30.75-40.00, 31.00-40.00, 31.25-40.00, 31.50-40.00, 31.75-40.00, 32.00-40.00, 32.25-40.00, 32.50-40.00, 32.75-40.00, 33.00-40.00, 33.25-40.00, 33.50-40.00, 33.75-40.00, 34.00-40.00, 34.25-40.00, 34.50-40.00, 34.75-40.00, 35.00-40.00, 35.25-40.00, 35.50-40.00, 35.75-40.00, 36.00-40.00, 36.25-40.00, 36.50-40.00, 36.75-40.00, 37.00-40.00, 37.25-40.00, 37.50-40.00, 37.75-40.00, 38.00-40.00, 38.25-40.00, 38.50-40.00, 38.75-40.00, 39.00-40.00, 39.25-40.00, 39.50-40.00 or 39.75-40.00 mol% of the sterol (such as cholesterol) to the total lipid amountof the lipid nanoparticle. In some embodiments, the lipid nanoparticle A comprises 1.00-3.00, 1.25-3.00, 1.50-3.00, 1.75-3.00, 2.00-3.00, 2.25-3.00, 2.50-3.00, 2.75-3.00 mol% of DOPE to the total lipid amount of the lipid nanoparticle. In some embodiments, the lipid nanoparticle A comprises 1.00-5.00, 1.25-5.00, 1.50-5.00, 1.75-5.00, 2.00-5.00, 2.25-5.00, 2.50-5.00, 2.75-5.00, 3.00-5.00, 3.25-5.00, 3.50-5.00, 3.75-5.00, 4.00-5.00, 4.25-5.00, 4.50-5.00,4.75-5.00, 1.00-4.00, 1.25-4.00, 1.50-4.00, 1.75-4.00, 2.00-4.00, 2.25-4.00, 2.50-4.00, 2.75-4.00, 3.00-4.00, 3.25-4.00, 3.50-4.00, 3.75-4.00, 1.00-3.00, 1.25-3.00, 1.50-3.00, 1.75-3.00, 2.00-3.00, 2.25-3.00, 2.50-3.00 or 2.75-3.00 mol% of DMG-PEG (such as DMG-PEG2000) to the total lipid amount of the lipid nanoparticle.

[0042] The molar ratio of the cationic lipid, the sterol, DOPE, and DMG-PEG in the lipid nanoparticle A (cationic lipid / sterol / DOPE / DMG-PEG) includes, but is not limited to, (55.00 / 41.00 / 1.50 / 2.50), (55.25 / 40.75 / 1.50 / 2.50), (55.50 / 40.50 / 1.50 / 2.50), (55.75 / 40.25 / 1.50 / 2.50), (56.00 / 40.00 / 1.50 / 2.50), (56.25 / 39.75 / 1.50 / 2.50), (56.50 / 39.50 / 1.50 / 2.50), (56.75 / 39.25 / 1.50 / 2.50), (57.00 / 39.00 / 1.50 / 2.50), (57.25 / 38.75 / 1.50 / 2.50), (57.50 / 38.50 / 1.50 / 2.50), (57.75 / 38.25 / 1.50 / 2.50), (58.00 / 38.00 / 1.50 / 2.50), (58.25 / 37.75 / 1.50 / 2.50), (58.50 / 37.50 / 1.50 / 2.50), (58.75 / 37.25 / 1.50 / 2.50), (59.00 / 37.00 / 1.50 / 2.50), (59.25 / 36.75 / 1.50 / 2.50), (59.50 / 36.50 / 1.50 / 2.50), (59.75 / 36.25 / 1.50 / 2.50), (60.00 / 36.00 / 1.50 / 2.50), (60.25 / 35.75 / 1.50 / 2.50), (60.50 / 35.50 / 1.50 / 2.50), (60.75 / 35.25 / 1.50 / 2.50), (61.00 / 35.00 / 1.50 / 2.50), (61.25 / 34.75 / 1.50 / 2.50), (61.50 / 34.50 / 1.50 / 2.50), (61.75 / 34.25 / 1.50 / 2.50), (62.00 / 34.00 / 1.50 / 2.50), (62.25 / 33.75 / 1.50 / 2.50), (62.50 / 33.50 / 1.50 / 2.50), (62.75 / 33.25 / 1.50 / 2.50), (63.00 / 33.00 / 1.50 / 2.50), (63.25 / 32.75 / 1.50 / 2.50), (63.50 / 32.50 / 1.50 / 2.50), (63.75 / 32.25 / 1.50 / 2.50), (64.00 / 32.00 / 1.50 / 2.50), (64.25 / 31.75 / 1.50 / 2.50), (64.50 / 31.50 / 1.50 / 2.50), (64.75 / 31.25 / 1.50 / 2.50), (65.00 / 31.00 / 1.50 / 2.50), (55.00 / 41.00 / 1.75 / 2.25), (55.25 / 40.75 / 1.75 / 2.25), (55.50 / 40.50 / 1.75 / 2.25), (55.75 / 40.25 / 1.75 / 2.25), (56.00 / 40.00 / 1.75 / 2.25), (56.25 / 39.75 / 1.75 / 2.25), (56.50 / 39.50 / 1.75 / 2.25), (56.75 / 39.25 / 1.75 / 2.25), (57.00 / 39.00 / 1.75 / 2.25), (57.25 / 38.75 / 1.75 / 2.25), (57.50 / 38.50 / 1.75 / 2.25), (57.75 / 38.25 / 1.75 / 2.25), (58.00 / 38.00 / 1.75 / 2.25), (58.25 / 37.75 / 1.75 / 2.25), (58.50 / 37.50 / 1.75 / 2.25), (58.75 / 37.25 / 1.75 / 2.25), (59.00 / 37.00 / 1.75 / 2.25), (59.25 / 36.75 / 1.75 / 2.25), (59.50 / 36.50 / 1.75 / 2.25), (59.75 / 36.25 / 1.75 / 2.25), (60.00 / 36.00 / 1.75 / 2.25), (60.25 / 35.75 / 1.75 / 2.25), (60.50 / 35.50 / 1.75 / 2.25), (60.75 / 35.25 / 1.75 / 2.25), (61.00 / 35.00 / 1.75 / 2.25), (61.25 / 34.75 / 1.75 / 2.25), (61.50 / 34.50 / 1.75 / 2.25), (61.75 / 34.25 / 1.75 / 2.25),(62.00 / 34.00 / 1.75 / 2.25), (62.25 / 33.75 / 1.75 / 2.25), (62.50 / 33.50 / 1.75 / 2.25), (62.75 / 33.25 / 1.75 / 2.25), (63.00 / 33.00 / 1.75 / 2.25), (63.25 / 32.75 / 1.75 / 2.25), (63.50 / 32.50 / 1.75 / 2.25), (63.75 / 32.25 / 1.75 / 2.25), (64.00 / 32.00 / 1.75 / 2.25), (64.25 / 31.75 / 1.75 / 2.25), (64.50 / 31.50 / 1.75 / 2.25), (64.75 / 31.25 / 1.75 / 2.25), (65.00 / 31.00 / 1.75 / 2.25), (55.00 / 41.00 / 2.00 / 2.00), (55.25 / 40.75 / 2.00 / 2.00), (55.50 / 40.50 / 2.00 / 2.00), (55.75 / 40.25 / 2.00 / 2.00), (56.00 / 40.00 / 2.00 / 2.00), (56.25 / 39.75 / 2.00 / 2.00), (56.50 / 39.50 / 2.00 / 2.00), (56.75 / 39.25 / 2.00 / 2.00), (57.00 / 39.00 / 2.00 / 2.00), (57.25 / 38.75 / 2.00 / 2.00), (57.50 / 38.50 / 2.00 / 2.00), (57.75 / 38.25 / 2.00 / 2.00), (58.00 / 38.00 / 2.00 / 2.00), (58.25 / 37.75 / 2.00 / 2.00), (58.50 / 37.50 / 2.00 / 2.00), (58.75 / 37.25 / 2.00 / 2.00), (59.00 / 37.00 / 2.00 / 2.00), (59.25 / 36.75 / 2.00 / 2.00), (59.50 / 36.50 / 2.00 / 2.00), (59.75 / 36.25 / 2.00 / 2.00), (60.00 / 36.00 / 2.00 / 2.00), (60.25 / 35.75 / 2.00 / 2.00), (60.50 / 35.50 / 2.00 / 2.00), (60.75 / 35.25 / 2.00 / 2.00), (61.00 / 35.00 / 2.00 / 2.00), (61.25 / 34.75 / 2.00 / 2.00), (61.50 / 34.50 / 2.00 / 2.00), (61.75 / 34.25 / 2.00 / 2.00), (62.00 / 34.00 / 2.00 / 2.00), (62.25 / 33.75 / 2.00 / 2.00), (62.50 / 33.50 / 2.00 / 2.00), (62.75 / 33.25 / 2.00 / 2.00), (63.00 / 33.00 / 2.00 / 2.00), (63.25 / 32.75 / 2.00 / 2.00), (63.50 / 32.50 / 2.00 / 2.00), (63.75 / 32.25 / 2.00 / 2.00), (64.00 / 32.00 / 2.00 / 2.00), (64.25 / 31.75 / 2.00 / 2.00), (64.50 / 31.50 / 2.00 / 2.00), (64.75 / 31.25 / 2.00 / 2.00), (65.00 / 31.00 / 2.00 / 2.00), (55.00 / 41.00 / 2.25 / 1.75), (55.25 / 40.75 / 2.25 / 1.75), (55.50 / 40.50 / 2.25 / 1.75), (55.75 / 40.25 / 2.25 / 1.75), (56.00 / 40.00 / 2.25 / 1.75), (56.25 / 39.75 / 2.25 / 1.75), (56.50 / 39.50 / 2.25 / 1.75), (56.75 / 39.25 / 2.25 / 1.75), (57.00 / 39.00 / 2.25 / 1.75), (57.25 / 38.75 / 2.25 / 1.75), (57.50 / 38.50 / 2.25 / 1.75), (57.75 / 38.25 / 2.25 / 1.75), (58.00 / 38.00 / 2.25 / 1.75), (58.25 / 37.75 / 2.25 / 1.75), (58.50 / 37.50 / 2.25 / 1.75), (58.75 / 37.25 / 2.25 / 1.75), (59.00 / 37.00 / 2.25 / 1.75), (59.25 / 36.75 / 2.25 / 1.75), (59.50 / 36.50 / 2.25 / 1.75), (59.75 / 36.25 / 2.25 / 1.75), (60.00 / 36.00 / 2.25 / 1.75), (60.25 / 35.75 / 2.25 / 1.75), (60.50 / 35.50 / 2.25 / 1.75), (60.75 / 35.25 / 2.25 / 1.75), (61.00 / 35.00 / 2.25 / 1.75), (61.25 / 34.75 / 2.25 / 1.75), (61.50 / 34.50 / 2.25 / 1.75), (61.75 / 34.25 / 2.25 / 1.75), (62.00 / 34.00 / 2.25 / 1.75), (62.25 / 33.75 / 2.25 / 1.75), (62.50 / 33.50 / 2.25 / 1.75), (62.75 / 33.25 / 2.25 / 1.75), (63.00 / 33.00 / 2.25 / 1.75), (63.25 / 32.75 / 2.25 / 1.75), (63.50 / 32.50 / 2.25 / 1.75), (63.75 / 32.25 / 2.25 / 1.75), (64.00 / 32.00 / 2.25 / 1.75), (64.25 / 31.75 / 2.25 / 1.75), (64.50 / 31.50 / 2.25 / 1.75), (64.75 / 31.25 / 2.25 / 1.75), (65.00 / 31.00 / 2.25 / 1.75), (55.00 / 41.00 / 2.50 / 1.50), (55.25 / 40.75 / 2.50 / 1.50), (55.50 / 40.50 / 2.50 / 1.50), (55.75 / 40.25 / 2.50 / 1.50), (56.00 / 40.00 / 2.50 / 1.50), (56.25 / 39.75 / 2.50 / 1.50), (56.50 / 39.50 / 2.50 / 1.50),(56.75 / 39.25 / 2.50 / 1.50), (57.00 / 39.00 / 2.50 / 1.50), (57.25 / 38.75 / 2.50 / 1.50), (57.50 / 38.50 / 2.50 / 1.50), (57.75 / 38.25 / 2.50 / 1.50), (58.00 / 38.00 / 2.50 / 1.50), (58.25 / 37.75 / 2.50 / 1.50), (58.50 / 37.50 / 2.50 / 1.50), (58.75 / 37.25 / 2.50 / 1.50), (59.00 / 37.00 / 2.50 / 1.50), (59.25 / 36.75 / 2.50 / 1.50), (59.50 / 36.50 / 2.50 / 1.50), (59.75 / 36.25 / 2.50 / 1.50), (60.00 / 36.00 / 2.50 / 1.50), (60.25 / 35.75 / 2.50 / 1.50), (60.50 / 35.50 / 2.50 / 1.50), (60.75 / 35.25 / 2.50 / 1.50), (61.00 / 35.00 / 2.50 / 1.50), (61.25 / 34.75 / 2.50 / 1.50), (61.50 / 34.50 / 2.50 / 1.50), (61.75 / 34.25 / 2.50 / 1.50), (62.00 / 34.00 / 2.50 / 1.50), (62.25 / 33.75 / 2.50 / 1.50), (62.50 / 33.50 / 2.50 / 1.50), (62.75 / 33.25 / 2.50 / 1.50), (63.00 / 33.00 / 2.50 / 1.50), (63.25 / 32.75 / 2.50 / 1.50), (63.50 / 32.50 / 2.50 / 1.50), (63.75 / 32.25 / 2.50 / 1.50), (64.00 / 32.00 / 2.50 / 1.50), (64.25 / 31.75 / 2.50 / 1.50), (64.50 / 31.50 / 2.50 / 1.50), (64.75 / 31.25 / 2.50 / 1.50), (65.00 / 31.00 / 2.50 / 1.50), (55.00 / 41.00 / 2.75 / 1.25), (55.25 / 40.75 / 2.75 / 1.25), (55.50 / 40.50 / 2.75 / 1.25), (55.75 / 40.25 / 2.75 / 1.25), (56.00 / 40.00 / 2.75 / 1.25), (56.25 / 39.75 / 2.75 / 1.25), (56.50 / 39.50 / 2.75 / 1.25), (56.75 / 39.25 / 2.75 / 1.25), (57.00 / 39.00 / 2.75 / 1.25), (57.25 / 38.75 / 2.75 / 1.25), (57.50 / 38.50 / 2.75 / 1.25), (57.75 / 38.25 / 2.75 / 1.25), (58.00 / 38.00 / 2.75 / 1.25), (58.25 / 37.75 / 2.75 / 1.25), (58.50 / 37.50 / 2.75 / 1.25), (58.75 / 37.25 / 2.75 / 1.25), (59.00 / 37.00 / 2.75 / 1.25), (59.25 / 36.75 / 2.75 / 1.25), (59.50 / 36.50 / 2.75 / 1.25), (59.75 / 36.25 / 2.75 / 1.25), (60.00 / 36.00 / 2.75 / 1.25), (60.25 / 35.75 / 2.75 / 1.25), (60.50 / 35.50 / 2.75 / 1.25), (60.75 / 35.25 / 2.75 / 1.25), (61.00 / 35.00 / 2.75 / 1.25), (61.25 / 34.75 / 2.75 / 1.25), (61.50 / 34.50 / 2.75 / 1.25), (61.75 / 34.25 / 2.75 / 1.25), (62.00 / 34.00 / 2.75 / 1.25), (62.25 / 33.75 / 2.75 / 1.25), (62.50 / 33.50 / 2.75 / 1.25), (62.75 / 33.25 / 2.75 / 1.25), (63.00 / 33.00 / 2.75 / 1.25), (63.25 / 32.75 / 2.75 / 1.25), (63.50 / 32.50 / 2.75 / 1.25), (63.75 / 32.25 / 2.75 / 1.25), (64.00 / 32.00 / 2.75 / 1.25), (64.25 / 31.75 / 2.75 / 1.25), (64.50 / 31.50 / 2.75 / 1.25), (64.75 / 31.25 / 2.75 / 1.25), (65.00 / 31.00 / 2.75 / 1.25), (55.00 / 41.00 / 3.00 / 1.00), (55.25 / 40.75 / 3.00 / 1.00), (55.50 / 40.50 / 3.00 / 1.00), (55.75 / 40.25 / 3.00 / 1.00), (56.00 / 40.00 / 3.00 / 1.00), (56.25 / 39.75 / 3.00 / 1.00), (56.50 / 39.50 / 3.00 / 1.00), (56.75 / 39.25 / 3.00 / 1.00), (57.00 / 39.00 / 3.00 / 1.00), (57.25 / 38.75 / 3.00 / 1.00), (57.50 / 38.50 / 3.00 / 1.00), (57.75 / 38.25 / 3.00 / 1.00), (58.00 / 38.00 / 3.00 / 1.00), (58.25 / 37.75 / 3.00 / 1.00), (58.50 / 37.50 / 3.00 / 1.00), (58.75 / 37.25 / 3.00 / 1.00), (59.00 / 37.00 / 3.00 / 1.00), (59.25 / 36.75 / 3.00 / 1.00), (59.50 / 36.50 / 3.00 / 1.00), (59.75 / 36.25 / 3.00 / 1.00), (60.00 / 36.00 / 3.00 / 1.00), (60.25 / 35.75 / 3.00 / 1.00), (60.50 / 35.50 / 3.00 / 1.00), (60.75 / 35.25 / 3.00 / 1.00), (61.00 / 35.00 / 3.00 / 1.00), (61.25 / 34.75 / 3.00 / 1.00), (61.50 / 34.50 / 3.00 / 1.00),(61.75 / 34.25 / 3.00 / 1.00), (62.00 / 34.00 / 3.00 / 1.00), (62.25 / 33.75 / 3.00 / 1.00), (62.50 / 33.50 / 3.00 / 1.00), (62.75 / 33.25 / 3.00 / 1.00), (63.00 / 33.00 / 3.00 / 1.00), (63.25 / 32.75 / 3.00 / 1.00), (63.50 / 32.50 / 3.00 / 1.00), (63.75 / 32.25 / 3.00 / 1.00), (64.00 / 32.00 / 3.00 / 1.00), (64.25 / 31.75 / 3.00 / 1.00), (64.50 / 31.50 / 3.00 / 1.00), (64.75 / 31.25 / 3.00 / 1.00), (65.00 / 31.00 / 3.00 / 1.00), or (58.25 / 39.25 / 2.50 / 1.50).

[0043] In some embodiments, the cationic lipid in the lipid nanoparticle A is a compound of Formula I (for example, a compound of Formula IA or a pharmaceutically acceptable salt thereof or Formula IB or a pharmaceutically acceptable salt thereof). In some embodiments, the cationic lipid of the lipid nanoparticle A is a compound of Formula II (for example, a compound of Formula HA or a pharmaceutically acceptable salt thereof).

[0044] In some embodiments, the lipid nanoparticle according to the present disclosure comprises:• 55-65 mol% of a cationic lipid to the total lipid amount of the lipid nanoparticle • 30-50 mol% of a sterol to the total lipid amount of the lipid nanoparticle;• 4-6 mol% of DOPC to the total lipid amount of the lipid nanoparticle; and• 1-5 mol% of DMG-PEG to the total lipid amount of the lipid nanoparticle.

[0045] This lipid nanoparticle may be referred to as “lipid nanoparticle B” hereinbelow.

[0046] In some embodiments, the lipid nanoparticle B comprises 55.00-65.00, 55.25-65.00, 55.50-65.00, 55.75-65.00, 56.00-65.00, 56.25-65.00, 56.50-65.00, 56.75-65.00, 57.00-65.00, 57.25-65.00, 57.50-65.00, 57.75-65.00, 58.00-65.00, 58.25-65.00, 58.50-65.00, 58.75-65.00, 59.00-65.00, 59.25-65.00, 59.50-65.00, 59.75-65.00, 60.00-65.00, 60.25-65.00, 60.50-65.00, 60.75-65.00, 61.00-65.00, 61.25-65.00, 61.50-65.00, 61.75-65.00, 62.00-65.00, 62.25-65.00, 62.50-65.00, 62.75-65.00, 63.00-65.00, 63.25-65.00, 63.50-65.00, 63.75-65.00, 64.00-65.00, 64.25-65.00, 64.50-65.00 or 64.75-65.00 mol% of the cationic lipid to the total lipid amount of the lipid nanoparticle. In some embodiments, the lipid nanoparticle B comprises 30.00-50.00, 30.25-50.00, 30.50-50.00, 30.75-50.00, 31.00-50.00, 31.25-50.00, 31.50-50.00, 31.75-50.00, 32.00-50.00, 32.25-50.00, 32.50-50.00, 32.75-50.00, 33.00-50.00, 33.25-50.00, 33.50-50.00, 33.75-50.00, 34.00-50.00, 34.25-50.00, 34.50-50.00, 34.75-50.00, 35.00-50.00, 35.25-50.00, 35.50-50.00, 35.75-50.00, 36.00-50.00, 36.25-50.00, 36.50-50.00, 36.75-50.00, 37.00-50.00, 37.25-50.00, 37.50-50.00, 37.75-50.00, 38.00-50.00, 38.25-50.00, 38.50-50.00, 38.75-50.00, 39.00-50.00, 39.25-50.00, 39.50-50.00, 39.75-50.00, 40.00-50.00, 40.25-50.00, 40.50-50.00, 40.75-50.00, 41.00-50.00, 41.25-50.00, 41.50-50.00, 41.75-50.00,42.00-50.00, 42.25-50.00, 42.50-50.00, 42.75-50.00, 43.00-50.00, 43.25-50.00, 43.50-50.00, 43.75-50.00, 44.00-50.00, 44.25-50.00, 44.50-50.00, 44.75-50.00, 45.00-50.00, 45.25-50.00, 45.50-50.00, 45.75-50.00, 46.00-50.00, 46.25-50.00, 46.50-50.00, 46.75-50.00, 47.00-50.00, 47.25-50.00, 47.50-50.00, 47.75-50.00, 48.00-50.00, 48.25-50.00, 48.50-50.00, 48.75-50.00, 49.00-50.00, 49.25-50.00, 49.50-50.00, 49.75-50.00, 30.00-40.00, 30.25-40.00, 30.50-40.00, 30.75-40.00, 31.00-40.00, 31.25-40.00, 31.50-40.00, 31.75-40.00, 32.00-40.00, 32.25-40.00, 32.50-40.00, 32.75-40.00, 33.00-40.00, 33.25-40.00, 33.50-40.00, 33.75-40.00, 34.00-40.00, 34.25-40.00, 34.50-40.00, 34.75-40.00, 35.00-40.00, 35.25-40.00, 35.50-40.00, 35.75-40.00, 36.00-40.00, 36.25-40.00, 36.50-40.00, 36.75-40.00, 37.00-40.00, 37.25-40.00, 37.50-40.00, 37.75-40.00, 38.00-40.00, 38.25-40.00, 38.50-40.00, 38.75-40.00, 39.00-40.00, 39.25-40.00, 39.50-40.00 or 39.75-40.00 mol% of the sterol (such as cholesterol) to the total lipid amount of the lipid nanoparticle. In some embodiments, the lipid nanoparticle B comprises 4.00-6.00, 4.25-6.00, 4.50-6.00, 4.75-6.00, 5.00-6.00, 5.25-6.00, 5.50-6.00, 5.75-6.00, 4.00-5.00, 4.25- 5.00, 4.50-5.00, 4.75-5.00, 5.00-5.00, 5.25-5.00, 5.50-5.00 or 5.75-5.00 mol% of DOPC to the total lipid amount of the lipid nanoparticle. In some embodiments, the lipid nanoparticle B comprises 1.00-5.00, 1.25-5.00, 1.50-5.00, 1.75-5.00, 2.00-5.00, 2.25-5.00, 2.50-5.00, 2.75-5.00, 3.00-5.00, 3.25-5.00, 3.50-5.00, 3.75-5.00, 4.00-5.00, 4.25-5.00, 4.50-5.00,4.75-5.00, 1.00-4.00, 1.25-4.00, 1.50-4.00, 1.75-4.00, 2.00-4.00, 2.25-4.00, 2.50-4.00, 2.75-4.00, 3.00- 4.00, 3.25-4.00, 3.50-4.00, 3.75-4.00, 1.00-3.00, 1.25-3.00, 1.50-3.00, 1.75-3.00, 2.00-3.00, 2.25-3.00, 2.50-3.00 or 2.75-3.00 mol% of DMG-PEG (such as DMG-PEG2000) to the total lipid amount of the lipid nanoparticle.

[0047] The molar ratio of the cationic lipid, the sterol, DOPE, and DMG-PEG in the lipid nanoparticle B (cationic lipid / sterol / DOPC / DMG-PEG) includes, but is not limited to, (55.00 / 38.00 / 6.00 / 1.00), (55.25 / 37.75 / 6.00 / 1.00), (55.50 / 37.50 / 6.00 / 1.00), (55.75 / 37.25 / 6.00 / 1.00), (56.00 / 37.00 / 6.00 / 1.00), (56.25 / 36.75 / 6.00 / 1.00), (56.50 / 36.50 / 6.00 / 1.00), (56.75 / 36.25 / 6.00 / 1.00), (57.00 / 36.00 / 6.00 / 1.00), (57.25 / 35.75 / 6.00 / 1.00), (57.50 / 35.50 / 6.00 / 1.00), (57.75 / 35.25 / 6.00 / 1.00), (58.00 / 35.00 / 6.00 / 1.00), (58.25 / 34.75 / 6.00 / 1.00), (58.50 / 34.50 / 6.00 / 1.00), (58.75 / 34.25 / 6.00 / 1.00), (59.00 / 34.00 / 6.00 / 1.00), (59.25 / 33.75 / 6.00 / 1.00), (59.50 / 33.50 / 6.00 / 1.00), (59.75 / 33.25 / 6.00 / 1.00), (60.00 / 33.00 / 6.00 / 1.00), (60.25 / 32.75 / 6.00 / 1.00), (60.50 / 32.50 / 6.00 / 1.00), (60.75 / 32.25 / 6.00 / 1.00), (61.00 / 32.00 / 6.00 / 1.00), (61.25 / 31.75 / 6.00 / 1.00), (61.50 / 31.50 / 6.00 / 1.00), (61.75 / 31.25 / 6.00 / 1.00), (62.00 / 31.00 / 6.00 / 1.00), (62.25 / 30.75 / 6.00 / 1.00), (62.50 / 30.50 / 6.00 / 1.00), (62.75 / 30.25 / 6.00 / 1.00), (63.00 / 30.00 / 6.00 / 1.00),(55.00 / 38.00 / 5.75 / 1.25), (55.25 / 37.75 / 5.75 / 1.25), (55.50 / 37.50 / 5.75 / 1.25), (55.75 / 37.25 / 5.75 / 1.25), (56.00 / 37.00 / 5.75 / 1.25), (56.25 / 36.75 / 5.75 / 1.25), (56.50 / 36.50 / 5.75 / 1.25), (56.75 / 36.25 / 5.75 / 1.25), (57.00 / 36.00 / 5.75 / 1.25), (57.25 / 35.75 / 5.75 / 1.25), (57.50 / 35.50 / 5.75 / 1.25), (57.75 / 35.25 / 5.75 / 1.25), (58.00 / 35.00 / 5.75 / 1.25), (58.25 / 34.75 / 5.75 / 1.25), (58.50 / 34.50 / 5.75 / 1.25), (58.75 / 34.25 / 5.75 / 1.25), (59.00 / 34.00 / 5.75 / 1.25), (59.25 / 33.75 / 5.75 / 1.25), (59.50 / 33.50 / 5.75 / 1.25), (59.75 / 33.25 / 5.75 / 1.25), (60.00 / 33.00 / 5.75 / 1.25), (60.25 / 32.75 / 5.75 / 1.25), (60.50 / 32.50 / 5.75 / 1.25), (60.75 / 32.25 / 5.75 / 1.25), (61.00 / 32.00 / 5.75 / 1.25), (61.25 / 31.75 / 5.75 / 1.25), (61.50 / 31.50 / 5.75 / 1.25), (61.75 / 31.25 / 5.75 / 1.25), (62.00 / 31.00 / 5.75 / 1.25), (62.25 / 30.75 / 5.75 / 1.25), (62.50 / 30.50 / 5.75 / 1.25), (62.75 / 30.25 / 5.75 / 1.25), (63.00 / 30.00 / 5.75 / 1.25), (55.00 / 38.00 / 5.50 / 1.50), (55.25 / 37.75 / 5.50 / 1.50), (55.50 / 37.50 / 5.50 / 1.50), (55.75 / 37.25 / 5.50 / 1.50), (56.00 / 37.00 / 5.50 / 1.50), (56.25 / 36.75 / 5.50 / 1.50), (56.50 / 36.50 / 5.50 / 1.50), (56.75 / 36.25 / 5.50 / 1.50), (57.00 / 36.00 / 5.50 / 1.50), (57.25 / 35.75 / 5.50 / 1.50), (57.50 / 35.50 / 5.50 / 1.50), (57.75 / 35.25 / 5.50 / 1.50), (58.00 / 35.00 / 5.50 / 1.50), (58.25 / 34.75 / 5.50 / 1.50), (58.50 / 34.50 / 5.50 / 1.50), (58.75 / 34.25 / 5.50 / 1.50), (59.00 / 34.00 / 5.50 / 1.50), (59.25 / 33.75 / 5.50 / 1.50), (59.50 / 33.50 / 5.50 / 1.50), (59.75 / 33.25 / 5.50 / 1.50), (60.00 / 33.00 / 5.50 / 1.50), (60.25 / 32.75 / 5.50 / 1.50), (60.50 / 32.50 / 5.50 / 1.50), (60.75 / 32.25 / 5.50 / 1.50), (61.00 / 32.00 / 5.50 / 1.50), (61.25 / 31.75 / 5.50 / 1.50), (61.50 / 31.50 / 5.50 / 1.50), (61.75 / 31.25 / 5.50 / 1.50), (62.00 / 31.00 / 5.50 / 1.50), (62.25 / 30.75 / 5.50 / 1.50), (62.50 / 30.50 / 5.50 / 1.50), (62.75 / 30.25 / 5.50 / 1.50), (63.00 / 30.00 / 5.50 / 1.50), (55.00 / 38.00 / 5.25 / 1.75), (55.25 / 37.75 / 5.25 / 1.75), (55.50 / 37.50 / 5.25 / 1.75), (55.75 / 37.25 / 5.25 / 1.75), (56.00 / 37.00 / 5.25 / 1.75), (56.25 / 36.75 / 5.25 / 1.75), (56.50 / 36.50 / 5.25 / 1.75), (56.75 / 36.25 / 5.25 / 1.75), (57.00 / 36.00 / 5.25 / 1.75), (57.25 / 35.75 / 5.25 / 1.75), (57.50 / 35.50 / 5.25 / 1.75), (57.75 / 35.25 / 5.25 / 1.75), (58.00 / 35.00 / 5.25 / 1.75), (58.25 / 34.75 / 5.25 / 1.75), (58.50 / 34.50 / 5.25 / 1.75), (58.75 / 34.25 / 5.25 / 1.75), (59.00 / 34.00 / 5.25 / 1.75), (59.25 / 33.75 / 5.25 / 1.75), (59.50 / 33.50 / 5.25 / 1.75), (59.75 / 33.25 / 5.25 / 1.75), (60.00 / 33.00 / 5.25 / 1.75), (60.25 / 32.75 / 5.25 / 1.75), (60.50 / 32.50 / 5.25 / 1.75), (60.75 / 32.25 / 5.25 / 1.75), (61.00 / 32.00 / 5.25 / 1.75), (61.25 / 31.75 / 5.25 / 1.75), (61.50 / 31.50 / 5.25 / 1.75), (61.75 / 31.25 / 5.25 / 1.75), (62.00 / 31.00 / 5.25 / 1.75), (62.25 / 30.75 / 5.25 / 1.75), (62.50 / 30.50 / 5.25 / 1.75), (62.75 / 30.25 / 5.25 / 1.75), (63.00 / 30.00 / 5.25 / 1.75), (55.00 / 38.00 / 5.00 / 2.00), (55.25 / 37.75 / 5.00 / 2.00), (55.50 / 37.50 / 5.00 / 2.00),(55.75 / 37.25 / 5.00 / 2.00), (56.00 / 37.00 / 5.00 / 2.00), (56.25 / 36.75 / 5.00 / 2.00), (56.50 / 36.50 / 5.00 / 2.00), (56.75 / 36.25 / 5.00 / 2.00), (57.00 / 36.00 / 5.00 / 2.00), (57.25 / 35.75 / 5.00 / 2.00), (57.50 / 35.50 / 5.00 / 2.00), (57.75 / 35.25 / 5.00 / 2.00), (58.00 / 35.00 / 5.00 / 2.00), (58.25 / 34.75 / 5.00 / 2.00), (58.50 / 34.50 / 5.00 / 2.00), (58.75 / 34.25 / 5.00 / 2.00), (59.00 / 34.00 / 5.00 / 2.00), (59.25 / 33.75 / 5.00 / 2.00), (59.50 / 33.50 / 5.00 / 2.00), (59.75 / 33.25 / 5.00 / 2.00), (60.00 / 33.00 / 5.00 / 2.00), (60.25 / 32.75 / 5.00 / 2.00), (60.50 / 32.50 / 5.00 / 2.00), (60.75 / 32.25 / 5.00 / 2.00), (61.00 / 32.00 / 5.00 / 2.00), (61.25 / 31.75 / 5.00 / 2.00), (61.50 / 31.50 / 5.00 / 2.00), (61.75 / 31.25 / 5.00 / 2.00), (62.00 / 31.00 / 5.00 / 2.00), (62.25 / 30.75 / 5.00 / 2.00), (62.50 / 30.50 / 5.00 / 2.00), (62.75 / 30.25 / 5.00 / 2.00), or (63.00 / 30.00 / 5.00 / 2.00).

[0048] In some embodiments, the cationic lipid in the lipid nanoparticle A is a compound of Formula I (for example, a compound of Formula IA or Formula IB or a pharmaceutically acceptable salt thereof). In some embodiments, the cationic lipid of the lipid nanoparticle A is a compound of Formula II (for example, a compound of Formula HA or a pharmaceutically acceptable salt thereof).

[0049] In some embodiments, the lipid nanoparticle according to the present disclosure comprises:• 40-60 mol% of a cationic lipid of Formula I or II to the total lipid amount of the lipid nanoparticle• 30-50 mol% of a sterol to the total lipid amount of the lipid nanoparticle;• 5-15 mol% of the phospholipid selected from the group consisting of DSPC, DOPC, and DOPE to the total lipid amount of the lipid nanoparticle; and• 1 -5 mol% of DMG-PEG to the total lipid amount of the lipid nanoparticle.

[0050] This lipid nanoparticle may be referred to as “lipid nanoparticle C” hereinbelow.

[0051] In some embodiments, the lipid nanoparticle C comprises 40-60 (such as 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5 or 60) mol% of the cationic lipid of Formula I or II to the total lipid amount of the lipid nanoparticle; 30-50 (such as 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5 or 50) mol% of the sterol (such as cholesterol) to the total lipid amount of the lipid nanoparticle; 5-15 (such as 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15) mol% of any one of DSPC, DOPC, andDOPE to the total lipid amount of the lipid nanoparticle; and 1-5 (such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5) mol% of DMG-PEG (such as DMG-PEG2000) to the total lipid amount of the lipid nanoparticle. The molar ratio of the cationic lipids, the sterol, the phospholipid selected from the group consisting of DSPC, DOPC, and DOPE, and DMG-PEG in the lipid nanoparticle C (cationic lipid / sterol / phospholipid / DMG-PEG) includes, but is not limited to, (60 / 31 / 8 / 1), (60 / 31 / 7.5 / 1.5), (60 / 31 / 7 / 2), (60 / 31 / 6.5 / 2.5), (60 / 31 / 6 / 3), (60 / 31 / 5.5 / 3.5), (60 / 31 / 5 / 4), (60 / 31 / 4.5 / 4.5), (60 / 31 / 4 / 5), (60 / 31.5 / 7.5 / 1), (60 / 30.5 / 7.5 / 2), (60 / 30 / 7.5 / 2.5), (60 / 29.5 / 7.5 / 3), (60 / 29 / 7.5 / 3.5), (60 / 28.5 / 7.5 / 4), (60 / 28 / 7.5 / 4.5), (60 / 27.5 / 7.5 / 5), (59 / 32 / 7.5 / 1.5), (58 / 33 / 7.5 / 1.5), (57 / 34 / 7.5 / 1.5), (56 / 35 / 7.5 / 1.5), (55 / 36 / 7.5 / 1.5), (60 / 29 / 10 / 1), (60 / 28.5 / 10 / 1.5), (60 / 28 / 10 / 2), (60 / 27.5 / 10 / 2.5), (60 / 27 / 10 / 3), (60 / 26.5 / 10 / 3.5), (60 / 26 / 10 / 4), (60 / 25.5 / 10 / 4.5), (60 / 25 / 10 / 5), (60 / 27 / 12 / 1), (60 / 27 / 11.5 / 1.5), (60 / 27 / 11 / 2), (60 / 27 / 10.5 / 2.5), (60 / 27 / 9.5 / 3.5), (60 / 27 / 9 / 4), (60 / 27 / 8.5 / 4.5), (60 / 27 / 8 / 5), (59 / 28 / 10 / 3), (58 / 29 / 10 / 3), (57 / 30 / 10 / 3), (56 / 31 / 10 / 3), (55 / 32 / 10 / 3), (50 / 39 / 10 / 1), (50 / 38.5 / 10 / 1.5), (50 / 38 / 10 / 2), (50 / 37.5 / 10 / 2.5), (50 / 37 / 10 / 3), (50 / 36.5 / 10 / 3.5), (50 / 36 / 10 / 4), (50 / 35.5 / 10 / 4.5), (50 / 35 / 10 / 5), (50 / 38.5 / 10.5 / 1), (50 / 38.5 / 9.5 / 2), (50 / 38.5 / 9 / 2.5), (50 / 38.5 / 8.5 / 3), (50 / 38.5 / 8 / 3.5), (50 / 38.5 / 7.5 / 4), (50 / 38.5 / 7 / 4.5), (50 / 38.5 / 6.5 / 5), (51 / 37.5 / 10 / 1.5), (52 / 36.5 / 10 / 1.5), (53 / 35.5 / 10 / 1.5), (54 / 34.5 / 10 / 1.5), (55 / 33.5 / 10 / 1.5), (49 / 39.5 / 10 / 1.5), (48 / 40.5 / 10 / 1.5), (47 / 41.5 / 10 / 1.5), (46 / 42.5 / 10 / 1.5), (45 / 43.5 / 10 / 1.5), (51 / 34 / 10 / 5), (52 / 33 / 10 / 5), (53 / 32 / 10 / 5), (54 / 31 / 10 / 5), (55 / 30 / 10 / 5), (40 / 44 / 15 / 1), (40 / 43.5 / 15 / 1.5), (40 / 43 / 15 / 2), (40 / 42.5 / 15 / 2.5), (40 / 42 / 15 / 3), (40 / 41.5 / 15 / 3.5), (40 / 41 / 15 / 4), (40 / 40.5 / 15 / 4.5), (40 / 40 / 15 / 5), (41 / 39 / 15 / 5), (42 / 38 / 15 / 5), (43 / 37 / 15 / 5), (44 / 36 / 15 / 5), (45 / 35 / 15 / 5), (41 / 41 / 14 / 4), (42 / 42 / 13 / 3), (43 / 43 / 12 / 2), and (44 / 44 / 11 / 1).

[0052] The lipid nanoparticles according to the present disclosure can be subjected to appropriate surface modification, as necessary. The lipid nanoparticles according to the present disclosure can be modified on the surface with hydrophilic polymers, etc. to enhance blood retention. Surface modification may be able to be achieved by using lipids modified with these modifying groups as constituent lipid of the lipid nanoparticles.

[0053] In the production of lipid nanoparticles according to the present disclosure, for example, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, and polyglycerol phospholipid derivatives, etc. can be used as lipid derivatives to enhance blood retention. In addition, dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin,pectin and carrageenan, etc., other than polyalkylene glycol, can be used for surface modification, as hydrophilic polymers to enhance blood retention.

[0054] In addition, in order to facilitate nuclear translocation of the lipid nanoparticles according to the present disclosure, for example, lipid nanoparticles can be surface-modified with oligosaccharide compounds with three or more saccharides. The type of oligosaccharide compounds with three or more saccharides is not particularly limited, but for example, oligosaccharide compounds in which approximately between 3 and 10 saccharide units are bound can be used, preferably oligosaccharide compounds in which approximately between 3 and 6 saccharide units are bound can be used. Among them, preferably, oligosaccharide compounds with trimer or hexamer of glucose can be used, and, further preferably, oligosaccharide compounds with trimer or tetramer of glucose can be used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, or maltohexaose can be preferably used, among which maltotriose, maltotetraose, maltopentaose, or maltohexaose with a1-4 bound glucose are further preferable. Particularly preferred are maltotriose or maltotetraose, and most preferred is maltotriose. Surface modification amount of lipid nanoparticles by oligosaccharide compound is not particularly limited, but, for example, it is approximately between 1 and 30 mol%, preferably approximately between 2 and 20 mol%, and more preferably approximately between 5 and 10 mol% to the total lipid amount.

[0055] The method for surface modifying lipid nanoparticles with oligosaccharide compound is not particularly limited, but, for example, liposomes in which lipid nanoparticles are surface modified with monosaccharides such as galactose and mannose (PCT Publication No. WO 2007 / 102481) are known, so the method for the surface modification described in the publication can be employed. The surface modification method described in this publication can be adopted. All of the disclosures in above publication shall be included by reference as the disclosures in the specification of this application.

[0056] In addition, the lipid nanoparticles according to the present disclosure can also be imparted any one or more functions such as temperature change sensitive function, membrane permeability function, gene expression function, and pH-sensitive function. Adding these functions appropriately can improve the retention of lipid nanoparticles in the blood and allow the lipid nanoparticles to efficiently escape from endosomes after endocytosis in target cells.

[0057] The lipid nanoparticles according to the present disclosure may comprise one or more substances selected from the group consisting of anti-oxidizing agents such astocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charged substances, and membrane polypeptides, etc. Charged substances which impart positive charges can include, for example, saturated or unsaturated aliphatic amines such as stearylamine and oleylamine, and charged substances which impart negative charges can include, for example, dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, phosphatidic acid, etc. Membrane polypeptides include, for example, membrane extrinsic polypeptide or membrane intrinsic polypeptide, etc. The amount of these substances is not particularly limited and can be appropriately selected according to the purpose.

[0058] The average particle size of the lipid nanoparticles according to the present disclosure is, for example, 400 nm or less, 300 nm or less, 200 nm or less, or 150 nm or less.

[0059] The polydispersity index (PDI) of the lipid nanoparticles according to the present disclosure is, for example, approximately between 0.01 and 0.7, preferably approximately between 0.01 and 0.6, further preferably approximately between 0.03 and 0.3. The zeta potential at pH 7.4 can be in the range of -50 mV-5 mV, preferably -45 mV-5 mV.

[0060] The morphology of the lipid nanoparticles according to the present disclosure is not particularly limited, but can include, for example, unilamellar liposome, multilayer liposome, spherical micelle, or unshaped layered structure as morphology dispersed in aqueous solvent. The lipid nanoparticles according to the present disclosure are preferably unilamellar liposome, multilayer liposome.

[0061] The lipid nanoparticles according to the present disclosure preferably encapsulate components for the purpose of being delivered into the target cells inside the particle covered with lipid membranes. The components which the lipid nanoparticles according to the present disclosure encapsulate inside the particles are not limited as long as they are sized available to be encapsulated. The lipid nanoparticles according to the present disclosure can encapsulate any component such as nucleic acids, saccharides, peptides, low molecularweight compounds, and metallic compounds. In some embodiment, the component is an active pharmaceutical ingredient.

[0062] The component encapsulated in the lipid nanoparticles according to the present disclosure is preferably a nucleic acid. The nucleic acid may be DNA, or may be RNA, or also may be analogs or derivatives thereof (e.g., peptide nucleic acid (PNA) or phosphorothioate DNA, etc.). The nucleic acids to be encapsulated in the lipid nanoparticles according to the present disclosure may be single-stranded nucleic acids, may be double¬ stranded nucleic acids, also may be linear, or cyclic.

[0063] In some embodiments, the nucleic acids to be encapsulated in the lipid nanoparticles according to the present disclosure comprise a foreign gene to be expressed in the target cell, preferably they are nucleic acids which functions to express the foreign gene in the cell by being taken up into the cell. The foreign genes may be genes originally comprised in the genomic DNA of the target cells, or they may be genes not comprised in the genomic DNA. Such nucleic acids include gene expression vectors comprising nucleic acids consisting of base sequences encoding genes of interest to be expressed. The gene expression vectors may be present as extrachromosomal genes in the introduced cell, or it may be taken up into the genomic DNA by homologous recombination.

[0064] The gene expression vectors to be encapsulated in the lipid nanoparticles according to the present disclosure are not particularly limited, and vectors generally used in gene therapy, etc. can be used. The gene expression vectors to be encapsulated in the lipid nanoparticles according to the present disclosure are preferably nucleic acid vectors such as plasmid vectors. The plasmid vectors may remain in a circular form or may be encapsulated in the lipid nanoparticles according to the present disclosure in a in a pre-cut linear form. The gene expression vectors can be designed by usual method using commonly used molecular biological tools based on the base sequence information of the gene of the target to be expressed and can be produced by various known methods.

[0065] The nucleic acids to be encapsulated in the lipid nanoparticles according to the present disclosure are also preferably functional nucleic acids which control the expression of target genes present in the target cells. The functional nucleic acids include antisense oligonucleotide, antisense oligonucleotide (including antisense DNA and antisense RNA), siRNA, microRNA(miRNA), and mRNA, etc. Also, they may be plasmid DNA (pDNA) becoming siRNA expression vectors which express siRNA in the cells. The siRNA expression vectors can be prepared from commercially available siRNA expression vectors, also which may be appropriately modified. In one embodiment of the present disclosure, the lipid nanoparticles according to the present disclosure comprise cationic lipids of the present disclosure and mRNA.

[0066] When the nucleic acids are mRNAs, the N / P ratio may be, for example, in the range of 3.0 to 12.0. In some embodiments, the N / P ratio is about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, about 11.0 or about 12.0.

[0067] The production method of lipid nanoparticles according to the present disclosure is not particularly limited, and any method available to those skilled in the art can be adopted. As an example, they can be produced by, after forming a lipid film by dissolvingall lipid components in an organic solvent such as chloroform and then drying under reduced pressure by an evaporator or spray drying by a spray dryer, adding components to be encapsulated into the lipid nanoparticles (for example, aqueous solvent comprising nucleic acids, etc.) to dried above mixture, then emulsifying by emulsifier such as homogenizer, ultrasonic emulsifier, or high pressure jet spray emulsifier, etc. They can also be produced by a well-known method for producing liposomes, for example, reversed-phase evaporation method. If the size of the lipid nanoparticles is to be controlled, extrusion (extruding filtration) may be carried out under high pressure using membrane filter with uniform pore size, etc.

[0068] The composition of the aqueous solvents (dispersion media) is not particularly limited, but can include, for example, buffer solutions such as phosphate buffer solution, citrate buffer solution, and phosphate buffered physiological saline, physiological saline, and culture media for cell culture. These aqueous solvents (dispersion media) can stably disperse lipid nanoparticles, but they may furthermore be added saccharides (aqueous solution) such as: monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and meredinose; polysaccharides such as cyclodextrin; sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, maltitol; and polyalcohols (aqueous solution) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol. In order to stably store the lipid nanoparticles dispersed in this aqueous solvent for a long time, it is desirable to eliminate electrolytes in the aqueous solvent as much as possible in terms of physical stability such as aggregation control, etc. In addition, in terms of chemical stability of the lipids, it is desirable to set the pH of the aqueous solvent between weak acidity and near neutral (approximately between pH 3.0 and 8.0) and / or to remove dissolved oxygen by nitrogen bubbling, etc.

[0069] The lipid nanoparticles according to the present disclosure also can be produced by alcohol dilution method using flow channel. The method is a method for producing lipid nanoparticles by introducing a solution in which lipid components are dissolved in alcohol solvent and a solution in which water-soluble components to be included in lipid nanoparticles are dissolved in aqueous solvent from different flow channels and merging them together. By using microchannel with built-in three-dimensional micromixer which can achieve instantaneous mixing of two liquids, lipid nanoparticles with a diameter of about 30 nm can beproduced at high reproducibility (see Leung et al., Journal of Physical Chemistry C Nanomater Interfaces, 2012, vol.116(34), p.18440-18450).

[0070] When obtained aqueous dispersions of lipid nanoparticles is lyophilized or spray dried, the stability may be able to be improved using, for example, saccharide (aqueous solution) such as: monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and meredinose; polysaccharides such as cyclodextrin; sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, maltitol. In addition, when freezing above aqueous dispersions, the stability may be able to be improved using, for example, aforementioned saccharides and polyalcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol.

[0071] In one embodiment of the present disclosure, the lipid nanoparticles according to the present disclosure are lyophilized.

[0072] In some embodiments, the lipid nanoparticles according to the present disclosure are synthesized by injecting ethanol solution of lipids into a buffer solution including a nucleic acid in the same manner as described in U. S. Publication No. 2013 / 0022665, PCT Publication No. WO2019 / 090359, and PCT Publication No. W02020 / 102668. In some embodiments, the lipid nanoparticles according to the present disclosure are synthesized by combining a lipid solution with a nucleic acid using a microfluidic mixing device such as NanoAssemblr ™ (Precision Nano Systems).

[0073] In some embodiments, the lipid nanoparticles according to the present disclosure have excellent stability. The lipid nanoparticles of the present disclosure are, for example, stable for at least 1 week when kept at -80°C.

[0074] An apparent pKa values of the lipid nanoparticles according to the present disclosure is not particularly limited, but can be selected, for example, in the range of approximately between 4.0 and 10.0, preferably approximately between 4.5 and 9.5, if it can be obtained. The pKa values are determined by using 2-(p-toluidino)-6-napthalene sulfonic acid (TNS) (for example, see PCT Publication No. WO2022 / 071582)Method of delivering a nucleic acid to a cell or a subject

[0075] In one aspect, the present disclosure relates to a method of delivering a nucleic acid to a cell, comprising contacting the lipid nanoparticle according to the presentdisclosure that encapsulates the nucleic acid with the cell. In certain embodiments, the cell is in vitro. In certain embodiments, the cell is in vivo. In certain embodiments, the cell is ex vivo.

[0076] In one aspect, the present disclosure relates to a method of delivering a nucleic acid to a subject in need thereof, comprising administering the lipid nanoparticle according to the present disclosure that encapsulates the nucleic acid to the subject. The subject may be human or non-human animals. The non-human animals include mammals such as cattle, pig, horse, sheep, goat, monkey, dog, cat, rabbit, mouse, rat, hamster, and guinea pig, and birds such as chicken, quail, and duck, etc. The lipid nanoparticle may be administered by any means known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, or airway (aerosol) administration.EXAMPLESSynthesis of cationic lipids

[0077] Compounds IA and IB were prepared according to the procedures described in U. S. Publication No. 2024 / 0024252. Compound III was obtained from Echelon Biosciences Inc. The chemical structures of Compounds IA, IB, and III are shown below:•OHNCompound IAOHCompound IBCompound IIISynthesis of Compound II A

[0078] Step 1: N-Boc-diethanolamine (19.4 g, 94.7 mmol, 1.0 eq.), 2- hexyldecanoic acid (53.4 g, 208 mmol, 2.2 eq.) and 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) HCI salt (45.4 g, 237 mmol, 2.5 eq.) were dissolved in dichloromethane (DCM) (1000 ml) in a round-bottom flask (RBF) (2000 ml) equipped with a magnetic stir bar to obtain a solution. 4-Dimethylaminopyridine (DMAP) (4.63 g, 37.9 mmol, 0.4 eq.) was added to the solution, and the clear / colorless solution was stirred at ambient temperature under Nitrogen gas. Next day, in-process control (I PC) (liquid chromatographymass spectrometry (LCMS)) showed that conversion to a product was completed in the reaction. The obtained mixture was washed with H2O (500 ml) and saturated brine (500 ml). The aqueous solution was washed back with DCM (150-200 ml). The obtained organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (77.4 g). The obtained crude oil was purified by ISCO’s medium pressure liquid chromatography (MPLC) using a Si column (80 g), solid load cartridge (SLC) (32 g) and a (0-20)% hexane / ethyl acetate gradient. The obtained fractions were pooled and concentrated via rotary evaporator to yield Intermediate 1 as clear colorless oil (60.31 g, 93.3 % yield).

[0079] Step 2: Intermediate 1 (4.00 g, 5.86 mmol, 1.0 eq) was dissolved in DCM (50 ml) and methane sulfonic acid (MSA, 1.14 ml, 17.6 mmol, 3.0 eq) was added to the solution in a RBF (200 ml) equipped with a magnetic stir bar. The clear / colorless solution was stirred overnight at ambient temperature under N2gas. Next day, I PC (LCMS) confirmed that the reaction was complete. The RBF was placed into an ice-bath, 2-((2-(dimethylamino)ethyl)thio)acetic acid (S104 head group fragment, 1.20 g, 7.33 mmol, 1.25 eq.), EDC HCI salt (1.69 g, 8.79 mmol, 1.50 eq.) and DMAP (143 mg, 1.17 mmol, 0.2 eq.) were then added to the solution followed by dropwise addition of N, N-Diisopropylethylamine (DIEA) (2.56 ml, 14.7 mmol, 2.5 eq.). When the addition was complete, the ice bath was warmed to ambient temperature. Next day, IPC (LCMS) confirmed that the reaction was complete. The obtained mixture was washed with H2O (75 ml) and saturated brine (75 ml). The aqueous solution was washed back with DCM (25-30 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (5.4 g). The obtained crude oil was purified by ISCO MPLC using a Si column (80 g), SLC (32 g) and a (0-100) % hexane / ethyl acetate gradient. The obtained fractions were pooled and concentrated via rotary evaporator to yield Compound HA as clear colorless oil (4.03 g, 94.6 % yield).Preparation of LNP formulations

[0080] LNP formulations were prepared by injecting ethanol solution of lipids into an mRNA buffer solution, in the same manner as described in U. S. Publication No.2013 / 0022665, PCT Publication No. WO2019 / 090359, and WO2020 / 102668, which are hereby expressly incorporated by reference in its entirely. Particle size (PS) and polydispersity index (PDI) were obtained by using Malvern Zetasizer Nano-ZS ZEN 3600. The ratio of the lipid components in the reference LNPs (i.e., 50 mol% of cationic lipid, 38.5 mol% of cholesterol, 10 mol% of DSPC and 1.5 mol% of DMG-PEG2000) is disclosed in, for example, U. S. Patent No.8, 158, 601.Example 1 hEPO expression study

[0081] LNPs shown in Table 1 in which hEPO mRNAs were encapsulated were prepared according to the above procedure. BALB / c mice (n=3) were administered intravenously with the LNPs at 0.5 mg / kg. Six hours after administration, plasma was collected and stored at -80 °C until analysis. hEPO protein level was measured by Human EPO ELISA Kit (Invitrogen) according to the manufacturer's protocol. hEPO protein level was calculatedusing a four-parameter logistic curve fit analysis in Graphpad Prism 6. The results are shown in Fig. 1. The plasma hEPO levels of the mice administered LNPs 1a or 1 were higher than the mice administered LNP R1a. DOPE is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPC is 1,2-dioleoyl-sn-glycero-3-phosphocholine, DSPC is 1,2-distearoyl-sn-glycero-3-phosphocholine, and DMG-PEG2000 is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 in Table 1-3 and 5 below.TABLE 1LNP Lipid composition PS (nm) PDI 1a Compound IA / Cholesterol / DOPE / DMG-PEG2000 = 57.5 / 38.5 / 2.5 / 1.5 104.0 0.029 1b Compound IA / Cholesterol / DOPC / DMG-PEG2000 = 59 / 34 / 5.5 / 1.5 108.5 0.082R1a Compound IA / Cholesterol / DSPC / DMG-PEG2000 = 50 / 38.5 / 10 / 1.5 96.6 0.056Example 2 LNP distribution study

[0082] LNPs shown in Table 1 in which Flue mRNAs were encapsulated were prepared according to the above procedure. BALB / c mice (n=3) were injected intravenously with the LNPs at a dose of 0.1 mg kg1. Six hours after administration, 1.5 mg of VivoGlo Luciferin In Vivo Grade (Promega, US) dissolved in PBS (-) was injected into the tail vein. Three minutes later, lung, liver, spleen, heart, and kidney were collected from the mice and bioluminescence was captured using the IVIS Imaging System. Avg Radiance [p / s / cm2 / sr] of the ROI for each organ was calculated. The results are summarized in Figs. 2 and 3. The LNPs of the present disclosure (LNPs 2a-1, 2b- 1, 2b-2, 2c-1, 2c-2) were selectively delivered to the liver compared to the reference LNPs (LNPs R2a, R2b, R2c).TABLE 2LNP Lipid composition PS (nm) PDI 2a-1 Compound III / Cholesterol / DOPE / DMG-PEG2000 = 57.5 / 38.5 / 2.5 / 1.5 93.2 0.021 2b-1 Compound IA / Cholesterol / DOPE / DMG-PEG2000 = 57.5 / 38.5 / 2.5 / 1.5 104.2 0.033 2b-2 Compound IA / Cholesterol / DOPC / DMG-PEG2000 = 59 / 34 / 5.5 / 1.5 109.8 0.051 2c-1 Compound IB / Cholesterol / DOPE / DMG-PEG2000 = 57.5 / 38.5 / 2.5 / 1.5 104.7 0.041 2c-2 Compound IB / Cholesterol / DOPC / DMG-PEG2000 = 59 / 34 / 5.5 / 1.5 116.4 0.012 R2a Compound III / Cholesterol / DSPC / DMG-PEG2000 = 50 / 38.5 / 10 / 1.5 67.5 0.066 R2b Compound IA / Cholesterol / DSPC / DMG-PEG2000 = 50 / 38.5 / 10 / 1.5 96.2 0.029R2c Compound IB / Cholesterol / DSPC / DMG-PEG2000 = 50 / 38.5 / 10 / 1.5 93.0 0.012Example 3 LNP distribution study

[0083] LNPs shown in Table 3 in which Flue mRNAs were encapsulated were prepared according to the above procedure.TABLE 3LNP Lipid composition PS (nm) PDI 3a Compound IIA / Cholesterol / DOPE / DMG-PEG2000 = 58.25 / 39.25 / 2.5 / 1.5 89 0.067R3a Compound IIA / Cholesterol / DSPC / DMG-PEG2000 = 50 / 38 / 10 / 2 97 0.101

[0084] BALB / c mice (n=4) were administered 0.5 mpk LNP in HEPES-sucrose solution via tail vein injection. After six hours, mice were sacrificed and the liver, lung, spleen, heart, and kidney were removed and flash-frozen in liquid nitrogen. Tissues were weighed and then homogenized in 1x Reporter Lysis Buffer (Promega) to a final concentration of 1g tissue per mL using a Qiagen TissueLyser III apparatus for ten minutes at maximum speed. Tissues were then placed on ice and 20uL of homogenate or dilutions thereof were added to a white 96-well plate in duplicate. Plates were placed in a Spectramax L microplate reader (Molecular Devices) and 100 uL of Luciferase Assay Reagent (Promega E1500) was added using the plate readers injector immediately prior to well luminescence reading (2 second delay after reagent injection, 10 second signal integration time). Readings converted to relative light units (RLU) per organ by multiplying the RLU reading by the sample dilution factor and the total lysate volume per sample, then divided by the sample volume in the plate (i.e.20uL). The total RLU from liver are shown in the following table. The LNP of the present disclosure (LNP 3a) was delivered to the liver more than the reference LNP (LNP R3a).TABLE 4Injected LNP Total RLU from Liver3a 2.78E+10R3a 8.21 E+9Example 4 Toxicity study

[0085] LNPs shown in Table 5 in which Flue mRNAs were encapsulated were prepared according to the above procedure. ICR mice (n=3) were administered intravenously with the LNPs at 1 mg / kg. Twenty-four hours after administration, plasma was collected and ALT (alanine aminotransferase), AST (aspartate aminotransferase), and MCP-1 (monocyte chemotactic protein-1) levels were determined. ALT and AST were quantified byTransaminase Cii Test Wako Kits (FUJIFILM Wako Pure Chemical Corporation) and MCP-1 was quantified by Mouse CCL2 / JE / MCP-1 Quantikine ELISA Kit 2nd Gen (R& D Systems, #MJE00B). The results are summarizedin Figs. 4 to 7. The LNP of the present disclosure (LNP 4a) showed lower toxicity than the reference LNP (LNP R4a).TABLE 5LNP Lipid composition PS (nm) PDI 4a Compound IA / Cholesterol / DCPE / DMG-PEG2000 = 57.5 / 38.5 / 2.5 / 1.5 98.7 0.028R4a Compound IA / Cholesterol / DSPC / DMG-PEG2000 = 50 / 38.5 / 10 / 1.5 124.5 0.023

[0086] The above description discloses several methods and materials of the present disclosure. The present disclosure is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of the present disclosure. Consequently, it is not intended that the present disclosure be limited to the specific embodiments disclosed herein, but that it covers all modifications and alternatives coming within the true scope and spirit of the present disclosure.

[0087] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

Claims

AMENDED CLAIMSreceived by the International Bureau on 18 June 2026 (18.06.2026)We claim:

1. A lipid nanoparticle, comprising:55-65 mol% of a cationic lipid to a total lipid amount of the lipid nanoparticle; 30-40 mol% of a sterol to the total lipid amount of the lipid nanoparticle,1-3 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to the total lipid amount of the lipid nanoparticle; and1-5 mol% of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG- PEG) to the total lipid amount of the lipid nanoparticle.

2. A lipid nanoparticle, comprising:55-65 mol% of a cationic lipid to a total lipid amount of the lipid nanoparticle; 30-40 mol% of a sterol to the total lipid amount of the lipid nanoparticle;4-6 mol% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to the total lipid amount of the lipid nanoparticle; and1-5 mol% of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG- PEG) to the total lipid amount of the lipid nanoparticle.

3. The lipid nanoparticle of claim 1 or 2, wherein the cationic lipid is a compound represented by Formula II or a pharmaceutically acceptable salt thereof:R1O<IMG file=null he=null id=imgf000001_0001 img-content=null img-format=null inline=null orientation=null wi=null>Formula IIwherein:R1and R3are each independently a linear Cs-Cts alkyl group,R2and R4are each independently a hydrogen or a linear Cs-Cis alkyl group, provided that R2and R4are not hydrogen at the same time,nl and n2 are each independently 2 or 3,A<IMG file=null he=null id=imgf000002_0001 img-content=null img-format=null inline=null orientation=null wi=null>l is R12R17the arrow' indicates a connection to the carbonyl group,R11and R12are the same or different and are a hydrogen or a hydroxy group, R13, R15, R16, and R17are the same or different and are a linear C1to C4alkyl group, R14is a linear C1to C4alkyl group or —(CH2)2—OH, andX’ is a pharmaceutically acceptable counter anion.

4. The iipid nanoparticle of claim 3, wherein R? and R’ are n-octyl group, and R2and R4are n-hexyl group.

5. The lipid nanoparticle of claim 3, wherein nl and n2 are 2.

6. The lipid nanoparticle of claim 3, wherein the cationic lipid is a compound represented by Formula IIA or a pharmaceutically acceptable salt thereof:N<IMG file=null he=null id=imgf000002_0002 img-content=null img-format=null inline=null orientation=null wi=null>Formula IIA7. The lipid nanoparticle of claim 3, wherein the cationic lipid is a compound represented by Formula III or a pharmaceutically acceptable salt thereof:HO.<IMG file=null he=null id=imgf000003_0001 img-content=null img-format=null inline=null orientation=null wi=null>Compound III8. The lipid nanoparticle of claim 1 or 2, wherein the cationic lipid is a compound represented by Formula I or a pharmaceutically acceptable salt thereof:<IMG file=null he=null id=imgf000003_0002 img-content=null img-format=null inline=null orientation=null wi=null>Formula Iwherein:a represents an integer of 3-5;b represents 0 or 1;R1and R2each independently represent a group represented by Formula A:(R11)(R12)-CH-(CO-O)c-(CH2)v-Formula Awherein:R11and R12each independently represent a linear or branched Cs-15 alkyl group; each c represents 0 or 1;each v represents an integer of 4-12; andX represents a 5- to 7-membered non-aromatic heterocyclic group, wherein a carbon atom of said heterocyclic group is bound to (O-CO)b- and one or two hydrogen atoms of said heterocyclic group may optionally be replaced with a CM alkyl group or C2- 4 alkenyl group, orX represents a group represented by Formula B:-(CH2)d-N(R3)(R4)Formula Bwherein:d represents an integer of 0-3; andR3and R4each independently represent a C1-4alkyl group or C2-4alkenyl group, or R3and R4are bound to each other to form a 5- to 7-membered non-aromatic heterocyclic group, wherein one or two hydrogen atoms of said heterocyclic group may optionally be replaced with a Ci-4 alkyl group or C2-4 alkenyl group.

9. The lipid nanoparticle of claim 8, wherein the cationic lipid is a compound represented by Formula IA or a pharmaceutically acceptable salt thereof, or Formula IB or a pharmaceutically acceptable salt thereof<IMG file=null he=null id=imgf000004_0001 img-content=null img-format=null inline=null orientation=null wi=null>Formula IB10. The lipid nanoparticle of claim 1 or 2, wherein a nucleic acid is encapsulated in the lipid nanoparticle.

11. A method of delivering the nucleic acid to a cell, comprising contacting the cell with the lipid nanoparticle of claim 10.

12. A method of delivering the nucleic acid to a subject in need thereof, comprising administering to the subject the lipid nanoparticle of claim 10.

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