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WO2025143232A1 - Agent for delivering nucleic acid to immune cells and method for delivering nucleic acid to immune cells - Google Patents

Agent for delivering nucleic acid to immune cells and method for delivering nucleic acid to immune cells Download PDF

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Publication number
WO2025143232A1
WO2025143232A1 PCT/JP2024/046426 JP2024046426W WO2025143232A1 WO 2025143232 A1 WO2025143232 A1 WO 2025143232A1 JP 2024046426 W JP2024046426 W JP 2024046426W WO 2025143232 A1 WO2025143232 A1 WO 2025143232A1
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Prior art keywords
bis
dioxo
dioxa
pentylheptyl
triazatricosane
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French (fr)
Japanese (ja)
Inventor
達也 二田原
直人 中村
彩也子 梅谷
広文 福永
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Fujifilm Corp
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Fujifilm Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to a nucleic acid delivery agent for immune cells, which contains a lipid.
  • the present invention further relates to a method for delivering a nucleic acid to an immune cell using the above-mentioned nucleic acid delivery agent.
  • lipid composition containing an ionizable lipid which is a compound represented by the following formula (1) or a salt thereof, a nonionizable lipid, a lipid having a nonionic polymer, and a nucleic acid can deliver nucleic acid to both activated and unactivated immune cells, thereby completing the present invention.
  • ionizable lipid which is a compound represented by the following formula (1) or a salt thereof
  • nonionizable lipid a lipid having a nonionic polymer
  • nucleic acid can deliver nucleic acid to both activated and unactivated immune cells, thereby completing the present invention.
  • the present invention provides the following:
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ; R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R 17 , R 17 represents a hydrocarbon group having 1 to 12 carbon atoms; R 5 and R 6 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted; Substituents on the optionally substituted hydrocarbon group having
  • ⁇ 2> The agent for delivering a nucleic acid to an immune cell according to ⁇ 1>, wherein the ionizable lipid is present in an amount of 20 to 60 mol % in terms of a molar ratio relative to the total lipids in the lipid composition.
  • ⁇ 3> The agent for delivering a nucleic acid to an immune cell according to ⁇ 1> or ⁇ 2>, wherein the non-ionized lipid comprises a sterol or a derivative thereof, and a phospholipid.
  • ⁇ 4> The agent for delivering a nucleic acid to an immune cell according to ⁇ 3>, wherein the phospholipid is selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine.
  • ⁇ 5> The agent for delivering a nucleic acid to an immune cell according to ⁇ 3> or ⁇ 4>, wherein the sterol or a derivative thereof is present in an amount of 30 to 70 mol % in terms of a molar ratio relative to the total lipids in the lipid composition.
  • lipid having a polyethylene glycol chain is selected from dimyristoyl-rac-glycerol polyethylene glycol, distearoyl-rac-glycerol polyethylene glycol, and distearoylphosphatidylethanolamine polyethylene glycol.
  • lipid having the nonionic polymer is present in an amount of 0.1 to 3 mol % in terms of a molar ratio relative to the total lipid in the lipid composition.
  • ⁇ 15> The method according to ⁇ 13>, comprising the step of adding (i) an apolipoprotein, and/or (ii) a protein comprising a cell-binding domain and a heparin-binding domain to the nucleic acid delivery agent or the immune cells before contacting the nucleic acid delivery agent with the immune cells.
  • ⁇ 16> A step of preparing a lipid particle not containing nucleic acid by using an ionizable lipid which is a compound represented by formula (1) or a salt thereof, a nonionizable lipid, and a lipid having a nonionic polymer; Mixing the nucleic acid-free lipid particles with nucleic acid, ⁇ 12> A method for producing a nucleic acid delivery agent according to any one of ⁇ 1> to ⁇ 12>.
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ; R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R 17 , R 17 represents a hydrocarbon group having 1 to 12 carbon atoms; R 5 and R 6 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted; Substituents on the optionally substituted hydrocarbon group having
  • nucleic acids it is possible to deliver nucleic acids to both activated and unactivated immune cells.
  • FIG. 1 shows the results of measuring nucleic acid delivery to activated T cells.
  • FIG. 2 shows the results of measuring nucleic acid delivery to non-activated T cells.
  • FIG. 3 shows the results of measuring nucleic acid delivery to activated T cells in an activation medium to which ApoE3 has not been added.
  • FIG. 4 shows the results of measuring nucleic acid delivery to non-activated T cells in an activation medium without the addition of ApoE3.
  • Figure 5 shows the results of measuring TCR KO efficiency in activated T cells.
  • FIG. 6 shows the results of measuring nucleic acid delivery to activated T cells under culture conditions in which various proteins were added.
  • FIG. 7 shows the results of measuring nucleic acid delivery to long-term cultured T cells.
  • the nucleic acid delivery agent for immune cells of the present invention comprises a lipid composition comprising an ionizable lipid that is a compound represented by formula (1) or a salt thereof, a non-ionizable lipid, a lipid having a non-ionic polymer, and a nucleic acid.
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ; R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R 17 , R 17 represents a hydrocarbon group having 1 to 12 carbon atoms; R 5 and R 6 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted; Substituents on the optionally substituted hydrocarbon group having
  • the nucleic acid delivery agent for immune cells of the present invention can be used to create immune cells with modified gene expression, and can deliver target nucleic acids even to inactive immune cells, preventing cell exhaustion and improving cell therapy performance.
  • the hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon group having 1 to 18 carbon atoms, the hydrocarbon group having 1 to 12 carbon atoms, and the hydrocarbon group having 1 to 8 carbon atoms are preferably an alkyl group, an alkenyl group, or an alkynyl group, respectively.
  • the alkyl group may be linear or branched, and may be linear or cyclic. Specific examples of the alkyl group include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, 2-butylhex
  • the alkenyl group may be linear or branched, and may be linear or cyclic. Specifically, it is an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably, a (Z)-2-nonenyl group or a (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably, a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), a pentadecenyl group (preferably, a (Z)-pentadecenyl-8-enyl group).
  • hexadecenyl group (preferably, (Z)-hexadecanyl group), hexadecadienyl group, heptadecenyl group (preferably, (Z)-heptadecanyl group), heptadecadienyl group (preferably, (8Z,11Z)-heptadecanyl group, octadecenyl group (preferably, (Z)-octadecane-9-enyl group), octadecadienyl group (preferably, (9Z,12Z)-octadecanyl group, 12-octadecanyl group), etc.
  • the alkynyl group may be straight-chained or branched, and may be linear or cyclic. Specific examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, and octadecynyl groups.
  • each of the above alkenyl groups has one or two double bonds, and it is preferable that each of the alkynyl groups has one or two triple bonds.
  • the hydrocarbon group having 2 to 8 carbon atoms represented by R 7 , R 8 and R 9 is preferably an alkylene group, an alkenylene group or an alkynylene group.
  • the alkylene group, alkenylene group or alkynylene group having 2 to 8 carbon atoms may be straight-chain or branched, and may be linear or cyclic. Specific examples include an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group.
  • aryl groups having 6 to 20 carbon atoms aryl groups having 6 to 18 carbon atoms are preferred, and aryl groups having 6 to 10 carbon atoms are even more preferred.
  • Specific examples include phenyl groups, naphthyl groups, anthracenyl groups, and phenanthrenyl groups.
  • Heterocyclic group means a heteroaryl group or a heteroaliphatic ring group.
  • heteroaryl group refers to an aromatic heterocyclic group, which may be an aromatic heterocyclic group, an aromatic hydrocarbon ring, a heteroaliphatic ring, or an aromatic heterocyclic group condensed with an aliphatic hydrocarbon ring, and is preferably a monocyclic nitrogen-containing heteroaryl group, a monocyclic oxygen-containing heteroaryl group, a monocyclic sulfur-containing heteroaryl group, a monocyclic nitrogen-containing oxygen-containing heteroaryl group, a monocyclic nitrogen-containing sulfur-containing heteroaryl group, a bicyclic nitrogen-containing heteroaryl group, a bicyclic oxygen-containing heteroaryl group, a bicyclic sulfur-containing heteroaryl group, a bicyclic nitrogen-containing oxygen-containing heteroaryl group, or a bicyclic nitrogen-containing sulfur-containing heteroaryl group.
  • a five-membered heteroaryl group is a monocyclic heteroaryl group having five atoms constituting the ring.
  • an aromatic heterocycle means an aromatic ring having a heteroatom as a ring member, and may be a condensed aromatic heterocycle, aromatic hydrocarbon ring, heteroaliphatic ring, or aliphatic hydrocarbon ring, and is preferably a monocyclic nitrogen-containing aromatic heterocycle, a monocyclic oxygen-containing aromatic heterocycle, a monocyclic sulfur-containing aromatic heterocycle, a monocyclic nitrogen-containing oxygen-containing aromatic heterocycle, a monocyclic nitrogen-containing sulfur-containing aromatic heterocycle, a bicyclic nitrogen-containing aromatic heterocycle, a bicyclic oxygen-containing aromatic heterocycle, a bicyclic sulfur-containing aromatic heterocycle, a bicyclic nitrogen-containing oxygen-containing aromatic heterocycle, or a bicyclic nitrogen-containing sulfur-containing aromatic heterocycle.
  • the term "monocyclic nitrogen-containing heteroaryl group” refers to a heteroaryl group (which may be partially saturated) in which the ring containing at least one nitrogen atom has aromaticity, such as pyrrolinyl, pyrrolyl, tetrahydropyridyl, pyridyl, imidazolinyl, imidazolyl, pyrazolinyl, pyrazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazolyl, and tetrazolyl groups.
  • This heteroaryl group may be further condensed with another aromatic ring or an aliphatic ring.
  • the term "monocyclic oxygen-containing heteroaryl group” refers to a heteroaryl group (which may be partially saturated) in which the ring containing at least one oxygen atom has aromaticity, such as a furanyl or pyranyl group, which may further be condensed with another aromatic ring or an aliphatic ring.
  • the monocyclic nitrogen-containing and oxygen-containing heteroaryl group means an oxazolyl, isoxazolyl, oxadiazolyl group, etc. This heteroaryl group may be further condensed with another aromatic ring or an aliphatic ring.
  • the monocyclic nitrogen-containing sulfur-containing heteroaryl group means a thiazolyl, isothiazolyl or thiadiazolyl group, etc. This heteroaryl group may be further condensed with another aromatic ring or an aliphatic ring.
  • the bicyclic nitrogen-containing heteroaryl group is indolyl, isoindolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pyrrolopyridyl, imidazopyridyl, pyrazolopyridyl, pyridopyrazyl, purinyl, pteridinyl, 5,6,7,8-tetrahydrophthalazinyl, 5,6,7,8-tetrahydrocinnolinyl, 1,2,3,4-tetrahydropyrido[2,3-d]pyridazinyl, 5,6,7,8-tetrahydro-[1,
  • bicyclic oxygen-containing heteroaryl group refers to a bicyclic heteroaryl group in which the ring containing at least one oxygen atom has aromaticity, such as benzofuranyl, isobenzofuranyl, and chromenyl groups (this heteroaryl group may be partially saturated).
  • the bicyclic nitrogen-containing and oxygen-containing heteroaryl group is benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, dihydropyranopyridyl, dihydrodioxinopyridyl, dihydropyridoxadienyl, 3,4-dihydro-2H-pyrano[2,3-d]pyridazinyl, 7,8-dihydro-5H-pyrano[3,4-d]pyridazinyl, 7,8-dihydro-6H-pyrano[3,2-c]pyridazinyl, 7,8-dihydro-5H-pyrano[4,3-c]pyridazinyl, It means a bicyclic heteroaryl group in which the ring containing at least one nitrogen atom and at least one oxygen atom has aromaticity, such as 2,3-dihydrofuro[2,3-d]pyridazinyl, 5,7-dihydrofuro
  • the heteroaliphatic ring group means a nitrogen-containing heteroaliphatic ring group, an oxygen-containing heteroaliphatic ring group, a sulfur-containing heteroaliphatic ring group, a nitrogen-containing oxygen-containing heteroaliphatic ring group, a nitrogen-containing sulfur-containing heteroaliphatic ring group, a heterobridged ring group, or a heterospiro ring group.
  • heteroaliphatic ring means an aliphatic ring having a heteroatom as a ring member, and preferred examples thereof include a nitrogen-containing heteroaliphatic ring, an oxygen-containing heteroaliphatic ring, a sulfur-containing heteroaliphatic ring, a nitrogen-containing oxygen-containing heteroaliphatic ring, a nitrogen-containing sulfur-containing heteroaliphatic ring, a heterobridged ring, and a heterospiro ring.
  • the nitrogen-containing heteroaliphatic ring group refers to a heteroaliphatic ring group in which the ring containing at least one nitrogen atom does not have aromaticity, such as azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, octahydroazocinyl, imidazolidinyl, pyrazolidinyl, piperazinyl, and homopiperazinyl groups, etc.
  • This nitrogen-containing heteroaliphatic ring group may be further condensed with another aromatic ring or an aliphatic ring.
  • the oxygen-containing heteroaliphatic ring group means a tetrahydrofuranyl, tetrahydropyranyl, oxetanyl, 1,3-dioxanyl group, etc. This oxygen-containing heteroaliphatic ring group may be further condensed with another aromatic ring or an aliphatic ring.
  • the nitrogen-containing, oxygen-containing heteroaliphatic ring group means a morpholinyl or 1,4-oxazepanyl group, etc. This nitrogen-containing, oxygen-containing heteroaliphatic ring group may be further condensed with another aromatic ring or an aliphatic ring.
  • the heteroaliphatic ring C 1-8 alkyl group means a straight-chain, branched-chain or cyclic C 1-8 alkyl group bonded to a heteroaliphatic ring group such as a pyrrolidinylmethyl group, a pyrrolidinylethyl group, a pyrrolidinylpropyl group, a pyrrolidinyloctyl group, a piperidinylmethyl group or a tetrahydrofuranylmethyl group.
  • a heteroaliphatic ring group such as a pyrrolidinylmethyl group, a pyrrolidinylethyl group, a pyrrolidinylpropyl group, a pyrrolidinyloctyl group, a piperidinylmethyl group or a tetrahydrofuranylmethyl group.
  • R 1 represents -R 1a -L 1 -R 1b
  • R 1a represents a hydrocarbon group having 1 to 18 carbon atoms
  • L 1 represents -C(O)O-, -OC(O)-, -OC(O)O-, or -S-S-
  • R 1b represents a hydrocarbon group having 1 to 18 carbon atoms
  • R 3 represents -R 3a -L 3 -R 3b
  • R 3a represents a hydrocarbon group having 1 to 18 carbon atoms
  • L3 represents -C(O)O-, -OC(O)-, -OC(O)O-, or -S-S-
  • R 3b represents a hydrocarbon group having 1 to 18 carbon atoms
  • R 2 and R 4 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted, and the substituents on the hydrocarbon group having 1 to 18 carbon atoms which may be substituted represented by R 2 and R 4 each independently represent -C
  • R 1 represents -R 1a -L 1 -R 1b
  • R 1a represents a hydrocarbon group having 1 to 18 carbon atoms
  • L 1 represents -C(O)O- or -OC(O)-
  • R 1b represents a hydrocarbon group having 1 to 18 carbon atoms
  • R 3 represents -R 3a -L 3 -R 3b
  • R 3a represents a hydrocarbon group having 1 to 18 carbon atoms
  • L 3 represents -C(O)O- or -OC(O)-
  • R 3b represents a hydrocarbon group having 1 to 18 carbon atoms
  • R 2 and R 4 each independently represent a hydrocarbon group having 1 to 10 carbon atoms
  • R 5 and R 6 each independently represent a hydrocarbon group having 1 to 6 carbon atoms which may be substituted
  • Substituents on the optionally substituted hydrocarbon group having 1 to 6 carbon atoms represented by R 5 and R 6 each independently represent -OH, -O-R 26 , -
  • R 1 represents -R 1a -L 1 -R 1b
  • R 1a represents a hydrocarbon group having 1 to 5 carbon atoms
  • L 1 represents -C(O)O-
  • R 1b represents a hydrocarbon group having 7 to 14 carbon atoms
  • R 3 represents -R 3a -L 3 -R 3b
  • R 3a represents a hydrocarbon group having 1 to 5 carbon atoms
  • L 3 represents -C(O)O-
  • R 3b represents a hydrocarbon group having 7 to 14 carbon atoms
  • R 2 and R 4 each independently represent a hydrocarbon group having 3 to 8 carbon atoms
  • R5 and R6 each independently represent a hydrocarbon group having 1 to 4 carbon atoms which may be substituted, and the substituents on the hydrocarbon group having 1 to 4 carbon atoms which may be substituted represented by R5 and R6 each independently are preferably an —OH group.
  • R 7 , R 8 and R 9 each independently represent --(CH 2 ) n
  • the compound represented by formula (1) may form a salt.
  • salts of basic groups include salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.
  • mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid
  • organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid,
  • salts of acidic groups include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl- ⁇ -phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine.
  • preferred salts include pharmacologically acceptable salts.
  • Compounds 1 to 7 and 31 to 74 are novel compounds. According to the present invention, compounds 1 to 7 and 31 to 74 are provided.
  • the amount of the compound represented by formula (1) or its salt is, in terms of molar ratio to the total lipids in the lipid composition, preferably 20 mol% to 60 mol%, more preferably 30 mol% to 60 mol%, and even more preferably 40 mol% to 60 mol%.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 have the same meanings as above;
  • R 8a , R 9a and R A represent a hydrocarbon group having 1 to 7 carbon atoms.
  • the compound of formula [3A] can be produced by reacting the compound of formula [2] in the presence of water and an acid, in the presence or absence of a solvent.
  • the acid used in this reaction may be an inorganic acid or an organic acid, preferably an organic acid, specifically formic acid, acetic acid, trifluoroacetic acid, 4-toluenesulfonic acid, methanesulfonic acid, etc., more preferably formic acid.
  • the amount of the acid used may be 1 to 100 times (v/w), preferably 1 to 10 times (v/w), relative to the amount of the compound of the formula [2].
  • the amount of water used may be 0.1 to 100 times (v/w), preferably 0.1 to 10 times (v/w), relative to the amount of the compound of the formula [2].
  • the solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination.
  • the amount of the solvent used is not particularly limited, but may be 0.1 to 50 times (v/w) the amount of the compound of the formula [2]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.
  • the compound of formula [1] can be prepared by reacting a compound of formula [3A] with a compound of formula [4] in the presence of a reducing agent.
  • a reducing agent for example, N,N-diethylethylenediamine and N,N-diethyl-1,3-diaminopropane.
  • the solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, alcohols, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include esters, with ethyl acetate being more preferred.
  • the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [3A].
  • the reducing agent used in this reaction includes sodium borohydride, sodium cyanoborohydride, pyridine borane, 2-picoline borane, and sodium triacetoxyborohydride, with sodium triacetoxyborohydride being more preferred.
  • the amount of the reducing agent used may be 1 to 100 times, preferably 1 to 10 times, the molar amount of the compound of the formula [3A].
  • the amount of the compound of the formula [4] used may be 0.1 to 1 mole per mole of the compound of the formula [3A]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.
  • the compound of formula [5] can be prepared by reacting a compound of formula [3A] with a compound of formula [4] in the presence of a reducing agent. This reaction may be carried out in accordance with the production method (1-2), and the compound of the formula [4] may be used in an amount of 1 to 10 times by mole relative to the compound of the formula [3A].
  • the compound of formula [1] can be prepared by reacting a compound of formula [3B] with a compound of formula [5] in the presence of a reducing agent. This reaction may be carried out in accordance with the production method (1-2), and the compound of the formula [3B] may be used in an amount of 1 to 10 times by mole relative to the compound of the formula [5].
  • R 1 , R 2 , R 9a and R A have the same meanings as above;
  • X 1 and X 2 represent leaving groups.
  • leaving groups include a chloro group, a fluoro group, a bromo group, a trichloromethoxy group, a 4-nitro-phenoxy group, a 2,4-dinitrophenoxy group, a 2,4,6-trichlorophenoxy group, a pentafluorophenoxy group, a 2,3,5,6-tetrafluorophenoxy group, an imidazolyl group, a triazolyl group, a 3,5-dioxo-4-methyl-1,2,4-oxadiazolidyl group, and an N-hydroxysuccinimidyl group.
  • the compound of formula [9] can be prepared by reacting a compound of formula [7] with a compound of formula [8] in the presence or absence of a base.
  • a compound of the formula [8] include 1,1'-carbonyldi(1,2,4-triazole), 1,1'-carbonyldiimidazole, 4-nitrophenyl chloroformate, triphosgene, and phosgene.
  • the solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination.
  • Preferred solvents include ethers, with tetrahydrofuran being more preferred.
  • the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [7].
  • the base used in this reaction may be an inorganic base or an organic base, preferably an organic base, such as triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, or N,N-dimethylaminopyridine.
  • the amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar amount of the compound of the formula [7].
  • the amount of the compound of the formula [8] used is not particularly limited, but may be 1 to 10 times the molar amount of the compound of the formula [7]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.
  • the compound of formula [2] can be prepared by reacting a compound of formula [6] with a compound of formula [9] in the presence of a base.
  • a compound of the formula [6] for example, dioctylamine is known.
  • the solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides and aromatic hydrocarbons, and these solvents may be used in combination. Preferred solvents include nitriles, with acetonitrile being more preferred.
  • the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [6].
  • the base used in this reaction may be an inorganic base or an organic base, such as potassium carbonate, sodium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, lithium phosphate, triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, or N,N-dimethylaminopyridine.
  • the amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar amount of the compound of the formula [6].
  • the amount of the compound of the formula [9] used is not particularly limited, but may be 0.1 to 10 times the molar amount of the compound of the formula [6]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.
  • the compound of formula [12A] can be produced by reacting a compound of formula [10A] with a compound of formula [11A] in the presence or absence of an acid, in the presence or absence of a condensing agent or an acid halide, and in the presence or absence of a base.
  • Known examples of the compound of the formula [10A] include 5-bromovaleric acid and chloroacetyl chloride.
  • Known examples of the compound of the formula [11A] include 2-butyl-1-octanol, 2-pentyl-1-heptanol, and 1-decanol.
  • the solvent used in this reaction is not particularly limited as long as it does not affect the reaction.
  • the solvent examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include aromatic hydrocarbons and ethers, with toluene and tetrahydrofuran being more preferred.
  • the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [10A].
  • the acid used in this reaction may be an inorganic acid or an organic acid.
  • the acid is preferably a sulfonic acid, specifically, sulfuric acid, 4-toluenesulfonic acid, methanesulfonic acid, etc.
  • Condensing agents used in this reaction include, for example, carbodiimides such as N,N'-dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; carbonyls such as carbonyldiimidazole; acid azides such as diphenylphosphoryl azide; acid cyanides such as diethylphosphoryl cyanide; 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; uroniums such as O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.
  • carbodiimides such as N,N'-dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethyla
  • the acid halide used in this reaction includes, for example, carboxylic acid halides such as acetyl chloride and trifluoroacetyl chloride; sulfonic acid halides such as methanesulfonyl chloride and tosyl chloride; chloroformates such as ethyl chloroformate and isobutyl chloroformate; and the like.
  • the base used in this reaction may be an inorganic base or an organic base, preferably an organic base, such as triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, or N,N-dimethylaminopyridine.
  • the amount of the base used may be 1 to 50 moles, preferably 1 to 10 moles, based on the amount of the compound of the formula [10A].
  • the amount of the compound of formula [11A] used is not particularly limited, but may be 0.8 to 10 times (v/w) the amount of the compound of formula [10A]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.
  • the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [12A].
  • the base used in this reaction may be an inorganic base or an organic base, specifically, potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, lithium phosphate, triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, or N,N-dimethylaminopyridine, and potassium carbonate is more preferred.
  • the amount of the base used may be 1 to 50 moles, preferably 1 to 10 moles, based on the amount of the compound of the formula [12A].
  • the amount of the compound of formula [13] used is not particularly limited, but may be 1 to 10 times the molar amount of the compound of formula [12A].
  • Specific examples of additives used in this reaction include lithium iodide, sodium iodide, potassium iodide, benzyltriethylammonium iodide, and benzyltriethylammonium bromide.
  • the amount of the additive used may be 0.1 to 10 times the molar amount of the compound of the formula [12A]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.
  • R 2 , R 1a , R 1b , X 3 and X 4 have the same meanings as above.
  • the compound of formula [12B] can be produced in the same manner as in Production Method (3-1), except that the compound of formula [10B] is used in place of formula [10A] and the compound of formula [11B] is used in place of formula [11A].
  • R 1a , R 1b and X 3 have the same meaning as above; R B represents an amino protecting group.
  • the compound of formula [15] can be produced by reacting a compound of formula [10B] with a compound of formula [14] in the presence or absence of an acid, in the presence or absence of a condensing agent or an acid halide, and in the presence or absence of a base.
  • Known examples of the compound of the formula [10B] include decanoic acid and decanoic acid chloride.
  • Known examples of the compound of the formula [15] include tert-butyl bis(2-hydroxyethyl)carbamate. This reaction may be carried out according to the production method (3-1).
  • the compound of formula [6C] can be prepared by deprotecting the compound of formula [15]. This reaction may be carried out, for example, according to the method described in T. W. Greene et al., Protective Groups in Organic Synthesis, 4th Edition, pp. 696-926, 2007, John Wiley & Sons, INC.
  • compounds having an amino group, a hydroxyl group, a carboxyl group, or the like can have these groups protected in advance with a conventional protecting group, and after the reaction, these protecting groups can be removed by a method known per se.
  • the compounds obtained by the above-mentioned production methods can be derived into other compounds by subjecting them to reactions known per se, such as condensation, addition, oxidation, reduction, rearrangement, substitution, halogenation, dehydration, or hydrolysis, or by appropriately combining these reactions.
  • the lipid composition of the present invention preferably contains a sterol or a derivative thereof as a non-ionized lipid.
  • a sterol in the lipid composition, the membrane fluidity can be reduced and the lipid composition can be stabilized.
  • sterols include, but are not limited to, cholesterol, phytosterols (sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol, etc.), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, etc.
  • cholesterol is preferred.
  • the amount of sterol or its derivative is preferably 30 to 70 mol%, more preferably 30 mol% to 65 mol%, and even more preferably 30 mol% to 60 mol%, in terms of the molar ratio to the total lipids in the lipid composition.
  • the lipid composition of the present invention preferably contains phospholipid as non-ionized lipid.
  • the phospholipid is not particularly limited, but may be phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, etc., and phosphatidylcholine is preferred.
  • the phospholipid may be a single lipid or a combination of multiple different neutral lipids.
  • the phospholipid is preferably selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine.
  • the amount of phospholipids is preferably 1 to 30 mol %, more preferably 1 to 20 mol %, based on the molar ratio of the total lipids in the lipid composition.
  • the lipid composition of the present invention may contain a lipid having a nonionic hydrophilic polymer.
  • a lipid having a nonionic hydrophilic polymer by containing a lipid having a nonionic hydrophilic polymer, a dispersion stabilization effect of the lipid composition can be obtained.
  • nonionic hydrophilic polymers include, but are not limited to, nonionic vinyl polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, nonionic modified natural polymers, and block polymers or graft copolymers having two or more of these polymers as constituent units.
  • nonionic hydrophilic polymers preferred are nonionic polyethers, nonionic polyesters, nonionic polyamino acids or nonionic synthetic polypeptides, more preferred are nonionic polyethers or nonionic polyesters, even more preferred are nonionic polyethers or nonionic monoalkoxy polyethers, and particularly preferred are polyethylene glycols (hereinafter, polyethylene glycols are also referred to as PEG). That is, the lipid having a nonionic polymer is preferably a lipid having a polyethylene glycol chain.
  • Lipids having a non-ionic hydrophilic polymer include, but are not limited to, PEG-modified phosphoethanolamine, diacylglycerol PEG derivatives, monoacylglycerol PEG derivatives, dialkylglycerol PEG derivatives, cholesterol PEG derivatives, and ceramide PEG derivatives. Among these, monoacylglycerol PEG or diacylglycerol PEG is preferred.
  • the lipid having a polyethylene glycol chain is particularly preferably selected from dimyristoyl-rac-glycerol polyethylene glycol, distearoyl-rac-glycerol polyethylene glycol, and distearoylphosphatidylethanolamine polyethylene glycol.
  • the weight average molecular weight of the PEG chain of the nonionic hydrophilic polymer derivative is preferably 500 to 5,000, and more preferably 750 to 3,000.
  • the non-ionic hydrophilic polymer chain may be branched and may have a substituent such as a hydroxymethyl group.
  • the amount of lipid having a nonionic hydrophilic polymer chain is preferably 0.1 to 3 mol %, more preferably 0.3 to 3 mol %, and even more preferably 0.5 to 3 mol %, in terms of molar ratio to the total lipid in the lipid composition.
  • the lipid composition comprises a nucleic acid.
  • nucleic acids include circular double-stranded DNA (plasmid DNA, small circular double-stranded DNA without drug resistance genes, etc.), single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (micro RNA), mRNA, antisense oligonucleotide (also called ASO), ribozyme, aptamer, saRNA, sgRNA, etc., and any of them may be included. Two or more types of nucleic acids may be used. In addition, modified nucleic acids may be included. As the nucleic acid, circular double-stranded DNA or RNA is particularly preferred, and plasmid DNA or mRNA is most preferred. The number of bases is preferably 5 to 20,000 bases.
  • the nucleic acid may be an mRNA encoding a DNA nuclease, which is a sequence for gene editing.
  • the nucleic acid may be a guide RNA, which is a sequence for gene editing.
  • the nucleic acid may be a nucleic acid mixture that is a sequence for gene editing and includes an mRNA encoding Cas nuclease and a guide RNA. That is, the nucleic acid may be a nucleic acid for gene editing that includes an mRNA encoding Cas nuclease and a guide RNA.
  • the mixture may further include any donor DNA.
  • the nucleic acid may be a nucleic acid mixture containing an mRNA encoding a deaminase and a mutant Cas nuclease, and a guide RNA, which is a sequence for single base editing.
  • the nucleic acid may be a nucleic acid mixture containing an mRNA encoding a fusion protein of an artificial reverse transcriptase and a Cas9 endonuclease, the nucleic acid being a sequence for replacing a target DNA nucleotide, and a prime editing guide RNA.
  • the nucleic acid may be a sequence for guide RNA-dependent gene transcription suppression (such as the CRISPR interference system), and may be a nucleic acid mixture containing an mRNA encoding a fusion protein of a transcription suppressor (such as KRAB) and a mutant Cas nuclease, and a guide RNA.
  • the nucleic acid may be an mRNA or DNA encoding a DNA recombinase, and may further be a nucleic acid mixture optionally containing a donor DNA.
  • the nucleic acid may be an mRNA or DNA encoding a DNA recombinase, or may be a nucleic acid mixture optionally containing a donor DNA.
  • the DNA recombinase may include, but is not limited to, transposase (e.g., Sleeping beauty transposase, piggyBac transposase, Tol2, etc.), Cre recombinase, serine integrase, etc.
  • the nucleic acid may be a nucleic acid mixture that contains a sequence for inserting a foreign gene into the host cell genome, an mRNA encoding a reverse transcriptase, and, optionally, a donor RNA.
  • the nucleic acid may be an mRNA encoding a sequence for expressing a foreign gene, or a DNA containing the foreign gene, a promoter sequence, and a terminal sequence.
  • the nucleic acid may be a sequence for gene editing or gene transcription inhibition of a gene (target gene) present in immune cells.
  • Target genes include, but are not limited to, T cell receptor genes (TRAC, TRBC), MHC-I (or HLA-I), MHC-II, B2M, genes related to self-antigens, genes related to inhibitory receptors or their ligands (PDCD1, CD274 (or PD-L1), PDCD1LG2, LAG3, CTLA4), genes related to cytotoxicity (TGFBR2, PGE2, EP2, EP4, FAS, FASLG), genes related to cell exhaustion or differentiation (SOCS1, ZC3H12A, NR4A1, NR4A2, NR4A3, PRDM1, BLIMP1, TIM3), cell death-related genes (CASP3, CASP6, CASP7), genes related to inflammatory responses (CGAS, STING, TBK1), etc.
  • T cell receptor genes TRAC, TRBC
  • MHC-I or HLA-I
  • methylation genes TET1, TET2, DNMT3A
  • genes involved in immune evasion CD47, NKG2A
  • others DGK, EZH2, CSF2, PAX5, LDLR, MAP4K1, CISH, CD5, CD52, ADORA2A, CD39, CD73, CD5, MCM3AP, EIF3D, CAD, HGS, RPL19, MAK16, PDG FRA, NRF1, EP400, CBLB, RPS7, CPSF4, IL2RG, RPL38, IL2RB, JAK3, MCM2, SNRPC, PSMD4, MAP4K1, BRD9, RNF20, RNF40, NFKB2, NMT1, MYB, TSC1, EIF3K, RPL19, TBX21, PRDM1, SUV39H1, ARID1A), etc.
  • the nucleic acid may be a sequence for expressing a chimeric antigen receptor gene, a T cell receptor gene, an antibody, a bispecific antibody, a multispecific antibody, a single chain Fv (scFv), a nanobody, or a bispecific T cell-inducing antibody (BiTE).
  • the nucleic acid may be a sequence for expressing a foreign gene involved in the initialization (or reprogramming) of a cell. Examples of the foreign gene include, but are not limited to, Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28.
  • the mass ratio of the total lipids to the nucleic acids in the lipid composition is preferably 5:1 to 1000:1, more preferably 5:1 to 500:1, even more preferably 7:1 to 200:1, and particularly preferably 7:1 to 100:1.
  • the method for producing the lipid composition is not limited, but the lipid composition can be produced by dissolving all or a part of the oil-soluble components of the lipid composition in an organic solvent or the like to form an oil phase, dissolving the water-soluble components in water to form an aqueous phase, and mixing the oil phase and the aqueous phase.
  • a micromixer may be used for mixing, or the lipid composition may be emulsified using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • An example of a method for producing a lipid composition containing nucleic acid is Step (a): dissolving the components of the lipid composition containing the compound represented by formula (1) in an organic solvent to obtain an oil phase, and dissolving the nucleic acid in an aqueous solvent to obtain an aqueous phase; Step (b): mixing the oil phase and aqueous phase obtained in step (a) to obtain a lipid particle dispersion; Step (c): diluting the lipid particle dispersion obtained in step (b); Step (d): removing the organic solvent from the lipid particle dispersion; and Step (e): adjusting the concentration of the lipid particle dispersion.
  • components such as a buffer component for pH adjustment and an antioxidant can be added.
  • the pH of the aqueous phase is preferably 2.0 to 7.0, more preferably 3.0 to 6.0.
  • acetic acid, citric acid, malic acid, phosphoric acid, MES, HEPES, etc. are preferably used as buffer components, and if necessary, salts such as sodium chloride and potassium chloride may be added to adjust the salt strength, and sugars or sugar alcohols such as sucrose, trehalose, and mannitol may be added to adjust the osmotic pressure.
  • the oil phase and the aqueous phase may be mixed by any method, including a batch method or an in-line method using a flow path device.
  • a micro flow path device is preferably used, and examples of the micro flow path device that can be used include a Y-shaped mixer, a T-shaped mixer, a herringbone mixer, a ring micro mixer, and an impingement jet mixer.
  • the mixing ratio (volume ratio) of the aqueous phase to the oil phase is preferably 5:1 to 1:1, and more preferably 4:1 to 2:1.
  • the lipid particle dispersion is mixed with a diluent solution to reduce the organic solvent content and stabilize the lipid particles.
  • the diluent solution may be water, but may also include adjusting the pH or salt strength.
  • the components contained in the diluent solution may be selected arbitrarily depending on the purpose.
  • a buffer solution e.g., citrate buffer, citrate buffered saline, acetate buffer, acetate buffered saline, phosphate buffered saline, Tris buffer, MES buffer, HEPES buffer, etc.
  • a buffer solution e.g., citrate buffer, citrate buffered saline, acetate buffer, acetate buffered saline, phosphate buffered saline, Tris buffer, MES buffer, HEPES buffer, etc.
  • multiple dilution steps may be performed consecutively, and the interval between one dilution step and the next dilution step may be set arbitrarily, for example, the interval may be 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours or 24 hours.
  • the pH of the lipid particle dispersion after step (c) is preferably pH 3.0 to pH 10.0, more preferably pH 3.5 to pH 9.0, and particularly preferably pH 4.0 to pH 8.5.
  • the lipid composition may be subjected to sizing as necessary.
  • the method of sizing is not particularly limited, but the particle size may be reduced using an extruder or the like.
  • the dispersion containing the lipid composition can be subjected to freezing or lyophilization by a general method.
  • the method for removing the organic solvent from the lipid particle dispersion is not particularly limited, and a general method can be used.
  • a pH buffer solution such as phosphate buffered saline or Tris buffer can be used as the dialysis fluid, and additives such as any salt or sugar can be added as necessary to adjust the osmotic pressure or protect the dispersion from freezing.
  • step (e) the concentration of the lipid particle dispersion obtained in step (d) can be adjusted.
  • a solution such as phosphate buffered saline, saline, Tris buffer, or sucrose-containing Tris buffer can be used as a diluent to dilute to an appropriate concentration.
  • the dispersion obtained in step (d) can be concentrated by ultrafiltration using an ultrafiltration membrane. The concentrated dispersion can be used as is, or it can be concentrated and then adjusted to the desired concentration using the above diluent.
  • the organic solvent removal step (step (d)) and the concentration adjustment step (step (e)) can be carried out continuously using tangential flow filtration (TFF).
  • TDF tangential flow filtration
  • the organic solvent removal step and the concentration adjustment step may be carried out in any order. If necessary, the organic solvent removal step and the concentration adjustment step may each be carried out multiple times.
  • the solution that can be used for dialysis in step (d) and dilution in step (e) may contain excipients, cryoprotectants, buffers, and antioxidants.
  • excipients and cryoprotectants include, but are not limited to, sugars and sugar alcohols.
  • sugars include sucrose, trehalose, maltose, glucose, lactose, and fructose
  • sugar alcohols include mannitol, sorbitol, inositol, and xylitol.
  • buffers include, but are not limited to, ACES, BES, Bicine, CAPS, CHES, DIPSO, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, TAPS, TAPSO, TES, Tricine, Tris, phosphoric acid, acetic acid, and citric acid.
  • antioxidants include EDTA, ascorbic acid, and tocopherol.
  • the lipid particle dispersion may be sterilized by filtration.As a filtration method, hollow fiber membrane, reverse osmosis membrane, membrane filter, etc. can be used to remove insoluble matter from the lipid particle dispersion.In the present invention, although not particularly limited, it is preferable to filter by a filter with a pore size that can be sterilized (preferably a 0.2 ⁇ m filter sterilization filter). In addition, it is preferable to carry out the sterile filtration after step (d) or step (e). Furthermore, if necessary, the lipid particle dispersion liquid may be subjected to freezing or lyophilization. The lipid particle dispersion liquid may be subjected to freezing or lyophilization by a general method, and the method is not particularly limited.
  • a micromixer may be used for mixing, or the emulsion may be produced using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • the lipid-containing solution can be dried under reduced pressure using an evaporator or spray-dried using a spray dryer to prepare a dried mixture containing lipids, and this mixture can be added to an aqueous solvent and further emulsified using the emulsifier described above.
  • the oil phase and the aqueous phase may be mixed by any method, including a batch method and an in-line method using a flow path device.
  • a micro flow path device is preferably used, and examples of the micro flow path device that can be used include a Y-shaped mixer, a T-shaped mixer, a herringbone mixer, a ring micro mixer, and an impingement jet mixer.
  • the mixing ratio (volume ratio) of the aqueous phase and the oil phase is preferably 5:1 to 1:1, and more preferably 4:1 to 2:1.
  • the lipid particle dispersion is mixed with a diluent solution to reduce the organic solvent content and stabilize the lipid particles.
  • the diluent solution may be water, but may also include adjusting the pH or salt strength.
  • the components contained in the diluent solution may be selected arbitrarily depending on the purpose.
  • a buffer solution e.g., citrate buffer, citrate buffered saline, acetate buffer, acetate buffered saline, phosphate buffered saline, Tris buffer, MES buffer, HEPES buffer, etc.
  • a buffer solution e.g., citrate buffer, citrate buffered saline, acetate buffer, acetate buffered saline, phosphate buffered saline, Tris buffer, MES buffer, HEPES buffer, etc.
  • sodium chloride, potassium chloride, sucrose, trehalose, fructose, mannitol, etc. may be included for the purpose of adjusting the salt strength or osmotic pressure, and the above buffer solutions to which these additives have been further added may also be used.
  • the lipid particle dispersion and the diluted solution may be mixed by any method, including a batch method and an in-line method using a flow path device.
  • the flow path device used during mixing may be a Y-shaped mixer, a T-shaped mixer, or the like.
  • the time from mixing the oil phase and the aqueous phase to mixing the diluted solution is not particularly limited, but it is preferable to perform the dilution within 30 seconds, and more preferably within 10 seconds, of mixing the oil phase and the aqueous phase.
  • the mixing ratio (liquid volume ratio) of the lipid particle dispersion and the dilution solution is preferably 1:0.5 to 1:10, and more preferably 1:1 to 1:5.
  • the lipid particle dispersion in step (C), may be mixed with the dilution solution multiple times depending on the purpose.
  • the dilution solutions used may be the same or different.
  • the particle size of the lipid particles may change depending on the pH, so adjusting the pH of the dispersion is important. Therefore, for example, in order to adjust the pH of the lipid particle dispersion after mixing with the dilution solution, a buffer solution having an appropriate concentration and pH, or a buffer solution containing other components, may be used.
  • multiple dilution steps may be performed consecutively, and the interval between one dilution step and the next dilution step may be set arbitrarily, for example, the interval may be 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours or 24 hours.
  • the pH of the lipid particle dispersion after step (C) is preferably pH 3.0 to pH 10.0, more preferably pH 3.5 to pH 9.0, and particularly preferably pH 4.0 to pH 8.5.
  • step (D) the method for removing the organic solvent from the lipid particle dispersion is not particularly limited, and a general method can be used.
  • a pH buffer solution such as phosphate buffered saline or Tris buffer can be used as the dialysis fluid, and additives such as any salt or sugar can be added as necessary to adjust the osmotic pressure or protect against freezing.
  • step (E) the concentration of the lipid particle dispersion obtained in step (D) can be adjusted.
  • a solution such as phosphate buffered saline, saline, Tris buffer, or sucrose-containing Tris buffer can be used as a diluent to dilute to an appropriate concentration.
  • the dispersion obtained in step (D) can be concentrated by ultrafiltration using an ultrafiltration membrane. The concentrated dispersion can be used as is, or it can be concentrated and then adjusted to the desired concentration using the diluent.
  • the organic solvent removal step (step (D)) and the concentration adjustment step (step (E)) can be carried out continuously using tangential flow filtration (TFF).
  • TDF tangential flow filtration
  • the organic solvent removal step and the concentration adjustment step may be carried out in any order. If necessary, the organic solvent removal step and the concentration adjustment step may each be carried out multiple times.
  • the solution that can be used for dialysis in step (D) and dilution in step (E) may contain excipients, cryoprotectants, buffers, and antioxidants.
  • excipients and cryoprotectants include, but are not limited to, sugars and sugar alcohols.
  • sugars include sucrose, trehalose, maltose, glucose, lactose, and fructose
  • sugar alcohols include mannitol, sorbitol, inositol, and xylitol.
  • buffers include, but are not limited to, ACES, BES, Bicine, CAPS, CHES, DIPSO, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, TAPS, TAPSO, TES, Tricine, Tris, phosphoric acid, acetic acid, and citric acid.
  • antioxidants include EDTA, ascorbic acid, and tocopherol.
  • the lipid particle dispersion may be subjected to sterile filtration.
  • a filtration method a hollow fiber membrane, a reverse osmosis membrane, a membrane filter, or the like may be used to remove insoluble matter from the lipid particle dispersion.
  • the dispersion of lipid particles not containing nucleic acid can be frozen or lyophilized.
  • the dispersion of lipid particles can be frozen or lyophilized by a general method, and the method is not particularly limited.
  • the lipid particles not containing nucleic acid are frozen and stored, and the frozen lipid particles not containing nucleic acid can be thawed before mixing with the nucleic acid.
  • the process of mixing the lipid particles that do not contain nucleic acid prepared as above with nucleic acid is not particularly limited, but can be carried out by any of the following methods: mixing liquid using a flow path, mixing liquid in a reciprocating direction in a container, mixing with a pipette, mixing with a stirrer in a batch container, mixing liquid in the container by rotating, or mixing with a flask.
  • the step of mixing the nucleic acid-free lipid particles with the nucleic acid may preferably include a step of incubating the nucleic acid-free lipid particles with the aqueous solution containing the nucleic acid at 0°C to 30°C for 0.1 to 120 minutes, and a step of adjusting the pH of the mixture obtained above to 6.5 to 8.5.
  • An aqueous solution containing nucleic acid can be obtained by dissolving nucleic acid in water or a buffer solution.
  • the concentration of nucleic acid is not particularly limited, but is preferably 1 to 2000 ⁇ g/mL, more preferably 10 to 1000 ⁇ g/mL. If necessary, a buffer component for adjusting pH or a component such as an antioxidant can be added.
  • the mass ratio of the lipid concentration to the nucleic acid concentration in the solution after mixing is preferably 5:1 to 1000:1, more preferably 5:1 to 500:1, even more preferably 7:1 to 200:1, and particularly preferably 7:1 to 100:1.
  • the lipid composition may be a lipid particle.
  • the lipid particle means a particle composed of lipid, and includes a composition having any structure selected from lipid aggregates, micelles, liposomes, lipid nanoparticles (LNPs), and lipoplexes.
  • the liposomes include liposomes having a lipid bilayer structure, an aqueous phase inside, and a single-layer bilayer membrane, and multilayer liposomes having multiple layers.
  • the present invention may include either type of liposome.
  • the lipid particle is preferably a lipid nanoparticle (LNP).
  • non-activated cells refer to cells cultured in the presence of cytokine signals such as IL-2, IL-7, and IL-15 without carrying out the above-mentioned activation treatment, although the method is not limited to those mentioned here.
  • the immune cells may preferably be selected from peripheral blood mononuclear cells (PBMC), lymphocytes, T cells, CD4 + cells, CD8 + cells, memory T cells, naive T cells, or stem cell memory T cells.
  • PBMC peripheral blood mononuclear cells
  • the immune cells may be primary cells or cells derived from pluripotent stem cells.
  • the recombinant protein containing a cell-binding domain and a heparin-binding domain is a cell adhesion protein (such as fibronectin or vitronectin) or a recombinant protein containing only a cell-binding domain and a heparin-binding domain derived from a cell adhesion protein.
  • a cell adhesion protein such as fibronectin or vitronectin
  • a recombinant protein containing only a cell-binding domain and a heparin-binding domain derived from a cell adhesion protein Preferably, it is a recombinant protein containing only a cell-binding domain and a heparin-binding domain, and more preferably, it is retronectin.
  • purification by column chromatography was performed using an automatic purification system ISOLERA (Biotage), a medium pressure fractionation purification system Purif-al-2 (Shoko Science Co., Ltd.), or a medium pressure liquid chromatograph YFLC W-prep 2XY (Yamazen Corporation).
  • the NMR spectrum was measured using tetramethylsilane as an internal standard with a Bruker AVNEO400 (manufactured by Bruker Corporation), and all ⁇ values are shown in ppm. MS spectra were measured using an ACQUITY SQD LC/MS System (Waters).
  • Potassium carbonate (1.3 g) was added to a mixture of 2-hexyloctyl 5-bromopentanoate (1.2 g), n-octylamine (1.2 g) and 1-methyl-2-pyrrolidone (6 mL), and the mixture was stirred at 60°C for 5 hours. After the reaction mixture was cooled to room temperature, ethyl acetate (12 mL) and water (6 mL) were added, and the organic layer was separated. The organic layer was washed with saturated saline, dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure.
  • the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2-hexyloctyl 5-(((2,2-diethoxyethoxy)carbonyl)(octyl)amino)pentanoic acid (0.83 g) as a pale yellow oil.
  • the obtained residue was purified by silica gel column chromatography (methanol-ethyl acetate-hexane) to obtain bis(2-hexyloctyl) 11-(2-(diethylamino)ethyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (referred to as Compound 1) (0.39 g) as a pale yellow oil.
  • Compound 1 bis(2-hexyloctyl) 11-(2-(diethylamino)ethyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate
  • 1,1'-carbonyldi(1,2,4-triazole) (18.3 g) was added to a solution of 2,2-diethoxyethanol (10.0 g) in tetrahydrofuran (100 mL), heated to 30°C, and stirred for 1 hour. After cooling the reaction mixture to room temperature, hexane (100 mL) and saturated sodium bicarbonate water (100 mL) were added, and the organic layer was separated. The obtained organic layer was washed with water (50 mL) and saturated saline, then dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure.
  • Decanoic acid chloride (11 mL) was added dropwise to a solution of tert-butyl N,N-bis(2-hydroxyethyl)carbamate (5.00 g) and triethylamine (8.15 mL) in tetrahydrofuran (50 mL) under ice cooling, and the mixture was then stirred at room temperature for 4 hours. Hexane (50 mL) and water (50 mL) were added to the reaction mixture, and the organic layer was separated. The obtained organic layer was washed with water (50 mL) and saturated saline, and then dried by adding anhydrous sodium sulfate.
  • the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain a colorless oily substance, (((2,2-diethoxyethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) (0.27 g).
  • Potassium carbonate (1.45 g) was added to a mixture of 2-pentylheptyl 5-bromopentanoic acid (1.2 g), isopropylamine (0.65 g) and acetonitrile (6 mL), and the mixture was stirred at 50°C for 1 hour. After the reaction mixture was cooled to room temperature, ethyl acetate (24 mL) and water (12 mL) were added, and the organic layer was separated. The organic layer was washed with water and saturated saline, and then dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure.
  • Trifluoroacetic acid (5 mL) was added to tert-butyl (2-(ethyl(4-hydroxybutyl)amino)ethyl)carbamate (IIA) (650 mg) and stirred at room temperature for 30 minutes. Trifluoroacetic acid was removed under reduced pressure, and the resulting residue was desalted using an ion exchange resin (Diaion SA10A (Mitsubishi Chemical), regenerated to OH type) and azeotropically dehydrated with ethanol to obtain 4-((2-aminoethyl)(ethyl)amino)butan-1-ol (IIA- NH2 ).
  • GFP mRNA product name: CleanCap GFP mRNA (5moU); manufactured by TriLink
  • This dispersion was dialyzed against 20 mmol/L Tris buffer pH 7.4 containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to remove the ethanol, yielding mRNA-encapsulated lipid particles.
  • the prepared samples were stored frozen at -70°C until use.
  • GFP pDNA (GenScript, custom-synthesized plasmid DNA) was diluted with 50 mmol/L citrate buffer at pH 4 so that the weight ratio of the total lipid concentration to the mRNA concentration after mixing of the oil and aqueous phases was as shown in Table 4 to obtain an aqueous phase.
  • the aqueous phase and the oil phase were then mixed using a NanoAssemblr (Precision NanoSystems) so that the volume ratio of the aqueous phase to the oil phase was 3:1, and the mixture was diluted 2-fold with water to obtain a dispersion of mRNA lipid particles.
  • This dispersion was dialyzed against 20 mmol/L Tris buffer pH 7.4 containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to remove ethanol, and GFP pDNA-encapsulated lipid particles were obtained.
  • the prepared samples were frozen and stored at -70°C until use.
  • This dispersion was dialyzed against 20 mmol/L MES buffer pH 6.0 containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to remove ethanol, and a concentration step was performed using an ultrafiltration filter (Amicon ultra 100 kDa, Merck) as necessary to obtain lipid particles that do not contain nucleic acids (empty LNPs).
  • the empty LNPs were stored frozen at -70°C until use.
  • ⁇ Evaluation of Nucleic Acid Encapsulation Rate> (Quantitative determination of total nucleic acid concentration)
  • the nucleic acid was diluted with MilliQ water to prepare diluted samples in a 2-fold dilution series from 100 ⁇ g/mL to 3.1 ⁇ g/mL, and a calibration curve solution was prepared. 50 ⁇ L of the calibration curve solution or lipid particles were mixed with 450 ⁇ L of methanol to prepare a measurement solution.
  • the absorbance of each measurement solution at 260 nm and 330 nm was measured using a UV plate reader (Multiskan Go, Thermo Fisher Scientific), and the absorbance at 330 nm was subtracted from the absorbance at 260 nm to obtain the absorbance of each measurement solution.
  • the total nucleic acid concentration was calculated from the calibration curve using the absorbance of each sample measurement solution.
  • the nucleic acid concentration in the external aqueous phase was quantified by the standard addition method using Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific).
  • the 20x TE buffer included in the above kit was diluted with water to obtain 1x TE buffer.
  • TE stands for Tris/EDTA (ethylenediaminetetraacetic acid).
  • the nucleic acid was diluted with TE buffer to a final concentration of 0 to 400 ng/mL to prepare a nucleic acid dilution series.
  • nucleic acid encapsulation rate (total nucleic acid concentration - nucleic acid concentration in external aqueous phase) ⁇ total nucleic acid concentration x 100 The results are shown in Table 7.
  • the medium used for culturing T cells under activation culture conditions consists of TexMACStm Medium (Miltenyi biotech, 130-097-196) and 5 ng/ml human interleukin-2 (IL-2, Roche, 11147528001) (hereinafter, activation medium).
  • T cells Frozen T cells derived from peripheral blood of healthy human donors (Human PB Pan-T, Cryo, STEMCELL Technologies, ST-70024) were thawed by placing in a water bath at 37° C. for several minutes. The thawed T cells were resuspended in TexMACS Medium containing 1% BSA (bovine serum albumin) (SIGMA, A9576) and 20 U/ml DNase I (Worthington Biochemical, LS002139), further washed by centrifugation, and resuspended in activation medium.
  • BSA bovine serum albumin
  • the T cells were adjusted to a cell concentration of 1.0 x 10 6 cells/ml using this medium, and Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher DB11131) was added to a concentration of 1.0 x 10 6 beads/ml.
  • the cells were seeded in a 24-well cell culture plate and activated by culturing for 3 days in a 37°C 5% CO 2 incubator. On the third day of activation, the Dynabeads were removed from the T cell culture medium. The T cells thus pretreated were used as activated T cells.
  • the medium used for culturing T cells under non-activation culture conditions consists of TexMACStm Medium (Miltenyi biotech, 130-097-196), 5 ng/ml human interleukin-2 (IL-2, Roche, 11147528001), 5 ng/ml human IL-7 (Miltenyi biotech 130-095-367), and 5 ng/ml human IL-15 (Miltenyi biotech, 130-095-760) (hereinafter, non-activation medium).
  • Frozen T cells Human PB Pan-T, Cryo, STEMCELL Technologies, ST-70024
  • Frozen T cells derived from peripheral blood of healthy human donors were thawed by placing them in a water bath at 37°C for several minutes.
  • the thawed T cells were resuspended in TexMACS Medium containing 1% BSA and 20 U/ml DNaseI, washed by centrifugation, and resuspended in non-activation medium.
  • the T cells were adjusted to a cell concentration of 1.0 x 10 6 cells/ml using this medium, seeded on a 24-well plate for cell culture, and cultured for 3 days in a 37°C, 5% CO 2 incubator.
  • the T cells thus pretreated were used as non-activated T cells.
  • T cells were stained for dead cells with BD Horizon TM Fixable Viability Stain (FVS) Reagents (BD, 565388). After staining, the cells were fixed and washed, and the cell status was analyzed using an Attune device (Thermo Fisher). The analysis data was analyzed using Flowjo software. After gating T cells by size, single cells, and live cells, the ratio of GFP-positive cells and median fluorescence intensity (MFI) were analyzed.
  • FVS Fixable Viability Stain
  • Test Example 2-1 Nucleic acid delivery to activated T cells
  • the required number of activated T cells were collected, centrifuged, and the supernatant was removed.
  • the activated T cells were adjusted to a concentration of 1.0 x 106 cells/ml in an activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 ⁇ g/ml, which had been prepared just before use, and seeded on a 96-well plate.
  • ApoE3 recombinant human apolipoprotein E3
  • the GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5 were added at 1.0 ⁇ g (total RNA amount) per 1.0 ⁇ 10 6 cells, and cultured in a 37°C, 5% CO 2 incubator. 24 hours after the addition of LNP, the cells were collected, and the GFP-positive cell ratio and MFI of T cells treated with each LNP were measured by flow cytometry to evaluate the GFP mRNA introduction efficiency. The results are shown in Figure 1. All of the LNPs of Examples 1 to 5 were capable of highly efficient delivery of mRNA to activated T cells.
  • the cells were harvested 24 hours after the addition of LNPs, and the GFP-positive cell ratio and MFI of T cells treated with each LNP were measured by flow cytometry to evaluate the efficiency of GFP mRNA transfer.
  • the results are shown in FIG. It was demonstrated that all of the LNPs of Examples 1 to 5 were capable of delivering mRNA to non-activated T cells, and that the LNPs of Examples 1, 3, and 4 in particular were highly efficient.
  • the GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5 were added at 1.0 ⁇ g (total RNA amount) per 1.0 ⁇ 10 6 cells, and cultured in a 37° C., 5% CO 2 incubator. 24 hours after the addition of LNP, the cells were collected, and the GFP-positive cell ratio and MFI of T cells treated with each LNP were measured by flow cytometry to evaluate the GFP mRNA introduction efficiency.
  • FIG. 3 As a reference example, data in the presence of ApoE3 shown in FIG. 1 are also shown.
  • the LNPs of Examples 1, 3, and 4 were capable of highly efficient delivery of mRNA to activated T cells even in an environment in which ApoE3 activation was not present.
  • the non-activated T cells were adjusted to a concentration of 1.0 x 106 cells/ml using an activation medium that does not contain ApoE3, and seeded on a 96-well plate.
  • 1.0 ⁇ g (total RNA amount) was added per 1.0 x 106 cells, and the cells were cultured in a 37°C, 5% CO2 incubator.
  • the GFP mRNA-encapsulated LNP prepared in Examples 1, 3, and 4 was added to 1.0 x 10 6 cells at 4.0 ⁇ g (total RNA amount) and cultured in a 37°C 5% CO 2 incubator. 24 hours after the addition of LNP, the cells were collected, and the GFP positive cell ratio of T cells treated with each LNP was measured by flow cytometry to evaluate the GFP mRNA introduction efficiency. The results are shown in Figure 7 (Day 9 MC_Day 10 TF condition, Day 9 OptiMEM_Day 10 TF condition).

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Abstract

The present invention addresses the problem of providing: an agent for delivering a nucleic acid to immune cells, which is capable of delivering a nucleic acid to both activated immune cells and non-activated immune cells; and a method for delivering a nucleic acid to immune cells using the agent for delivering a nucleic acid to immune cells. The present invention provides an agent for delivering a nucleic acid to immune cells, the agent comprising a lipid composition comprising: an ionizable lipid that is a compound represented by formula (1) or a salt thereof; a non-ionizable lipid; a lipid having a nonionic polymer; and a nucleic acid. In the formula, R1 to R9 are as defined in the description.

Description

免疫細胞への核酸送達剤、および免疫細胞に核酸を送達する方法Agent for delivering nucleic acid to immune cells and method for delivering nucleic acid to immune cells

　本発明は、脂質を含む免疫細胞への核酸送達剤に関する。本発明はさらに、上記の核酸送達剤を用いた、免疫細胞に核酸を送達する方法に関する。 The present invention relates to a nucleic acid delivery agent for immune cells, which contains a lipid. The present invention further relates to a method for delivering a nucleic acid to an immune cell using the above-mentioned nucleic acid delivery agent.

　キメラ抗原受容体（ＣＡＲ）Ｔ細胞療法をはじめとする免疫細胞療法では、ＣＡＲ等の治療用外来遺伝子を発現させることや内在性の遺伝子発現を変動させるように遺伝子改変した免疫細胞が用いられる。免疫細胞の遺伝子改変では、一般的にウイルスベクター法が用いられるがウイルスによる安全性懸念や高コスト等の課題がある。また、非ウイルス法としては電気穿孔法が従来から用いられているが、電気穿孔による細胞傷害性やＤＮＡ損傷が生じ増殖遅延や染色体異常に繋がる問題がある。 Immune cell therapies, including chimeric antigen receptor (CAR) T cell therapy, use immune cells that have been genetically modified to express foreign therapeutic genes such as CAR or to alter endogenous gene expression. The viral vector method is generally used to genetically modify immune cells, but there are issues such as safety concerns and high costs due to viruses. In addition, electroporation has traditionally been used as a non-viral method, but there are problems with electroporation causing cytotoxicity and DNA damage, which can lead to growth retardation and chromosomal abnormalities.

　脂質ナノ粒子（ＬＮＰ）を用いた免疫細胞への核酸送達については、特許文献１には、複数箇所のゲノム編集を行うために、Ｃａｓ９　ｍＲＮＡとｇＲＮＡを別々のＬＮＰに内包し、これらを用いたＴ細胞のゲノム編集を行うことが記載されている。また、特許文献２には、核酸（ｍＲＮＡ）を内包したＬＮＰを使用してＴ細胞へ核酸を送達することが記載されている。 Regarding the delivery of nucleic acids to immune cells using lipid nanoparticles (LNPs), Patent Document 1 describes the use of separate LNPs to encapsulate Cas9 mRNA and gRNA for genome editing at multiple sites, and the use of these to edit the genome of T cells. Patent Document 2 describes the delivery of nucleic acids to T cells using LNPs encapsulating nucleic acids (mRNA).

国際公開ＷＯ２０２１／２２２２８７号公報International Publication No. WO2021/222287 国際公開ＷＯ２０２０／２１０９０１号公報International Publication No. WO2020/210901

　特許文献１および特許文献２に記載の脂質ナノ粒子においては、免疫細胞を十分に活性化させる処理が必要であり、使用可能な培養方法に制限があった。免疫細胞の活性化処理は、免疫細胞の治療における効能を低減させることが知られていることから、活性化処理は行わないことが望ましい。 The lipid nanoparticles described in Patent Documents 1 and 2 require a process to fully activate immune cells, and there are limitations to the culture methods that can be used. Since immune cell activation processes are known to reduce the efficacy of immune cells in treatment, it is desirable not to perform the activation process.

　本発明は、かかる状況に鑑み、活性化処理した免疫細胞及び活性化処理を施していない免疫細胞のいずれにも核酸を送達することが可能である、免疫細胞への核酸送達剤を提供することを解決すべき課題とした。さらに本発明は、上記の免疫細胞への核酸送達剤を用いた、免疫細胞に核酸を送達する方法を提供することを解決すべき課題とした。 In view of the above circumstances, the present invention aims to provide a nucleic acid delivery agent for immune cells that is capable of delivering nucleic acid to both activated and unactivated immune cells. Furthermore, the present invention aims to provide a method for delivering nucleic acid to immune cells using the above-mentioned nucleic acid delivery agent for immune cells.

　本発明者らは、上記課題を解決するために鋭意検討した結果、下記式（１）で表される化合物またはその塩であるイオン化可能な脂質と、非イオン化脂質と、非イオン性高分子を有する脂質と、核酸とを含む脂質組成物が、活性化処理した免疫細胞及び活性化処理を施していない免疫細胞のいずれにも核酸を送達できることを見出し、本発明を完成するに至った。本発明によれば、以下の発明が提供される。 The present inventors conducted extensive research to solve the above problems and discovered that a lipid composition containing an ionizable lipid, which is a compound represented by the following formula (1) or a salt thereof, a nonionizable lipid, a lipid having a nonionic polymer, and a nucleic acid can deliver nucleic acid to both activated and unactivated immune cells, thereby completing the present invention. The present invention provides the following:

＜１＞　　式（１）で表される化合物またはその塩であるイオン化可能な脂質と、非イオン化脂質と、非イオン性高分子を有する脂質と、核酸とを含む脂質組成物を含む、免疫細胞への核酸送達剤。
式中、Ｒ１、Ｒ２、Ｒ３およびＲ４はそれぞれ独立に、水素原子、置換されていてもよい炭素数１～２４の炭化水素基を示し、
　Ｒ１、Ｒ２、Ｒ３およびＲ４が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基はそれぞれ独立に、－Ｃ（Ｏ）Ｏ－Ｒ１１、－ＯＣ（Ｏ）－Ｒ１２、－Ｏ－Ｒ１３、－ＣＯ－Ｒ１４、－ＯＣ（Ｏ）Ｏ－Ｒ１５、または－Ｓ－Ｓ－Ｒ１６を示し、
　Ｒ１１、Ｒ１２、Ｒ１３、Ｒ１４、Ｒ１５およびＲ１６はそれぞれ独立に、－Ｓ－Ｒ１７で置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ１７は、炭素数１～１２の炭化水素基を示し、
　Ｒ５およびＲ６はそれぞれ独立に、置換されていてもよい炭素数１～１８の炭化水素基を示し、
　Ｒ５およびＲ６が示す置換されていてもよい炭素数１～１８の炭化水素基上の置換基は
それぞれ独立に、－ＯＨ、－ＣＯＯＨ、－ＮＲ２１Ｒ２２、－ＯＣ（Ｏ）Ｏ－Ｒ２３、－Ｃ（Ｏ）Ｏ－Ｒ２４、－ＯＣ（Ｏ）－Ｒ２５、－Ｏ－Ｒ２６、－Ｃ（Ｏ）ＮＲ２７Ｒ２８、－ＮＲ２９Ｃ（Ｏ）Ｒ３０、－Ｎ（Ｒ３１）Ｓ（Ｏ）２Ｒ３２、－Ｎ（Ｒ３３）Ｃ（Ｏ）Ｎ（Ｒ３４）Ｒ３５、－Ｎ（Ｒ３６）Ｃ（Ｓ）Ｎ（Ｒ３７）Ｒ３８、－ＯＣ（Ｏ）Ｎ（Ｒ３９）Ｒ４０、または－Ｎ（Ｒ４１）Ｃ（Ｏ）ＯＲ４２を示し、
　Ｒ２１およびＲ２２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２はそれぞれ独立に、水素原子、または置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基は、炭素数６～２０のアリール基、ヘテロ環基、－ＯＨ、－ＣＯＯＨ、または－ＮＲ５１Ｒ５２を示し、Ｒ５１およびＲ５２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ７、Ｒ８、およびＲ９はそれぞれ独立に、炭素数２～８の炭化水素基を示し、
　Ｒ５とＲ６、またはＲ５とＲ７は一緒になって４～７員環を形成してもよい。
＜２＞　上記イオン化可能な脂質が、脂質組成物中の全脂質に対するモル比で、２０～６０モル％である、＜１＞に記載の免疫細胞への核酸送達剤。
＜３＞　上記非イオン化脂質が、ステロールまたはその誘導体、およびリン脂質を含む、＜１＞または＜２＞に記載の免疫細胞への核酸送達剤。
＜４＞　上記リン脂質が、ジステアロイルホスファチジルコリン、ジオレオイルホスファチジルコリン、およびジオレオイルホスファチジルエタノールアミンからなる群から選択される、＜３＞に記載の免疫細胞への核酸送達剤。
＜５＞　上記ステロールまたはその誘導体が、脂質組成物中の全脂質に対するモル比で、３０～７０モル％である、＜３＞または＜４＞に記載の免疫細胞への核酸送達剤。
＜６＞　上記リン脂質が、脂質組成物中の全脂質に対するモル比で、１～３０モル％である、＜３＞から＜５＞の何れか一に記載の免疫細胞への核酸送達剤。
＜７＞　上記非イオン性高分子を有する脂質が、ポリエチレングリコール鎖を有する脂質である、＜１＞から＜６＞の何れか一に記載の免疫細胞への核酸送達剤。
＜８＞　上記ポリエチレングリコール鎖を有する脂質が、ジミリストイル－ｒａｃ－グリセロールポリエチレングリコール、ジステアロイル－ｒａｃ－グリセロールポリエチレングリコール、およびジステアロイルホスファチジルエタノールアミンポリエチレングリコールから選択される、＜７＞に記載の免疫細胞への核酸送達剤。
＜９＞　上記非イオン性高分子を有する脂質が、脂質組成物中の全脂質に対するモル比で、０．１～３モル％である、＜１＞から＜８＞の何れか一に記載の免疫細胞への核酸送達剤。
＜１０＞　上記脂質組成物の全脂質と核酸の質量比が７：１～１０００：１である、＜１＞から＜９＞の何れか一に記載の免疫細胞への核酸送達剤。
＜１１＞　上記免疫細胞が、活性化細胞であるか、または非活性化細胞である、＜１＞から＜１０＞の何れか一に記載の免疫細胞への核酸送達剤。
＜１２＞　上記イオン化可能な脂質が以下の化合物のうちの１つ以上である、＜１＞から＜１１＞の何れか一に記載の免疫細胞への核酸送達剤。


















＜１３＞　＜１＞から＜１２＞の何れか一に記載の免疫細胞への核酸送達剤を、免疫細胞と接触させることを含む、免疫細胞に核酸を送達する方法（ただし、インビボでの送達方法を除く）。
＜１４＞　上記免疫細胞が、活性化細胞または非活性化細胞である、＜１３＞に記載の方法。
＜１５＞　上記核酸送達剤を免疫細胞に接触させる前に、核酸送達剤もしくは免疫細胞に対して（ｉ）アポリポタンパク、及び／又は（ｉｉ）細胞結合ドメインとヘパリン結合ドメインとを含むタンパク質を添加する工程を含む、＜１３＞に記載の方法。
＜１６＞　式（１）で表される化合物またはその塩であるイオン化可能な脂質と、非イオン化脂質と、非イオン性高分子を有する脂質とを用いて、核酸を含まない脂質粒子を調製する工程と、
上記の核酸を含まない脂質粒子と核酸とを混合する工程とを含む、
＜１＞から＜１２＞の何れか一に記載の核酸送達剤の製造方法。
式中、Ｒ１、Ｒ２、Ｒ３およびＲ４はそれぞれ独立に、水素原子、置換されていてもよい炭素数１～２４の炭化水素基を示し、
　Ｒ１、Ｒ２、Ｒ３およびＲ４が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基はそれぞれ独立に、－Ｃ（Ｏ）Ｏ－Ｒ１１、－ＯＣ（Ｏ）－Ｒ１２、－Ｏ－Ｒ１３、－ＣＯ－Ｒ１４、－ＯＣ（Ｏ）Ｏ－Ｒ１５、または－Ｓ－Ｓ－Ｒ１６を示し、
　Ｒ１１、Ｒ１２、Ｒ１３、Ｒ１４、Ｒ１５およびＲ１６はそれぞれ独立に、－Ｓ－Ｒ１７で置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ１７は、炭素数１～１２の炭化水素基を示し、
　Ｒ５およびＲ６はそれぞれ独立に、置換されていてもよい炭素数１～１８の炭化水素基を示し、
　Ｒ５およびＲ６が示す置換されていてもよい炭素数１～１８の炭化水素基上の置換基はそれぞれ独立に、－ＯＨ、－ＣＯＯＨ、－ＮＲ２１Ｒ２２、－ＯＣ（Ｏ）Ｏ－Ｒ２３、－Ｃ（Ｏ）Ｏ－Ｒ２４、－ＯＣ（Ｏ）－Ｒ２５、－Ｏ－Ｒ２６、－Ｃ（Ｏ）ＮＲ２７Ｒ２８、－ＮＲ２９Ｃ（Ｏ）Ｒ３０、－Ｎ（Ｒ３１）Ｓ（Ｏ）２Ｒ３２、－Ｎ（Ｒ３３）Ｃ（Ｏ）Ｎ（Ｒ３４）Ｒ３５、－Ｎ（Ｒ３６）Ｃ（Ｓ）Ｎ（Ｒ３７）Ｒ３８、－ＯＣ（Ｏ）Ｎ（Ｒ３９）Ｒ４０、または－Ｎ（Ｒ４１）Ｃ（Ｏ）ＯＲ４２を示し、
　Ｒ２１およびＲ２２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２はそれぞれ独立に、水素原子、または置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基は、炭素数６～２０のアリール基、ヘテロ環基、－ＯＨ、－ＣＯＯＨ、または－ＮＲ５１Ｒ５２を示し、Ｒ５１およびＲ５２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ７、Ｒ８、およびＲ９はそれぞれ独立に、炭素数２～８の炭化水素基を示し、
　Ｒ５とＲ６、またはＲ５とＲ７は一緒になって４～７員環を形成してもよい。
＜１７>　下記の化合物からなる群から選択される少なくとも１つの化合物またはその塩。
ビス（２－ヘキシロクチル）　１１－（２－（ジエチルアミノ）エチル）－６，１６－ジオクチル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
２－（２－（２－（ビス（２－デカノイルオキシエチル）カルバモイルオキシ）エチル－（２－（ジエチルアミノ）エチル）アミノ）エトキシカルボニル－（２－デカノイルオキシエチル）アミノ）エチル　デカノエート
ビス（２－ペンチルヘプチル）　１１－（３－（ジエチルアミノ）プロピル）－６，１６－ジイソプロピル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
ビス（２－ペンチルヘプチル）　１１－（３－（ジエチルアミノ）プロピル）－７，１５－ジオキソ－６，１６－ジプロピル－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
ビス（２－ヘキシルオクチル）　６，１６－ジブチル－１１－（３－（ジエチルアミノ）プロピル）－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
ジトリデシル　８－（２－（ジエチルアミノ）エチル）－４，１２－ジオキソ－３，１３－ビス（２－オキソ－２－（トリデシルオキシ）エチル）－５，１１－ジオキサ－３，８，１３－トリアザペンタデカンジオエート
ビス（２－ヘキシルオクチル）　１１－（３－（ジエチルアミノ）プロピル）－６，１６－ジオクチル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
ビス（２－ペンチルヘプチル）　１２－（４－（ジエチルアミノ）ブチル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（ジメチルアミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－メチルピペリジン－４－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（１－エチルピペリジン－４－イル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－イソプロピルピペリジン－４－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（４－ヒドロキシブチル）アミノ）エチル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（２－（（４－ヒドロキシブチル）（メチル）アミノ）エチル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（（４－ヒドロキシブチル）（メチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（３－（（４－ヒドロキシブチル）（メチル）アミノ）プロピル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（（４－ヒドロキシブチル）（メチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（４－ヒドロキシブチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（４－ヒドロキシブチル）アミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（４－ヒドロキシブチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（２－（（３－ヒドロキシプロピル）（メチル）アミノ）エチル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（（３－ヒドロキシプロピル）（メチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（３－（（３－ヒドロキシプロピル）（メチル）アミノ）プロピル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（（３－ヒドロキシプロピル）（メチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（３－ヒドロキシプロピル）アミノ）エチル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（３－ヒドロキシプロピル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（３－ヒドロキシプロピル）アミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（３－ヒドロキシプロピル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（２－（（２－ヒドロキシエチル）（メチル）アミノ）エチル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（（２－ヒドロキシエチル）（メチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（３－（（２－ヒドロキシエチル）（メチル）アミノ）プロピル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（（２－ヒドロキシエチル）（メチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（２－ヒドロキシエチル）アミノ）エチル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（２－ヒドロキシエチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（２－ヒドロキシエチル）アミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（２－ヒドロキシエチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（８－メチル－８－アザビシクロ［３．２．１］オクタン－３－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２、　１７－トリアザトリコサン二酸
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－８，１６－ジオキソ－１２－（１，２，２，６，６－ペンタメチルピペリジン－４－イル）－９，１５－ジオキサ－７，１２，１７－トリアザトリコサン二酸塩
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－メチルアゼチジン－３－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－メチルピロリジン－３－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－メチルアゼパン－４－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（（１ｒ，４ｒ）－４－（ジメチルアミノ）シクロヘキシル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（（１ｓ，４ｓ）－４－（ジメチルアミノ）シクロヘキシル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－（２－ヒドロキシエチル）ピペリジン－４－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－８，１６－ジオキソ－１２－（２－（ピロリジン－１－イル）エチル）－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－８，１６－ジオキソ－１２－（２－（ピペリジン－１－イル）エチル）－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（１－メチルピペリジン－４－イル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（ビス（２－ヒドロキシエチル）アミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（ジエチルアミノ）エチル）－８，１６－ジオキソ－７，１７－ジプロピル－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（ジエチルアミノ）プロピル）－８，１６－ジオキソ－７，１７－ジプロピル－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジブチル－１２－（３－（ジエチルアミノ）プロピル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート <1> A nucleic acid delivery agent for immune cells, comprising a lipid composition including an ionizable lipid that is a compound represented by formula (1) or a salt thereof, a nonionizable lipid, a lipid having a nonionic polymer, and a nucleic acid.
In the formula, R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms;
Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ;
R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R 17 , R 17 represents a hydrocarbon group having 1 to 12 carbon atoms;
R 5 and R 6 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted;
Substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by R 5 and R 6 are each independently -OH, -COOH, -NR 21 R 22 , -OC(O)O-R 23 , -C(O)O-R 24 , -OC(O)-R 25 , -O-R 26 , -C(O)NR 27 R 28 , -NR 29 C(O)R 30 , -N(R 31 )S(O) 2 R 32 , -N(R 33 )C(O)N(R 34 ) R 35 , -N(R 36 )C(S)N(R 37 )R 38 , -OC(O)N(R 39 )R 40 , or -N(R 41 )C(O)OR Showing 42 ,
R 21 and R 22 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , and R 42 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 40 , R 41 , and R 42 represents an aryl group, heterocyclic group, -OH, -COOH, or -NR 51 R 52 having 6 to 20 carbon atoms, and R 51 and R 52 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 7 , R 8 , and R 9 each independently represent a hydrocarbon group having 2 to 8 carbon atoms;
R 5 and R 6 , or R 5 and R 7 may join together to form a 4- to 7-membered ring.
<2> The agent for delivering a nucleic acid to an immune cell according to <1>, wherein the ionizable lipid is present in an amount of 20 to 60 mol % in terms of a molar ratio relative to the total lipids in the lipid composition.
<3> The agent for delivering a nucleic acid to an immune cell according to <1> or <2>, wherein the non-ionized lipid comprises a sterol or a derivative thereof, and a phospholipid.
<4> The agent for delivering a nucleic acid to an immune cell according to <3>, wherein the phospholipid is selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine.
<5> The agent for delivering a nucleic acid to an immune cell according to <3> or <4>, wherein the sterol or a derivative thereof is present in an amount of 30 to 70 mol % in terms of a molar ratio relative to the total lipids in the lipid composition.
<6> The agent for delivering a nucleic acid to an immune cell according to any one of <3> to <5>, wherein the phospholipid is present in an amount of 1 to 30 mol % based on the total lipids in the lipid composition.
<7> The agent for delivering a nucleic acid to an immune cell according to any one of <1> to <6>, wherein the lipid having a nonionic polymer is a lipid having a polyethylene glycol chain.
<8> The agent for delivering a nucleic acid to an immune cell according to <7>, wherein the lipid having a polyethylene glycol chain is selected from dimyristoyl-rac-glycerol polyethylene glycol, distearoyl-rac-glycerol polyethylene glycol, and distearoylphosphatidylethanolamine polyethylene glycol.
<9> The agent for delivering a nucleic acid to an immune cell according to any one of <1> to <8>, wherein the lipid having the nonionic polymer is present in an amount of 0.1 to 3 mol % in terms of a molar ratio relative to the total lipid in the lipid composition.
<10> The agent for delivering nucleic acid to an immune cell according to any one of <1> to <9>, wherein a mass ratio of the total lipids to the nucleic acid in the lipid composition is 7:1 to 1000:1.
<11> The agent for delivering a nucleic acid to an immune cell according to any one of <1> to <10>, wherein the immune cell is an activated cell or a non-activated cell.
<12> The agent for delivering a nucleic acid to an immune cell according to any one of <1> to <11>, wherein the ionizable lipid is one or more of the following compounds:


















<13> A method for delivering a nucleic acid to an immune cell, comprising contacting the immune cell with the nucleic acid delivery agent for immune cells according to any one of <1> to <12> (excluding in vivo delivery methods).
<14> The method according to <13>, wherein the immune cells are activated cells or non-activated cells.
<15> The method according to <13>, comprising the step of adding (i) an apolipoprotein, and/or (ii) a protein comprising a cell-binding domain and a heparin-binding domain to the nucleic acid delivery agent or the immune cells before contacting the nucleic acid delivery agent with the immune cells.
<16> A step of preparing a lipid particle not containing nucleic acid by using an ionizable lipid which is a compound represented by formula (1) or a salt thereof, a nonionizable lipid, and a lipid having a nonionic polymer;
Mixing the nucleic acid-free lipid particles with nucleic acid,
<12> A method for producing a nucleic acid delivery agent according to any one of <1> to <12>.
In the formula, R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms;
Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ;
R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R 17 , R 17 represents a hydrocarbon group having 1 to 12 carbon atoms;
R 5 and R 6 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted;
Substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by R 5 and R 6 are each independently -OH, -COOH, -NR 21 R 22 , -OC(O)O-R 23 , -C(O)O-R 24 , -OC(O)-R 25 , -O-R 26 , -C(O)NR 27 R 28 , -NR 29 C(O)R 30 , -N(R 31 )S(O) 2 R 32 , -N(R 33 )C(O)N(R 34 ) R 35 , -N(R 36 )C(S)N(R 37 )R 38 , -OC(O)N(R 39 )R 40 , or -N(R 41 )C(O)OR Showing 42 ,
R 21 and R 22 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , and R 42 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 40 , R 41 , and R 42 represents an aryl group, heterocyclic group, -OH, -COOH, or -NR 51 R 52 having 6 to 20 carbon atoms, and R 51 and R 52 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 7 , R 8 , and R 9 each independently represent a hydrocarbon group having 2 to 8 carbon atoms;
R 5 and R 6 , or R 5 and R 7 may join together to form a 4- to 7-membered ring.
<17> At least one compound selected from the group consisting of the following compounds or a salt thereof:
Bis(2-hexyloctyl) 11-(2-(diethylamino)ethyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate
2-(2-(2-(bis(2-decanoyloxyethyl)carbamoyloxy)ethyl-(2-(diethylamino)ethyl)amino)ethoxycarbonyl-(2-decanoyloxyethyl)amino)ethyl decanoate
Bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-6,16-diisopropyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate
Bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-7,15-dioxo-6,16-dipropyl-8,14-dioxa-6,11,16-triazahenicosanedioate
Bis(2-hexyloctyl) 6,16-dibutyl-11-(3-(diethylamino)propyl)-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate
Ditridecyl 8-(2-(diethylamino)ethyl)-4,12-dioxo-3,13-bis(2-oxo-2-(tridecyloxy)ethyl)-5,11-dioxa-3,8,13-triazapentadecanedioate
Bis(2-hexyloctyl) 11-(3-(diethylamino)propyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate
Bis(2-pentylheptyl) 12-(4-(diethylamino)butyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(dimethylamino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(1-ethylpiperidin-4-yl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-isopropylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((4-hydroxybutyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-((4-hydroxybutyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((4-hydroxybutyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-((4-hydroxybutyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(4-hydroxybutyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(4-hydroxybutyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((3-hydroxypropyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-((3-hydroxypropyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((3-hydroxypropyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-((3-hydroxypropyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(3-hydroxypropyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(3-hydroxypropyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(3-hydroxypropyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(3-hydroxypropyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((2-hydroxyethyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-((2-hydroxyethyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(2-hydroxyethyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(2-hydroxyethyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)-8,16-dioxo-9,15-dioxa-7,12, 17-triazatricosane diacid
Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(1,2,2,6,6-pentamethylpiperidin-4-yl)-9,15-dioxa-7,12,17-triazatricosane diacid salt
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazetidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpyrrolidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazepan-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-((1r,4r)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-((1s,4s)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-(2-hydroxyethyl)piperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(pyrrolidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(piperidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(1-methylpiperidin-4-yl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(bis(2-hydroxyethyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(diethylamino)ethyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(diethylamino)propyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-dibutyl-12-(3-(diethylamino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate

　本発明によれば、活性化処理した免疫細胞及び活性化処理を施していない免疫細胞のいずれにも核酸を送達することが可能である。 According to the present invention, it is possible to deliver nucleic acids to both activated and unactivated immune cells.

図１は、活性化Ｔ細胞への核酸送達を測定した結果を示す。FIG. 1 shows the results of measuring nucleic acid delivery to activated T cells. 図２は、非活性化Ｔ細胞への核酸送達を測定した結果を示す。FIG. 2 shows the results of measuring nucleic acid delivery to non-activated T cells. 図３は、ＡｐｏＥ３を添加しない活性化用培地における活性化Ｔ細胞への核酸送達を測定した結果を示す。FIG. 3 shows the results of measuring nucleic acid delivery to activated T cells in an activation medium to which ApoE3 has not been added. 図４は、ＡｐｏＥ３を添加しない活性化用培地における非活性化Ｔ細胞への核酸送達を測定した結果を示す。FIG. 4 shows the results of measuring nucleic acid delivery to non-activated T cells in an activation medium without the addition of ApoE3. 図５は、活性化Ｔ細胞におけるＴＣＲ　ＫＯ効率を測定した結果を示す。Figure 5 shows the results of measuring TCR KO efficiency in activated T cells. 図６は、各種タンパクを添加した培養条件における活性化Ｔ細胞への核酸送達を測定した結果を示す。FIG. 6 shows the results of measuring nucleic acid delivery to activated T cells under culture conditions in which various proteins were added. 図７は、長期培養したＴ細胞への核酸送達を測定した結果を示す。FIG. 7 shows the results of measuring nucleic acid delivery to long-term cultured T cells.

　以下、本発明について詳細に説明する。
　本明細書において「～」は、その前後に記載される数値をそれぞれ最小値および最大値として含む範囲を示す。 The present invention will be described in detail below.
In this specification, the symbol "to" indicates a range that includes the numerical values before and after it as the minimum and maximum values, respectively.

［免疫細胞への核酸送達剤］
　本発明の免疫細胞への核酸送達剤は、式（１）で表される化合物またはその塩であるイオン化可能な脂質と、非イオン化脂質と、非イオン性高分子を有する脂質と、核酸とを含む脂質組成物を含む。
式中、Ｒ１、Ｒ２、Ｒ３およびＲ４はそれぞれ独立に、水素原子、置換されていてもよい炭素数１～２４の炭化水素基を示し、
　Ｒ１、Ｒ２、Ｒ３およびＲ４が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基はそれぞれ独立に、－Ｃ（Ｏ）Ｏ－Ｒ１１、－ＯＣ（Ｏ）－Ｒ１２、－Ｏ－Ｒ１３、－ＣＯ－Ｒ１４、－ＯＣ（Ｏ）Ｏ－Ｒ１５、または－Ｓ－Ｓ－Ｒ１６を示し、
　Ｒ１１、Ｒ１２、Ｒ１３、Ｒ１４、Ｒ１５およびＲ１６はそれぞれ独立に、－Ｓ－Ｒ１７で置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ１７は、炭素数１～１２の炭化水素基を示し、
　Ｒ５およびＲ６はそれぞれ独立に、置換されていてもよい炭素数１～１８の炭化水素基を示し、
　Ｒ５およびＲ６が示す置換されていてもよい炭素数１～１８の炭化水素基上の置換基はそれぞれ独立に、－ＯＨ、－ＣＯＯＨ、－ＮＲ２１Ｒ２２、－ＯＣ（Ｏ）Ｏ－Ｒ２３、－Ｃ（Ｏ）Ｏ－Ｒ２４、－ＯＣ（Ｏ）－Ｒ２５、－Ｏ－Ｒ２６、－Ｃ（Ｏ）ＮＲ２７Ｒ２８、－ＮＲ２９Ｃ（Ｏ）Ｒ３０、－Ｎ（Ｒ３１）Ｓ（Ｏ）２Ｒ３２、－Ｎ（Ｒ３３）Ｃ（Ｏ）Ｎ（Ｒ３４）Ｒ３５、－Ｎ（Ｒ３６）Ｃ（Ｓ）Ｎ（Ｒ３７）Ｒ３８、－ＯＣ（Ｏ）Ｎ（Ｒ３９）Ｒ４０、または－Ｎ（Ｒ４１）Ｃ（Ｏ）ＯＲ４２を示し、
　Ｒ２１およびＲ２２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２はそれぞれ独立に、水素原子、または置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基は、炭素数６～２０のアリール基、ヘテロ環基、－ＯＨ、－ＣＯＯＨ、または－ＮＲ５１Ｒ５２を示し、Ｒ５１およびＲ５２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ７、Ｒ８、およびＲ９はそれぞれ独立に、炭素数２～８の炭化水素基を示し、
　Ｒ５とＲ６、またはＲ５とＲ７は一緒になって４～７員環を形成してもよい。 [Nucleic acid delivery agent to immune cells]
The nucleic acid delivery agent for immune cells of the present invention comprises a lipid composition comprising an ionizable lipid that is a compound represented by formula (1) or a salt thereof, a non-ionizable lipid, a lipid having a non-ionic polymer, and a nucleic acid.
In the formula, R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms;
Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ;
R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R 17 , R 17 represents a hydrocarbon group having 1 to 12 carbon atoms;
R 5 and R 6 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted;
Substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by R 5 and R 6 are each independently -OH, -COOH, -NR 21 R 22 , -OC(O)O-R 23 , -C(O)O-R 24 , -OC(O)-R 25 , -O-R 26 , -C(O)NR 27 R 28 , -NR 29 C(O)R 30 , -N(R 31 )S(O) 2 R 32 , -N(R 33 )C(O)N(R 34 ) R 35 , -N(R 36 )C(S)N(R 37 )R 38 , -OC(O)N(R 39 )R 40 , or -N(R 41 )C(O)OR Showing 42 ,
R 21 and R 22 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , and R 42 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 40 , R 41 , and R 42 represents an aryl group, heterocyclic group, -OH, -COOH, or -NR 51 R 52 having 6 to 20 carbon atoms, and R 51 and R 52 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 7 , R 8 , and R 9 each independently represent a hydrocarbon group having 2 to 8 carbon atoms;
R 5 and R 6 , or R 5 and R 7 may join together to form a 4- to 7-membered ring.

　本発明の免疫細胞への核酸送達剤は、遺伝子発現を改変した免疫細胞の作製に利用可能であり、非活性化状態の免疫細胞にも目的核酸を送達することができるため、細胞の疲弊を防止することができ、細胞治療性能の向上に繋がる。 The nucleic acid delivery agent for immune cells of the present invention can be used to create immune cells with modified gene expression, and can deliver target nucleic acids even to inactive immune cells, preventing cell exhaustion and improving cell therapy performance.

＜式（１）で表される化合物またはその塩＞
　炭素数１～２４の炭化水素基、炭素数１～１８の炭化水素基、炭素数１～１２の炭化水素基、及び炭素数１～８の炭化水素基は、それぞれアルキル基、アルケニル基またはアルキニル基であることが好ましい。 <Compound represented by formula (1) or a salt thereof>
The hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon group having 1 to 18 carbon atoms, the hydrocarbon group having 1 to 12 carbon atoms, and the hydrocarbon group having 1 to 8 carbon atoms are preferably an alkyl group, an alkenyl group, or an alkynyl group, respectively.

　アルキル基は直鎖でも分岐であってもよく、鎖状でも環状であってもよい。アルキル基としては、具体的には、メチル基、エチル基、プロピル基、イソプロピル基、シクロプロピル基、ブチル基、イソブチル基、ｔｅｒｔ－ブチル基、シクロブチル基、ペンチル基、シクロペンチル基、ヘキシル基、シクロヘキシル基、ヘプチル基、オクチル基、ノニル基、デシル基、ウンデシル基、ドデシル基、トリデシル基、トリメチルドデシル基（好ましくは、３，７，１１－トリメチルドデシル基）、テトラデシル基、ペンタデシル基、ヘキサデシル基、テトラメチルヘキサデシル基（好ましくは、３，７，１１，１５－テトラメチルヘキサデシル基）、ヘプタデシル基、オクタデシル基、２－ブチルヘキシル基、２－ブチルオクチル基、１－ペンチルヘキシル基、２－ペンチルヘプチル基、３－ペンチルオクチル基、１－ヘキシルヘプチル基、１－ヘキシルノニル基、２－ヘキシルオクチル基、２－ヘキシルデシル基、３－ヘキシルノニル基、１－ヘプチルオクチル基、２－ヘプチルノニル基、２－ヘプチルウンデシル基、３－ヘプチルデシル基、１－オクチルノニル基、２－オクチルデシル基、２－オクチルドデシル基、３－オクチルウンデシル基、２－ノニルウンデシル基、３－ノニルドデシル基、２－デシルドデシル基、２－デシルテトラデシル基、３－デシルトリデシル基、２－（４，４－ジメチルペンタン－２－イル）－５，７，７－トリメチルオクチル基などが挙げられる。 The alkyl group may be linear or branched, and may be linear or cyclic. Specific examples of the alkyl group include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, 2-butylhexyl, and 2-butyloctyl. , 1-pentylhexyl group, 2-pentylheptyl group, 3-pentyloctyl group, 1-hexylheptyl group, 1-hexylnonyl group, 2-hexyloctyl group, 2-hexyldecyl group, 3-hexylnonyl group, 1-heptyloctyl group, 2-heptylnonyl group, 2-heptylundecyl group, 3-heptyldecyl group, 1-octylnonyl group, 2-octyldecyl group, 2-octyldodecyl group, 3-octylundecyl group, 2-nonylundecyl group, 3-nonyldodecyl group, 2-decyldodecyl group, 2-decyltetradecyl group, 3-decyltridecyl group, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, etc.

　アルケニル基は直鎖でも分岐であってもよく、鎖状でも環状であってもよい。具体的には、アリル基、プレニル基、ペンテニル基、ヘキセニル基、ヘプテニル基、オクテニル基、ノネニル基（好ましくは、（Ｚ）－２－ノネニル基または、（Ｅ）－２－ノネニル基）、デセニル基、ウンデセニル基、ドデセニル基、ドデカジエニル基、トリデセニル基（好ましくは、（Ｚ）－トリデカ－８－エニル基）、テトラデセニル基（好ましくは、テトラデカ－９－エニル基）、ペンタデセニル基（好ましくは、（Ｚ）－ペンタデカ－８－エニル基）、ヘキサデセニル基（好ましくは、（Ｚ）－ヘキサデカ－９－エニル基）、ヘキサデカジエニル基、ヘプタデセニル基（好ましくは、（Ｚ）－ヘプタデカ－８－エニル基）、ヘプタデカジエニル基（好ましくは、（８Ｚ，１１Ｚ）－ヘプタデカ－８，１１－ジエニル基）、オクタデセニル基（好ましくは、（Ｚ）－オクタデカ－９－エニル基）、オクタデカジエニル基（好ましくは、（９Ｚ，１２Ｚ）－オクタデカ－９，１２－ジエニル基）などが挙げられる。 The alkenyl group may be linear or branched, and may be linear or cyclic. Specifically, it is an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably, a (Z)-2-nonenyl group or a (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably, a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), a pentadecenyl group (preferably, a (Z)-pentadecenyl-8-enyl group). , hexadecenyl group (preferably, (Z)-hexadecanyl group), hexadecadienyl group, heptadecenyl group (preferably, (Z)-heptadecanyl group), heptadecadienyl group (preferably, (8Z,11Z)-heptadecanyl group, octadecenyl group (preferably, (Z)-octadecane-9-enyl group), octadecadienyl group (preferably, (9Z,12Z)-octadecanyl group, 12-octadecanyl group), etc.

　アルキニル基は直鎖でも分岐であってもよく、鎖状でも環状であってもよい。具体的には、プロパルギル基、ブチニル基、ペンチニル基、ヘキシニル基、ヘプチニル基、オクチニル基、ノニニル基、デシニル基、ウンデシニル基、ドデシニル基、テトラデシニル基、ペンタデシニル基、ヘキサデシニル基、ヘプタデシニル基、オクタデシニル基などが挙げられる。 The alkynyl group may be straight-chained or branched, and may be linear or cyclic. Specific examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, and octadecynyl groups.

　上記のアルケニル基はいずれも二重結合を１つまたは２つ有することが好ましく、アルキニル基はいずれも三重結合を１つまたは２つ有することが好ましい。 It is preferable that each of the above alkenyl groups has one or two double bonds, and it is preferable that each of the alkynyl groups has one or two triple bonds.

　Ｒ７、Ｒ８、およびＲ９が示す炭素数２～８の炭化水素基は、アルキレン基、アルケニレン基またはアルキニレン基であることが好ましい。
　炭素数２～８のアルキレン基、アルケニレン基またはアルキニレン基は、直鎖でも分岐であってもよく、鎖状でも環状であってもよい。
　具体的には、エチレン基、トリメチレン基、テトラメチレン基、ペンタメチレン基、ヘキサメチレン基、ヘプタメチレン基、オクタメチレン基などが挙げられる。 The hydrocarbon group having 2 to 8 carbon atoms represented by R 7 , R 8 and R 9 is preferably an alkylene group, an alkenylene group or an alkynylene group.
The alkylene group, alkenylene group or alkynylene group having 2 to 8 carbon atoms may be straight-chain or branched, and may be linear or cyclic.
Specific examples include an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group.

　炭素数６～２０のアリール基は、炭素数６～１８のアリール基が好ましく、炭素数６～１０のアリール基がより好ましい。具体的には、フェニル基、ナフチル基、アントラセニル基、フェナントレニル基などが挙げられる。 Of the aryl groups having 6 to 20 carbon atoms, aryl groups having 6 to 18 carbon atoms are preferred, and aryl groups having 6 to 10 carbon atoms are even more preferred. Specific examples include phenyl groups, naphthyl groups, anthracenyl groups, and phenanthrenyl groups.

　ヘテロ環基とは、ヘテロアリール基又はヘテロ脂肪族環基を意味する。 Heterocyclic group means a heteroaryl group or a heteroaliphatic ring group.

　ヘテロアリール基とは、芳香族ヘテロ環基を意味し、芳香族ヘテロ環、芳香族炭化水素環、ヘテロ脂肪族環又は脂肪族炭化水素環が縮環した芳香族ヘテロ環基であってもよく、単環の含窒素ヘテロアリール基、単環の含酸素ヘテロアリール基、単環の含硫黄ヘテロアリール基、単環の含窒素含酸素ヘテロアリール基、単環の含窒素含硫黄ヘテロアリール基、二環式の含窒素ヘテロアリール基、二環式の含酸素ヘテロアリール基、二環式の含硫黄ヘテロアリール基、二環式の含窒素含酸素ヘテロアリール基又は二環式の含窒素含硫黄ヘテロアリール基であることが好ましい。５員環のヘテロアリール基とは、単環のヘテロアリール基であって、環を構成する原子の数が５個のものである。 The term "heteroaryl group" refers to an aromatic heterocyclic group, which may be an aromatic heterocyclic group, an aromatic hydrocarbon ring, a heteroaliphatic ring, or an aromatic heterocyclic group condensed with an aliphatic hydrocarbon ring, and is preferably a monocyclic nitrogen-containing heteroaryl group, a monocyclic oxygen-containing heteroaryl group, a monocyclic sulfur-containing heteroaryl group, a monocyclic nitrogen-containing oxygen-containing heteroaryl group, a monocyclic nitrogen-containing sulfur-containing heteroaryl group, a bicyclic nitrogen-containing heteroaryl group, a bicyclic oxygen-containing heteroaryl group, a bicyclic sulfur-containing heteroaryl group, a bicyclic nitrogen-containing oxygen-containing heteroaryl group, or a bicyclic nitrogen-containing sulfur-containing heteroaryl group. A five-membered heteroaryl group is a monocyclic heteroaryl group having five atoms constituting the ring.

　また、芳香族ヘテロ環とは、環員にヘテロ原子を有する芳香環を意味し、芳香族ヘテロ環、芳香族炭化水素環、ヘテロ脂肪族環又は脂肪族炭化水素環が縮環していてもよく、単環の含窒素芳香族ヘテロ環、単環の含酸素芳香族ヘテロ環、単環の含硫黄芳香族ヘテロ環、単環の含窒素含酸素芳香族ヘテロ環、単環の含窒素含硫黄芳香族ヘテロ環、二環式の含窒素芳香族ヘテロ環、二環式の含酸素芳香族ヘテロ環、二環式の含硫黄芳香族ヘテロ環、二環式の含窒素含酸素芳香族ヘテロ環又は二環式の含窒素含硫黄芳香族ヘテロ環であることが好ましい。 Furthermore, an aromatic heterocycle means an aromatic ring having a heteroatom as a ring member, and may be a condensed aromatic heterocycle, aromatic hydrocarbon ring, heteroaliphatic ring, or aliphatic hydrocarbon ring, and is preferably a monocyclic nitrogen-containing aromatic heterocycle, a monocyclic oxygen-containing aromatic heterocycle, a monocyclic sulfur-containing aromatic heterocycle, a monocyclic nitrogen-containing oxygen-containing aromatic heterocycle, a monocyclic nitrogen-containing sulfur-containing aromatic heterocycle, a bicyclic nitrogen-containing aromatic heterocycle, a bicyclic oxygen-containing aromatic heterocycle, a bicyclic sulfur-containing aromatic heterocycle, a bicyclic nitrogen-containing oxygen-containing aromatic heterocycle, or a bicyclic nitrogen-containing sulfur-containing aromatic heterocycle.

　単環の含窒素ヘテロアリール基とは、ピロリニル、ピロリル、テトラヒドロピリジル、ピリジル、イミダゾリニル、イミダゾリル、ピラゾリニル、ピラゾリル、ピラジニル、ピリダジニル、ピリミジニル、トリアゾリル及びテトラゾリル基などの少なくとも１つの窒素原子を含む環が芳香族性を有するヘテロアリール基（このヘテロアリール基は、一部飽和されていてもよい。）を意味する。このヘテロアリール基は、更に他の芳香族環又は脂肪族環と縮合していてもよい。
　単環の含酸素ヘテロアリール基とは、フラニル又はピラニル基などの少なくとも１つの酸素原子を含む環が芳香族性を有するヘテロアリール基（このヘテロアリール基は、一部飽和されていてもよい。）を意味する。このヘテロアリール基は、更に他の芳香族環又は脂肪族環と縮合していてもよい。
　単環の含窒素含酸素ヘテロアリール基とは、オキサゾリル、イソオキサゾリル又はオキサジアゾリル基などを意味する。このヘテロアリール基は、更に他の芳香族環又は脂肪族環と縮合していてもよい。
　単環の含窒素含硫黄ヘテロアリール基とは、チアゾリル、イソチアゾリル又はチアジアゾリル基などを意味する。このヘテロアリール基は、更に他の芳香族環又は脂肪族環と縮合していてもよい。 The term "monocyclic nitrogen-containing heteroaryl group" refers to a heteroaryl group (which may be partially saturated) in which the ring containing at least one nitrogen atom has aromaticity, such as pyrrolinyl, pyrrolyl, tetrahydropyridyl, pyridyl, imidazolinyl, imidazolyl, pyrazolinyl, pyrazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazolyl, and tetrazolyl groups. This heteroaryl group may be further condensed with another aromatic ring or an aliphatic ring.
The term "monocyclic oxygen-containing heteroaryl group" refers to a heteroaryl group (which may be partially saturated) in which the ring containing at least one oxygen atom has aromaticity, such as a furanyl or pyranyl group, which may further be condensed with another aromatic ring or an aliphatic ring.
The monocyclic nitrogen-containing and oxygen-containing heteroaryl group means an oxazolyl, isoxazolyl, oxadiazolyl group, etc. This heteroaryl group may be further condensed with another aromatic ring or an aliphatic ring.
The monocyclic nitrogen-containing sulfur-containing heteroaryl group means a thiazolyl, isothiazolyl or thiadiazolyl group, etc. This heteroaryl group may be further condensed with another aromatic ring or an aliphatic ring.

　二環式の含窒素ヘテロアリール基とは、インドリル、イソインドリル、ベンズイミダゾリル、インダゾリル、ベンゾトリアゾリル、キノリル、イソキノリル、テトラヒドロキノリル、テトラヒドロイソキノリル、キノリジニル、シンノリニル、フタラジニル、キナゾリニル、キノキサリニル、ナフチリジニル、ピロロピリジル、イミダゾピリジル、ピラゾロピリジル、ピリドピラジル、プリニル、プテリジニル、５，６，７，８－テトラヒドロフタラジニル、５，６，７，８－テトラヒドロシンノリニル、１，２，３，４－テトラヒドロピリド［２，３－ｄ］ピリダジニル、５，６，７，８－テトラヒドロ－［１，２，４］トリアゾロ［４，３－ａ］ピラジニル、５，６，７，８－テトラヒドロピリド［３，４－ｄ］ピリダジニル、５，６，７，８－テトラヒドロピリド［３，２－ｃ］ピリダジニル、５，６，７，８－テトラヒドロピリド［４，３－ｃ］ピリダジニル、６，７－ジヒドロ－５Ｈ－シクロペンタ［ｄ］ピリダジニル、６，７－ジヒドロ－５Ｈ－シクロペンタ［ｃ］ピリダジニル、２，３－ジヒドロ－１Ｈ－ピロロ［２，３－ｄ］ピリダジニル、６，７－ジヒドロ－５Ｈ－ピロロ［３，４－ｄ］ピリダジニル、６，７－ジヒドロ－５Ｈ－ピロロ［３，２－ｃ］ピリダジニル、６，７－ジヒドロ－５Ｈ－ピロロ［３，４－ｃ］ピリダジニル及び６，７－ジヒドロ－５Ｈ－ピロロ［２，３－ｃ］ピリダジニル基などの少なくとも１つの窒素原子を含む環が芳香族性を有する二環式のヘテロアリール基（このヘテロアリール基は、一部飽和されていてもよい。）を意味する。 　The bicyclic nitrogen-containing heteroaryl group is indolyl, isoindolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pyrrolopyridyl, imidazopyridyl, pyrazolopyridyl, pyridopyrazyl, purinyl, pteridinyl, 5,6,7,8-tetrahydrophthalazinyl, 5,6,7,8-tetrahydrocinnolinyl, 1,2,3,4-tetrahydropyrido[2,3-d]pyridazinyl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, 5,6,7,8-tetrahydropyrido[3,4-d]pyridazinyl, 5,6 ,7,8-tetrahydropyrido[3,2-c]pyridazinyl, 5,6,7,8-tetrahydropyrido[4,3-c]pyridazinyl, 6,7-dihydro-5H-cyclopenta[d]pyridazinyl, 6,7-dihydro-5H-cyclopenta[c]pyridazinyl, 2,3-dihydro-1H-pyrrolo[2,3-d]pyridazinyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyridazinyl This means a bicyclic heteroaryl group in which the ring containing at least one nitrogen atom has aromaticity, such as ridazinyl, 6,7-dihydro-5H-pyrrolo[3,2-c]pyridazinyl, 6,7-dihydro-5H-pyrrolo[3,4-c]pyridazinyl, and 6,7-dihydro-5H-pyrrolo[2,3-c]pyridazinyl groups (this heteroaryl group may be partially saturated).

　二環式の含酸素ヘテロアリール基とは、ベンゾフラニル、イソベンゾフラニル及びクロメニル基などの少なくとも１つの酸素原子を含む環が芳香族性を有する二環式のヘテロアリール基（このヘテロアリール基は、一部飽和されていてもよい。）を意味する。 The term "bicyclic oxygen-containing heteroaryl group" refers to a bicyclic heteroaryl group in which the ring containing at least one oxygen atom has aromaticity, such as benzofuranyl, isobenzofuranyl, and chromenyl groups (this heteroaryl group may be partially saturated).

　二環式の含窒素含酸素ヘテロアリール基とは、ベンゾオキサゾリル、ベンゾイソオキサゾリル、ベンゾオキサジアゾリル、ジヒドロピラノピリジル、ジヒドロジオキシノピリジル、ジヒドロピリドオキサジエニル、３，４－ジヒドロ－２Ｈ－ピラノ［２，３－ｄ］ピリダジニル、７，８－ジヒドロ－５Ｈ－ピラノ［３，４－ｄ］ピリダジニル、７，８－ジヒドロ－６Ｈ－ピラノ［３，２－ｃ］ピリダジニル、７，８－ジヒドロ－５Ｈ－ピラノ［４，３－ｃ］ピリダジニル、２，３－ジヒドロフロ［２，３－ｄ］ピリダジニル、５，７－ジヒドロフロ［３，４－ｄ］ピリダジニル、６，７－ジヒドロフロ［３，２－ｃ］ピリダジニル、５，７－ジヒドロフロ［３，４－ｃ］ピリダジニル及び５，６－ジヒドロフロ［２，３－ｃ］ピリダジニル基などの少なくとも１つの窒素原子と少なくとも１つの酸素原子とを含む環が芳香族性を有する二環式のヘテロアリール基（このヘテロアリール基は、一部飽和されていてもよい。）を意味する。 The bicyclic nitrogen-containing and oxygen-containing heteroaryl group is benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, dihydropyranopyridyl, dihydrodioxinopyridyl, dihydropyridoxadienyl, 3,4-dihydro-2H-pyrano[2,3-d]pyridazinyl, 7,8-dihydro-5H-pyrano[3,4-d]pyridazinyl, 7,8-dihydro-6H-pyrano[3,2-c]pyridazinyl, 7,8-dihydro-5H-pyrano[4,3-c]pyridazinyl, It means a bicyclic heteroaryl group in which the ring containing at least one nitrogen atom and at least one oxygen atom has aromaticity, such as 2,3-dihydrofuro[2,3-d]pyridazinyl, 5,7-dihydrofuro[3,4-d]pyridazinyl, 6,7-dihydrofuro[3,2-c]pyridazinyl, 5,7-dihydrofuro[3,4-c]pyridazinyl, and 5,6-dihydrofuro[2,3-c]pyridazinyl (this heteroaryl group may be partially saturated).

　ヘテロ脂肪族環基とは、含窒素ヘテロ脂肪族環基、含酸素ヘテロ脂肪族環基、含硫黄ヘテロ脂肪族環基、含窒素含酸素ヘテロ脂肪族環基、含窒素含硫黄ヘテロ脂肪族環基、ヘテロ架橋環基又はヘテロスピロ環基を意味する。
　また、ヘテロ脂肪族環とは、環員にヘテロ原子を有する脂肪族環を意味し、含窒素ヘテロ脂肪族環、含酸素ヘテロ脂肪族環、含硫黄ヘテロ脂肪族環、含窒素含酸素ヘテロ脂肪族環、含窒素含硫黄ヘテロ脂肪族環、ヘテロ架橋環、及び、ヘテロスピロ環が好ましいものとして挙げられる。 The heteroaliphatic ring group means a nitrogen-containing heteroaliphatic ring group, an oxygen-containing heteroaliphatic ring group, a sulfur-containing heteroaliphatic ring group, a nitrogen-containing oxygen-containing heteroaliphatic ring group, a nitrogen-containing sulfur-containing heteroaliphatic ring group, a heterobridged ring group, or a heterospiro ring group.
Further, the heteroaliphatic ring means an aliphatic ring having a heteroatom as a ring member, and preferred examples thereof include a nitrogen-containing heteroaliphatic ring, an oxygen-containing heteroaliphatic ring, a sulfur-containing heteroaliphatic ring, a nitrogen-containing oxygen-containing heteroaliphatic ring, a nitrogen-containing sulfur-containing heteroaliphatic ring, a heterobridged ring, and a heterospiro ring.

　含窒素ヘテロ脂肪族環基とは、アゼチジニル、ピロリジニル、ピペリジニル、ホモピペリジニル、オクタヒドロアゾシニル、イミダゾリジニル、ピラゾリジニル、ピペラジニル及びホモピペラジニル基などの少なくとも１つの窒素原子を含む環が芳香族性を有さないヘテロ脂肪族環基を意味する。この含窒素ヘテロ脂肪族環基は、更に他の芳香族環又は脂肪族環と縮合していてもよい。
　含酸素ヘテロ脂肪族環基とは、テトラヒドロフラニル、テトラヒドロピラニル、オキセタニル又は１，３－ジオキサニル基などを意味する。この含酸素ヘテロ脂肪族環基は、更に他の芳香族環又は脂肪族環と縮合していてもよい。
　含窒素含酸素ヘテロ脂肪族環基とは、モルホリニル又は１，４－オキサゼパニル基などを意味する。この含窒素含酸素ヘテロ脂肪族環基は、更に他の芳香族環又は脂肪族環と縮合していてもよい。 The nitrogen-containing heteroaliphatic ring group refers to a heteroaliphatic ring group in which the ring containing at least one nitrogen atom does not have aromaticity, such as azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, octahydroazocinyl, imidazolidinyl, pyrazolidinyl, piperazinyl, and homopiperazinyl groups, etc. This nitrogen-containing heteroaliphatic ring group may be further condensed with another aromatic ring or an aliphatic ring.
The oxygen-containing heteroaliphatic ring group means a tetrahydrofuranyl, tetrahydropyranyl, oxetanyl, 1,3-dioxanyl group, etc. This oxygen-containing heteroaliphatic ring group may be further condensed with another aromatic ring or an aliphatic ring.
The nitrogen-containing, oxygen-containing heteroaliphatic ring group means a morpholinyl or 1,4-oxazepanyl group, etc. This nitrogen-containing, oxygen-containing heteroaliphatic ring group may be further condensed with another aromatic ring or an aliphatic ring.

　ヘテロ脂肪族環Ｃ１－８アルキル基とは、ピロリジニルメチル基、ピロリジニルエチル基、ピロリジニルプロピル基、ピロリジニルオクチル基、ピペリジニルメチル基、テトラヒドロフラニルメチル基などのヘテロ脂肪族環基が結合した直鎖状、分枝鎖状又は環状のＣ１－８アルキル基を意味する。 The heteroaliphatic ring C 1-8 alkyl group means a straight-chain, branched-chain or cyclic C 1-8 alkyl group bonded to a heteroaliphatic ring group such as a pyrrolidinylmethyl group, a pyrrolidinylethyl group, a pyrrolidinylpropyl group, a pyrrolidinyloctyl group, a piperidinylmethyl group or a tetrahydrofuranylmethyl group.

　式（１）において、好ましくは、
　Ｒ１が－Ｒ１ａ－Ｌ１－Ｒ１ｂを示し、Ｒ１ａが炭素数１～１８の炭化水素基を示し、
Ｌ１が－Ｃ（Ｏ）Ｏ－、－ＯＣ（Ｏ）－、－ＯＣ（Ｏ）Ｏ－、または－Ｓ－Ｓ－を示し、
Ｒ１ｂが炭素数１～１８の炭化水素基を示し、
　Ｒ３が－Ｒ３ａ－Ｌ３－Ｒ３ｂを示し、Ｒ３ａが炭素数１～１８の炭化水素基を示し、
Ｌ３が－Ｃ（Ｏ）Ｏ－、－ＯＣ（Ｏ）－、－ＯＣ（Ｏ）Ｏ－、または－Ｓ－Ｓ－を示し、
Ｒ３ｂが炭素数１～１８の炭化水素基を示し、
　Ｒ２およびＲ４がそれぞれ独立に、置換されていてもよい炭素数１～１８の炭化水素基を示し、Ｒ２およびＲ４が示す置換されていてもよい炭素数１～１８の炭化水素基上の置換基はそれぞれ独立に、－Ｃ（Ｏ）Ｏ－Ｒ１１、－ＯＣ（Ｏ）－Ｒ１２、－Ｏ－Ｒ１３、－ＣＯ－Ｒ１４、－ＯＣ（Ｏ）Ｏ－Ｒ１５、または－Ｓ－Ｓ－Ｒ１６を示し、
　Ｒ１１、Ｒ１２、Ｒ１３、Ｒ１４、Ｒ１５およびＲ１６はそれぞれ独立に、炭素数１～１８の炭化水素基を示し、
　Ｒ５およびＲ６がそれぞれ独立に、置換されていてもよい炭素数１～１２の炭化水素基を示し、
　Ｒ５およびＲ６が示す置換されていてもよい炭素数１～１２の炭化水素基上の置換基はそれぞれ独立に、－ＯＨ、－Ｏ－Ｒ２６、－Ｃ（Ｏ）ＮＲ２７Ｒ２８、または－ＮＲ２９Ｃ（Ｏ）Ｒ３０を示し、
　Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、およびＲ３０はそれぞれ独立に、水素原子、または置換されていてもよい炭素数１～１２の炭化水素基を示し、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、およびＲ３０が示す置換されていてもよい炭素数１～１２の炭化水素基上の置換基は、炭素数６～１０のアリール基、またはヘテロ環基を示し、
　Ｒ７、Ｒ８およびＲ９はそれぞれ独立に、－（ＣＨ２）ｎ－を示し、ｎは２～８の整数を示す。
　Ｒ５とＲ６、またはＲ５とＲ７は一緒になって４～７員環を形成してもよい。 In the formula (1), preferably,
R 1 represents -R 1a -L 1 -R 1b , R 1a represents a hydrocarbon group having 1 to 18 carbon atoms,
L 1 represents -C(O)O-, -OC(O)-, -OC(O)O-, or -S-S-;
R 1b represents a hydrocarbon group having 1 to 18 carbon atoms;
R 3 represents -R 3a -L 3 -R 3b , R 3a represents a hydrocarbon group having 1 to 18 carbon atoms,
L3 represents -C(O)O-, -OC(O)-, -OC(O)O-, or -S-S-;
R 3b represents a hydrocarbon group having 1 to 18 carbon atoms;
R 2 and R 4 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted, and the substituents on the hydrocarbon group having 1 to 18 carbon atoms which may be substituted represented by R 2 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 , or -S-S-R 16 ;
R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 18 carbon atoms;
R 5 and R 6 each independently represent a hydrocarbon group having 1 to 12 carbon atoms which may be substituted;
Substituents on the optionally substituted hydrocarbon group having 1 to 12 carbon atoms represented by R 5 and R 6 each independently represent -OH, -O-R 26 , -C(O)NR 27 R 28 , or -NR 29 C(O)R 30 ;
R 26 , R 27 , R 28 , R 29 , and R 30 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 12 carbon atoms, and a substituent on the optionally substituted hydrocarbon group having 1 to 12 carbon atoms represented by R 26 , R 27 , R 28 , R 29 , and R 30 represents an aryl group or heterocyclic group having 6 to 10 carbon atoms;
R 7 , R 8 and R 9 each independently represent —(CH 2 ) n —, where n is an integer of 2 to 8.
R 5 and R 6 , or R 5 and R 7 may join together to form a 4- to 7-membered ring.

　式（１）においてさらに好ましくは、
　Ｒ１が－Ｒ１ａ－Ｌ１－Ｒ１ｂを示し、Ｒ１ａが炭素数１～１８の炭化水素基を示し、
Ｌ１が－Ｃ（Ｏ）Ｏ－、または－ＯＣ（Ｏ）－を示し、Ｒ１ｂが炭素数１～１８の炭化水素基を示し、
　Ｒ３が－Ｒ３ａ－Ｌ３－Ｒ３ｂを示し、Ｒ３ａが炭素数１～１８の炭化水素基を示し、
Ｌ３が－Ｃ（Ｏ）Ｏ－、または－ＯＣ（Ｏ）－を示し、Ｒ３ｂが炭素数１～１８の炭化水素基を示し、
　Ｒ２およびＲ４がそれぞれ独立に、炭素数１～１０の炭化水素基を示し、
　Ｒ５およびＲ６がそれぞれ独立に、置換されていてもよい炭素数１～６の炭化水素基を示し、
　Ｒ５およびＲ６が示す置換されていてもよい炭素数１～６の炭化水素基上の置換基はそれぞれ独立に、－ＯＨ、－Ｏ－Ｒ２６、－Ｃ（Ｏ）ＮＲ２７Ｒ２８、または－ＮＲ２９Ｃ（Ｏ）Ｒ３０を示し、
　Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、およびＲ３０はそれぞれ独立に、水素原子、または置換されていてもよい炭素数１～１２の炭化水素基を示し、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、およびＲ３０が示す置換されていてもよい炭素数１～１２の炭化水素基上の置換基は、炭素数６～１０のアリール基を示し、
　Ｒ７、Ｒ８およびＲ９はそれぞれ独立に、－（ＣＨ２）ｎ－を示し、ｎは２～８の整数を示す。 In the formula (1), more preferably,
R 1 represents -R 1a -L 1 -R 1b , R 1a represents a hydrocarbon group having 1 to 18 carbon atoms,
L 1 represents -C(O)O- or -OC(O)-; R 1b represents a hydrocarbon group having 1 to 18 carbon atoms;
R 3 represents -R 3a -L 3 -R 3b , R 3a represents a hydrocarbon group having 1 to 18 carbon atoms,
L 3 represents -C(O)O- or -OC(O)-; R 3b represents a hydrocarbon group having 1 to 18 carbon atoms;
R 2 and R 4 each independently represent a hydrocarbon group having 1 to 10 carbon atoms;
R 5 and R 6 each independently represent a hydrocarbon group having 1 to 6 carbon atoms which may be substituted;
Substituents on the optionally substituted hydrocarbon group having 1 to 6 carbon atoms represented by R 5 and R 6 each independently represent -OH, -O-R 26 , -C(O)NR 27 R 28 , or -NR 29 C(O)R 30 ;
R 26 , R 27 , R 28 , R 29 , and R 30 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 12 carbon atoms, and a substituent on the optionally substituted hydrocarbon group having 1 to 12 carbon atoms represented by R 26 , R 27 , R 28 , R 29 , and R 30 represents an aryl group having 6 to 10 carbon atoms;
R 7 , R 8 and R 9 each independently represent —(CH 2 ) n —, where n is an integer of 2 to 8.

　式（１）において最も好ましくは、
　Ｒ１が－Ｒ１ａ－Ｌ１－Ｒ１ｂを示し、Ｒ１ａが炭素数１～５の炭化水素基を示し、Ｌ１が－Ｃ（Ｏ）Ｏ－を示し、Ｒ１ｂが炭素数７～１４の炭化水素基を示し、
　Ｒ３が－Ｒ３ａ－Ｌ３－Ｒ３ｂを示し、Ｒ３ａが炭素数１～５の炭化水素基を示し、Ｌ３が－Ｃ（Ｏ）Ｏ－を示し、Ｒ３ｂが炭素数７～１４の炭化水素基を示し、
　Ｒ２およびＲ４がそれぞれ独立に、炭素数３～８の炭化水素基を示し、
　Ｒ５およびＲ６がそれぞれ独立に置換されてもよい炭素数１～４の炭化水素基を示し、Ｒ５およびＲ６が示す置換されてもよい炭素数１～４の炭化水素基上の置換基はそれぞれ独立に、－ＯＨ基が好ましい。
　Ｒ７、Ｒ８およびＲ９はそれぞれ独立に、－（ＣＨ２）ｎ－を示し、ｎは２～４の整数を示す。
　Ｒ５とＲ６、またはＲ５とＲ７は一緒になって５～７員環を形成してもよい。 In the formula (1), most preferably,
R 1 represents -R 1a -L 1 -R 1b , R 1a represents a hydrocarbon group having 1 to 5 carbon atoms, L 1 represents -C(O)O-, and R 1b represents a hydrocarbon group having 7 to 14 carbon atoms;
R 3 represents -R 3a -L 3 -R 3b , R 3a represents a hydrocarbon group having 1 to 5 carbon atoms, L 3 represents -C(O)O-, and R 3b represents a hydrocarbon group having 7 to 14 carbon atoms;
R 2 and R 4 each independently represent a hydrocarbon group having 3 to 8 carbon atoms;
R5 and R6 each independently represent a hydrocarbon group having 1 to 4 carbon atoms which may be substituted, and the substituents on the hydrocarbon group having 1 to 4 carbon atoms which may be substituted represented by R5 and R6 each independently are preferably an —OH group.
R 7 , R 8 and R 9 each independently represent --(CH 2 ) n --, where n is an integer of 2 to 4.
R 5 and R 6 , or R 5 and R 7 may join together to form a 5- to 7-membered ring.

　式（１）で表される化合物は塩を形成していてもよい。
　塩基性基における塩としては、例えば、塩酸、臭化水素酸、硝酸および硫酸などの鉱酸との塩；ギ酸、酢酸、クエン酸、シュウ酸、フマル酸、マレイン酸、コハク酸、リンゴ酸、酒石酸、アスパラギン酸、トリクロロ酢酸およびトリフルオロ酢酸などの有機カルボン酸との塩；並びにメタンスルホン酸、ベンゼンスルホン酸、ｐ－トルエンスルホン酸、メシチレンスルホン酸およびナフタレンスルホン酸などのスルホン酸との塩が挙げられる。
　酸性基における塩としては、例えば、ナトリウムおよびカリウムなどのアルカリ金属との塩；カルシウムおよびマグネシウムなどのアルカリ土類金属との塩；アンモニウム塩；並びにトリメチルアミン、トリエチルアミン、トリブチルアミン、ピリジン、Ｎ，Ｎ－ジメチルアニリン、Ｎ－メチルピペリジン、Ｎ－メチルモルホリン、ジエチルアミン、ジシクロヘキシルアミン、プロカイン、ジベンジルアミン、Ｎ－ベンジル－β－フェネチルアミン、１－エフェナミンおよびＮ，Ｎ’－ジベンジルエチレンジアミンなどの含窒素有機塩基との塩などが挙げられる。
　上記した塩の中で、好ましい塩としては、薬理学的に許容される塩が挙げられる。 The compound represented by formula (1) may form a salt.
Examples of salts of basic groups include salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.
Examples of salts of acidic groups include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine.
Among the above-mentioned salts, preferred salts include pharmacologically acceptable salts.

　好ましくは、イオン化可能な脂質は、以下の化合物のうちの１つ以上であるが、本発明がこれにより限定して解釈されるものではない。


 Preferably, the ionizable lipid is one or more of the following compounds, although the invention is not intended to be limited thereto:


　化合物１～７および３１～７４は新規化合物である。本発明によれば、化合物１～７および３１～７４が提供される。 Compounds 1 to 7 and 31 to 74 are novel compounds. According to the present invention, compounds 1 to 7 and 31 to 74 are provided.

　脂質組成物において、式（１）で示される化合物またはその塩の配合量は、脂質組成物中の全脂質に対するモル比で、２０モル％～６０モル％であることが好ましく、３０モル％～６０モル％であることがより好ましく、４０モル％～６０モル％であることがさらに好ましい。 In the lipid composition, the amount of the compound represented by formula (1) or its salt is, in terms of molar ratio to the total lipids in the lipid composition, preferably 20 mol% to 60 mol%, more preferably 30 mol% to 60 mol%, and even more preferably 40 mol% to 60 mol%.

＜式（１）で表される化合物の製造方法＞
　式（１）で表される化合物の製造法について説明する。
　式（１）で表される化合物は、公知の方法を組み合わせることにより製造することができるが、例えば、以下に示す製造法に従い、製造することができる。 <Method for producing the compound represented by formula (1)>
A method for producing the compound represented by formula (1) will now be described.
The compound represented by formula (1) can be produced by combining known methods, for example, according to the production method shown below.

［製造法１］
式［２］の化合物から式［１］の化合物を製造する方法。
 [Manufacturing method 1]
A method for producing a compound of formula [1] from a compound of formula [2].

式中、Ｒ１、Ｒ２、Ｒ３、Ｒ４、Ｒ５、Ｒ６、Ｒ７、Ｒ８およびＲ９は上記と同じ意味を有し；Ｒ８ａ、Ｒ９ａおよびＲＡは炭素数１～７の炭化水素基を意味する。 In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 have the same meanings as above; R 8a , R 9a and R A represent a hydrocarbon group having 1 to 7 carbon atoms.

（１－１）
　式［３Ａ］の化合物は、式［２］の化合物を、水および酸の存在下、溶媒の存在下もしくは不存在下に反応させることにより製造することができる。
　この反応に用いられる酸としては、無機酸または有機酸が挙げられる。有機酸が好ましく、具体的には、ギ酸、酢酸、トリフルオロ酢酸、４－トルエンスルホン酸、メタンスルホン酸などが挙げられ、ギ酸がより好ましい。
　酸の使用量は、式［２］の化合物に対して、１～１００倍量（ｖ／ｗ）、好ましくは、１～１０倍量（ｖ／ｗ）であればよい。
　水の使用量は、式［２］の化合物に対して、０．１～１００倍量（ｖ／ｗ）、好ましくは、０．１～１０倍量（ｖ／ｗ）であればよい。
　この反応に使用される溶媒としては、反応に影響を及ぼさないものであれば特に限定されないが、例えば、ハロゲン炭化水素類、エーテル類、エステル類、アミド類、ニトリル類、スルホキシド類、芳香族炭化水素類が挙げられ、これらの溶媒は混合して使用してもよい。
　溶媒の使用量は、特に限定されないが、式［２］の化合物に対して、０．１～５０倍量
（ｖ／ｗ）であればよい。
　この反応は、－３０～１５０℃、好ましくは０～１００℃で５分間～４８時間実施すればよい。 (1-1)
The compound of formula [3A] can be produced by reacting the compound of formula [2] in the presence of water and an acid, in the presence or absence of a solvent.
The acid used in this reaction may be an inorganic acid or an organic acid, preferably an organic acid, specifically formic acid, acetic acid, trifluoroacetic acid, 4-toluenesulfonic acid, methanesulfonic acid, etc., more preferably formic acid.
The amount of the acid used may be 1 to 100 times (v/w), preferably 1 to 10 times (v/w), relative to the amount of the compound of the formula [2].
The amount of water used may be 0.1 to 100 times (v/w), preferably 0.1 to 10 times (v/w), relative to the amount of the compound of the formula [2].
The solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination.
The amount of the solvent used is not particularly limited, but may be 0.1 to 50 times (v/w) the amount of the compound of the formula [2].
This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

（１－２）
　式［１］の化合物は、還元剤の存在下、式［３Ａ］の化合物を式［４］の化合物と反応させることにより製造することができる。
　式［４］の化合物として、例えば、Ｎ，Ｎ－ジエチルエチレンジアミン、およびＮ，Ｎ－ジエチル－１，３－ジアミノプロパンなどが知られている。
　この反応に使用される溶媒としては、反応に影響を及ぼさないものであれば特に限定されないが、例えば、ハロゲン炭化水素類、アルコール類、エーテル類、エステル類、アミド類、ニトリル類、スルホキシド類、芳香族炭化水素類が挙げられ、これらの溶媒は混合して使用してもよい。
　好ましい溶媒としては、エステル類が挙げられ、酢酸エチルがより好ましい。
　溶媒の使用量は、特に限定されないが、式［３Ａ］の化合物に対して、１～５００倍量（ｖ／ｗ）であればよい。
　この反応に用いられる還元剤としては、水素化ホウ素ナトリウム、シアノ水素化ホウ素ナトリウム、ピリジンボラン、２－ピコリンボラン、およびトリアセトキシ水素化ホウ素ナトリウムなどが挙げられ、トリアセトキシ水素化ホウ素ナトリウムがより好ましい。
還元剤の使用量は、式［３Ａ］の化合物に対して、１～１００倍モル、好ましくは、１～１０倍モルであればよい。
　式［４］の化合物の使用量は、式［３Ａ］の化合物に対して、０．１～１倍モルであればよい。
　この反応は、－３０～１５０℃、好ましくは０～１００℃で５分間～４８時間実施すればよい。 (1-2)
The compound of formula [1] can be prepared by reacting a compound of formula [3A] with a compound of formula [4] in the presence of a reducing agent.
Known examples of the compound of the formula [4] include N,N-diethylethylenediamine and N,N-diethyl-1,3-diaminopropane.
The solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, alcohols, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination.
Preferred solvents include esters, with ethyl acetate being more preferred.
The amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [3A].
The reducing agent used in this reaction includes sodium borohydride, sodium cyanoborohydride, pyridine borane, 2-picoline borane, and sodium triacetoxyborohydride, with sodium triacetoxyborohydride being more preferred.
The amount of the reducing agent used may be 1 to 100 times, preferably 1 to 10 times, the molar amount of the compound of the formula [3A].
The amount of the compound of the formula [4] used may be 0.1 to 1 mole per mole of the compound of the formula [3A].
This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

（１－３）
　式［５］の化合物は、還元剤の存在下、式［３Ａ］の化合物を式［４］の化合物と反応させることにより製造することができる。
　この反応は、製造法（１－２）に準じて行えばよく、式［４］の化合物を式［３Ａ］の化合物に対して、１～１０倍モル使用すればよい。 (1-3)
The compound of formula [5] can be prepared by reacting a compound of formula [3A] with a compound of formula [4] in the presence of a reducing agent.
This reaction may be carried out in accordance with the production method (1-2), and the compound of the formula [4] may be used in an amount of 1 to 10 times by mole relative to the compound of the formula [3A].

（１－４）
　式［１］の化合物は、還元剤の存在下、式［３Ｂ］の化合物を式［５］の化合物と反応させることにより製造することができる。
　この反応は、製造法（１－２）に準じて行えばよく、式［３Ｂ］の化合物を式［５］の化合物に対して、１～１０倍モル使用すればよい。 (1-4)
The compound of formula [1] can be prepared by reacting a compound of formula [3B] with a compound of formula [5] in the presence of a reducing agent.
This reaction may be carried out in accordance with the production method (1-2), and the compound of the formula [3B] may be used in an amount of 1 to 10 times by mole relative to the compound of the formula [5].

［製造法２］
式［６］および式［７］の化合物から式［２］の化合物を製造する方法。
 [Manufacturing method 2]
A method for producing a compound of formula [2] from compounds of formula [6] and formula [7].

式中、Ｒ１、Ｒ２、Ｒ９ａおよびＲＡは上記と同じ意味を有し；Ｘ１およびＸ２は脱離基を意味する。
　脱離基として、例えば、クロロ基、フルオロ基、ブロモ基、トリクロロメトキシ基、４－ニトロ－フェノキシ基、２，４－ジニトロフェノキシ基、２，４，６－トリクロロフェノキシ基、ペンタフルオロフェノキシ基、２，３，５，６－テトラフルオロフェノキシ基、イミダゾリル基、トリアゾリル基、３，５－ジオキソ－４－メチル－１，２，４－オキサジアゾリジル基、Ｎ－ヒドロキシスクシンイミジル基などが挙げられる。 In the formula, R 1 , R 2 , R 9a and R A have the same meanings as above; X 1 and X 2 represent leaving groups.
Examples of leaving groups include a chloro group, a fluoro group, a bromo group, a trichloromethoxy group, a 4-nitro-phenoxy group, a 2,4-dinitrophenoxy group, a 2,4,6-trichlorophenoxy group, a pentafluorophenoxy group, a 2,3,5,6-tetrafluorophenoxy group, an imidazolyl group, a triazolyl group, a 3,5-dioxo-4-methyl-1,2,4-oxadiazolidyl group, and an N-hydroxysuccinimidyl group.

（２－１）
　式［９］の化合物は、塩基の存在下もしくは不存在下、式［７］の化合物を式［８］の化合物と反応させることにより製造することができる。
　式［８］の化合物として、例えば、１，１'－カルボニルジ（１，２，４－トリアゾール）、１，１'－カルボニルジイミダゾール、クロロギ酸４－ニトロフェニル、トリホスゲンおよびホスゲンなどが知られている。
　この反応に使用される溶媒としては、反応に影響を及ぼさないものであれば特に限定されないが、例えば、ハロゲン炭化水素類、エーテル類、エステル類、アミド類、ニトリル類、スルホキシド類および芳香族炭化水素類が挙げられ、これらの溶媒は混合して使用してもよい。
　好ましい溶媒としては、エーテル類が挙げられ、テトラヒドロフランがより好ましい。
　溶媒の使用量は、特に限定されないが、式［７］の化合物に対して、１～５００倍量（ｖ／ｗ）であればよい。
　この反応に用いられる塩基としては、無機塩基または有機塩基が挙げられる。塩基は有機塩基が好ましく、具体的には、トリエチルアミン、Ｎ，Ｎ－ジイソプロピルエチルアミン、４－メチルモルホリン、 ピリジン、１，８－ジアザビシクロ［５．４．０］－７－ウンデセン、またはＮ，Ｎ－ジメチルアミノピリジンなどが挙げられる。
　塩基の使用量は、式［７］の化合物に対して、１～５０倍モル、好ましくは、１～１０倍モルであればよい。
　式［８］の化合物の使用量は、特に限定されないが、式［７］の化合物に対して、１～１０倍モルであればよい。
　この反応は、－３０～１５０℃、好ましくは０～１００℃で５分間～４８時間実施すればよい。 (2-1)
The compound of formula [9] can be prepared by reacting a compound of formula [7] with a compound of formula [8] in the presence or absence of a base.
Known examples of the compound of the formula [8] include 1,1'-carbonyldi(1,2,4-triazole), 1,1'-carbonyldiimidazole, 4-nitrophenyl chloroformate, triphosgene, and phosgene.
The solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination.
Preferred solvents include ethers, with tetrahydrofuran being more preferred.
The amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [7].
The base used in this reaction may be an inorganic base or an organic base, preferably an organic base, such as triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, or N,N-dimethylaminopyridine.
The amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar amount of the compound of the formula [7].
The amount of the compound of the formula [8] used is not particularly limited, but may be 1 to 10 times the molar amount of the compound of the formula [7].
This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

（２－２）
　式［２］の化合物は、塩基の存在下、式［６］の化合物を式［９］の化合物と反応させることにより製造することができる。
　式［６］の化合物として、例えば、ジオクチルアミンなどが知られている。
　この反応に使用される溶媒としては、反応に影響を及ぼさないものであれば特に限定されないが、例えば、ハロゲン炭化水素類、エーテル類、エステル類、アミド類、ニトリル類、スルホキシド類および芳香族炭化水素類が挙げられ、これらの溶媒は混合して使用してもよい。
　好ましい溶媒としては、ニトリル類が挙げられ、アセトニトリルがより好ましい。
　溶媒の使用量は、特に限定されないが、式［６］の化合物に対して、１～５００倍量（ｖ／ｗ）であればよい。
　この反応に用いられる塩基としては、無機塩基または有機塩基が挙げられる。具体的には、炭酸カリウム、炭酸ナトリウム、炭酸リチウム、リン酸カリウム、リン酸ナトリウム、リン酸リチウム、トリエチルアミン、Ｎ，Ｎ－ジイソプロピルエチルアミン、４－メチルモルホリン、 ピリジン、１，８－ジアザビシクロ［５．４．０］－７－ウンデセン、またはＮ，Ｎ－ジメチルアミノピリジンなどが挙げられる。
　塩基の使用量は、式［６］の化合物に対して、１～５０倍モル、好ましくは、１～１０倍モルであればよい。
　式［９］の化合物の使用量は、特に限定されないが、式［６］の化合物に対して、０．１～１０倍モルであればよい。
　この反応は、－３０～１５０℃、好ましくは０～１００℃で５分間～４８時間実施すればよい。 (2-2)
The compound of formula [2] can be prepared by reacting a compound of formula [6] with a compound of formula [9] in the presence of a base.
As a compound of the formula [6], for example, dioctylamine is known.
The solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides and aromatic hydrocarbons, and these solvents may be used in combination.
Preferred solvents include nitriles, with acetonitrile being more preferred.
The amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [6].
The base used in this reaction may be an inorganic base or an organic base, such as potassium carbonate, sodium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, lithium phosphate, triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, or N,N-dimethylaminopyridine.
The amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar amount of the compound of the formula [6].
The amount of the compound of the formula [9] used is not particularly limited, but may be 0.1 to 10 times the molar amount of the compound of the formula [6].
This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

［製造法３］
式［６Ａ］の化合物を製造する方法。
 [Manufacturing method 3]
A method for producing a compound of formula [6A].

式中、Ｒ２、Ｒ１aおよびＲ１bは上記と同じ意味を有し；Ｘ３は水酸基および脱離基を；Ｘ４は脱離基を意味し；脱離基は上記と同じ意味を有する。 In the formula, R 2 , R 1a and R 1b have the same meanings as above; X 3 represents a hydroxyl group and a leaving group; X 4 represents a leaving group; the leaving group has the same meaning as above.

（３－１）
　式［１２Ａ］の化合物は、酸の存在下もしくは不存在下、縮合剤または酸ハロゲン化物の存在下もしくは不存在下、塩基の存在下もしくは不存在下、式［１０Ａ］の化合物を式［１１Ａ］の化合物と反応させることにより製造することができる。
　式［１０Ａ］の化合物として、例えば、５－ブロモ吉草酸、クロロアセチルクロリドなどが知られている。
　式［１１Ａ］の化合物として、例えば、２－ブチル－１－オクタノール、２－ペンチル－１－ヘプタノール、１－デカノールなどが知られている。
　この反応に使用される溶媒としては、反応に影響を及ぼさないものであれば特に限定されないが、例えば、ハロゲン炭化水素類、エーテル類、エステル類、アミド類、ニトリル類、スルホキシド類および芳香族炭化水素類が挙げられ、これらの溶媒は混合して使用してもよい。
　好ましい溶媒としては、芳香族炭化水素類およびエーテル類が挙げられ、トルエンおよびテトラヒドロフランがより好ましい。
　溶媒の使用量は、特に限定されないが、式［１０Ａ］の化合物に対して、１～５００倍量（ｖ／ｗ）であればよい。
　この反応に用いられる酸としては、無機酸または有機酸が挙げられる。酸はスルホン酸類が好ましく、具体的には、硫酸、４－トルエンスルホン酸、メタンスルホン酸などが挙げられる。
　この反応に使用される縮合剤としては、例えば、Ｎ，Ｎ’－ジシクロヘキシルカルボジイミドおよび１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミドなどのカルボジイミド類；カルボニルジイミダゾールなどのカルボニル類；ジフェニルホスホリルアジドなどの酸アジド類；ジエチルホスホリルシアニドなどの酸シアニド類；２－エトキシ－１－エトキシカルボニル－１，２－ジヒドロキノリン；Ｏ－ベンゾトリアゾール－１－イル－１，１，３，３－テトラメチルウロニウム＝ヘキサフルオロホスフェートならびにＯ－（７－アザベンゾトリアゾール－１－イル）－１，１，３，３－テトラメチルウロニウム＝ヘキサフルオロホスフェートなどのウロニウム類などが挙げられる。
　この反応に使用される酸ハロゲン化物としては、例えば、塩化アセチルおよびトリフルオロアセチルクロリドなどのカルボン酸ハロゲン化物類；塩化メタンスルホニルおよび塩化トシルなどのスルホン酸ハロゲン化物類；クロロギ酸エチルおよびクロロギ酸イソブチルなどのクロロギ酸エステル類などが挙げられる。
　この反応に用いられる塩基としては、無機塩基または有機塩基が挙げられる。有機塩基が好ましく、具体的には、トリエチルアミン、Ｎ，Ｎ－ジイソプロピルエチルアミン、４－メチルモルホリン、 ピリジン、１，８－ジアザビシクロ［５．４．０］－７－ウンデセン、またはＮ，Ｎ－ジメチルアミノピリジンなどが挙げられる。
　塩基の使用量は、式［１０Ａ］の化合物に対して、１～５０倍モル、好ましくは、１～１０倍モルであればよい。
　式［１１Ａ］の化合物の使用量は、特に限定されないが、式［１０Ａ］の化合物に対して、０．８～１０倍量（ｖ／ｗ）であればよい。
　この反応は、－３０～１５０℃、好ましくは０～１００℃で５分間～４８時間実施すればよい。 (3-1)
The compound of formula [12A] can be produced by reacting a compound of formula [10A] with a compound of formula [11A] in the presence or absence of an acid, in the presence or absence of a condensing agent or an acid halide, and in the presence or absence of a base.
Known examples of the compound of the formula [10A] include 5-bromovaleric acid and chloroacetyl chloride.
Known examples of the compound of the formula [11A] include 2-butyl-1-octanol, 2-pentyl-1-heptanol, and 1-decanol.
The solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination.
Preferred solvents include aromatic hydrocarbons and ethers, with toluene and tetrahydrofuran being more preferred.
The amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [10A].
The acid used in this reaction may be an inorganic acid or an organic acid. The acid is preferably a sulfonic acid, specifically, sulfuric acid, 4-toluenesulfonic acid, methanesulfonic acid, etc.
Condensing agents used in this reaction include, for example, carbodiimides such as N,N'-dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; carbonyls such as carbonyldiimidazole; acid azides such as diphenylphosphoryl azide; acid cyanides such as diethylphosphoryl cyanide; 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; uroniums such as O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.
The acid halide used in this reaction includes, for example, carboxylic acid halides such as acetyl chloride and trifluoroacetyl chloride; sulfonic acid halides such as methanesulfonyl chloride and tosyl chloride; chloroformates such as ethyl chloroformate and isobutyl chloroformate; and the like.
The base used in this reaction may be an inorganic base or an organic base, preferably an organic base, such as triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, or N,N-dimethylaminopyridine.
The amount of the base used may be 1 to 50 moles, preferably 1 to 10 moles, based on the amount of the compound of the formula [10A].
The amount of the compound of formula [11A] used is not particularly limited, but may be 0.8 to 10 times (v/w) the amount of the compound of formula [10A].
This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

（３－２）
　式［６Ａ］の化合物は、塩基の存在下もしくは不存在下、添加剤の存在下もしくは不存在下、式［１２Ａ］の化合物を式［１３］の化合物と反応させることにより製造することができる。
　式［１３］の化合物として、例えば、１－ブチルアミン、１－ヘキシルアミンなどが知られている。
　この反応に使用される溶媒としては、反応に影響を及ぼさないものであれば特に限定されないが、例えば、ハロゲン炭化水素類、エーテル類、エステル類、アミド類、ニトリル類、スルホキシド類および芳香族炭化水素類が挙げられ、これらの溶媒は混合して使用してもよい。
　好ましい溶媒としては、ニトリル類およびエーテル類が挙げられ、アセトニトリルおよびテトラヒドロフランがより好ましい。
　溶媒の使用量は、特に限定されないが、式［１２Ａ］の化合物に対して、１～５００倍量（ｖ／ｗ）であればよい。
　この反応に用いられる塩基としては、無機塩基または有機塩基が挙げられる。具体的には、水酸化カリウム、水酸化ナトリム、水酸化リチウム、炭酸カリウム、炭酸ナトリウム、炭酸リチウム、リン酸カリウム、リン酸ナトリウム、リン酸リチウム、トリエチルアミン、Ｎ，Ｎ－ジイソプロピルエチルアミン、４－メチルモルホリン、 ピリジン、１，８－ジアザビシクロ［５．４．０］－７－ウンデセン、またはＮ，Ｎ－ジメチルアミノピリジンなどが挙げられ、炭酸カリウムがより好ましい。
　塩基の使用量は、式［１２Ａ］の化合物に対して、１～５０倍モル、好ましくは、１～１０倍モルであればよい。
　式［１３］の化合物の使用量は、特に限定されないが、式［１２Ａ］の化合物に対して、１～１０倍モルであればよい。
　この反応に用いられる添加剤としては、具体的には、ヨウ化リチウム、ヨウ化ナトリウム、ヨウ化カリウム、ヨウ化ベンジルトリエチルアンモニウム、臭化ベンジルトリエチルアンモニウムなどが挙げられる。
　添加剤の使用量は、式［１２Ａ］の化合物に対して、０．１～１０倍モルであればよい。
　この反応は、－３０～１５０℃、好ましくは０～１００℃で５分間～４８時間実施すればよい。 (3-2)
The compound of formula [6A] can be produced by reacting a compound of formula [12A] with a compound of formula [13] in the presence or absence of a base and in the presence or absence of an additive.
Known examples of the compound of the formula [13] include 1-butylamine and 1-hexylamine.
The solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides and aromatic hydrocarbons, and these solvents may be used in combination.
Preferred solvents include nitriles and ethers, with acetonitrile and tetrahydrofuran being more preferred.
The amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of the formula [12A].
The base used in this reaction may be an inorganic base or an organic base, specifically, potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, lithium phosphate, triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, or N,N-dimethylaminopyridine, and potassium carbonate is more preferred.
The amount of the base used may be 1 to 50 moles, preferably 1 to 10 moles, based on the amount of the compound of the formula [12A].
The amount of the compound of formula [13] used is not particularly limited, but may be 1 to 10 times the molar amount of the compound of formula [12A].
Specific examples of additives used in this reaction include lithium iodide, sodium iodide, potassium iodide, benzyltriethylammonium iodide, and benzyltriethylammonium bromide.
The amount of the additive used may be 0.1 to 10 times the molar amount of the compound of the formula [12A].
This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

［製造法４］
式［６Ｂ］の化合物を製造する方法。
 [Manufacturing method 4]
A method for producing a compound of formula [6B].

式中、Ｒ２、Ｒ１ａ、Ｒ１ｂ、Ｘ３およびＸ４は上記と同じ意味を有する。 In the formula, R 2 , R 1a , R 1b , X 3 and X 4 have the same meanings as above.

（４－１）
　式［１２Ｂ］の化合物は、式［１０Ａ］の代わりに式［１０Ｂ］の化合物を用い、式［１１Ａ］の代わりに式［１１Ｂ］の化合物を用いることで、製造法（３－１）と同様の方法により製造することができる。 (4-1)
The compound of formula [12B] can be produced in the same manner as in Production Method (3-1), except that the compound of formula [10B] is used in place of formula [10A] and the compound of formula [11B] is used in place of formula [11A].

（４－２）
　式［６Ｂ］の化合物は、式［１２Ａ］の代わりに式［１２Ｂ］の化合物を用いることで、製造法（３－２）と同様の方法により製造することができる。 (4-2)
The compound of formula [6B] can be produced in the same manner as in Production Method (3-2), except that the compound of formula [12B] is used in place of the compound of formula [12A].

［製造法５］
式［６Ｃ］の化合物を製造する方法。
 [Manufacturing method 5]
A method for producing a compound of formula [6C].

式中、Ｒ１ａ、Ｒ１ｂおよびＸ３は上記と同じ意味を有し；ＲＢはアミノ保護基を意味する。 In the formula, R 1a , R 1b and X 3 have the same meaning as above; R B represents an amino protecting group.

（５－１）
　式［１５］の化合物は、酸の存在下もしくは不存在下、縮合剤または酸ハロゲン化物の存在下もしくは不存在下、塩基の存在下もしくは不存在下、式［１０Ｂ］の化合物を式［１４］の化合物と反応させることにより製造することができる。
　式［１０Ｂ］の化合物として、例えば、デカン酸、デカン酸クロリドなどが知られている。
　式［１５］の化合物として、例えば、ｔｅｒｔ－ブチルビス（２－ヒドロキシエチル）カルバメートなどが知られている。
　この反応は、製造法（３－１）に準じて行えばよい。 (5-1)
The compound of formula [15] can be produced by reacting a compound of formula [10B] with a compound of formula [14] in the presence or absence of an acid, in the presence or absence of a condensing agent or an acid halide, and in the presence or absence of a base.
Known examples of the compound of the formula [10B] include decanoic acid and decanoic acid chloride.
Known examples of the compound of the formula [15] include tert-butyl bis(2-hydroxyethyl)carbamate.
This reaction may be carried out according to the production method (3-1).

（５－２）
　式［６Ｃ］の化合物は、式[１５]の化合物を脱保護することにより製造することができる。
　この反応は、例えば、Ｔ．Ｗ．グリーン（Ｔ．Ｗ．Ｇｒｅｅｎｅ）ら、プロテクティブ・グループス・イン・オーガニック・シンセシス（Protective Groups in Organic Synthesis）第4版、第696～926頁、2007年、ジョン・ワイリー・アンド・サンズ社（John Wiley & Sons,INC.）に記載の方法に準じて行えばよい。 (5-2)
The compound of formula [6C] can be prepared by deprotecting the compound of formula [15].
This reaction may be carried out, for example, according to the method described in T. W. Greene et al., Protective Groups in Organic Synthesis, 4th Edition, pp. 696-926, 2007, John Wiley & Sons, INC.

［製造法６］
式［６Ｄ］の化合物を製造する方法。
 [Manufacturing method 6]
A method for producing a compound of formula [6D].

式中、Ｒ１ａ、Ｒ１ｂおよびＲＢは上記と同じ意味を有する。 In the formula, R 1a , R 1b and R B have the same meanings as above.

（６－１）
　式［６Ｄ］の化合物は、酸の存在下もしくは不存在下、縮合剤または酸ハロゲン化物の存在下もしくは不存在下、塩基の存在下もしくは不存在下、式［１１］の化合物を式［１６］の化合物と反応させることにより製造することができる。
　式［１１］の化合物は、例えば、１－デカノールなどが知られている。
　式［１６］の化合物は、例えば、Ｎ－（ｔｅｒｔ－ブトキシカルボニル）イミノ二酢酸などが知られている。
　この反応は、製造法（３－１）に準じて行えばよい。 (6-1)
The compound of formula [6D] can be produced by reacting a compound of formula [11] with a compound of formula [16] in the presence or absence of an acid, in the presence or absence of a condensing agent or an acid halide, and in the presence or absence of a base.
An example of the compound of the formula [11] is 1-decanol.
An example of the compound of the formula [16] is N-(tert-butoxycarbonyl)iminodiacetic acid.
This reaction may be carried out according to the production method (3-1).

（６－２）
　式［６Ｄ］の化合物は、式[１７]の化合物を脱保護することにより製造することができる。
　この反応は、製造法（３－２）に準じて行えばよい。 (6-2)
The compound of formula [6D] can be prepared by deprotecting the compound of formula [17].
This reaction may be carried out according to the production method (3-2).

　上記した製造法で使用される化合物において、異性体（例えば、光学異性体、幾何異性体および互変異性体など）が存在する場合、これらの異性体も使用することができる。
　また、溶媒和物、水和物および種々の形状の結晶が存在する場合、これらの溶媒和物、水和物および種々の形状の結晶も使用することができる。 In the compounds used in the above-mentioned production methods, when isomers (for example, optical isomers, geometric isomers, tautomers, etc.) exist, these isomers can also be used.
In addition, when solvates, hydrates and various forms of crystals exist, these solvates, hydrates and various forms of crystals can also be used.

　上記した製造法で使用される化合物において、例えば、アミノ基、ヒドロキシル基またはカルボキシル基などを有している化合物は、予めこれらの基を通常の保護基で保護しておき、反応後、自体公知の方法でこれらの保護基を脱離することができる。
　上記した製造法で得られる化合物は、例えば、縮合、付加、酸化、還元、転位、置換、ハロゲン化、脱水もしくは加水分解などの自体公知の反応に付すことにより、または、それらの反応を適宜組み合わせることにより、他の化合物に誘導することができる。 In the compounds used in the above-mentioned production methods, for example, compounds having an amino group, a hydroxyl group, a carboxyl group, or the like can have these groups protected in advance with a conventional protecting group, and after the reaction, these protecting groups can be removed by a method known per se.
The compounds obtained by the above-mentioned production methods can be derived into other compounds by subjecting them to reactions known per se, such as condensation, addition, oxidation, reduction, rearrangement, substitution, halogenation, dehydration, or hydrolysis, or by appropriately combining these reactions.

＜ステロール＞
　本発明における脂質組成物は、非イオン化脂質としてステロールまたはその誘導体を含むことが好ましい。脂質組成物において、ステロールを含むことで、膜流動性を低下させ、脂質組成物の安定化効果を得ることができる。
　ステロールとしては、特に限定されないが、コレステロール、フィトステロール（シトステロール、スチグマステロール、フコステロール、スピナステロール、ブラシカステロールなど）、エルゴステロール、コレスタノン、コレステノン、コプロスタノール、コレステリル－２’－ヒドロキシエチルエーテル、コレステリル－４’－ヒドロキシブチルエーテルなどを挙げることができる。これらの中でも、コレステロールが好ましい。 <Sterol>
The lipid composition of the present invention preferably contains a sterol or a derivative thereof as a non-ionized lipid. By containing a sterol in the lipid composition, the membrane fluidity can be reduced and the lipid composition can be stabilized.
Examples of sterols include, but are not limited to, cholesterol, phytosterols (sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol, etc.), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, etc. Among these, cholesterol is preferred.

　脂質組成物において、ステロールまたはその誘導体の配合量は、脂質組成物中の全脂質に対するモル比で、３０～７０モル％であることが好ましく、３０モル％～６５モル％であることがより好ましく、３０モル％～６０モル％であることがさらに好ましい。 In the lipid composition, the amount of sterol or its derivative is preferably 30 to 70 mol%, more preferably 30 mol% to 65 mol%, and even more preferably 30 mol% to 60 mol%, in terms of the molar ratio to the total lipids in the lipid composition.

＜リン脂質＞
　本発明における脂質組成物は、非イオン化脂質としてリン脂質を含むことが好ましい。リン脂質としては、特に限定されないが、ホスファチジルコリン、ホスファチジルエタノールアミン、スフィンゴミエリン、セラミドなどが挙げられ、ホスファチジルコリンが好ましい。また、リン脂質としては、単独でも、複数の異なる中性脂質を組み合わせても良い。 <Phospholipids>
The lipid composition of the present invention preferably contains phospholipid as non-ionized lipid. The phospholipid is not particularly limited, but may be phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, etc., and phosphatidylcholine is preferred. The phospholipid may be a single lipid or a combination of multiple different neutral lipids.

　ホスファチジルコリンとしては、特に限定されないが、大豆レシチン（ＳＰＣ）、水添大豆レシチン（ＨＳＰＣ）、卵黄レシチン（ＥＰＣ）、水添卵黄レシチン（ＨＥＰＣ）、ジミリストイルホスファチジルコリン（ＤＭＰＣ）、ジパルミトイルホスファチジルコリン（ＤＰＰＣ）、ジステアロイルホスファチジルコリン（ＤＳＰＣ）、１－パルミトイル－２－オレオイルホスファチジルコリン（ＰＯＰＣ）、ジオレオイルホスファチジルコリン（ＤＯＰＣ）などが挙げられる。 Phosphatidylcholines include, but are not limited to, soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated egg yolk lecithin (HEPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), dioleoyl phosphatidylcholine (DOPC), etc.

　ホスファチジルエタノールアミンとしては特に限定されないが、ジミリストイルホスファチジルエタノールアミン（ＤＭＰＥ）、ジパルミトイルホスファチジルエタノールアミン（ＤＰＰＥ）、ジステアロイルホスファチジルエタノールアミン（ＤＳＰＥ）、ジオレオイルホスファチジルエタノールアミン（ＤＯＰＥ）、ジリノレオイルホスファチジルエタノールアミン（ＤＬｏＰＥ）、ジフィタノイルホスファチジルエタノールアミン（Ｄ（Ｐｈｙ）ＰＥ）、１－パルミトイル－２－オレオイルホスファチジルエタノールアミン（ＰＯＰＥ）、ジテトラデシルホスファチジルエタノールアミン、ジヘキサデシルホスファチジルエタノールアミン、ジオクタデシルホスファチジルエタノールアミン、ジフィタニルホスファチジルエタノールアミンなどが挙げられる。 Phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, dioctadecylphosphatidylethanolamine, diphytanylphosphatidylethanolamine, etc.

　スフィンゴミエリンとしては、特に限定されないが、卵黄由来スフィンゴミエリン、牛乳由来スフィンゴミエリンなどが挙げられる。
　セラミドとしては、特に限定されないが、卵黄由来セラミド、牛乳由来セラミドなどが挙げられる。 Examples of sphingomyelin include, but are not limited to, egg yolk-derived sphingomyelin and milk-derived sphingomyelin.
Examples of ceramides include, but are not limited to, egg yolk-derived ceramide and milk-derived ceramide.

　リン脂質は、好ましくは、ジステアロイルホスファチジルコリン、ジオレオイルホスファチジルコリン、およびジオレオイルホスファチジルエタノールアミンからなる群から選択される。 The phospholipid is preferably selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine.

　脂質組成物において、リン脂質の配合量は、脂質組成物中の全脂質に対するモル比で、１～３０モル％であることが好ましく、１～２０モル％であることがより好ましい。 In the lipid composition, the amount of phospholipids is preferably 1 to 30 mol %, more preferably 1 to 20 mol %, based on the molar ratio of the total lipids in the lipid composition.

＜非イオン性親水性高分子鎖を有する脂質＞
　本発明における脂質組成物は、非イオン性親水性高分子を有する脂質を含んでもよい。本発明において、非イオン性親水性高分子を有する脂質を含むことで、脂質組成物の分散安定化効果を得ることができる。
　非イオン性親水性高分子の例としては、特に限定されないが、非イオン性のビニル系高分子、非イオン性ポリアミノ酸、非イオン性ポリエステル、非イオン性ポリエーテル、非イオン性天然高分子、非イオン性改変天然高分子、これらの２種以上の高分子を構成単位とするブロック重合体またはグラフト共重合体が挙げられる。
　これらの非イオン性親水性高分子のうち、好ましくは非イオン性ポリエーテル、非イオン性ポリエステル、非イオン性ポリアミノ酸もしくは非イオン性合成ポリペプチドであり、さらに好ましくは非イオン性ポリエーテルまたは非イオン性ポリエステル、よりさらに好ましくは非イオン性ポリエーテルまたは非イオン性モノアルコキシポリエーテルであり、特に好ましくはポリエチレングリコール（ポリエチレングリコールは、以下においてＰＥＧとも称する）である。即ち、非イオン性高分子を有する脂質は、好ましくは、ポリエチレングリコール鎖を有する脂質である。 <Lipids having non-ionic hydrophilic polymer chains>
The lipid composition of the present invention may contain a lipid having a nonionic hydrophilic polymer. In the present invention, by containing a lipid having a nonionic hydrophilic polymer, a dispersion stabilization effect of the lipid composition can be obtained.
Examples of nonionic hydrophilic polymers include, but are not limited to, nonionic vinyl polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, nonionic modified natural polymers, and block polymers or graft copolymers having two or more of these polymers as constituent units.
Among these nonionic hydrophilic polymers, preferred are nonionic polyethers, nonionic polyesters, nonionic polyamino acids or nonionic synthetic polypeptides, more preferred are nonionic polyethers or nonionic polyesters, even more preferred are nonionic polyethers or nonionic monoalkoxy polyethers, and particularly preferred are polyethylene glycols (hereinafter, polyethylene glycols are also referred to as PEG). That is, the lipid having a nonionic polymer is preferably a lipid having a polyethylene glycol chain.

　非イオン性親水性高分子を有する脂質としては、特に限定されないが、ＰＥＧ修飾ホスホエタノールアミン、ジアシルグリセロールＰＥＧ誘導体、モノアシルグリセロールＰＥＧ誘導体、ジアルキルグリセロールＰＥＧ誘導体、コレステロールＰＥＧ誘導体、セラミドＰＥＧ誘導体などが挙げられる。これらの中でも、モノアシルグリセロールＰＥＧもしくはジアシルグリセロールＰＥＧが好ましい。 Lipids having a non-ionic hydrophilic polymer include, but are not limited to, PEG-modified phosphoethanolamine, diacylglycerol PEG derivatives, monoacylglycerol PEG derivatives, dialkylglycerol PEG derivatives, cholesterol PEG derivatives, and ceramide PEG derivatives. Among these, monoacylglycerol PEG or diacylglycerol PEG is preferred.

　ポリエチレングリコール鎖を有する脂質は、特に好ましくは、ジミリストイル－ｒａｃ－グリセロールポリエチレングリコール、ジステアロイル－ｒａｃ－グリセロールポリエチレングリコール、およびジステアロイルホスファチジルエタノールアミンポリエチレングリコールから選択される。 The lipid having a polyethylene glycol chain is particularly preferably selected from dimyristoyl-rac-glycerol polyethylene glycol, distearoyl-rac-glycerol polyethylene glycol, and distearoylphosphatidylethanolamine polyethylene glycol.

　上記非イオン性親水性高分子誘導体のＰＥＧ鎖の重量平均分子量は、５００～５０００が好ましく、７５０～３０００がより好ましい。
　非イオン性親水性高分子鎖は分岐していてもよく、ヒドロキシメチル基のような置換基を有していてもよい。 The weight average molecular weight of the PEG chain of the nonionic hydrophilic polymer derivative is preferably 500 to 5,000, and more preferably 750 to 3,000.
The non-ionic hydrophilic polymer chain may be branched and may have a substituent such as a hydroxymethyl group.

　脂質組成物において、非イオン性親水性高分子鎖を有する脂質の配合量は、脂質組成物中の全脂質に対するモル比で、０．１～３モル％であることが好ましく、０．３～３モル％であることがより好ましく、０．５～３モル％であることがさらに好ましい。 In the lipid composition, the amount of lipid having a nonionic hydrophilic polymer chain is preferably 0.1 to 3 mol %, more preferably 0.3 to 3 mol %, and even more preferably 0.5 to 3 mol %, in terms of molar ratio to the total lipid in the lipid composition.

＜核酸＞
　脂質組成物は、核酸を含む。
　核酸としては環状２本鎖ＤＮＡ（プラスミドＤＮＡ、薬剤耐性遺伝子を持たない小型環状２本鎖ＤＮＡなど）、１本鎖ＤＮＡ、２本鎖ＤＮＡ、ｓｉＲＮＡ（ｓｍａｌｌ　ｉｎｔｅｒｆｅｒｉｎｇ　ＲＮＡ） 、ｍｉＲＮＡ（ｍｉｃｒｏ　ＲＮＡ）、ｍＲＮＡ、アンチセンスオリゴヌクレオチド(ＡＳＯとも言う)、リボザイム、アプタマー、ｓａＲＮＡ、ｓｇＲＮＡ等が挙げられ、いずれを含んでもよい。２種類以上の核酸を使用してもよい。また、修飾化された核酸を含んでもよい。核酸としては、環状２本鎖ＤＮＡもしくはＲＮＡが特に好ましく、プラスミドＤＮＡもしくはｍＲＮＡが最も好ましい。塩基数としては５～２００００塩基が好ましい。 <Nucleic acid>
The lipid composition comprises a nucleic acid.
Examples of nucleic acids include circular double-stranded DNA (plasmid DNA, small circular double-stranded DNA without drug resistance genes, etc.), single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (micro RNA), mRNA, antisense oligonucleotide (also called ASO), ribozyme, aptamer, saRNA, sgRNA, etc., and any of them may be included. Two or more types of nucleic acids may be used. In addition, modified nucleic acids may be included. As the nucleic acid, circular double-stranded DNA or RNA is particularly preferred, and plasmid DNA or mRNA is most preferred. The number of bases is preferably 5 to 20,000 bases.

　核酸としては、遺伝子編集のための配列であって、ＤＮＡヌクレアーゼをコードするｍＲＮＡでもよい。
　核酸としては、遺伝子編集のための配列であってガイドＲＮＡでもよい。
　核酸としては、遺伝子編集のための配列であって、ＣａｓヌクレアーゼをコードするｍＲＮＡと、ガイドＲＮＡを含む核酸混合物でもよい。即ち、核酸は、ＣａｓヌクレアーゼをコードするｍＲＮＡとガイドＲＮＡとを含む、遺伝子編集のための核酸であってもよい。上記混合物は、さらに、任意のドナーＤＮＡを含んでいてもよい。
　核酸としては、一塩基編集のための配列であって、デアミナーゼおよび変異型ＣａｓヌクレアーゼをコードするｍＲＮＡと、ガイドＲＮＡを含む核酸混合物でもよい。
　核酸としては、標的ＤＮＡヌクレオチドを置換するための配列であって、人工逆転写酵素とＣａｓ９エンドヌクレアーゼの融合タンパク質をコードするｍＲＮＡと、プライム編集ガイドＲＮＡとを含む核酸混合物でもよい。 The nucleic acid may be an mRNA encoding a DNA nuclease, which is a sequence for gene editing.
The nucleic acid may be a guide RNA, which is a sequence for gene editing.
The nucleic acid may be a nucleic acid mixture that is a sequence for gene editing and includes an mRNA encoding Cas nuclease and a guide RNA. That is, the nucleic acid may be a nucleic acid for gene editing that includes an mRNA encoding Cas nuclease and a guide RNA. The mixture may further include any donor DNA.
The nucleic acid may be a nucleic acid mixture containing an mRNA encoding a deaminase and a mutant Cas nuclease, and a guide RNA, which is a sequence for single base editing.
The nucleic acid may be a nucleic acid mixture containing an mRNA encoding a fusion protein of an artificial reverse transcriptase and a Cas9 endonuclease, the nucleic acid being a sequence for replacing a target DNA nucleotide, and a prime editing guide RNA.

　核酸としては、ガイドＲＮＡ依存的な遺伝子転写活性化のための配列（ＣＲＩＳＰＲ　ａｃｔｉｖａｔｏｒシステム等）であって、アクティベータータンパク質（ＶＰ６４、ｐ６５、Ｒｔａ）と変異型Ｃａｓヌクレアーゼの融合タンパクをコードするｍＲＮＡと、ガイドＲＮＡを含む核酸混合物でもよい。もしくは、アクティベータータンパク質（ＭＳ２、ｐ６５、ＨＳＦ１）をコードするｍＲＮＡと、ＶＰ６４と変異型Ｃａｓヌクレアーゼの融合タンパク質をコードするｍＲＮＡと、ガイドＲＮＡを含む核酸混合物でもよい。
　核酸としては、ガイドＲＮＡ依存的な遺伝子転写抑制のための配列（ＣＲＩＳＰＲ　ｉｎｔｅｒｆｅｒｅｎｃｅシステム等）であって、転写抑制因子（ＫＲＡＢ等）と変異型Ｃａｓヌクレアーゼの融合タンパクをコードするｍＲＮＡと、ガイドＲＮＡを含む核酸混合物でもよい。
　核酸としては、ＤＮＡ組換え酵素をコードするｍＲＮＡもしくはＤＮＡであり、さらに任意でドナーＤＮＡを含む核酸混合物でもよい。
　核酸としては、ＤＮＡ組換え酵素をコードするｍＲＮＡもしくはＤＮＡであり、さらに任意でドナーＤＮＡを含む核酸混合物でもよい。ＤＮＡ組換え酵素としては、特に限定されるものではないが、トランスポザーゼ（例えば、Sleeping beauty transposase、piggyBac transposase、Tol2など）、Ｃｒｅリコンビナーゼ、セリン・インテグラーゼなどが挙げられる。
　核酸としては、外来遺伝子を宿主細胞ゲノムに挿入するための配列であって、逆転写酵素をコードするｍＲＮＡであり、さらに任意でドナーとなるＲＮＡを含む核酸混合物でもよい。
　核酸としては、外来遺伝子を発現させるための配列をコードするｍＲＮＡ、もしくは先の外来遺伝子とプロモーター配列とターミナル配列とを含むＤＮＡであってもよい。 The nucleic acid may be a nucleic acid mixture containing a sequence for guide RNA-dependent gene transcription activation (CRISPR activator system, etc.), an mRNA encoding a fusion protein of an activator protein (VP64, p65, Rta) and a mutant Cas nuclease, and a guide RNA. Alternatively, the nucleic acid mixture may be a nucleic acid mixture containing an mRNA encoding an activator protein (MS2, p65, HSF1), an mRNA encoding a fusion protein of VP64 and a mutant Cas nuclease, and a guide RNA.
The nucleic acid may be a sequence for guide RNA-dependent gene transcription suppression (such as the CRISPR interference system), and may be a nucleic acid mixture containing an mRNA encoding a fusion protein of a transcription suppressor (such as KRAB) and a mutant Cas nuclease, and a guide RNA.
The nucleic acid may be an mRNA or DNA encoding a DNA recombinase, and may further be a nucleic acid mixture optionally containing a donor DNA.
The nucleic acid may be an mRNA or DNA encoding a DNA recombinase, or may be a nucleic acid mixture optionally containing a donor DNA. The DNA recombinase may include, but is not limited to, transposase (e.g., Sleeping beauty transposase, piggyBac transposase, Tol2, etc.), Cre recombinase, serine integrase, etc.
The nucleic acid may be a nucleic acid mixture that contains a sequence for inserting a foreign gene into the host cell genome, an mRNA encoding a reverse transcriptase, and, optionally, a donor RNA.
The nucleic acid may be an mRNA encoding a sequence for expressing a foreign gene, or a DNA containing the foreign gene, a promoter sequence, and a terminal sequence.

　核酸としては、免疫細胞に存在する遺伝子（標的遺伝子）を遺伝子編集もしくは遺伝子転写抑制するための配列であってもよい。標的遺伝子としては、特に限定されるものではないが、Ｔ細胞受容体遺伝子（ＴＲＡＣ、ＴＲＢＣ）、ＭＨＣ－Ｉ（もしくはＨＬＡ－Ｉ）、ＭＨＣ－ＩＩ、　Ｂ２Ｍ、自己抗原に関連する遺伝子、抑制性受容体またはそのリガンドに関する遺伝子（ＰＤＣＤ１、ＣＤ２７４（もしくはＰＤ－Ｌ１）、ＰＤＣＤ１ＬＧ２、ＬＡＧ３、ＣＴＬＡ４）、細胞毒性に関連する遺伝子（ＴＧＦＢＲ２、ＰＧＥ２、ＥＰ２、ＥＰ４、ＦＡＳ、ＦＡＳＬＧ）、細胞疲弊もしくは分化に関する遺伝子（ＳＯＣＳ１、ＺＣ３Ｈ１２Ａ、ＮＲ４Ａ１、ＮＲ４Ａ２、ＮＲ４Ａ３、ＰＲＤＭ１、ＢＬＩＭＰ１、ＴＩＭ３）、細胞死関連遺伝子（ＣＡＳＰ３、ＣＡＳＰ６、ＣＡＳＰ７）、炎症応答に関わる遺伝子（ＣＧＡＳ、ＳＴＩＮＧ、ＴＢＫ１など）、メチル化遺伝子（ＴＥＴ１、ＴＥＴ２、ＤＮＭＴ３Ａ）、免疫回避に関わる遺伝子（ＣＤ４７、ＮＫＧ２Ａ）、その他（ＤＧＫ、ＥＺＨ２、ＣＳＦ２、ＰＡＸ５、ＬＤＬＲ、ＭＡＰ４Ｋ１、ＣＩＳＨ、ＣＤ５、ＣＤ５２、ＡＤＯＲＡ２Ａ、ＣＤ３９、ＣＤ７３、ＣＤ５、ＭＣＭ３ＡＰ、ＥＩＦ３Ｄ、ＣＡＤ、ＨＧＳ、ＲＰＬ１９、ＭＡＫ１６、ＰＤＧＦＲＡ、ＮＲＦ１、ＥＰ４００、ＣＢＬＢ、ＲＰＳ７、ＣＰＳＦ４、ＩＬ２ＲＧ、ＲＰＬ３８、ＩＬ２ＲＢ、ＪＡＫ３、ＭＣＭ２、ＳＮＲＰＣ、ＰＳＭＤ４、ＭＡＰ４Ｋ１、ＢＲＤ９、ＲＮＦ２０、ＲＮＦ４０、ＮＦＫＢ２、ＮＭＴ１、ＭＹＢ、ＴＳＣ１、ＥＩＦ３Ｋ、ＲＰＬ１９、ＴＢＸ２１、ＰＲＤＭ１、ＳＵＶ３９Ｈ１、ＡＲＩＤ１Ａ）等が挙げられる。 The nucleic acid may be a sequence for gene editing or gene transcription inhibition of a gene (target gene) present in immune cells. Target genes include, but are not limited to, T cell receptor genes (TRAC, TRBC), MHC-I (or HLA-I), MHC-II, B2M, genes related to self-antigens, genes related to inhibitory receptors or their ligands (PDCD1, CD274 (or PD-L1), PDCD1LG2, LAG3, CTLA4), genes related to cytotoxicity (TGFBR2, PGE2, EP2, EP4, FAS, FASLG), genes related to cell exhaustion or differentiation (SOCS1, ZC3H12A, NR4A1, NR4A2, NR4A3, PRDM1, BLIMP1, TIM3), cell death-related genes (CASP3, CASP6, CASP7), genes related to inflammatory responses (CGAS, STING, TBK1), etc. etc.), methylation genes (TET1, TET2, DNMT3A), genes involved in immune evasion (CD47, NKG2A), others (DGK, EZH2, CSF2, PAX5, LDLR, MAP4K1, CISH, CD5, CD52, ADORA2A, CD39, CD73, CD5, MCM3AP, EIF3D, CAD, HGS, RPL19, MAK16, PDG FRA, NRF1, EP400, CBLB, RPS7, CPSF4, IL2RG, RPL38, IL2RB, JAK3, MCM2, SNRPC, PSMD4, MAP4K1, BRD9, RNF20, RNF40, NFKB2, NMT1, MYB, TSC1, EIF3K, RPL19, TBX21, PRDM1, SUV39H1, ARID1A), etc.

　核酸としては、免疫細胞を機能強化させる外来遺伝子を発現させるための配列、もしくは免疫細胞に存在する遺伝子（標的遺伝子）の転写活性化するための配列であってもよい。外来遺伝子または転写活性化の標的遺伝子としては、特に限定されるものではないが、サイトカイン（ＩＦＮＧ、ＩＬ２、ＩＬ７、ＩＬ１２、ＩＬ１５、ＩＬ１８、ＩＬ１９、ＩＬ２１）、サイトカイン受容体（ＩＬ２ＲＡ、ＩＬ２ＲＢ、ＩＬ２ＲＧ、ＩＬ１２Ｂ１、ＩＬ１２Ｂ２）、共刺激因子（ＣＤ２８、ＴＮＦＲＳＦ９）、免疫回避に関わる遺伝子（ＣＤ４７、ＨＬＡ－Ｅ）、代謝関連遺伝子（ＧＬＵＴ１、ＰＰＡＲＧＣ１Ａ）、疲弊抑制に関する遺伝子（ＦＯＸＯ１、ＴＣＦ７、ＬＥＦ１）、細胞生存に関わる遺伝子（ＢＣＬ２）、その他（ドミナントネガティブフォームＴＧＦＢＲ２、ＡＫＴ１、ＬＴＢＲ、ＡＨＣＹ、ＤＵＰＤ１、ＡＫＲ１Ｃ４、ＡＴＦ６Ｂ、ＩＴＭ２Ａ、ＡＨＮＡＫ、ＢＡＴＦ、ＧＰＤ１、ＣＤＫ２、ＣＤＫ１、ＧＰＮ３、ＭＲＰＬ５１、ＤＢＩ、ＣＡＬＭＬ２、ＩＬ１２Ｂ、ＩＦＮＬ２、ＣＬＩＣ１、ＨＯＭＥＲ１、ＡＤＡ、ＣＹＰ２７Ａ１、ＭＲＰＬ１８、ＲＡＮ、ＳＬＣ１０Ａ７、ＣＲＬＦ２、ＶＡＶ１、ＴＲＩＭ２１、ＬＨＸ６、ＦＯＸＯ４、ＩＲＸ４、ＦＯＸＱ１、ＯＴＵＤ７Ｂ、ＬＣＰ２、ＦＯＳＢ、ＲＡＣ２、ＦＯＳＬ１、ＡＰＯＢＥＣ３Ｄ、ＲＩＰＫ３、ＥＭＰ１、ＡＮＸＡ２Ｒ、ＣＤＫＮ２Ｃ、ＯＴＵＤ７Ａ、ＣＤ２、ＬＡＴ、ＬＣＰ２、ＴＢＸ２１、ＥＯＭＥＳ）、炎症を惹起させる分泌タンパクをコードする配列が挙げられる。
　核酸としては、キメラ抗原受容体遺伝子、Ｔ細胞受容体遺伝子、抗体、バイスペシフィック抗体、マルチスペシフィック抗体、ｓｉｎｇｌｅ　ｃｈａｉｎ　Ｆｖ(ｓｃＦｖ)、ナノボディ、二重特異性T細胞誘導抗体(ＢｉＴＥ)を発現させるための配列であってもよい。
　核酸としては、細胞の初期化（もしくはリプログラミング）に関わる外来遺伝子を発現させるための配列であってもよい。外来遺伝子としては、特に限定されるものではないが、Ｏｃｔ３／４、Ｓｏｘ２、Ｋｌｆ４、Ｃ－Ｍｙｃ、Ｎａｎｏｇ、Ｌｉｎ２８挙げられる　。 The nucleic acid may be a sequence for expressing a foreign gene that enhances the function of immune cells, or a sequence for activating the transcription of a gene (target gene) present in immune cells. The foreign gene or the target gene for transcription activation is not particularly limited, but may be any of cytokines (IFNG, IL2, IL7, IL12, IL15, IL18, IL19, IL21), cytokine receptors (IL2RA, IL2RB, IL2RG, IL12B1, IL12B2), costimulatory factors (CD28, TNFRSF9), genes involved in immune evasion (CD47, HLA-E), metabolism-related genes (GLUT1, PPARGC1A), genes involved in exhaustion suppression (FOXO1, TCF7, LEF1), genes involved in cell survival (BCL2), and others (dominant negative form TGFBR2, AKT1, LTBR, AHCY, DUPD1). , AKR1C4, ATF6B, ITM2A, AHNAK, BATF, GPD1, CDK2, CDK1, GPN3, MRPL51, DBI, CALML2, IL1 2B, IFNL2, CLIC1, HOMER1, ADA, CYP27A1, MRPL18, RAN, SLC10A7, CRLF2, VAV1, TRIM21, L HX6, FOXO4, IRX4, FOXQ1, OTUD7B, LCP2, FOSB, RAC2, FOSL1, APOBEC3D, RIPK3, EMP1, ANXA2R, CDKN2C, OTUD7A, CD2, LAT, LCP2, TBX21, EOMES), and sequences encoding secreted proteins that induce inflammation.
The nucleic acid may be a sequence for expressing a chimeric antigen receptor gene, a T cell receptor gene, an antibody, a bispecific antibody, a multispecific antibody, a single chain Fv (scFv), a nanobody, or a bispecific T cell-inducing antibody (BiTE).
The nucleic acid may be a sequence for expressing a foreign gene involved in the initialization (or reprogramming) of a cell. Examples of the foreign gene include, but are not limited to, Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28.

　脂質組成物において、脂質組成物の全脂質と核酸の質量比が５：１～１０００：１であることが好ましく、５：１～５００：１であることがより好ましく、７：１～２００：１であることがさらに好ましく、７：１～１００：１であることが特に好ましい。 In the lipid composition, the mass ratio of the total lipids to the nucleic acids in the lipid composition is preferably 5:1 to 1000:1, more preferably 5:1 to 500:1, even more preferably 7:1 to 200:1, and particularly preferably 7:1 to 100:1.

＜脂質組成物の製造方法＞
　脂質組成物の製造方法について説明する。
　脂質組成物の製造方法は限定されないが、脂質組成物の構成成分全てまたは一部の油溶性成分を有機溶媒等に溶解させ油相とし、水溶性成分を水に溶解させ水相とし、油相と水相を混合して製造することができる。混合にはマイクロミキサーを使用してもよく、ホモジナイザー等の乳化機、超音波乳化機、高圧噴射乳化機等により乳化してもよい。
　あるいは、脂質を含む溶液をエバポレータなどによる減圧乾固または噴霧乾燥機などによる噴霧乾燥などにより脂質を含む乾燥した混合物を調製し、この混合物を水系溶媒に添加し、さらに前述の乳化機などで乳化することで製造することもできる。 <Method of producing lipid composition>
A method for producing the lipid composition will now be described.
The method for producing the lipid composition is not limited, but the lipid composition can be produced by dissolving all or a part of the oil-soluble components of the lipid composition in an organic solvent or the like to form an oil phase, dissolving the water-soluble components in water to form an aqueous phase, and mixing the oil phase and the aqueous phase. A micromixer may be used for mixing, or the lipid composition may be emulsified using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
Alternatively, the lipid-containing solution can be dried under reduced pressure using an evaporator or spray-dried using a spray dryer to prepare a dried mixture containing lipids, and this mixture can be added to an aqueous solvent and further emulsified using the emulsifier described above to produce the lipid-containing solution.

　核酸を含む脂質組成物の製造方法の一例としては、
　工程（ａ）；式（１）で表される化合物を含む脂質組成物の構成成分を有機溶媒に溶解して油相を、核酸を水性溶媒に溶解して水相を、それぞれ得る工程
　工程（ｂ）；工程（ａ）で得た油相と水相を混合して脂質粒子の分散液を得る工程
　工程（ｃ）；工程（ｂ）で得た脂質粒子の分散液を希釈する工程
　工程（ｄ）；脂質粒子の分散液から上記有機溶媒を除去する工程
　工程（ｅ）；脂質粒子の分散液の濃度を調節する工程
　を含む方法が挙げられる。 An example of a method for producing a lipid composition containing nucleic acid is
Step (a): dissolving the components of the lipid composition containing the compound represented by formula (1) in an organic solvent to obtain an oil phase, and dissolving the nucleic acid in an aqueous solvent to obtain an aqueous phase; Step (b): mixing the oil phase and aqueous phase obtained in step (a) to obtain a lipid particle dispersion; Step (c): diluting the lipid particle dispersion obtained in step (b); Step (d): removing the organic solvent from the lipid particle dispersion; and Step (e): adjusting the concentration of the lipid particle dispersion.

　工程（ａ）においては、式（１）で表される化合物を含む脂質組成物の構成成分を、有機溶媒（エタノールなどのアルコール、またはエステルなど）に溶解させる。総脂質濃度は特に限定されないが、一般的には１ｍｍｏｌ／Ｌ～１００ｍｍｏｌ／Ｌであり、好ましくは５ｍｍｏｌ／Ｌ～８０ｍｍｏｌ／Ｌであり、より好ましくは１０ｍｍｏｌ／Ｌ～７０ｍｍｏｌ／Ｌである。
　水相は、核酸（例えば、ｍＲＮＡなど）を、水または緩衝液に溶解することで得ることができる。核酸の濃度は特に限定されないが、１～１０００μｇ／ｍＬが好ましく、１０～５００μｇ／ｍＬがより好ましい。必要に応じてｐＨ調整のための緩衝成分や酸化防止剤などの成分を添加することができる。水相のｐＨは２．０～７．０であることが好ましく、３．０～６．０であることがより好ましい。上記ｐＨに調整するために緩衝成分として酢酸、クエン酸、リンゴ酸、リン酸、ＭＥＳ、ＨＥＰＥＳなどが好ましく用いられ、必要に応じて塩強度を調整することを目的に、塩化ナトリウム、塩化カリウムなどの塩や、浸透圧を調整することを目的に、スクロース、トレハロース、マンニトールなどの糖や糖アルコールを添加してもよい。 In step (a), the components of the lipid composition containing the compound represented by formula (1) are dissolved in an organic solvent (an alcohol such as ethanol, or an ester, etc.). The total lipid concentration is not particularly limited, but is generally 1 mmol/L to 100 mmol/L, preferably 5 mmol/L to 80 mmol/L, and more preferably 10 mmol/L to 70 mmol/L.
The aqueous phase can be obtained by dissolving nucleic acid (e.g., mRNA, etc.) in water or a buffer solution. The concentration of the nucleic acid is not particularly limited, but is preferably 1 to 1000 μg/mL, more preferably 10 to 500 μg/mL. If necessary, components such as a buffer component for pH adjustment and an antioxidant can be added. The pH of the aqueous phase is preferably 2.0 to 7.0, more preferably 3.0 to 6.0. To adjust the pH to the above range, acetic acid, citric acid, malic acid, phosphoric acid, MES, HEPES, etc. are preferably used as buffer components, and if necessary, salts such as sodium chloride and potassium chloride may be added to adjust the salt strength, and sugars or sugar alcohols such as sucrose, trehalose, and mannitol may be added to adjust the osmotic pressure.

　工程（ｂ）において、油相と水相は任意の方法で混合してよく、バッチ式でも流路デバイスを用いたインライン方式でもよい。インライン方式としてはマイクロ流路デバイスを用いることが好ましく、用いるマイクロ流路デバイスとしては、Ｙ字ミキサー、Ｔ字ミキサー、ヘリンボーンミキサー、リングマイクロミキサー、インピンジメントジェットミキサーなどを用いることができる。水相と油相を混合する比率（体積比）は、５：１～１：１が好ましく、４：１～２：１がより好ましい。 In step (b), the oil phase and the aqueous phase may be mixed by any method, including a batch method or an in-line method using a flow path device. For the in-line method, a micro flow path device is preferably used, and examples of the micro flow path device that can be used include a Y-shaped mixer, a T-shaped mixer, a herringbone mixer, a ring micro mixer, and an impingement jet mixer. The mixing ratio (volume ratio) of the aqueous phase to the oil phase is preferably 5:1 to 1:1, and more preferably 4:1 to 2:1.

　工程（ｃ）において、脂質粒子の分散液を希釈溶液と混合することにより、有機溶媒の含率を下げ、脂質粒子を安定化することができる。希釈溶液は、水でもよいが、ｐＨや塩強度を調整することを含んでもよい。希釈溶液に含まれる成分は目的に応じて任意に選択することができる。例えば、ｐＨを調整する目的で、緩衝液（例えばクエン酸緩衝液、クエン酸緩衝生理食塩水、酢酸緩衝液、酢酸緩衝生理食塩水、リン酸緩衝生理食塩水、トリス緩衝液、ＭＥＳ緩衝液、ＨＥＰＥＳ緩衝液など）を用いてもよい。また、塩強度や浸透圧を調整することを目的として塩化ナトリウム、塩化カリウム、スクロース、トレハロース、フルクトースまたはマンニトールなどを含んでもよく、上記緩衝液にこれらの添加剤をさらに添加したものも使用できる。 In step (c), the lipid particle dispersion is mixed with a diluent solution to reduce the organic solvent content and stabilize the lipid particles. The diluent solution may be water, but may also include adjusting the pH or salt strength. The components contained in the diluent solution may be selected arbitrarily depending on the purpose. For example, a buffer solution (e.g., citrate buffer, citrate buffered saline, acetate buffer, acetate buffered saline, phosphate buffered saline, Tris buffer, MES buffer, HEPES buffer, etc.) may be used for the purpose of adjusting the pH. In addition, sodium chloride, potassium chloride, sucrose, trehalose, fructose, mannitol, etc. may be included for the purpose of adjusting the salt strength or osmotic pressure, and the above buffer solutions to which these additives have been further added may also be used.

　脂質粒子の分散液と希釈溶液は任意の方法で混合してよく、バッチ式でも流路デバイスを用いたインライン方式でもよい。混合時に用いる流路デバイスは、Ｙ字ミキサー、Ｔ字ミキサーなどを用いることができる。また、油相と水相を混合してから希釈溶液を混合するまでの時間は特に限定されないが、油相及び水相を混合した３０秒以内に希釈を実施することが好ましく、１０秒以内に希釈を実施することがより好ましい。
　脂質粒子の分散液と希釈溶液を混合する比率（液量比）は、１：０．５～１：１０が好ましく、１：１～１：５がより好ましい。 The lipid particle dispersion and the diluted solution may be mixed by any method, including a batch method and an in-line method using a flow path device. The flow path device used during mixing may be a Y-shaped mixer, a T-shaped mixer, or the like. The time from mixing the oil phase and the aqueous phase to mixing the diluted solution is not particularly limited, but it is preferable to perform the dilution within 30 seconds of mixing the oil phase and the aqueous phase, and more preferably within 10 seconds.
The mixing ratio (liquid volume ratio) of the lipid particle dispersion and the dilution solution is preferably 1:0.5 to 1:10, and more preferably 1:1 to 1:5.

　いくつかの実施形態では、工程（ｃ）において脂質粒子の分散液は目的に応じて複数回希釈溶液と混合してもよい。また用いる希釈溶液は同一であっても異なっていてもよい。
脂質粒子の分散液では、ｐＨによって脂質粒子の粒径が変化することがあり、分散液のｐＨ調整は重要となる。そのため、例えば希釈溶液との混合後の脂質粒子の分散液のｐＨを調整するために、それに適した濃度およびｐＨを有する緩衝液や、さらに他の成分を含む緩衝液を用いてもよい。 In some embodiments, in step (c), the lipid particle dispersion may be mixed with a dilution solution multiple times as desired, and the dilution solutions used may be the same or different.
In the dispersion of lipid particles, the particle size of lipid particles may change depending on pH, so that pH adjustment of the dispersion is important.Therefore, for example, in order to adjust the pH of the dispersion of lipid particles after mixing with dilution solution, the buffer solution having suitable concentration and pH, or the buffer solution containing other components may be used.

　さらに、複数の希釈工程は連続的に実施してもよく、希釈工程と次の希釈工程の間隔を任意に設定することができ、例えば、その間隔は１０秒、３０秒、１分、５分、１０分、３０分、１時間、２時間、３時間、４時間、６時間、１２時間または２４時間であってもよい。 Furthermore, multiple dilution steps may be performed consecutively, and the interval between one dilution step and the next dilution step may be set arbitrarily, for example, the interval may be 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours or 24 hours.

　また、工程（ｃ）を行った後の脂質粒子の分散液のｐＨはｐＨ３．０～１０．０が好ましく、ｐＨ３．５～ｐＨ９．０がより好ましく、ｐＨ４．０～ｐＨ８．５が特に好ましい。 Furthermore, the pH of the lipid particle dispersion after step (c) is preferably pH 3.0 to pH 10.0, more preferably pH 3.5 to pH 9.0, and particularly preferably pH 4.0 to pH 8.5.

　脂質組成物には、必要に応じてサイジングを施すことができる。サイジングの方法は、特に限定されないが、エクストルーダーなどを用いて粒子径を小さくすることができる。
　また、脂質組成物を含む分散液には、一般的な方法により、凍結や凍結乾燥を施すこと
ができる。 The lipid composition may be subjected to sizing as necessary. The method of sizing is not particularly limited, but the particle size may be reduced using an extruder or the like.
Furthermore, the dispersion containing the lipid composition can be subjected to freezing or lyophilization by a general method.

　工程（ｄ）において、脂質粒子の分散液から有機溶媒を除去する方法としては、特に限定されず、一般的な手法を使用することができる。例えば、透析液としてはリン酸緩衝生理食塩水、トリス緩衝液などのｐＨ緩衝液を用いることができ、必要に応じて浸透圧の調整や凍結からの保護を目的として任意の塩や糖などの添加剤を加えることができる。 In step (d), the method for removing the organic solvent from the lipid particle dispersion is not particularly limited, and a general method can be used. For example, a pH buffer solution such as phosphate buffered saline or Tris buffer can be used as the dialysis fluid, and additives such as any salt or sugar can be added as necessary to adjust the osmotic pressure or protect the dispersion from freezing.

　工程（ｅ）において、工程（ｄ）で得られた脂質粒子の分散液の濃度を調整することができる。希釈する場合は、リン酸緩衝生理食塩水、生理食塩水、トリス緩衝液、スクロース含有トリス緩衝液などの溶液を希釈液として用いて適切な濃度に希釈することができる。濃縮する場合は、工程（ｄ）で得られた分散液を、限外ろ過膜を用いた限外ろ過などにより濃縮することができる。濃縮した分散液をそのまま用いても好ましく、濃縮した後に上記希釈液を用いて所望の濃度に調整しても好ましい。 In step (e), the concentration of the lipid particle dispersion obtained in step (d) can be adjusted. When diluting, a solution such as phosphate buffered saline, saline, Tris buffer, or sucrose-containing Tris buffer can be used as a diluent to dilute to an appropriate concentration. When concentrating, the dispersion obtained in step (d) can be concentrated by ultrafiltration using an ultrafiltration membrane. The concentrated dispersion can be used as is, or it can be concentrated and then adjusted to the desired concentration using the above diluent.

　また、いくつかの実施形態では、接線流ろ過（ＴＦＦ）を用いて有機溶媒除去工程（工程（ｄ））、濃度調整工程（工程（ｅ））を連続的に行うことができる。本工程において有機溶媒除去工程と濃度調整工程を任意の順番で実施してもよい。必要に応じて有機溶媒除去工程と濃度調整工程をそれぞれ複数回ずつ実施してもよい。 In some embodiments, the organic solvent removal step (step (d)) and the concentration adjustment step (step (e)) can be carried out continuously using tangential flow filtration (TFF). In this process, the organic solvent removal step and the concentration adjustment step may be carried out in any order. If necessary, the organic solvent removal step and the concentration adjustment step may each be carried out multiple times.

　工程（ｄ）における透析や、工程（ｅ）における希釈において用いることのできる溶液としては、賦形剤、凍結保護剤、緩衝剤や酸化防止剤を添加してもよい。賦形剤や凍結保護剤としては、特に限定されないが、糖類や糖アルコール類が挙げられる。糖類としては、例えばスクロース、トレハロース、マルトース、グルコース、ラクトース、フルクトースなどが、糖アルコール類としては、例えばマンニトール、ソルビトール、イノシトール、キシリトールなどが挙げられる。緩衝剤としては、特に限定されないが、例えば、ＡＣＥＳ、ＢＥＳ、Ｂｉｃｉｎｅ、ＣＡＰＳ、ＣＨＥＳ、ＤＩＰＳＯ、ＥＰＰＳ、ＨＥＰＥＳ、ＨＥＰＰＳＯ、ＭＥＳ、ＭＯＰＳ、ＭＯＰＳＯ、ＴＡＰＳ、ＴＡＰＳＯ、ＴＥＳ、Ｔｒｉｃｉｎｅ、トリス、リン酸、酢酸、クエン酸などが挙げられる。酸化防止剤としては、ＥＤＴＡ、アスコルビン酸、トコフェロールなどが挙げられる。 The solution that can be used for dialysis in step (d) and dilution in step (e) may contain excipients, cryoprotectants, buffers, and antioxidants. Examples of excipients and cryoprotectants include, but are not limited to, sugars and sugar alcohols. Examples of sugars include sucrose, trehalose, maltose, glucose, lactose, and fructose, and examples of sugar alcohols include mannitol, sorbitol, inositol, and xylitol. Examples of buffers include, but are not limited to, ACES, BES, Bicine, CAPS, CHES, DIPSO, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, TAPS, TAPSO, TES, Tricine, Tris, phosphoric acid, acetic acid, and citric acid. Examples of antioxidants include EDTA, ascorbic acid, and tocopherol.

　脂質粒子の分散液について、無菌ろ過を行ってもよい。ろ過の方法としては、中空糸膜、逆浸透膜、メンブレンフィルターなどを用いて、脂質粒子の分散液から不溶なものを除去することができる。本発明では、特に限定されないが、滅菌できる孔径を有するフィルター（好ましくは０．２μｍのろ過滅菌フィルター）によってろ過することが好ましい。
また、無菌ろ過を行うのは、工程（ｄ）または工程（ｅ）の後が好ましい。
　さらに必要に応じて、脂質粒子の分散液を、凍結や凍結乾燥を施すことができる。脂質粒子の分散液は、一般的な方法により凍結や凍結乾燥を施すことができ、その手法は特に限定されない。 The lipid particle dispersion may be sterilized by filtration.As a filtration method, hollow fiber membrane, reverse osmosis membrane, membrane filter, etc. can be used to remove insoluble matter from the lipid particle dispersion.In the present invention, although not particularly limited, it is preferable to filter by a filter with a pore size that can be sterilized (preferably a 0.2 μm filter sterilization filter).
In addition, it is preferable to carry out the sterile filtration after step (d) or step (e).
Furthermore, if necessary, the lipid particle dispersion liquid may be subjected to freezing or lyophilization. The lipid particle dispersion liquid may be subjected to freezing or lyophilization by a general method, and the method is not particularly limited.

　また、核酸送達剤の製造方法としては特に限定されないが、式（１）で表される化合物またはその塩であるイオン化可能な脂質と、非イオン化脂質と、非イオン性高分子を有する脂質とを用いて、核酸を含まない脂質粒子を調製する工程と、前記の核酸を含まない脂質粒子と核酸とを混合する工程とを含む、方法が挙げられる。
　核酸を含まない脂質粒子は、核酸を含まない脂質粒子の構成成分全てまたは一部の油溶性成分を有機溶媒等に溶解させ油相とし、これに水相を混合して製造することができる。混合にはマイクロミキサーを使用してもよく、ホモジナイザー等の乳化機、超音波乳化機、高圧噴射乳化機等により乳化してもよい。
　あるいは、脂質を含む溶液をエバポレータなどによる減圧乾固または噴霧乾燥機などによる噴霧乾燥などにより脂質を含む乾燥した混合物を調製し、この混合物を水系溶媒に添加し、さらに前述の乳化機などで乳化することで製造することもできる。 In addition, the method for producing a nucleic acid delivery agent is not particularly limited, but may include a method comprising the steps of preparing lipid particles that do not contain nucleic acid using an ionizable lipid that is a compound represented by formula (1) or a salt thereof, a nonionizable lipid, and a lipid having a nonionic polymer, and mixing the lipid particles that do not contain nucleic acid with the nucleic acid.
The lipid particles not containing nucleic acid can be produced by dissolving all or some of the oil-soluble components of the lipid particles not containing nucleic acid in an organic solvent or the like to form an oil phase, and then mixing this with an aqueous phase. A micromixer may be used for mixing, or the emulsion may be produced using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
Alternatively, the lipid-containing solution can be dried under reduced pressure using an evaporator or spray-dried using a spray dryer to prepare a dried mixture containing lipids, and this mixture can be added to an aqueous solvent and further emulsified using the emulsifier described above.

　核酸を含まない脂質粒子の製造方法の一例としては、
　工程（Ａ）；核酸を含まない脂質粒子の構成成分を有機溶媒に溶解して油相を調製する工程
　工程（Ｂ）；工程（Ａ）で得た油相と水相を混合して脂質粒子の分散液を得る工程
　工程（Ｃ）；工程（Ｂ）で得た脂質粒子の分散液を希釈する工程
　工程（Ｄ）；脂質粒子の分散液から上記有機溶媒を除去する工程
　工程（Ｅ）；脂質粒子の分散液の濃度を調節する工程
　を含む方法が挙げられる。 An example of a method for producing lipid particles that do not contain nucleic acids is
Step (A): Dissolving components of lipid particles not containing nucleic acid in an organic solvent to prepare an oil phase; Step (B): Mixing the oil phase obtained in step (A) with an aqueous phase to obtain a lipid particle dispersion; Step (C): Diluting the lipid particle dispersion obtained in step (B); Step (D): Removing the organic solvent from the lipid particle dispersion; Step (E): Adjusting the concentration of the lipid particle dispersion.

　工程（Ａ）においては、核酸を含まない脂質粒子の構成成分を、有機溶媒（エタノールなどのアルコール、またはエステルなど）に溶解させる。総脂質濃度は特に限定されないが、一般的には１ｍｍｏｌ／Ｌ～１００ｍｍｏｌ／Ｌであり、好ましくは５ｍｍｏｌ／Ｌ～５０ｍｍｏｌ／Ｌであり、より好ましくは１０ｍｍｏｌ／Ｌ～３０ｍｍｏｌ／Ｌである。 In step (A), the components of the lipid particles that do not contain nucleic acids are dissolved in an organic solvent (an alcohol such as ethanol, or an ester, etc.). The total lipid concentration is not particularly limited, but is generally 1 mmol/L to 100 mmol/L, preferably 5 mmol/L to 50 mmol/L, and more preferably 10 mmol/L to 30 mmol/L.

　工程（Ｂ）において、油相と水相は任意の方法で混合してよく、バッチ式でも流路デバイスを用いたインライン方式でもよい。インライン方式としてはマイクロ流路デバイスを用いることが好ましく、用いるマイクロ流路デバイスとしては、Ｙ字ミキサー、Ｔ字ミキサー、ヘリンボーンミキサー、リングマイクロミキサー、インピンジメントジェットミキサーなどを用いることができる。水相と油相を混合する比率（体積比）は、５：１～１：１が好ましく、４：１～２：１がより好ましい。 In step (B), the oil phase and the aqueous phase may be mixed by any method, including a batch method and an in-line method using a flow path device. For the in-line method, a micro flow path device is preferably used, and examples of the micro flow path device that can be used include a Y-shaped mixer, a T-shaped mixer, a herringbone mixer, a ring micro mixer, and an impingement jet mixer. The mixing ratio (volume ratio) of the aqueous phase and the oil phase is preferably 5:1 to 1:1, and more preferably 4:1 to 2:1.

　水相には、必要に応じてｐＨ調整のための緩衝成分や酸化防止剤などの成分を添加することができる。水相のｐＨは２．０～７．０であることが好ましく、３．０～６．０であることがより好ましい。上記ｐＨに調整するために緩衝成分として酢酸、クエン酸、リンゴ酸、リン酸、ＭＥＳ、ＨＥＰＥＳなどが好ましく用いられ、必要に応じて塩強度を調整することを目的に、塩化ナトリウム、塩化カリウムなどの塩や、浸透圧を調整することを目的に、スクロース、トレハロース、マンニトールなどの糖や糖アルコールを添加してもよい。 If necessary, components such as buffer components for pH adjustment and antioxidants can be added to the aqueous phase. The pH of the aqueous phase is preferably 2.0 to 7.0, and more preferably 3.0 to 6.0. To adjust the pH to the above range, buffer components such as acetic acid, citric acid, malic acid, phosphoric acid, MES, and HEPES are preferably used. If necessary, salts such as sodium chloride and potassium chloride can be added to adjust the salt strength, and sugars and sugar alcohols such as sucrose, trehalose, and mannitol can be added to adjust the osmotic pressure.

　工程（Ｃ）において、脂質粒子の分散液を希釈溶液と混合することにより、有機溶媒の含率を下げ、脂質粒子を安定化することができる。希釈溶液は、水でもよいが、ｐＨや塩強度を調整することを含んでもよい。希釈溶液に含まれる成分は目的に応じて任意に選択することができる。例えば、ｐＨを調整する目的で、緩衝液（例えばクエン酸緩衝液、クエン酸緩衝生理食塩水、酢酸緩衝液、酢酸緩衝生理食塩水、リン酸緩衝生理食塩水、トリス緩衝液、ＭＥＳ緩衝液、ＨＥＰＥＳ緩衝液など）を用いてもよい。また、塩強度や浸透圧を調整することを目的として塩化ナトリウム、塩化カリウム、スクロース、トレハロース、フルクトースまたはマンニトールなどを含んでもよく、上記緩衝液にこれらの添加剤をさらに添加したものも使用できる。 In step (C), the lipid particle dispersion is mixed with a diluent solution to reduce the organic solvent content and stabilize the lipid particles. The diluent solution may be water, but may also include adjusting the pH or salt strength. The components contained in the diluent solution may be selected arbitrarily depending on the purpose. For example, a buffer solution (e.g., citrate buffer, citrate buffered saline, acetate buffer, acetate buffered saline, phosphate buffered saline, Tris buffer, MES buffer, HEPES buffer, etc.) may be used for the purpose of adjusting the pH. In addition, sodium chloride, potassium chloride, sucrose, trehalose, fructose, mannitol, etc. may be included for the purpose of adjusting the salt strength or osmotic pressure, and the above buffer solutions to which these additives have been further added may also be used.

　脂質粒子の分散液と希釈溶液は任意の方法で混合してよく、バッチ式でも流路デバイスを用いたインライン方式でもよい。混合時に用いる流路デバイスは、Ｙ字ミキサー、Ｔ字ミキサーなどを用いることができる。また、油相と水相を混合してから希釈溶液を混合するまでの時間は特に限定されないが、油相及び水相を混合した３０秒以内に希釈を実施することが好ましく、１０秒以内に希釈を実施することがより好ましい。
　脂質粒子の分散液と希釈溶液を混合する比率（液量比）は、１：０．５～１：１０が好ましく、１：１～１：５がより好ましい。 The lipid particle dispersion and the diluted solution may be mixed by any method, including a batch method and an in-line method using a flow path device. The flow path device used during mixing may be a Y-shaped mixer, a T-shaped mixer, or the like. The time from mixing the oil phase and the aqueous phase to mixing the diluted solution is not particularly limited, but it is preferable to perform the dilution within 30 seconds, and more preferably within 10 seconds, of mixing the oil phase and the aqueous phase.
The mixing ratio (liquid volume ratio) of the lipid particle dispersion and the dilution solution is preferably 1:0.5 to 1:10, and more preferably 1:1 to 1:5.

　いくつかの実施形態では、工程（Ｃ）において脂質粒子の分散液は目的に応じて複数回希釈溶液と混合してもよい。また用いる希釈溶液は同一であっても異なっていてもよい。脂質粒子の分散液では、ｐＨによって脂質粒子の粒径が変化することがあり、分散液のｐＨ調整は重要となる。そのため、例えば希釈溶液との混合後の脂質粒子の分散液のｐＨを調整するために、それに適した濃度およびｐＨを有する緩衝液や、さらに他の成分を含む緩衝液を用いてもよい。 In some embodiments, in step (C), the lipid particle dispersion may be mixed with the dilution solution multiple times depending on the purpose. The dilution solutions used may be the same or different. In the lipid particle dispersion, the particle size of the lipid particles may change depending on the pH, so adjusting the pH of the dispersion is important. Therefore, for example, in order to adjust the pH of the lipid particle dispersion after mixing with the dilution solution, a buffer solution having an appropriate concentration and pH, or a buffer solution containing other components, may be used.

　さらに、複数の希釈工程は連続的に実施してもよく、希釈工程と次の希釈工程の間隔を任意に設定することができ、例えば、その間隔は１０秒、３０秒、１分、５分、１０分、３０分、１時間、２時間、３時間、４時間、６時間、１２時間または２４時間であってもよい。 Furthermore, multiple dilution steps may be performed consecutively, and the interval between one dilution step and the next dilution step may be set arbitrarily, for example, the interval may be 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours or 24 hours.

　また、工程（Ｃ）を行った後の脂質粒子の分散液のｐＨはｐＨ３．０～１０．０が好ましく、ｐＨ３．５～ｐＨ９．０がより好ましく、ｐＨ４．０～ｐＨ８．５が特に好ましい。 Furthermore, the pH of the lipid particle dispersion after step (C) is preferably pH 3.0 to pH 10.0, more preferably pH 3.5 to pH 9.0, and particularly preferably pH 4.0 to pH 8.5.

　脂質粒子には、必要に応じてサイジングを施すことができる。サイジングの方法は、特に限定されないが、エクストルーダーなどを用いて粒子径を小さくすることができる。
　また、脂質組成物を含む分散液には、一般的な方法により、凍結や凍結乾燥を施すことができる。 The lipid particles can be subjected to sizing as necessary. The method of sizing is not particularly limited, but the particle size can be reduced by using an extruder or the like.
Furthermore, the dispersion containing the lipid composition can be subjected to freezing or lyophilization by a general method.

　工程（Ｄ）において、脂質粒子の分散液から有機溶媒を除去する方法としては、特に限定されず、一般的な手法を使用することができる。例えば、透析液としてはリン酸緩衝生理食塩水、トリス緩衝液などのｐＨ緩衝液を用いることができ、必要に応じて浸透圧の調整や凍結からの保護を目的として任意の塩や糖などの添加剤を加えることができる。 In step (D), the method for removing the organic solvent from the lipid particle dispersion is not particularly limited, and a general method can be used. For example, a pH buffer solution such as phosphate buffered saline or Tris buffer can be used as the dialysis fluid, and additives such as any salt or sugar can be added as necessary to adjust the osmotic pressure or protect against freezing.

　工程（Ｅ）において、工程（Ｄ）で得られた脂質粒子の分散液の濃度を調整することができる。希釈する場合は、リン酸緩衝生理食塩水、生理食塩水、トリス緩衝液、スクロース含有トリス緩衝液などの溶液を希釈液として用いて適切な濃度に希釈することができる。濃縮する場合は、工程（Ｄ）で得られた分散液を、限外ろ過膜を用いた限外ろ過などにより濃縮することができる。濃縮した分散液をそのまま用いても好ましく、濃縮した後に上記希釈液を用いて所望の濃度に調整しても好ましい。 In step (E), the concentration of the lipid particle dispersion obtained in step (D) can be adjusted. When diluting, a solution such as phosphate buffered saline, saline, Tris buffer, or sucrose-containing Tris buffer can be used as a diluent to dilute to an appropriate concentration. When concentrating, the dispersion obtained in step (D) can be concentrated by ultrafiltration using an ultrafiltration membrane. The concentrated dispersion can be used as is, or it can be concentrated and then adjusted to the desired concentration using the diluent.

　また、いくつかの実施形態では、接線流ろ過（ＴＦＦ）を用いて有機溶媒除去工程（工程（Ｄ））、濃度調整工程（工程（Ｅ））を連続的に行うことができる。本工程において有機溶媒除去工程と濃度調整工程を任意の順番で実施してもよい。必要に応じて有機溶媒除去工程と濃度調整工程をそれぞれ複数回ずつ実施してもよい。 In some embodiments, the organic solvent removal step (step (D)) and the concentration adjustment step (step (E)) can be carried out continuously using tangential flow filtration (TFF). In this process, the organic solvent removal step and the concentration adjustment step may be carried out in any order. If necessary, the organic solvent removal step and the concentration adjustment step may each be carried out multiple times.

　工程（Ｄ）における透析や、工程（Ｅ）における希釈において用いることのできる溶液としては、賦形剤、凍結保護剤、緩衝剤や酸化防止剤を添加してもよい。賦形剤や凍結保護剤としては、特に限定されないが、糖類や糖アルコール類が挙げられる。糖類としては、例えばスクロース、トレハロース、マルトース、グルコース、ラクトース、フルクトースなどが、糖アルコール類としては、例えばマンニトール、ソルビトール、イノシトール、キシリトールなどが挙げられる。緩衝剤としては、特に限定されないが、例えば、ＡＣＥＳ、ＢＥＳ、Ｂｉｃｉｎｅ、ＣＡＰＳ、ＣＨＥＳ、ＤＩＰＳＯ、ＥＰＰＳ、ＨＥＰＥＳ、ＨＥＰＰＳＯ、ＭＥＳ、ＭＯＰＳ、ＭＯＰＳＯ、ＴＡＰＳ、ＴＡＰＳＯ、ＴＥＳ、Ｔｒｉｃｉｎｅ、トリス、リン酸、酢酸、クエン酸などが挙げられる。酸化防止剤としては、ＥＤＴＡ、アスコルビン酸、トコフェロールなどが挙げられる。 The solution that can be used for dialysis in step (D) and dilution in step (E) may contain excipients, cryoprotectants, buffers, and antioxidants. Examples of excipients and cryoprotectants include, but are not limited to, sugars and sugar alcohols. Examples of sugars include sucrose, trehalose, maltose, glucose, lactose, and fructose, and examples of sugar alcohols include mannitol, sorbitol, inositol, and xylitol. Examples of buffers include, but are not limited to, ACES, BES, Bicine, CAPS, CHES, DIPSO, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, TAPS, TAPSO, TES, Tricine, Tris, phosphoric acid, acetic acid, and citric acid. Examples of antioxidants include EDTA, ascorbic acid, and tocopherol.

　脂質粒子の分散液について、無菌ろ過を行ってもよい。ろ過の方法としては、中空糸膜、逆浸透膜、メンブレンフィルターなどを用いて、脂質粒子の分散液から不溶なものを除去することができる。本発明では、特に限定されないが、滅菌できる孔径を有するフィルター（好ましくは０．２μｍのろ過滅菌フィルター）によってろ過することが好ましい。また、無菌ろ過を行うのは、工程（Ｄ）または工程（Ｅ）の後が好ましい。 The lipid particle dispersion may be subjected to sterile filtration. As a filtration method, a hollow fiber membrane, a reverse osmosis membrane, a membrane filter, or the like may be used to remove insoluble matter from the lipid particle dispersion. In the present invention, although there are no particular limitations, it is preferable to filter using a filter having a pore size that allows sterilization (preferably a 0.2 μm filtration sterilization filter). In addition, it is preferable to perform sterile filtration after step (D) or step (E).

　さらに必要に応じて、核酸を含まない脂質粒子の分散液を、凍結や凍結乾燥を施すことができる。脂質粒子の分散液は、一般的な方法により凍結や凍結乾燥を施すことができ、その手法は特に限定されない。 Furthermore, if necessary, the dispersion of lipid particles not containing nucleic acid can be frozen or lyophilized. The dispersion of lipid particles can be frozen or lyophilized by a general method, and the method is not particularly limited.

　好ましくは、本発明においては、核酸を含まない脂質粒子を凍結保存し、凍結保存した核酸を含まない脂質粒子を核酸との混合前に解凍することができる。 Preferably, in the present invention, the lipid particles not containing nucleic acid are frozen and stored, and the frozen lipid particles not containing nucleic acid can be thawed before mixing with the nucleic acid.

　本発明においては、上記のようにして調製した核酸を含まない脂質粒子と核酸とを混合する工程を行う。核酸を含まない脂質粒子と核酸との混合する工程は、特に限定されないが、流路を用いて液を混合する方法、容器内で往復方向に液を行き来させる混合、ピペット混合、バッチ容器でのスターラー混合、容器を回転させることで内容液を攪拌させる混合、またはフラスコ攪拌のいずれかによって行うことができる。
　核酸を含まない脂質粒子と核酸との混合する工程は、好ましくは、核酸を含まない脂質粒子と、核酸を含む水溶液とを、０℃～３０℃で０．１～１２０分間インキュベートする工程と、上記で得られた混合物のｐＨを６．５～８．５に調整する工程とを含んでいてもよい。
　核酸を含む水溶液は、核酸を水または緩衝液に溶解することで得ることができる。核酸の濃度は特に限定されないが、１～２０００μｇ／ｍＬが好ましく、１０～１０００μｇ／ｍＬがより好ましい。必要に応じてｐＨ調整のための緩衝成分や酸化防止剤などの成分を添加することができる。 In the present invention, carry out the process of mixing the lipid particles that do not contain nucleic acid prepared as above with nucleic acid.The process of mixing the lipid particles that do not contain nucleic acid with nucleic acid is not particularly limited, but can be carried out by any of the following methods: mixing liquid using a flow path, mixing liquid in a reciprocating direction in a container, mixing with a pipette, mixing with a stirrer in a batch container, mixing liquid in the container by rotating, or mixing with a flask.
The step of mixing the nucleic acid-free lipid particles with the nucleic acid may preferably include a step of incubating the nucleic acid-free lipid particles with the aqueous solution containing the nucleic acid at 0°C to 30°C for 0.1 to 120 minutes, and a step of adjusting the pH of the mixture obtained above to 6.5 to 8.5.
An aqueous solution containing nucleic acid can be obtained by dissolving nucleic acid in water or a buffer solution. The concentration of nucleic acid is not particularly limited, but is preferably 1 to 2000 μg/mL, more preferably 10 to 1000 μg/mL. If necessary, a buffer component for adjusting pH or a component such as an antioxidant can be added.

　核酸を含まない脂質粒子と核酸との混合する工程において、混合後の溶液中の脂質濃度と核酸濃度の質量比が５：１～１０００：１であることが好ましく、５：１～５００：１であることがより好ましく、７：１～２００：１であることがさらに好ましく、７：１～１００：１であることが特に好ましい。 In the step of mixing the nucleic acid-free lipid particles with the nucleic acid, the mass ratio of the lipid concentration to the nucleic acid concentration in the solution after mixing is preferably 5:1 to 1000:1, more preferably 5:1 to 500:1, even more preferably 7:1 to 200:1, and particularly preferably 7:1 to 100:1.

＜脂質組成物＞
　脂質組成物は脂質粒子でもよい。脂質粒子とは、脂質から構成される粒子を意味し、脂質が凝集している脂質凝集体、ミセル、リポソーム、脂質ナノ粒子（ＬＮＰ）、リポプレックスから選択されるいずれかの構造を有する組成物が含まれる。リポソームとしては、脂質二重層構造を有し、内部に水相を有し、二重膜が単層のリポソーム、多数層状に重なった多重層リポソームがある。本発明にはどちらのリポソームが含まれてもよい。脂質粒子としては、脂質ナノ粒子（ＬＮＰ）であることが好ましい。 <Lipid composition>
The lipid composition may be a lipid particle. The lipid particle means a particle composed of lipid, and includes a composition having any structure selected from lipid aggregates, micelles, liposomes, lipid nanoparticles (LNPs), and lipoplexes. The liposomes include liposomes having a lipid bilayer structure, an aqueous phase inside, and a single-layer bilayer membrane, and multilayer liposomes having multiple layers. The present invention may include either type of liposome. The lipid particle is preferably a lipid nanoparticle (LNP).

　脂質粒子の形態は、電子顕微鏡観察またはエックス線を用いた構造解析などにより確認できる。例えば、Ｃｒｙｏ透過型電子顕微鏡観察（ＣｒｙｏＴＥＭ法）を用いた方法により、リポソームのように脂質粒子が脂質二分子膜構造（ラメラ構造）および内水層を持つ構造であるか、粒子内部に電子密度が高いコアを持ち、脂質をはじめとする構成成分が詰まった構造を有しているか、などが確認できる。エックス線小角散乱（ＳＡＸＳ）測定によっても、脂質粒子についての脂質二分子膜構造（ラメラ構造）の有無を確認できる。 The morphology of lipid particles can be confirmed by electron microscopy or structural analysis using X-rays. For example, by using a cryo-transmission electron microscope (cryo-TEM) it can be confirmed whether the lipid particles have a lipid bilayer structure (lamellar structure) and an inner water layer like liposomes, or whether the particles have a core with high electron density inside and are packed with lipids and other components. Small angle X-ray scattering (SAXS) measurements can also be used to confirm whether lipid particles have a lipid bilayer structure (lamellar structure).

　脂質粒子の粒子径は特に限定されないが、好ましくは１０～１０００ｎｍであり、より好ましくは３０～５００ｎｍであり、さらに好ましくは５０～２５０ｎｍである。脂質粒子の粒子径は、一般的な方法（例えば、動的光散乱法、レーザー回折法など）により測定することができる。 The particle size of the lipid particles is not particularly limited, but is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and even more preferably 50 to 250 nm. The particle size of the lipid particles can be measured by a general method (e.g., dynamic light scattering method, laser diffraction method, etc.).

＜免疫細胞に核酸を送達する方法＞
　本発明によれば、上記した本発明の免疫細胞への核酸送達剤を、免疫細胞と接触させることを含む、免疫細胞に核酸を送達する方法が提供される。ただし、インビボでの送達方法を除いてもよい。即ち、脂質組成物を核酸と混合して、エクスビボ、インビトロ、またはインビボで免疫細胞にトランスフェクションをすることにより、免疫細胞に核酸などを導入することができる。 Methods for delivering nucleic acids to immune cells
According to the present invention, a method for delivering nucleic acid to immune cells is provided, which comprises contacting the above-mentioned nucleic acid delivery agent for immune cells of the present invention with immune cells. However, in vivo delivery methods may be excluded. That is, nucleic acid or the like can be introduced into immune cells by mixing a lipid composition with nucleic acid and transfecting immune cells ex vivo, in vitro, or in vivo.

＜医薬用途＞
　本発明によれば、上記した本発明の免疫細胞への核酸送達剤を、医薬用途で使用することができる。医薬用途で使用する場合には、本発明の核酸送達剤は単独でまたは薬学的に許容される担体と混合して生体に投与することができる。また、医薬用途で使用する場合、核酸送達剤を投与する場合の投与経路はとくに限定されず、任意の方法で投与することができる。 <Medicinal Use>
According to the present invention, the above-mentioned nucleic acid delivery agent for immune cells of the present invention can be used for medical purposes. When used for medical purposes, the nucleic acid delivery agent of the present invention can be administered to a living body alone or in combination with a pharma- ceutically acceptable carrier. In addition, when used for medical purposes, the administration route of the nucleic acid delivery agent is not particularly limited, and it can be administered by any method.

　本発明の核酸送達剤は、より免疫細胞に送達するために、脂質組成物の表面に免疫細胞を標的とする分子（以下、標的分子ともいう）を結合してもよい。標的分子としては、特に限定されないが、低分子、ペプチド、核酸及び抗体を使用することができる。
　また、脂質組成物の表面に標的分子を結合する場合、脂質組成物は、脂質組成物と標的分子とが化学的または電気的に結合するための修飾基を有する脂質を含んでいても良い。当該修飾基を有する脂質としては、例えば、マレイミド基とポリエチレングリコール鎖とを有する脂質等が挙げられる。 In order to more effectively deliver the nucleic acid delivery agent of the present invention to immune cells, a molecule that targets immune cells (hereinafter also referred to as a target molecule) may be bound to the surface of the lipid composition. The target molecule is not particularly limited, but a small molecule, a peptide, a nucleic acid, and an antibody can be used.
In addition, when a target molecule is bound to the surface of the lipid composition, the lipid composition may contain a lipid having a modifying group for chemically or electrically binding the lipid composition to the target molecule. Examples of the lipid having the modifying group include lipids having a maleimide group and a polyethylene glycol chain.

　免疫細胞は、活性化細胞または非活性化細胞のいずれでもよく、細胞の製造方法に応じて任意に選択することができる。
　活性化処理とは、例えばＴ細胞の場合は、抗ＣＤ３抗体および抗ＣＤ２８抗体等を用いたＴＣＲ／ＣＤ３複合体を介した主刺激シグナルとＣＤ２８を介した共刺激シグナル、もしくはレクチン経路を介した刺激等を行うことを指す。この活性化処理は、通常、ＩＬ－２、ＩＬ－７、ＩＬ－１５などのサイトカインシグナル存在下で行うことが多く、これら活性化処理により、ＩＬ－２Ｒなどのサイトカイン受容体の発現や活発な細胞増殖が誘導される。例えば、活性化Ｔ細胞とは、このような活性化処理を施したＴ細胞のことを意味する。
　また、非活性化細胞とは、例えばＴ細胞の場合は、上記の活性化処理を行わずにＩＬ－２、ＩＬ－７、ＩＬ－１５等のサイトカインシグナル存在下で培養を行うことを指す。ただし、ここに挙げた方法に限定はされない。 The immune cells may be either activated or non-activated cells, and can be arbitrarily selected depending on the method for producing the cells.
For example, in the case of T cells, the activation treatment refers to the stimulation of a main stimulatory signal via the TCR/CD3 complex and a costimulatory signal via CD28 using an anti-CD3 antibody and an anti-CD28 antibody, or the stimulation via the lectin pathway. This activation treatment is usually performed in the presence of cytokine signals such as IL-2, IL-7, and IL-15, and these activation treatments induce the expression of cytokine receptors such as IL-2R and active cell proliferation. For example, activated T cells refer to T cells that have been subjected to such an activation treatment.
In addition, in the case of T cells, for example, non-activated cells refer to cells cultured in the presence of cytokine signals such as IL-2, IL-7, and IL-15 without carrying out the above-mentioned activation treatment, although the method is not limited to those mentioned here.

　免疫細胞は、好ましくは哺乳類由来の細胞であり、より好ましくはヒト由来の細胞である。
　免疫細胞は、特に限定されないが、例えば、リンパ球（例えば、Ｔ細胞、Ｂ細胞、ナチュラルキラー細胞（ＮＫ細胞）、ＮＫＴ細胞、ｉＮＫＴ細胞）、単球、マクロファージ、肥満細胞、樹状細胞、顆粒球（例えば、好中球、好酸球、及び好塩基球）、造血幹・前駆細胞、初代免疫細胞、ＣＤ３＋細胞、ＣＤ４＋細胞、ＣＤ８＋Ｔ細胞、制御性Ｔ細胞（Ｔｒｅｇ）、Ｂ細胞、ＮＫ細胞、自然リンパ球、または樹状細胞（ＤＣ）から選択することができる。
　免疫細胞は、好ましくは、末梢血単核細胞（ＰＢＭＣ）、リンパ球、Ｔ細胞、ＣＤ４＋細胞、ＣＤ８＋細胞、メモリＴ細胞、ナイーブＴ細胞、または幹細胞メモリＴ細胞から選択してもよい。
　免疫細胞は、初代細胞でもよいし、または多能性幹細胞に由来する細胞でもよい。 The immune cells are preferably cells of mammalian origin, more preferably cells of human origin.
The immune cells are not particularly limited, and may be selected from, for example, lymphocytes (e.g., T cells, B cells, natural killer cells (NK cells), NKT cells, iNKT cells), monocytes, macrophages, mast cells, dendritic cells, granulocytes (e.g., neutrophils, eosinophils, and basophils), hematopoietic stem/progenitor cells, primary immune cells, CD3 + cells, CD4 + cells, CD8 + T cells, regulatory T cells (Treg), B cells, NK cells, innate lymphocytes, or dendritic cells (DCs).
The immune cells may preferably be selected from peripheral blood mononuclear cells (PBMC), lymphocytes, T cells, CD4 + cells, CD8 + cells, memory T cells, naive T cells, or stem cell memory T cells.
The immune cells may be primary cells or cells derived from pluripotent stem cells.

　核酸送達剤を免疫細胞に接触させる前に、核酸送達剤もしくは免疫細胞に対して（ｉ）アポリポタンパク、及び／又は（ｉｉ）細胞結合ドメインとヘパリン結合ドメインとを含むタンパク質を添加する工程を含めてもよい。 The method may include a step of adding (i) an apolipoprotein and/or (ii) a protein containing a cell-binding domain and a heparin-binding domain to the nucleic acid delivery agent or the immune cells before contacting the nucleic acid delivery agent with the immune cells.

　アポリポタンパクとは、リポタンパク質と結合し、リポタンパク質の認識や脂質代謝に関与する酵素群の活性化あるいは補酵素として働く一群のタンパク質である。アポリポタンパク質は、構造や機能によりアポリポタンパク質ＡからＥまでの５種に大別され、それらの一部は、アポリポプロテインＡ－ＩやＣ－ＩＩのようにサブクラスに分けられている。本発明においては、例えば、アポリポプロテインＥ、特にはアポリポプロテインＥ３を使用してもよい。アポリポプロテインの由来は特に限定されず、ヒトなどの哺乳動物のアポリポプロテインを使用することができる。 Apolipoproteins are a group of proteins that bind to lipoproteins and activate enzymes involved in lipoprotein recognition and lipid metabolism, or act as coenzymes. Apolipoproteins are broadly classified into five types, A to E, based on their structure and function, and some of them are divided into subclasses, such as apolipoprotein A-I and C-II. In the present invention, for example, apolipoprotein E, particularly apolipoprotein E3, may be used. The origin of the apolipoprotein is not particularly limited, and apolipoproteins from mammals such as humans may be used.

　細胞結合ドメインとヘパリン結合ドメインを含む組換えタンパク質とは、細胞接着タンパク（フィブロネクチンやビトロネクチンなど）、細胞接着タンパクに由来する細胞結合ドメインとヘパリン結合ドメインのみを含む組換えタンパクである。好ましくは、細胞結合ドメインとヘパリン結合ドメインのみを含む組換えタンパクであり、より好ましくはレトロネクチンである。 The recombinant protein containing a cell-binding domain and a heparin-binding domain is a cell adhesion protein (such as fibronectin or vitronectin) or a recombinant protein containing only a cell-binding domain and a heparin-binding domain derived from a cell adhesion protein. Preferably, it is a recombinant protein containing only a cell-binding domain and a heparin-binding domain, and more preferably, it is retronectin.

　次に本発明について実施例を挙げて説明するが、本発明はこれらに限定されるものではない。 The present invention will now be described with reference to examples, but the present invention is not limited to these.

　特に記載のない場合、カラムクロマトグラフィーによる精製は、自動精製装置ISOLERA（Biotage社）、中圧分取精製装置Purif-espoir-2（昭光サイエンス株式会社）または中圧液体クロマトグラフYFLC W-prep 2XY （山善株式会社）を使用した。 Unless otherwise specified, purification by column chromatography was performed using an automatic purification system ISOLERA (Biotage), a medium pressure fractionation purification system Purif-espoir-2 (Shoko Science Co., Ltd.), or a medium pressure liquid chromatograph YFLC W-prep 2XY (Yamazen Corporation).

　特に記載のない場合、シリカゲルカラムクロマトグラフィーにおける担体は、Chromatorex Q-Pack SI 50（富士シリシア化学株式会社）、ハイフラッシュカラムW001、W002、W003、W004またはW005（山善株式会社）を使用した。
　ＮＨシリカゲルは、Chromatorex Q-Pack NH 60（富士シリシア化学株式会社）を使用した。 Unless otherwise specified, the carrier used in silica gel column chromatography was Chromatorex Q-Pack SI 50 (Fuji Silysia Chemical Ltd.) or Hi-Flash Column W001, W002, W003, W004 or W005 (Yamazen Corporation).
The NH silica gel used was Chromatorex Q-Pack NH 60 (Fuji Silysia Chemical Ltd.).

　ＮＭＲスペクトルは、内部基準としてテトラメチルシランを用い、Ｂｒｕｋｅｒ ＡＶＮＥＯ４００（Ｂｒｕｋｅｒ社製）を用いて測定し、全δ値をｐｐｍで示した。
　ＭＳスペクトルは、ＡＣＱＵＩＴＹ ＳＱＤ ＬＣ／ＭＳ Ｓｙｓｔｅｍ（Ｗａｔｅｒｓ社製）を用いて測定した。 The NMR spectrum was measured using tetramethylsilane as an internal standard with a Bruker AVNEO400 (manufactured by Bruker Corporation), and all δ values are shown in ppm.
MS spectra were measured using an ACQUITY SQD LC/MS System (Waters).

［合成例１］
（１）
 [Synthesis Example 1]
(1)

　２，２－ジエトキシエタノール(5.0g)、テトラヒドロフラン(25mL)およびトリエチルアミン(15.6mL)の混合物に、氷冷下でクロロギ酸４－ニトロフェニル(11.3g)を２分割して添加し、氷冷下で1時間撹拌した。反応混合物に、氷冷下で水(25mL)およびヘキサン(25mL)を添加し、有機層を分取した。得られた有機層を、水(25mL)および飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、淡黄色油状物の２，２－ジエトキシエチル（４－ニトロフェニル）カーボネート(12.2g)を得た。
1H-NMR(CDCl3)δ: 8.30-8.26 (2H, m), 7.41-7.37 (2H, m), 4.79 (1H, t, J=5.3Hz), 4.28 (2H, d, J=5.3Hz), 3.80-3.72 (2H, m), 3.66-3.58 (2H, m), 1.26 (6H, t, J=7.0Hz). To a mixture of 2,2-diethoxyethanol (5.0 g), tetrahydrofuran (25 mL) and triethylamine (15.6 mL), 4-nitrophenyl chloroformate (11.3 g) was added in two portions under ice-cooling, and the mixture was stirred under ice-cooling for 1 hour. Water (25 mL) and hexane (25 mL) were added to the reaction mixture under ice-cooling, and the organic layer was separated. The obtained organic layer was washed with water (25 mL) and saturated saline, and then dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2,2-diethoxyethyl (4-nitrophenyl) carbonate (12.2 g) as a pale yellow oil.
1 H-NMR(CDCl 3 )δ: 8.30-8.26 (2H, m), 7.41-7.37 (2H, m), 4.79 (1H, t, J=5.3Hz), 4.28 (2H, d, J=5.3Hz), 3.80-3.72 (2H, m), 3.66-3.58 (2H, m), 1.26 (6H, t, J=7.0Hz).

（２）
 (2)

　２－ヘキシル－１－オクタノール(5.0g)、５-ブロモ吉草酸(4.6g)のトルエン(25mL)混合物に、硫酸(0.5mL)を添加し、110℃で5時間撹拌した。反応混合物を室温まで冷却した後に、シリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、無色油状物の２－ヘキシルオクチル５－ブロモペンタン酸(8.2g)を得た。
1H-NMR(CDCl3)δ: 3.98 (2H, d, J=5.7Hz), 3.42 (2H, t, J=6.5Hz), 2.34 (2H, t, 7.2Hz), 1.94-1.87 (2H, m), 1.82-1.74 (2H, m), 1.65-1.57 (1H, m), 1.32-1.23 (20H, m), 0.90-0.86 (6H, m). To a mixture of 2-hexyl-1-octanol (5.0 g), 5-bromovaleric acid (4.6 g) and toluene (25 mL), sulfuric acid (0.5 mL) was added and stirred for 5 hours at 110° C. The reaction mixture was cooled to room temperature and then purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2-hexyloctyl 5-bromopentanoate (8.2 g) as a colorless oil.
1 H-NMR(CDCl 3 )δ: 3.98 (2H, d, J=5.7Hz), 3.42 (2H, t, J=6.5Hz), 2.34 (2H, t, 7.2Hz), 1.94-1.87 (2H, m), 1.82-1.74 (2H, m), 1.65-1.57 (1H, m), 1.32-1.23 (20H, m), 0.90-0.86 (6H, m).

（３）
 (3)

　２－ヘキシルオクチル５－ブロモペンタン酸(1.2g)、ｎ－オクチルアミン(1.2g)および１－メチル－２－ピロリドン(6mL)の混合物に、炭酸カリウム(1.3g)を添加し、60℃で5時間撹拌した。反応混合物を室温まで冷却した後に、酢酸エチル(12mL)および水(6mL)を添加し、有機層を分取した。有機層を飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(ヘキサン－酢酸エチル－メタノール)で精製し、淡黄色油状物の２－ヘキシルオクチル　５－（オクチルアミノ）ペンタン酸(1.25g)を得た。
1H-NMR(CDCl3)δ: 3.96 (2H, d, J=5.8Hz), 2.63-2.52 (4H, m), 2.32 (2H, t, J=7.4Hz), 2.06-1.98 (1H, m), 1.70-1.40 (7H, m), 1.34-1.20 (30H, m), 0.90-0.87 (9H, m). Potassium carbonate (1.3 g) was added to a mixture of 2-hexyloctyl 5-bromopentanoate (1.2 g), n-octylamine (1.2 g) and 1-methyl-2-pyrrolidone (6 mL), and the mixture was stirred at 60°C for 5 hours. After the reaction mixture was cooled to room temperature, ethyl acetate (12 mL) and water (6 mL) were added, and the organic layer was separated. The organic layer was washed with saturated saline, dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane-ethyl acetate-methanol) to obtain 2-hexyloctyl 5-(octylamino)pentanoate (1.25 g) as a pale yellow oil.
1 H-NMR(CDCl 3 )δ: 3.96 (2H, d, J=5.8Hz), 2.63-2.52 (4H, m), 2.32 (2H, t, J=7.4Hz), 2.06-1.98 (1H, m), 1.70-1.40 (7H, m), 1.34-1.20 (30H, m), 0.90-0.87 (9H, m).

（４）
 (4)

　２－ヘキシルオクチル５－（オクチルアミノ）ペンタン酸(1.25g)、アセトニトリル(4mL)、２，２－ジエトキシエチル（４－ニトロフェニル）カーボネート(0.49g)およびトリエチルアミン(0.46mL)の混合物を、60℃で4時間撹拌した。室温まで冷却した反応混合物に、酢酸エチル(4mL)および水(4mL)を加え、有機層を分取した。得られた有機層を、水および飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、淡黄色油状物の２－ヘキシルオクチル５－（（（２，２－ジエトキシエトキシ）カルボニル）（オクチル）アミノ）ペンタン酸(0.83g)を得た。
1H-NMR(CDCl3)δ: 4.69 (1H, t, J=5.4Hz), 4.08 (2H, d, J=5.5Hz), 3.97 (2H, d, J=5.7Hz), 3.74-3.62 (2H, m), 3.60-3.52 (2H, m), 3.26-3.14 (4H, m), 2.36-2.30 (2H, m), 1.65-1.46 (7H, m), 1.33-1.19 (36H, m), 0.90-0.85 (9H, m). A mixture of 2-hexyloctyl 5-(octylamino)pentanoic acid (1.25 g), acetonitrile (4 mL), 2,2-diethoxyethyl (4-nitrophenyl) carbonate (0.49 g) and triethylamine (0.46 mL) was stirred at 60°C for 4 hours. Ethyl acetate (4 mL) and water (4 mL) were added to the reaction mixture cooled to room temperature, and the organic layer was separated. The organic layer obtained was washed with water and saturated saline, and then dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2-hexyloctyl 5-(((2,2-diethoxyethoxy)carbonyl)(octyl)amino)pentanoic acid (0.83 g) as a pale yellow oil.
1 H-NMR(CDCl 3 )δ: 4.69 (1H, t, J=5.4Hz), 4.08 (2H, d, J=5.5Hz), 3.97 (2H, d, J=5.7Hz), 3.74-3.62 (2H, m), 3.60-3.52 (2H, m), 3.26-3.14 (4H, m), 2.36-2.30 (2H, m), 1.65-1.46 (7H, m), 1.33-1.19 (36H, m), 0.90-0.85 (9H, m).

（５）
 (5)

　２－ヘキシルオクチル５－（（（２，２－ジエトキシエトキシ）カルボニル）（オクチル）アミノ）ペンタン酸(0.83g)、ギ酸(6mL)および水(1.5mL) の混合物を、50℃で3時間撹拌した後に、トルエンを添加し減圧留去した。再度トルエンを添加し、減圧留去する操作を２回繰り返し、淡黄色油状物の２－ヘキシルオクチル５－（オクチル（（２－オキソエトキシ）カルボニル）アミノ）ペンタン酸(0.94g)を粗体として得た。
1H-NMR(CDCl3)δ: 4.10 (4H, t, J=6.3Hz), 3.97 (4H, d, J=5.7Hz), 3.25-3.11 (8H, m), 2.79 (4H, t, J=6.4Hz), 2.69-2.63 (2H, m), 2.56-2.47 (6H, m), 2.36-2.29 (4H, m), 1.64-1.49 (14H, m), 1.32-1.21 (60H, m), 1.01 (6H, t, J=7.0Hz), 0.90-0.85 (18H, m). A mixture of 2-hexyloctyl 5-(((2,2-diethoxyethoxy)carbonyl)(octyl)amino)pentanoic acid (0.83 g), formic acid (6 mL) and water (1.5 mL) was stirred at 50°C for 3 hours, after which toluene was added and the mixture was distilled off under reduced pressure. Toluene was added again and the distillation under reduced pressure was repeated twice to obtain a pale yellow oily substance, 2-hexyloctyl 5-(octyl((2-oxoethoxy)carbonyl)amino)pentanoic acid (0.94 g), as a crude product.
1 H-NMR(CDCl 3 )δ: 4.10 (4H, t, J=6.3Hz), 3.97 (4H, d, J=5.7Hz), 3.25-3.11 (8H, m), 2.79 (4H, t, J=6.4Hz), 2.69-2.63 (2H, m), 2.56-2.47 (6H, m), 2.36-2.29 (4H, m), 1.64-1.49 (14H, m), 1.32-1.21 (60H, m), 1.01 (6H, t, J=7.0Hz), 0.90-0.85 (18H, m).

（６）
 (6)

　２－ヘキシルオクチル５－（オクチル（（２－オキソエトキシ）カルボニル）アミノ）ペンタン酸(0.73g)の酢酸エチル(8mL)溶液に、Ｎ，Ｎ－ジエチルエチレンジアミン(0.083g)、酢酸(43mg)およびトリアセトキシ水素化ホウ素ナトリウム(0.91g)を室温で添加し、室温で5時間撹拌した。反応混合物に20%炭酸カリウム水溶液(10mL)を添加した後、有機層を分取し、水および飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(メタノール－酢酸エチル－ヘキサン)で精製し、淡黄色油状物のビス（２－ヘキシルオクチル）１１－（２－（ジエチルアミノ）エチル）－６，１６－ジオクチル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート（化合物１と称する）(0.39g)を得た。
1H-NMR(CDCl3)δ: 4.10 (4H, t, J=6.3Hz), 3.97 (4H, d, J=5.7Hz), 3.25-3.11 (8H, m), 2.79 (4H, t, J=6.4Hz), 2.69-2.63 (2H, m), 2.56-2.47 (6H, m), 2.36-2.29 (4H, m), 1.64-1.49 (14H, m), 1.32-1.21 (60H, m), 1.01 (6H, t, J=7.0Hz), 0.90-0.85 (18H, m).
MS m/z(M+H):1108. To a solution of 2-hexyloctyl 5-(octyl((2-oxoethoxy)carbonyl)amino)pentanoic acid (0.73 g) in ethyl acetate (8 mL), N,N-diethylethylenediamine (0.083 g), acetic acid (43 mg) and sodium triacetoxyborohydride (0.91 g) were added at room temperature, and the mixture was stirred at room temperature for 5 hours. After adding a 20% aqueous potassium carbonate solution (10 mL) to the reaction mixture, the organic layer was separated and washed with water and saturated saline, and then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (methanol-ethyl acetate-hexane) to obtain bis(2-hexyloctyl) 11-(2-(diethylamino)ethyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (referred to as Compound 1) (0.39 g) as a pale yellow oil.
1 H-NMR(CDCl 3 )δ: 4.10 (4H, t, J=6.3Hz), 3.97 (4H, d, J=5.7Hz), 3.25-3.11 (8H, m), 2.79 (4H, t, J=6.4Hz), 2.69-2.63 (2H, m), 2.56-2.47 (6H, m), 2.36-2.29 (4H, m), 1.64-1.49 (14H, m), 1.32-1.21 (60H, m), 1.01 (6H, t, J=7.0Hz), 0.90-0.85 (18H, m).
MS m/z(M+H):1108.

［合成例２］
（１）
 [Synthesis Example 2]
(1)

　２，２－ジエトキシエタノール(10.0g)のテトラヒドロフラン(100mL)溶液に、１，１'－カルボニルジ（１，２，４－トリアゾール）(18.3g)を添加し、30℃に加熱し1時間撹拌した。反応混合物を室温まで冷却した後、ヘキサン(100mL)および飽和重曹水(100mL)を添加し、有機層を分取した。得られた有機層を、水(50mL)および飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、無色油状物の２，２－ジエトキシエチル１Ｈ－１，２，４－トリアゾール－１－カルボキシラート(10.4g)を得た。
1H-NMR(CDCl3)δ: 8.83 (1H, s), 8.09 (1H, s), 4.87 (1H, t, J=5.3Hz), 4.49 (2H, d, J=5.3Hz), 3.80-3.73 (2H, m), 3.66-3.56 (2H, m), 1.23 (6H, t, J=7.0Hz). 1,1'-carbonyldi(1,2,4-triazole) (18.3 g) was added to a solution of 2,2-diethoxyethanol (10.0 g) in tetrahydrofuran (100 mL), heated to 30°C, and stirred for 1 hour. After cooling the reaction mixture to room temperature, hexane (100 mL) and saturated sodium bicarbonate water (100 mL) were added, and the organic layer was separated. The obtained organic layer was washed with water (50 mL) and saturated saline, then dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2,2-diethoxyethyl 1H-1,2,4-triazole-1-carboxylate (10.4 g) as a colorless oil.
1 H-NMR(CDCl 3 )δ: 8.83 (1H, s), 8.09 (1H, s), 4.87 (1H, t, J=5.3Hz), 4.49 (2H, d, J=5.3Hz), 3.80-3.73 (2H, m), 3.66-3.56 (2H, m), 1.23 (6H, t, J=7.0Hz).

（２）
 (2)

　Ｎ，Ｎ－ビス（２－ヒドロキシエチル）カルバミン酸ｔｅｒｔ－ブチル(5.00g)、トリエチルアミン(8.15mL)のテトラヒドロフラン(50mL)溶液に、氷冷下でデカン酸クロリド(11mL)を滴下し、その後室温にて4時間撹拌した。反応混合物にヘキサン(50mL)および水(50mL)を添加し、有機層を分取した。得られた有機層を、水(50mL)および飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去し、黄色油状の（（ｔｅｒｔ－ブトキシカルボニル）アザンジイル）ビス（エタン－２，１－ジイル）ビス（デカン酸）(13.1g)を粗体として得た。
1H-NMR(CDCl3)δ: 4.21-4.13 (4H, m), 3.52-3.44 (4H, m), 2.30 (4H, t, J=7.5Hz), 1.67-1.55 (4H, m), 1.46 (9H, s), 1.33-1.23 (24H, m), 0.88 (6H, t, J=6.8Hz). Decanoic acid chloride (11 mL) was added dropwise to a solution of tert-butyl N,N-bis(2-hydroxyethyl)carbamate (5.00 g) and triethylamine (8.15 mL) in tetrahydrofuran (50 mL) under ice cooling, and the mixture was then stirred at room temperature for 4 hours. Hexane (50 mL) and water (50 mL) were added to the reaction mixture, and the organic layer was separated. The obtained organic layer was washed with water (50 mL) and saturated saline, and then dried by adding anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain a yellow oily product of ((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) (13.1 g) as a crude product.
1 H-NMR(CDCl 3 )δ: 4.21-4.13 (4H, m), 3.52-3.44 (4H, m), 2.30 (4H, t, J=7.5Hz), 1.67-1.55 (4H, m), 1.46 (9H, s), 1.33-1.23 (24H, m), 0.88 (6H, t, J=6.8Hz).

（３）
 (3)

　（（ｔｅｒｔ－ブトキシカルボニル）アザンジイル）ビス（エタン－２，１－ジイル）ビス（デカン酸）(13.0g)と水(1mL)の混合物に、室温にてトリフルオロ酢酸(20mL)を添加し1晩撹拌した。減圧留去し、残留物にヘキサン(60mL)、酢酸エチル(30mL)および20%炭酸カリウム水溶液(40mL)を添加し、有機層を分取した。得られた有機層に無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去し、残留物をシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、淡黄色油状物のアザンジイルビス（エタン－２，１－ジイル）ビス（デカン酸）(7.39g)を得た。
MS m/z(M+H):415. To a mixture of ((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) (13.0 g) and water (1 mL), trifluoroacetic acid (20 mL) was added at room temperature and stirred overnight. The mixture was evaporated under reduced pressure, and hexane (60 mL), ethyl acetate (30 mL) and 20% aqueous potassium carbonate solution (40 mL) were added to the residue, and the organic layer was separated. Anhydrous sodium sulfate was added to the resulting organic layer to dry it, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain a pale yellow oily substance, azanediylbis(ethane-2,1-diyl)bis(decanoic acid) (7.39 g).
MS m/z(M+H):415.

（４）
 (4)

アザンジイルビス（エタン－２，１－ジイル）ビス（デカン酸）(1.00g)、２，２－ジエトキシエチル１Ｈ－１，２，４－トリアゾール－１－カルボキシラート(0.55g)、アセトニトリル(4mL)およびトリエチルアミン(1.0mL)の混合物を、50℃で2時間撹拌した。室温まで冷却した反応混合物に、酢酸エチル(4mL)および水(4mL)を加え、有機層を分取した。得られた有機層に無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、無色油状物の（（（２，２－ジエトキシエトキシ）カルボニル）アザンジイル）ビス（エタン－２，１－ジイル）ビス（デカン酸）(0.27g)を得た。
1H-NMR(CDCl3)δ: 4.70 (1H, t, J=5.5Hz), 4.24-4.16 (4H, m), 4.10 (2H, d, J=5.5Hz), 3.77-3.47 (8H, m), 2.30 (4H, t, J=7.6Hz), 1.66-1.54 (4H, m), 1.35-1.18 (30H, m
), 0.92-0.83 (6H, m). A mixture of azanediylbis(ethane-2,1-diyl)bis(decanoic acid) (1.00 g), 2,2-diethoxyethyl 1H-1,2,4-triazole-1-carboxylate (0.55 g), acetonitrile (4 mL) and triethylamine (1.0 mL) was stirred at 50° C. for 2 hours. Ethyl acetate (4 mL) and water (4 mL) were added to the reaction mixture cooled to room temperature, and the organic layer was separated. Anhydrous sodium sulfate was added to the obtained organic layer to dry it, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain a colorless oily substance, (((2,2-diethoxyethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) (0.27 g).
1 H-NMR(CDCl 3 )δ: 4.70 (1H, t, J=5.5Hz), 4.24-4.16 (4H, m), 4.10 (2H, d, J=5.5Hz), 3.77-3.47 (8H, m), 2.30 (4H, t, J=7.6Hz), 1.66-1.54 (4H, m), 1.35-1.18 (30H, m
), 0.92-0.83 (6H, m).

（５）
　（（（２，２－ジエトキシエトキシ）カルボニル）アザンジイル）ビス（エタン－２，１－ジイル）ビス（デカン酸）(0.27g)、ギ酸(1.2mL)および水(0.3mL) の混合物を、30℃で2時間撹拌した後に、揮発成分を減圧留去した。得られた粗体の（（（２－オキソエトキシ）カルボニル）アザンジイル）ビス（エタン－２，１－ジイル）ビス（デカン酸）に、酢酸エチル(2.4mL)、Ｎ，Ｎ－ジエチルエチレンジアミン(27.6mg)およびトリアセトキシ水素化ホウ素ナトリウム(303mg)を室温で添加し、室温で2時間撹拌した。反応混合物に20%炭酸カリウム水溶液を添加した後、有機層を分取し、水および飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をＮＨシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、淡黄色油状物のビス（２－ヘキシルオクチル）１１－（２－（ジエチルアミノ）エチル）－６，１６－ジオクチル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート (0.14g) （化合物２と称する）を得た。
1H-NMR(CDCl3)δ: 4.22-4.11 (12H, m), 3.56-3.49 (8H, m), 2.79 (4H, t, J=6.4Hz), 2.68-2.63 (2H, m), 2.54-2.47 (6H, m), 2.30 (8H, t, J=7.5Hz), 1.64-1.54 (8H, m), 1.33-1.22 (48H, m), 1.01 (6H, t, J=7.1Hz), 0.88 (12H, t, J=6.8Hz).
MS m/z(M+H):1084. (5)
A mixture of (((2,2-diethoxyethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) (0.27 g), formic acid (1.2 mL) and water (0.3 mL) was stirred at 30° C. for 2 hours, and then the volatile components were distilled off under reduced pressure. Ethyl acetate (2.4 mL), N,N-diethylethylenediamine (27.6 mg) and sodium triacetoxyborohydride (303 mg) were added to the obtained crude (((2-oxoethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) at room temperature, and the mixture was stirred at room temperature for 2 hours. A 20% aqueous solution of potassium carbonate was added to the reaction mixture, and the organic layer was separated and washed with water and saturated saline, and then dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by NH silica gel column chromatography (ethyl acetate-hexane) to obtain a pale yellow oily substance, bis(2-hexyloctyl) 11-(2-(diethylamino)ethyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (0.14 g) (referred to as Compound 2).
1 H-NMR(CDCl 3 )δ: 4.22-4.11 (12H, m), 3.56-3.49 (8H, m), 2.79 (4H, t, J=6.4Hz), 2.68-2.63 (2H, m), 2.54-2.47 (6H, m), 2.30 (8H, t, J=7.5Hz), 1.64-1.54 (8H, m), 1.33-1.22 (48H, m), 1.01 (6H, t, J=7.1Hz), 0.88 (12H, t, J=6.8Hz).
MS m/z(M+H):1084.

［合成例３］
（１）
 [Synthesis Example 3]
(1)

　２－ペンチル－１－ヘプタノール(6.0g)、５-ブロモ吉草酸(6.4g)のトルエン(30mL)混合物に、４－トルエンスルホン酸・一水和物(168mg)を室温で添加した後、加熱還流下、ディーン・スターク装置で水を除きながら2時間撹拌した。反応混合物を室温まで冷却した後に、シリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、無色油状物の２－ペンチルヘプチル５－ブロモペンタン酸(10.8g)を得た。
1H-NMR(CDCl3)δ: 3.98 (2H, d, J=5.8Hz), 3.42 (2H, t, J=6.6Hz), 2.35 (2H, t, 7.2Hz), 1.94-1.87 (2H, m), 1.82-1.74 (2H, m), 1.65-1.59 (1H, m), 1.34-1.23 (16H, m), 0.89 (6H, t, J=6.9Hz). To a mixture of 2-pentyl-1-heptanol (6.0 g), 5-bromovaleric acid (6.4 g) and toluene (30 mL) was added 4-toluenesulfonic acid monohydrate (168 mg) at room temperature, and the mixture was stirred for 2 hours while removing water with a Dean-Stark apparatus under heating under reflux. The reaction mixture was cooled to room temperature and then purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2-pentylheptyl 5-bromopentanoic acid (10.8 g) as a colorless oil.
1 H-NMR(CDCl 3 )δ: 3.98 (2H, d, J=5.8Hz), 3.42 (2H, t, J=6.6Hz), 2.35 (2H, t, 7.2Hz), 1.94-1.87 (2H, m), 1.82-1.74 (2H, m), 1.65-1.59 (1H, m), 1.34-1.23 (16H, m), 0.89 (6H, t, J=6.9Hz).

（２）
 (2)

　２－ペンチルヘプチル５－ブロモペンタン酸(1.2g)、イソプロピルアミン(0.65g)およびアセトニトリル(6mL)の混合物に、炭酸カリウム(1.45g)を添加し、50℃で1時間撹拌した。反応混合物を室温まで冷却した後に、酢酸エチル(24mL)および水(12mL)を添加し、有機層を分取した。有機層を水、飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(ヘキサン－酢酸エチル－メタノール)で精製し、淡黄色油状物の２－ペンチルヘプチル５－（イソプロピルアミノ）ペンタン酸 (0.69g)を得た。
1H-NMR(CDCl3)δ: 3.97 (2H, d, J=5.8Hz), 2.78 (1H, sept, J=6.2Hz), 2.60 (2H, t, J=7.3Hz), 2.33 (2H, t, J=7.5Hz), 1.72-1.40 (6H, m), 1.36-1.20 (16H, m), 1.05 (6H, t, J=6.2Hz), 0.88 (6H, t, J=6.9Hz).
MS m/z(M+H):328. Potassium carbonate (1.45 g) was added to a mixture of 2-pentylheptyl 5-bromopentanoic acid (1.2 g), isopropylamine (0.65 g) and acetonitrile (6 mL), and the mixture was stirred at 50°C for 1 hour. After the reaction mixture was cooled to room temperature, ethyl acetate (24 mL) and water (12 mL) were added, and the organic layer was separated. The organic layer was washed with water and saturated saline, and then dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane-ethyl acetate-methanol) to obtain 2-pentylheptyl 5-(isopropylamino)pentanoic acid (0.69 g) as a pale yellow oil.
1 H-NMR(CDCl 3 )δ: 3.97 (2H, d, J=5.8Hz), 2.78 (1H, sept, J=6.2Hz), 2.60 (2H, t, J=7.3Hz), 2.33 (2H, t, J=7.5Hz), 1.72-1.40 (6H, m), 1.36-1.20 (16H, m), 1.05 (6H, t, J=6.2Hz), 0.88 (6H, t, J=6.9Hz).
MS m/z(M+H):328.

（３）
 (3)

　２－ペンチルヘプチル５－（イソプロピルアミノ）ペンタン酸 (0.38g)、２，２－ジエトキシエチル１Ｈ－１，２，４－トリアゾール－１－カルボキシラート(0.27g)、アセトニトリル(2mL)、トリエチルアミン(0.33mL)およびＮ，Ｎ－ジメチルアミノピリジン(10mg)の混合物を、60℃で3時間撹拌した。室温まで冷却した反応混合物に、酢酸エチル(20mL)および水(20mL)を加え、有機層を分取した。得られた有機層を、飽和塩化アンモニウム水および飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、無色油状物の２－ペンチルヘプチル５－（（（２，２－ジエトキシエトキシ）カルボニル）（イソプロピル）アミノ）ペンタン酸(0.55g)を得た。 A mixture of 2-pentylheptyl 5-(isopropylamino)pentanoic acid (0.38 g), 2,2-diethoxyethyl 1H-1,2,4-triazole-1-carboxylate (0.27 g), acetonitrile (2 mL), triethylamine (0.33 mL) and N,N-dimethylaminopyridine (10 mg) was stirred at 60°C for 3 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction mixture cooled to room temperature, and the organic layer was separated. The organic layer obtained was washed with saturated ammonium chloride solution and saturated saline, then dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue obtained was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2-pentylheptyl 5-(((2,2-diethoxyethoxy)carbonyl)(isopropyl)amino)pentanoic acid (0.55 g) as a colorless oil.

（４）
 (4)

　２－ペンチルヘプチル５－（（（２，２－ジエトキシエトキシ）カルボニル）（イソプロピル）アミノ）ペンタン酸(0.55g)、ギ酸(2.2mL)および水(0.55mL) の混合物を、40℃で2時間撹拌した。室温まで冷却した反応混合物に、酢酸エチルおよび水を加え、有機層を飽和重曹水で２回洗浄した。得られた有機層に無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた粗体の２－ペンチルヘプチル　５－（イソプロピル（（２－オキソエトキシ）カルボニル）アミノ）ペンタン酸(0.15g)に、酢酸エチル(1.5mL)、Ｎ，Ｎ－ジエチルプロパン－１，３－ジアミン(23mg)およびトリアセトキシ水素化ホウ素ナトリウム(0.23g)を室温で添加し、室温で1時間撹拌した。反応混合物に10%炭酸カリウム水溶液を添加した後、有機層を分取し、水および飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(ヘキサン－酢酸エチル－メタノール)、およびＮＨシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、無色油状物のビス（２－ペンチルヘプチル）　１１－（３－（ジエチルアミノ）プロピル）－６，１６－ジイソプロピル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート(0.108g) （化合物３と称する）を得た。
1H-NMR(CDCl3)δ:4.30-4.05 (2H, m), 4.11 (4H, t, J=6.4Hz), 3.97 (4H, d, J=5.8Hz), 3.16-3.00 (4H, m), 2.77 (4H, t, J=6.4Hz), 2.57-2.47 (6H, m), 2.43-2.39 (2H, m), 2.32 (4H, t, J=7.0Hz), 1.66-1.53 (12H, m), 1.35-1.21 (32H, m), 1.13 (12H, d, J=6.8Hz), 1.00 (6H, t, J=7.1Hz), 0.88 (12H, t, J=6.9Hz).
MS m/z(M+H):926. A mixture of 2-pentylheptyl 5-(((2,2-diethoxyethoxy)carbonyl)(isopropyl)amino)pentanoic acid (0.55 g), formic acid (2.2 mL) and water (0.55 mL) was stirred at 40° C. for 2 hours. Ethyl acetate and water were added to the reaction mixture cooled to room temperature, and the organic layer was washed twice with saturated sodium bicarbonate water. Anhydrous sodium sulfate was added to the obtained organic layer to dry it, and the solvent was distilled off under reduced pressure. Ethyl acetate (1.5 mL), N,N-diethylpropane-1,3-diamine (23 mg) and sodium triacetoxyborohydride (0.23 g) were added to the obtained crude 2-pentylheptyl 5-(isopropyl((2-oxoethoxy)carbonyl)amino)pentanoic acid (0.15 g) at room temperature, and the mixture was stirred at room temperature for 1 hour. After adding a 10% aqueous potassium carbonate solution to the reaction mixture, the organic layer was separated and washed with water and saturated saline, then anhydrous sodium sulfate was added for drying, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane-ethyl acetate-methanol) and NH silica gel column chromatography (ethyl acetate-hexane) to obtain a colorless oily substance, bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-6,16-diisopropyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (0.108 g) (referred to as compound 3).
1 H-NMR(CDCl 3 )δ:4.30-4.05 (2H, m), 4.11 (4H, t, J=6.4Hz), 3.97 (4H, d, J=5.8Hz), 3.16-3.00 (4H, m), 2.77 (4H, t, J=6.4Hz), 2.57-2.47 (6H, m), 2.43-2.39 (2H, m), 2.32 (4H, t, J=7.0Hz), 1.66-1.53 (12H, m), 1.35-1.21 (32H, m), 1.13 (12H, d, J=6.8Hz), 1.00 (6H, t, J=7.1Hz), 0.88 (12H, t, J=6.9Hz).
MS m/z(M+H):926.

［合成例４］
 [Synthesis Example 4]

　合成例３（２）において、イソプロピルアミンを用いた代わりにｎ－プロピルアミンを用いること以外は合成例３と同様の方法で、無色油状物のビス（２－ペンチルヘプチル）１１－（３－（ジエチルアミノ）プロピル）－７，１５－ジオキソ－６，１６－ジプロピル－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート（化合物４と称する）を得た。
1H-NMR(CDCl3)δ: 4.10 (4H, t, J=6.4Hz), 3.97 (4H, d, J=5.8Hz), 3.26-3.09 (8H, m), 2.76 (4H, t, J=6.4Hz), 2.57-2.48 (6H, m), 2.43-2.39 (2H, m), 2.34-2.31 (4H, m), 1.66-1.49 (16H, m), 1.36-1.21 (32H, m), 1.01 (6H, t, J=7.1Hz), 0.90-0.84 (18H, m).
MS m/z(M+H):926. A colorless oily substance, bis(2-pentylheptyl)-11-(3-(diethylamino)propyl)-7,15-dioxo-6,16-dipropyl-8,14-dioxa-6,11,16-triazahenicosanedioate (referred to as Compound 4), was obtained in the same manner as in Synthesis example 3, except that n-propylamine was used instead of isopropylamine in Synthesis example 3(2).
1 H-NMR(CDCl 3 )δ: 4.10 (4H, t, J=6.4Hz), 3.97 (4H, d, J=5.8Hz), 3.26-3.09 (8H, m), 2.76 (4H, t, J=6.4Hz), 2.57-2.48 (6H, m), 2.43-2.39 (2H, m), 2.34-2.31 (4H, m), 1.66-1.49 (16H, m), 1.36-1.21 (32H, m), 1.01 (6H, t, J=7.1Hz), 0.90-0.84 (18H, m).
MS m/z(M+H):926.

［合成例５］
 [Synthesis Example 5]

　合成例３（２）において、２－ペンチルヘプチル５－ブロモペンタン酸を用いた代わりに、２－ヘキシルオクチル５－ブロモペンタン酸を用い、イソプロピルアミンを用いた代わりにｎ－ブチルアミンを用いること以外は合成例３と同様の方法で、無色油状物のビス（２－ヘキシルオクチル）６，１６－ジブチル－１１－（３－（ジエチルアミノ）プロピル）－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート（化合物５と称する）を得た。
1H-NMR(CDCl3)δ: 4.09 (4H, t, J=6.4Hz), 3.96 (4H, d, J=5.8Hz), 3.26-3.12 (8H, m), 2.76 (4H, t, J=6.4Hz), 2.66-2.36 (8H, m), 2.32 (4H, t, J=6.2Hz), 1.66-1.43 (16H, m), 1.36-1.18 (44H, m), 1.08-0.95 (6H, m), 0.95-0.83(18H, m).
MS m/z(M+H):1010. A colorless oily substance, bis(2-hexyloctyl)6,16-dibutyl-11-(3-(diethylamino)propyl)-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (referred to as Compound 5), was obtained in the same manner as in Synthesis example 3, except that in Synthesis example 3(2), 2-hexyloctyl 5-bromopentanoic acid was used instead of 2-pentylheptyl 5-bromopentanoic acid, and n-butylamine was used instead of isopropylamine.
1 H-NMR(CDCl 3 )δ: 4.09 (4H, t, J=6.4Hz), 3.96 (4H, d, J=5.8Hz), 3.26-3.12 (8H, m), 2.76 (4H, t, J=6.4Hz), 2.66-2.36 (8H, m), 2.32 (4H, t, J=6.2Hz), 1.66-1.43 (16H, m), 1.36-1.18 (44H, m), 1.08-0.95 (6H, m), 0.95-0.83(18H, m).
MS m/z(M+H):1010.

［合成例６］
（１）
 [Synthesis Example 6]
(1)

　Ｎ－（ｔｅｒｔ－ブトキシカルボニル）イミノ二酢酸(2.00g)および１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩(1.97g)のジクロロメタン(20mL)混合物に、室温下で１－トリデカノール(3.44g)、トリエチルアミン(5.98mL)およびＮ，Ｎ－ジメチルアミノピリジン(1.05g)を添加し10分間撹拌した。この反応混合物に、１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩(1.97g)を追加し、40℃にて4時間撹拌した。反応混合物に水(20mL)を添加し、有機層を分取した。水層に酢酸エチル(20mL)を添加し、有機層を飽和食塩水で洗浄し、先に得られた有機層と合わせた後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。残留物をシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、無色油状物のジトリデシル２，２’－（（ｔｅｒｔ－ブトキシカルボニル）アザンジイル）ジアセテート (4.26g)を得た。
1H-NMR(CDCl3)δ: 4.17-3.94 (8H, m), 1.68-1.57 (4H, m), 1.44 (9H, s), 1.38-1.17 (40H, m), 0.92-0.84 (6H, m). To a mixture of N-(tert-butoxycarbonyl)iminodiacetic acid (2.00 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.97 g) in dichloromethane (20 mL), 1-tridecanol (3.44 g), triethylamine (5.98 mL) and N,N-dimethylaminopyridine (1.05 g) were added at room temperature and stirred for 10 minutes. To this reaction mixture, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.97 g) was added and stirred at 40° C. for 4 hours. Water (20 mL) was added to the reaction mixture, and the organic layer was separated. Ethyl acetate (20 mL) was added to the aqueous layer, the organic layer was washed with saturated saline, and the organic layer was combined with the organic layer obtained earlier, and then anhydrous sodium sulfate was added to dry, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain a colorless oily matter of ditridecyl 2,2'-((tert-butoxycarbonyl)azanediyl)diacetate (4.26 g).
1H -NMR( CDCl3 )δ: 4.17-3.94 (8H, m), 1.68-1.57 (4H, m), 1.44 (9H, s), 1.38-1.17 (40H, m), 0.92-0.84 (6H, m).

（２）
 (2)

　ジトリデシル２，２’－（（ｔｅｒｔ－ブトキシカルボニル）アザンジイル）ジアセテート(4.26g)、トルエン(2mL)および水(0.3mL)の混合物に、氷冷下トリフルオロ酢酸(6.0mL)を添加し、室温にて1時間撹拌して減圧留去した。残留物にトルエン(20mL)を添加し、減圧留去する操作を3回繰り返し、得られた残留物にヘキサン(40mL)を添加し、氷冷下撹拌した。析出した固体をろ取し、白色固体のジトリデシル２，２’－アザンジイルジアセテートのトリフルオロ酢酸塩(4.69g)を得た。
1H-NMR(CDCl3)δ: 5.57 (2H, brs), 4.22 (4H, t, J=6.8Hz), 4.00 (4H, s), 1.71-1.59 (4H, m), 1.39-1.16 (40H, m), 0.93-0.83 (6H, m). To a mixture of ditridecyl 2,2'-((tert-butoxycarbonyl)azanediyl)diacetate (4.26 g), toluene (2 mL) and water (0.3 mL), trifluoroacetic acid (6.0 mL) was added under ice cooling, the mixture was stirred at room temperature for 1 hour and then distilled off under reduced pressure. Toluene (20 mL) was added to the residue and the mixture was distilled off under reduced pressure, which was repeated three times. Hexane (40 mL) was added to the resulting residue and the mixture was stirred under ice cooling. The precipitated solid was collected by filtration to obtain a white solid trifluoroacetate salt of ditridecyl 2,2'-azanediyldiacetate (4.69 g).
1 H-NMR(CDCl 3 )δ: 5.57 (2H, brs), 4.22 (4H, t, J=6.8Hz), 4.00 (4H, s), 1.71-1.59 (4H, m), 1.39-1.16 (40H, m), 0.93-0.83 (6H, m).

（３）
 (3)

　ジトリデシル２，２’－アザンジイルジアセテートのトリフルオロ酢酸塩(1.50g)、２，２－ジエトキシエチル１Ｈ－１，２，４－トリアゾール－１－カルボキシラート(0.56g)、アセトニトリル(7.5mL)、トリエチルアミン(1.03mL)およびＮ，Ｎ－ジメチルアミノピリジン(0.30g)の混合物を、70℃で3時間、80℃で1時間撹拌した。室温まで冷却した反応混合物に、酢酸エチル(10mL)および水(5mL)を加え、有機層を分取した。得られた有機層を飽和食塩水で洗浄した後、無水硫酸ナトリウムを加えて乾燥させ、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィー(酢酸エチル－ヘキサン)で精製し、無色油状物のジトリデシル２，２’－（（（２，２－ジエトキシエトキシ）カルボニル）アザンジイル）ジアセテート(1.06g)を得た。
1H-NMR(CDCl3)δ: 4.66 (1H, t, J=5.5Hz), 4.18-4.06 (10H, m), 3.75-3.49 (4H, m), 1.68-1.57 (4H, m), 1.37-1.20 (40H, m), 1.21 (6H, t, J=7.1Hz), 0.92-0.84 (6H, m). A mixture of ditridecyl 2,2'-azanediyl diacetate trifluoroacetate (1.50 g), 2,2-diethoxyethyl 1H-1,2,4-triazole-1-carboxylate (0.56 g), acetonitrile (7.5 mL), triethylamine (1.03 mL) and N,N-dimethylaminopyridine (0.30 g) was stirred at 70°C for 3 hours and at 80°C for 1 hour. Ethyl acetate (10 mL) and water (5 mL) were added to the reaction mixture cooled to room temperature, and the organic layer was separated. The organic layer obtained was washed with saturated saline, and then dried by adding anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain a colorless oily substance, ditridecyl 2,2'-(((2,2-diethoxyethoxy)carbonyl)azanediyl)diacetate (1.06 g).
1 H-NMR(CDCl 3 )δ: 4.66 (1H, t, J=5.5Hz), 4.18-4.06 (10H, m), 3.75-3.49 (4H, m), 1.68-1.57 (4H, m), 1.37-1.20 (40H, m), 1.21 (6H, t, J=7.1Hz), 0.92-0.84 (6H, m).

（４）
 (4)

　合成例２（５）において、（（（２，２－ジエトキシエトキシ）カルボニル）アザンジイル）ビス（エタン－２，１－ジイル）ビス（デカン酸）を用いた代わりに、ジトリデシル２，２’－（（（２，２－ジエトキシエトキシ）カルボニル）アザンジイル）ジアセテートを用いること以外は合成例２（５）と同様の方法で、無色油状物のジトリデシル８－（２－（ジエチルアミノ）エチル）－４，１２－ジオキソ－３，１３－ビス（２－オキソ－２－（トリデシルオキシ）エチル）－５，１１－ジオキサ－３，８，１３－トリアザペンタデカンジオエート（化合物６と称する）を得た。
1H-NMR(CDCl3)δ: 4.19-4.06 (20H, m), 2.84-2.71 (4H, m), 2.68-2.56 (2H, m), 2.55-2.41 (6H, m), 1.69-1.49 (16H, m), 1.38-1.18 (72H, m), 1.07-0.95 (6H, m), 0.88 (12H, t, J=6.8Hz). A colorless oily substance, ditridecyl 8-(2-(diethylamino)ethyl)-4,12-dioxo-3,13-bis(2-oxo-2-(tridecyloxy)ethyl)-5,11-dioxa-3,8,13-triazapentadecanedioate (referred to as Compound 6), was obtained in the same manner as in Synthesis example 2(5), except that ditridecyl 2,2'-(((2,2-diethoxyethoxy)carbonyl)azanediyl)diacetate was used instead of (((2,2-diethoxyethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) in Synthesis example 2(5).
1 H-NMR(CDCl 3 )δ: 4.19-4.06 (20H, m), 2.84-2.71 (4H, m), 2.68-2.56 (2H, m), 2.55-2.41 (6H, m), 1.69-1.49 (16H, m), 1.38-1.18 (72H, m), 1.07-0.95 (6H, m), 0.88 (12H, t, J=6.8Hz).

［合成例７］
 [Synthesis Example 7]

　合成例３（３）において、２－ペンチルヘプチル５－（イソプロピルアミノ）ペンタン酸を用いた代わりに、２－ヘキシルオクチル５－（オクチルアミノ）ペンタン酸を用いること以外は　合成例３と同様の方法で、ビス（２－ヘキシルオクチル）１１－（３－（ジエチルアミノ）プロピル）－６，１６－ジオクチル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート（化合物７と称する）を得た。
1H-NMR(CDCl3)δ: 4.09 (4H, t, J=6.4Hz), 3.96 (4H, d, J=5.8Hz), 3.27-3.07 (8H, m), 2.76 (4H, t, J=6.4Hz), 2.67-2.36 (8H, m), 2.32 (4H, t, J=6.4Hz), 1.67-1.44 (16H, m), 1.36-1.16 (60H, m), 1.06-0.96 (6H, m), 0.92-0.82 (18H, m).
MS m/z(M+H):1123. Bis(2-hexyloctyl)11-(3-(diethylamino)propyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (referred to as Compound 7) was obtained in the same manner as in Synthesis example 3, except that 2-hexyloctyl 5-(octylamino)pentanoic acid was used instead of 2-pentylheptyl 5-(isopropylamino)pentanoic acid in Synthesis example 3(3).
1 H-NMR(CDCl 3 )δ: 4.09 (4H, t, J=6.4Hz), 3.96 (4H, d, J=5.8Hz), 3.27-3.07 (8H, m), 2.76 (4H, t, J=6.4Hz), 2.67-2.36 (8H, m), 2.32 (4H, t, J=6.4Hz), 1.67-1.44 (16H, m), 1.36-1.16 (60H, m), 1.06-0.96 (6H, m), 0.92-0.82 (18H, m).
MS m/z(M+H):1123.

［合成例８］
 [Synthesis Example 8]

　ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 11-(2-(ジエチルアミノ)エチル)-7,15-ジオキソ-6,16-ジプロピル-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物８）を合成した。 Bis(2-pentylheptyl) 11-(2-(diethylamino)ethyl)-7,15-dioxo-6,16-dipropyl-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 8) was synthesized according to the examples described in WO2024/158042.

［合成例９］
 [Synthesis Example 9]

　ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 6,16-ジブチル-11-(2-(ジエチルアミノ)エチル)-7,15-ジオキソ-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物９）を合成した。 Bis(2-pentylheptyl) 6,16-dibutyl-11-(2-(diethylamino)ethyl)-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 9) was synthesized according to the examples described in WO2024/158042.

［合成例１０］
 [Synthesis Example 10]

　ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 11-(2-(ジエチルアミノ)エチル)-7,15-ジオキソ-6,16-ジペンチル-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１０）を合成した。 Bis(2-pentylheptyl) 11-(2-(diethylamino)ethyl)-7,15-dioxo-6,16-dipentyl-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 10) was synthesized according to the examples described in WO2024/158042.

［合成例１１］
 [Synthesis Example 11]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 11-(2-(ジエチルアミノ)エチル)-6,16-ジオクチル-7,15-ジオキソ-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１１）を合成した。 Bis(2-pentylheptyl) 11-(2-(diethylamino)ethyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 11) was synthesized according to the examples described in WO2024/158042.

［合成例１２］
 [Synthesis Example 12]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 6,16-ジブチル-11-(3-(ジエチルアミノ)プロピル)-7,15-ジオキソ-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１２）を合成した。 Bis(2-pentylheptyl) 6,16-dibutyl-11-(3-(diethylamino)propyl)-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 12) was synthesized according to the examples described in WO2024/158042.

［合成例１３］
 [Synthesis Example 13]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 11-(3-(ジエチルアミノ)プロピル)-7,15-ジオキソ-6,16-ジペンチル-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１３）を合成した。 Bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-7,15-dioxo-6,16-dipentyl-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 13) was synthesized according to the examples described in WO2024/158042.

［合成例１４］
 [Synthesis Example 14]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 11-(3-(ジエチルアミノ)プロピル)-6,16-ジヘプチル-7,15-ジオキソ-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１４）を合成した。 Bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-6,16-diheptyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 14) was synthesized according to the examples described in WO2024/158042.

［合成例１５］
 [Synthesis Example 15]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 11-(3-(ジエチルアミノ)プロピル)-6,16-ジオクチル-7,15-ジオキソ-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１５）を合成した。 Bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 15) was synthesized according to the examples described in WO2024/158042.

［合成例１６］
 [Synthesis Example 16]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 11-(4-(ジエチルアミノ)ブチル)-6,16-ジオクチル-7,15-ジオキソ-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１６）を合成した。 Bis(2-pentylheptyl) 11-(4-(diethylamino)butyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 16) was synthesized according to the examples described in WO2024/158042.

［合成例１７］
 [Synthesis Example 17]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 11-(2-(ジエチルアミノ)エチル)-6,16-ジヘプチル-7,15-ジオキソ-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１７）を合成した。 Bis(2-pentylheptyl) 11-(2-(diethylamino)ethyl)-6,16-diheptyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 17) was synthesized according to the examples described in WO2024/158042.

［合成例１８］
 [Synthesis Example 18]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル) 11-(2-(ジエチルアミノ)エチル)-6,16-ジヘキシル-7,15-ジオキソ-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１８）を合成した。 Bis(2-pentylheptyl) 11-(2-(diethylamino)ethyl)-6,16-dihexyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 18) was synthesized according to the examples described in WO2024/158042.

［合成例１９］
 [Synthesis Example 19]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ヘキシルオクチル) 11-(4-(ジエチルアミノ)ブチル)-6,16-ジオクチル-7,15-ジオキソ-8,14-ジオキサ-6,11,16-トリアザヘニコサンジオエート（化合物１９）を合成した。 Bis(2-hexyloctyl) 11-(4-(diethylamino)butyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (compound 19) was synthesized according to the examples described in WO2024/158042.

［合成例２０］
 [Synthesis Example 20]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル)12-(2-(ジエチルアミノ)エチル)-7,17-ジオクチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオエート（化合物２０）を合成した。 Bis(2-pentylheptyl) 12-(2-(diethylamino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 20) was synthesized according to the examples described in WO2024/158042.

［合成例２１］
 [Synthesis Example 21]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル)12-(2-(ジエチルアミノ)エチル)-7,17-ジヘプチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオエート（化合物２１）を合成した。 Bis(2-pentylheptyl) 12-(2-(diethylamino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 21) was synthesized according to the examples described in WO2024/158042.

［合成例２２］
 [Synthesis Example 22]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル)12-(2-(ジエチルアミノ)エチル)-8,16-ジオキソ-7,17-ジペンチル-9,15-ジオキサ-7,12,17-トリアザトリコサンジオエート（化合物２２）を合成した。 Bis(2-pentylheptyl) 12-(2-(diethylamino)ethyl)-8,16-dioxo-7,17-dipentyl-9,15-dioxa-7,12,17-triazatricosane dioate (compound 22) was synthesized according to the examples described in WO2024/158042.

［合成例２３］
 [Synthesis Example 23]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ヘキシルオクチル)7,17-ジブチル-12-(2-(ジエチルアミノ)エチル)-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオエート（化合物２３）を合成した。 Bis(2-hexyloctyl) 7,17-dibutyl-12-(2-(diethylamino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 23) was synthesized according to the examples described in WO2024/158042.

［合成例２４］
 [Synthesis Example 24]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル)12-(3-(ジエチルアミノ)プロピル)-7,17-ジオクチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオエート（化合物２４）を合成した。 Bis(2-pentylheptyl)12-(3-(diethylamino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 24) was synthesized according to the examples described in WO2024/158042.

［合成例２５］
 [Synthesis Example 25]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル)12-(3-(ジエチルアミノ)プロピル)-7,17-ジヘプチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオエート（化合物２５）を合成した。 Bis(2-pentylheptyl) 12-(3-(diethylamino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 25) was synthesized according to the examples described in WO2024/158042.

［合成例２６］
 [Synthesis Example 26]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ビス(2-ペンチルヘプチル)12-(3-(ジエチルアミノ)プロピル)-8,16-ジオキソ-7,17-ジペンチル-9,15-ジオキサ-7,12,17-トリアザトリコサンジオエート（化合物２６）を合成した。 Bis(2-pentylheptyl)12-(3-(diethylamino)propyl)-8,16-dioxo-7,17-dipentyl-9,15-dioxa-7,12,17-triazatricosane dioate (compound 26) was synthesized according to the examples described in WO2024/158042.

［合成例２７］
 [Synthesis Example 27]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、2-(2-(2-(ビス(2-ドデカノイルオキシエチル)カルバモイルオキシ)エチル-(2-(ジエチルアミノ)エチル)アミノ)エトキシカルボニル-(2-ドデカノイルオキシエチル)アミノ)エチル ドデカノエート（化合物２７）を合成した。 2-(2-(2-(bis(2-dodecanoyloxyethyl)carbamoyloxy)ethyl-(2-(diethylamino)ethyl)amino)ethoxycarbonyl-(2-dodecanoyloxyethyl)amino)ethyl dodecanoate (compound 27) was synthesized according to the examples described in WO2024/158042.

［合成例２８］
 [Synthesis Example 28]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、デシル 2-(2-(2-(ビス(2-デコキシ-2-オキソ-エチル)カルバモイルオキシ)エチル-(2-(ジエチルアミノ)エチル)アミノ)エトキシカルボニル-(2-デコキシ-2-オキソ-エチル)アミノ）アセテート（化合物２８）を合成した。 Decyl 2-(2-(2-(bis(2-decoxy-2-oxo-ethyl)carbamoyloxy)ethyl-(2-(diethylamino)ethyl)amino)ethoxycarbonyl-(2-decoxy-2-oxo-ethyl)amino)acetate (compound 28) was synthesized according to the examples described in WO2024/158042.

［合成例２９］
 [Synthesis Example 29]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ジドデシル 8-(2-(ジエチルアミノ)エチル)-3,13-ビス(2-(ドデシルオキシ)-2-オキソエチル)-4,12-ジオキソ-5,11-ジオキサ-3,8,13-トリアザペンタデカンジオエート（化合物２９）を合成した。 Didodecyl 8-(2-(diethylamino)ethyl)-3,13-bis(2-(dodecyloxy)-2-oxoethyl)-4,12-dioxo-5,11-dioxa-3,8,13-triazapentadecanedioate (compound 29) was synthesized according to the examples described in WO2024/158042.

［合成例３０］
 [Synthesis Example 30]

ＷＯ２０２４／１５８０４２に記載の実施例に従って、ジウンデシル 8-(2-(ジエチルアミノ)エチル)-4,12-ジオキソ-3,13-ビス(2-オキソ-2-(ウンデシルオキシ)エチル)-5,11-ジオキサ-3,8,13-トリアザペンタデカンジオエート（化合物３０）を合成した。 Diundecyl 8-(2-(diethylamino)ethyl)-4,12-dioxo-3,13-bis(2-oxo-2-(undecyloxy)ethyl)-5,11-dioxa-3,8,13-triazapentadecanedioate (compound 30) was synthesized according to the examples described in WO2024/158042.

［合成例３１］
 [Synthesis Example 31]

ＷＯ２０２４／１５８０４２に記載の実施例に従って合成した6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチル(1.0g)の酢酸エチル(10mL)溶液を0℃に冷却し、4-(ジエチルアミノ)ブチルアミン(0.14g)、トリアセトキシボロヒドリドナトリウム(1.3g)を順次加えた。冷却媒を外して室温で1時間攪拌し、反応完結を確認した後に10%炭酸水素ナトリウム水溶液を加えて反応を停止した。有機層を10%炭酸水素ナトリウム水溶液、ついで飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥、溶媒を留去した。得られた残さをシリカゲルカラムクロマトグラフィー(酢酸エチル/メタノール)、ついでNHシリカゲルカラムクロマトグラフィー(ヘキサン/酢酸エチル)で精製し、ビス(2-ペンチルヘプチル) 12-(4-(ジエチルアミノ)ブチル)-7,17-ジヘプチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物３１）（594mg）を無色油状物として得た。
LC/MS
rt(min): 1.73
MS(ESI,m/z): 1080.3 [M+H]+ A solution of 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate (1.0 g) synthesized according to the example described in WO2024/158042 in ethyl acetate (10 mL) was cooled to 0° C., and 4-(diethylamino)butylamine (0.14 g) and sodium triacetoxyborohydride (1.3 g) were added in that order. The cooling medium was removed and the mixture was stirred at room temperature for 1 hour. After confirming the completion of the reaction, a 10% aqueous solution of sodium hydrogen carbonate was added to quench the reaction. The organic layer was washed with a 10% aqueous solution of sodium hydrogen carbonate and then with saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off. The obtained residue was purified by silica gel column chromatography (ethyl acetate/methanol) and then NH silica gel column chromatography (hexane/ethyl acetate) to obtain bis(2-pentylheptyl) 12-(4-(diethylamino)butyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosandioate (compound 31) (594 mg) as a colorless oil.
LC/MS
rt(min): 1.73
MS(ESI,m/z): 1080.3 [M+H] +

［合成例３２］
 [Synthesis Example 32]

6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび3-(ジメチルアミノ)プロピルアミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 12-(3-(ジメチルアミノ)プロピル)-7,17-ジヘプチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物３２）を合成した。
LC/MS
rt(min): 1.52
MS(ESI,m/z): 1038.3 [M+H]+ Bis(2-pentylheptyl) 12-(3-(dimethylamino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 32) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 3-(dimethylamino)propylamine.
LC/MS
rt(min): 1.52
MS(ESI,m/z): 1038.3 [M+H] +

［合成例３３］
 [Synthesis Example 33]

6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび1-メチルピペリジン-4-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-12-(1-メチルピペリジン-4-イル)-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物３３）を合成した。
LC/MS
rt(min): 1.77
MS(ESI,m/z): 1050.3 [M+H]+ Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 33) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 1-methylpiperidin-4-amine.
LC/MS
rt(min): 1.77
MS(ESI,m/z): 1050.3 [M+H] +

［合成例３４］
 [Synthesis Example 34]

6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび1-エチルピペリジン-4-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 12-(1-エチルピペリジン-4-イル)-7,17-ジヘプチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物３４）を合成した。
LC/MS
rt(min): 1.48
MS(ESI,m/z): 1065.2 [M+H]+ Bis(2-pentylheptyl) 12-(1-ethylpiperidin-4-yl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 34) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 1-ethylpiperidin-4-amine.
LC/MS
rt(min): 1.48
MS(ESI,m/z): 1065.2 [M+H] +

［合成例３５］
 [Synthesis Example 35]

6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび1-イソプロピルピペリジン-4-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-12-(1-イソプロピルピペリジン-4-イル)-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物３５）を合成した。
LC/MS
rt(min): 1.52
MS(ESI,m/z): 1079.3 [M+H]+ Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-isopropylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 35) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 1-isopropylpiperidin-4-amine.
LC/MS
rt(min): 1.52
MS(ESI,m/z): 1079.3 [M+H] +

［合成例３６］
 [Synthesis Example 36]

（１）
(2-ブロモエチル)カルバミン酸tert-ブチル（A）(1.12g)、炭酸カリウム(2.0g)のジメチルホルムアミド(10mL)溶液に、4-(エチルアミノ)ブタン-1-オール（II）(1.3mL)を加え、80℃で30分間攪拌した。反応液を酢酸エチル/１％塩酸で分液し、得られた水層を炭酸カリウム(5g)で塩基性にした後、酢酸エチルで抽出した。無水硫酸ナトリウムで乾燥し、溶媒を留去することで、tert-ブチル (2-(エチル(4-ヒドロキシブチル)アミノ)エチル)カルバメート（IIA）(1.08g)を無色油状物として得た。
LC/MS
rt(min): 0.63
MS(ESI,m/z): 261.3 [M+H]+ (1)
4-(ethylamino)butan-1-ol (II) (1.3 mL) was added to a solution of tert-butyl (2-bromoethyl)carbamate (A) (1.12 g) and potassium carbonate (2.0 g) in dimethylformamide (10 mL), and the mixture was stirred at 80° C. for 30 minutes. The reaction solution was separated with ethyl acetate/1% hydrochloric acid, and the resulting aqueous layer was made basic with potassium carbonate (5 g) and then extracted with ethyl acetate. The mixture was dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain tert-butyl (2-(ethyl(4-hydroxybutyl)amino)ethyl)carbamate (IIA) (1.08 g) as a colorless oil.
LC/MS
rt(min): 0.63
MS(ESI,m/z): 261.3 [M+H] +

（２）
tert-ブチル (2-(エチル(4-ヒドロキシブチル)アミノ)エチル)カルバメート（IIA）(650mg)にトリフルオロ酢酸(5mL)を加え、室温で30分間攪拌した。減圧下にてトリフルオロ酢酸を留去し、得られた残さをイオン交換樹脂(ダイヤイオンSA10A（三菱ケミカル）、OH型に再生)を用いて脱塩し、エタノールで共沸脱水することで、4-((2-アミノエチル)(エチル)アミノ)ブタン-1-オール（IIA-NH2）を得た。
LC/MS
rt(min): 0.19
MS(ESI,m/z): 161.2 [M+H]+ (2)
Trifluoroacetic acid (5 mL) was added to tert-butyl (2-(ethyl(4-hydroxybutyl)amino)ethyl)carbamate (IIA) (650 mg) and stirred at room temperature for 30 minutes. Trifluoroacetic acid was removed under reduced pressure, and the resulting residue was desalted using an ion exchange resin (Diaion SA10A (Mitsubishi Chemical), regenerated to OH type) and azeotropically dehydrated with ethanol to obtain 4-((2-aminoethyl)(ethyl)amino)butan-1-ol (IIA- NH2 ).
LC/MS
rt(min): 0.19
MS(ESI,m/z): 161.2 [M+H] +

（３）
6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチル(0.50g)および4-((2-アミノエチル)(エチル)アミノ)ブタン-1-オール（IIA-NH2）(93mg)を用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 12-(2-(エチル(4-ヒドロキシブチル)アミノ)エチル)-7,17-ジヘプチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート
（化合物３６）(375mg)を得た。
LC/MS
rt(min): 1.43
MS(ESI,m/z): 1097.2 [M+H]+ (3)
Using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate (0.50 g) and 4-((2-aminoethyl)(ethyl)amino)butan-1-ol (IIA-NH 2 ) (93 mg), bis(2-pentylheptyl) 12-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 36) (375 mg) was obtained in the same manner as in Synthesis Example 31.
LC/MS
rt(min): 1.43
MS(ESI,m/z): 1097.2 [M+H] +

［合成例３７～５９］
合成例３６（１）～（３）と同様の方法で、
4-(メチルアミノ)ブタン-1-オール（I）、
4-(エチルアミノ)ブタン-1-オール（II）、
3-(メチルアミノ)プロパン-1-オール（III）、
3-(エチルアミノ)プロパン-1-オール（IV）、
2-(メチルアミノ)エタン-1-オール（V）、
2-(エチルアミノ)エタン-1-オール（VI）、
および、
(2-ブロモエチル)カルバミン酸tert-ブチル（A）、
(3-ブロモプロピル)カルバミン酸tert-ブチル（B）、
を用いて、
tert-ブチル (2-((4-ヒドロキシブチル)(メチル)アミノ)エチル)カルバメート（IA）、
tert-ブチル (3-((4-ヒドロキシブチル)(メチル)アミノ)プロピル)カルバメート（IB）、
tert-ブチル (2-(エチル(4-ヒドロキシブチル)アミノ)エチル)カルバメート（IIA）、
tert-ブチル (3-(エチル(4-ヒドロキシブチル)アミノ)プロピル)カルバメート（IIB）、
tert-ブチル (2-((3-ヒドロキシプロピル)(メチル)アミノ)エチル)カルバメート（IIIA）、
tert-ブチル (3-((3-ヒドロキシプロピル)(メチル)アミノ)プロピル)カルバメート（IIIB）、
tert-ブチル (2-(エチル(3-ヒドロキシプロピル)アミノ)エチル)カルバメート（IVA）、
tert-ブチル (3-(エチル(3-ヒドロキシプロピル)アミノ)プロピル)カルバメート（IVB）、
tert-ブチル (2-((2-ヒドロキシエチル)(メチル)アミノ)エチル)カルバメート（VA）、
tert-ブチル (3-((2-ヒドロキシエチル)(メチル)アミノ)プロピル)カルバメート（VB）、
tert-ブチル (2-(エチル(2-ヒドロキシエチル)アミノ)エチル)カルバメート（VIA）、
および、
tert-ブチル (3-(エチル(2-ヒドロキシエチル)アミノ)プロピル)カルバメート（VIB）、
を合成し、次いでBOC基を脱保護することで、
4-((2-アミノエチル)(メチル)アミノ)ブタン-1-オール（IA-NH2）、
4-((3-アミノプロピル)(メチル)アミノ)ブタン-1-オール（IB-NH2）、
4-((2-アミノエチル)(エチル)アミノ)ブタン-1-オール（IIA-NH2）、
4-((3-アミノプロピル)(エチル)アミノ)ブタン-1-オール（IIB-NH2）、
3-((2-アミノエチル)(メチル)アミノ)プロパン-1-オール（IIIA-NH2）、
3-((3-アミノプロピル)(メチル)アミノ)プロパン-1-オール（IIIB-NH2）、
3-((2-アミノエチル)(エチル)アミノ)プロパン-1-オール（IVA-NH2）、
3-((3-アミノプロピル)(エチル)アミノ)プロパン-1-オール（IVB-NH2）、
2-((2-アミノエチル)(メチル)アミノ)エタン-1-オール（VA-NH2）、
2-((3-アミノプロピル)(メチル)アミノ)エタン-1-オール（VB-NH2）、
2-((2-アミノエチル)(エチル)アミノ)エタン-1-オール（VIA-NH2）、
および、
2-((3-アミノプロピル)(エチル)アミノ)エタン-1-オール（VIB-NH2）、
を合成し（表１）、これら得られたアミンと、
6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチル、または、6-(オクチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸 2-ペンチルを用いて、合成例３１と同様の方法により化合物（化合物３７－化合物５９）を合成した（表２）。 [Synthesis Examples 37 to 59]
In the same manner as in Synthesis Example 36 (1) to (3),
4-(methylamino)butan-1-ol (I),
4-(ethylamino)butan-1-ol (II),
3-(methylamino)propan-1-ol (III),
3-(ethylamino)propan-1-ol (IV),
2-(methylamino)ethan-1-ol (V),
2-(ethylamino)ethan-1-ol (VI),
and,
tert-Butyl (2-bromoethyl)carbamate (A),
tert-Butyl (3-bromopropyl)carbamate (B),
Using
tert-Butyl (2-((4-hydroxybutyl)(methyl)amino)ethyl)carbamate (IA),
tert-Butyl (3-((4-hydroxybutyl)(methyl)amino)propyl)carbamate (IB),
tert-Butyl (2-(ethyl(4-hydroxybutyl)amino)ethyl)carbamate (IIA),
tert-Butyl (3-(ethyl(4-hydroxybutyl)amino)propyl)carbamate (IIB),
tert-Butyl (2-((3-hydroxypropyl)(methyl)amino)ethyl)carbamate (IIIA),
tert-Butyl (3-((3-hydroxypropyl)(methyl)amino)propyl)carbamate (IIIB),
tert-Butyl (2-(ethyl(3-hydroxypropyl)amino)ethyl)carbamate (IVA),
tert-Butyl (3-(ethyl(3-hydroxypropyl)amino)propyl)carbamate (IVB),
tert-Butyl (2-((2-hydroxyethyl)(methyl)amino)ethyl)carbamate (VA),
tert-Butyl (3-((2-hydroxyethyl)(methyl)amino)propyl)carbamate (VB),
tert-Butyl (2-(ethyl(2-hydroxyethyl)amino)ethyl)carbamate (VIA),
and,
tert-Butyl (3-(ethyl(2-hydroxyethyl)amino)propyl)carbamate (VIB),
Then, the BOC group is deprotected to give
4-((2-aminoethyl)(methyl)amino)butan-1-ol (IA- NH2 ),
4-((3-aminopropyl)(methyl)amino)butan-1-ol (IB- NH2 ),
4-((2-aminoethyl)(ethyl)amino)butan-1-ol (IIA-NH 2 ),
4-((3-aminopropyl)(ethyl)amino)butan-1-ol (IIB-NH 2 ),
3-((2-aminoethyl)(methyl)amino)propan-1-ol (IIIA-NH 2 ),
3-((3-aminopropyl)(methyl)amino)propan-1-ol (IIIB-NH 2 ),
3-((2-aminoethyl)(ethyl)amino)propan-1-ol (IVA- NH2 ),
3-((3-aminopropyl)(ethyl)amino)propan-1-ol (IVB-NH 2 ),
2-((2-aminoethyl)(methyl)amino)ethan-1-ol (VA- NH2 ),
2-((3-aminopropyl)(methyl)amino)ethan-1-ol (VB- NH2 ),
2-((2-aminoethyl)(ethyl)amino)ethan-1-ol (VIA- NH2 ),
and,
2-((3-aminopropyl)(ethyl)amino)ethan-1-ol (VIB- NH2 ),
(Table 1) and the resulting amines and
Compounds (Compounds 37 to 59) were synthesized in the same manner as in Synthesis example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate or 2-pentyl 6-(octyl((2-oxoethoxy)carbonyl)amino)hexanoate (Table 2).









［合成例６０］
 [Synthesis Example 60]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび8-メチル-8-アザビシクロ[3.2.1]オクタン-3-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-12-(8-メチル-8-アザビシクロ[3.2.1]オクタン-3-イル)-8,16-ジオキソ-9,15-ジオキサ-7,12、 17-トリアザトリコサン二酸（化合物６０）を合成した。
LC/MS
rt(min): 1.52
MS(ESI,m/z): 1177.3 [M+H]+ Bis(2-pentylheptyl)7,17-diheptyl-12-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diacid (compound 60) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 8-methyl-8-azabicyclo[3.2.1]octan-3-amine.
LC/MS
rt(min): 1.52
MS(ESI,m/z): 1177.3 [M+H] +

［合成例６１］
 [Synthesis Example 61]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび1,2,2,6,6-ペンタメチルピペリジン-4-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-8,16-ジオキソ-12-(1,2,2,6,6-ペンタメチルピペリジン-4-イル)-9,15-ジオキサ-7,12,17-トリアザトリコサン二酸塩（化合物６１）を合成した。
LC/MS
rt(min): 1.55
MS(ESI,m/z): 1107.3 [M+H]+ Bis(2-pentylheptyl)7,17-diheptyl-8,16-dioxo-12-(1,2,2,6,6-pentamethylpiperidin-4-yl)-9,15-dioxa-7,12,17-triazatricosane diacid salt (compound 61) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 1,2,2,6,6-pentamethylpiperidin-4-amine.
LC/MS
rt(min): 1.55
MS(ESI,m/z): 1107.3 [M+H] +

［合成例６２］
 [Synthesis Example 62]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび1-メチルアゼチジン-3-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-12-(1-メチルアゼチジン-3-イル)-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物６２）を合成した。
LC/MS
rt(min): 1.46
MS(ESI,m/z): 1023.1 [M+H]+ Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazetidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 62) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 1-methylazetidin-3-amine.
LC/MS
rt(min): 1.46
MS(ESI,m/z): 1023.1 [M+H] +

［合成例６３］
 [Synthesis Example 63]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび1-メチルピロリジン-3-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-12-(1-メチルピロリジン-3-イル)-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物６３）を合成した。
LC/MS
rt(min): 1.51
MS(ESI,m/z): 1037.1 [M+H]+ Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpyrrolidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 63) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 1-methylpyrrolidin-3-amine.
LC/MS
rt(min): 1.51
MS(ESI,m/z): 1037.1 [M+H] +

［合成例６４］
 [Synthesis Example 64]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび1-メチルアゼパン-4-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-12-(1-メチルアゼパン-4-イル)-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物６４）を合成した。
LC/MS
rt(min): 1.45
MS(ESI,m/z): 1065.4 [M+H]+ Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazepan-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 64) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 1-methylazepan-4-amine.
LC/MS
rt(min): 1.45
MS(ESI,m/z): 1065.4 [M+H] +

［合成例６５、６６］
 [Synthesis Examples 65 and 66]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよびrac-N1,N1-ジメチルシクロヘキサン-1,4-ジアミンを用いて、合成例３１と同様の方法により縮合体を合成し、シリカゲルカラムクロマトグラフィーによって極性が異なる2化合物（低極性化合物：化合物６５、高極性化合物：化合物６６）を得た。 A condensation product was synthesized using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and rac-N1,N1-dimethylcyclohexane-1,4-diamine in the same manner as in Synthesis Example 31, and two compounds with different polarities (low polarity compound: Compound 65, high polarity compound: Compound 66) were obtained by silica gel column chromatography.

ビス(2-ペンチルヘプチル) 12-((1r,4r)-4-(ジメチルアミノ)シクロヘキシル)-7,17-ジヘプチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物６５）
LC/MS
rt(min): 1.46
MS(ESI,m/z): 1079.4 [M+H]+ Bis(2-pentylheptyl) 12-((1r,4r)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 65)
LC/MS
rt(min): 1.46
MS(ESI,m/z): 1079.4 [M+H] +

ビス(2-ペンチルヘプチル) 12-((1s,4s)-4-(ジメチルアミノ)シクロヘキシル)-7,17-ジヘプチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物６６）
LC/MS
rt(min): 1.12
MS(ESI,m/z): 1079.4 [M+H]+ Bis(2-pentylheptyl) 12-((1s,4s)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 66)
LC/MS
rt(min): 1.12
MS(ESI,m/z): 1079.4 [M+H] +

［合成例６７］
 [Synthesis Example 67]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび2-(4-アミノピペリジン-1-イル)エタン-1-オールを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-12-(1-(2-ヒドロキシエチル)ピペリジン-4-イル)-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物６７）を合成した。
LC/MS
rt(min): 1.49
MS(ESI,m/z): 1081.4 [M+H]+ Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-(2-hydroxyethyl)piperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 67) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 2-(4-aminopiperidin-1-yl)ethan-1-ol.
LC/MS
rt(min): 1.49
MS(ESI,m/z): 1081.4 [M+H] +

［合成例６８］
 [Synthesis Example 68]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび2-(ピロリジン-1-イル)エタン-1-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-8,16-ジオキソ-12-(2-(ピロリジン-1-イル)エチル)-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物６８）を合成した。
LC/MS
rt(min): 1.50
MS(ESI,m/z): 1051.1 [M+H]+ Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(pyrrolidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatricosane dioate (compound 68) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 2-(pyrrolidin-1-yl)ethan-1-amine.
LC/MS
rt(min): 1.50
MS(ESI,m/z): 1051.1 [M+H] +

［合成例６９］
 [Synthesis Example 69]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび2-(ピペリジン-1-イル)エタン-1-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 7,17-ジヘプチル-8,16-ジオキソ-12-(2-(ピペリジン-1-イル)エチル)-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物６９）を合成した。
LC/MS
rt(min): 1.52
MS(ESI,m/z): 1065.1 [M+H]+ Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(piperidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatricosane dioate (compound 69) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 2-(piperidin-1-yl)ethan-1-amine.
LC/MS
rt(min): 1.52
MS(ESI,m/z): 1065.1 [M+H] +

［合成例７０］
 [Synthesis Example 70]

　6-(オクチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸 2-ペンチルヘプチルおよび1-メチルピペリジン-4-アミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 12-(1-メチルピペリジン-4-イル)-7,17-ジオクチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物７０）を合成した。
LC/MS
rt(min): 1.69
MS(ESI,m/z): 1079.2 [M+H]+ Bis(2-pentylheptyl) 12-(1-methylpiperidin-4-yl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 70) was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(octyl((2-oxoethoxy)carbonyl)amino)hexanoate and 1-methylpiperidin-4-amine.
LC/MS
rt(min): 1.69
MS(ESI,m/z): 1079.2 [M+H] +

［合成例７１］
 [Synthesis Example 71]

　6-(ヘプチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸2-ペンチルヘプチルおよび2,2'-((3-アミノプロピル)アザンジイル)ビス(エタン-1-オール)を用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 12-(3-(ビス(2-ヒドロキシエチル)アミノ)プロピル)-7,17-ジヘプチル-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物７１）を合成した。
LC/MS
rt(min): 1.38
MS(ESI,m/z): 1099.4 [M+H]+ Bis(2-pentylheptyl) 12-(3-(bis(2-hydroxyethyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 71) was synthesized in the same manner as in Synthesis example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and 2,2'-((3-aminopropyl)azanediyl)bis(ethan-1-ol).
LC/MS
rt(min): 1.38
MS(ESI,m/z): 1099.4 [M+H] +

［合成例７２］
 [Synthesis Example 72]

　6-(((2-オキソエトキシ)カルボニル)(プロピル)アミノ)ヘキサン酸 2-ペンチルヘプチルおよび2-(ジエチルアミノ)エチルアミンを用いて、合成例３１と同様の方法により、
ビス(2-ペンチルヘプチル) 12-(2-(ジエチルアミノ)エチル)-8,16-ジオキソ-7,17-ジプロピル-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物７２）を合成した。
LC/MS
rt(min): 0.91
MS(ESI,m/z): 940.2 [M+H]+ Using 2-pentylheptyl 6-(((2-oxoethoxy)carbonyl)(propyl)amino)hexanoate and 2-(diethylamino)ethylamine, a similar procedure to that of Synthesis Example 31 was repeated to obtain
Bis(2-pentylheptyl) 12-(2-(diethylamino)ethyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatricosane dioate (compound 72) was synthesized.
LC/MS
rt(min): 0.91
MS(ESI,m/z): 940.2 [M+H] +

［合成例７３］
 [Synthesis Example 73]

　6-(((2-オキソエトキシ)カルボニル)(プロピル)アミノ)ヘキサン酸 2-ペンチルヘプチルおよび3-ジエチルアミノプロピルアミンを用いて、合成例３１と同様の方法により、ビス(2-ペンチルヘプチル) 12-(3-(ジエチルアミノ)プロピル)-8,16-ジオキソ-7,17-ジプロピル-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物７３）を合成した。
LC/MS
rt(min): 0.91
MS(ESI,m/z): 954.2 [M+H]+ Bis(2-pentylheptyl) 12-(3-(diethylamino)propyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatricosane dioate (compound 73) was synthesized in the same manner as in Synthesis example 31 using 2-pentylheptyl 6-(((2-oxoethoxy)carbonyl)(propyl)amino)hexanoate and 3-diethylaminopropylamine.
LC/MS
rt(min): 0.91
MS(ESI,m/z): 954.2 [M+H] +

［合成例７４］
 [Synthesis Example 74]

　6-(ブチル((2-オキソエトキシ)カルボニル)アミノ)ヘキサン酸 2-ペンチルヘプチルおよび3-ジエチルアミノプロピルアミンを用いて、合成例３１と同様の方法により、
ビス(2-ペンチルヘプチル) 7,17-ジブチル-12-(3-(ジエチルアミノ)プロピル)-8,16-ジオキソ-9,15-ジオキサ-7,12,17-トリアザトリコサンジオアート（化合物７４）を合成した。
LC/MS
rt(min): 0.99
MS(ESI,m/z): 982.3 [M+H]+ Using 2-pentylheptyl 6-(butyl((2-oxoethoxy)carbonyl)amino)hexanoate and 3-diethylaminopropylamine, a similar procedure to that of Synthesis Example 31 was repeated to obtain
Bis(2-pentylheptyl) 7,17-dibutyl-12-(3-(diethylamino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate (compound 74) was synthesized.
LC/MS
rt(min): 0.99
MS(ESI,m/z): 982.3 [M+H] +

［試験例１］
＜ＧＦＰ　ｍＲＮＡ脂質粒子の調製＞
　表３に記載のイオン化可能な脂質、DSPC（1,2-ジステアロイル-sn-グリセロ-3-ホスフォコリン　製品名：ＣＯＡＴＳＯＭＥ（Ｒ）ＭＣ－８０８０；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）、コレステロール、ＤＭＧ－ＰＥＧ２０００（１，２－ジミリストイル－ｒａｃ－グリセロ－３－メトキシポリエチレングリコール－２０００　製品名：ＳＵＮＢＲＩＧＨＴ（Ｒ）ＧＭ－０２０；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）を、表３に記載のモル比で、総脂質濃度が１２．５ｍｍｏｌ／Ｌとなるようにエタノールに溶解させ、油相を得た。 [Test Example 1]
<Preparation of GFP mRNA lipid particles>
The ionizable lipids shown in Table 3, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, product name: COATSOME® MC-8080; manufactured by NOF Corporation), cholesterol, and DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, product name: SUNBRIGHT® GM-020; manufactured by NOF Corporation) were dissolved in ethanol in the molar ratios shown in Table 3 so that the total lipid concentration was 12.5 mmol/L to obtain an oil phase.

　GFP　ｍＲＮＡ（製品名：ＣｌｅａｎＣａｐ　GFP　ｍＲＮＡ（5moU）；ＴｒｉＬｉｎｋ社製）を、ｐＨ４の５０ｍｍｏｌ／Ｌクエン酸バッファーで、油相・水相混合後の総脂質濃度のｍＲＮＡ濃度に対する重量比が表３記載のとおりになるように希釈して水相を得た。つづいて水相と油相の体積比が水相：油相＝３：１となるようにＮａｎｏＡｓｓｅｍｂｌｒ（Ｐｒｅｃｉｓｉｏｎ　ＮａｎｏＳｙｓｔｅｍｓ）を使用して混合し、混合液を水で２倍希釈してｍＲＮＡ脂質粒子の分散液を得た。この分散液を透析カセット（Ｓｌｉｄｅ－Ａ－Ｌｙｚｅｒ　Ｇ２、　ＭＷＣＯ：１０ｋＤ、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用いて８％スクロースを含む２０ｍｍｏｌ／Ｌ　Ｔｒｉｓ緩衝液ｐＨ７．４に対して透析することによりエタノールの除去を行い、ｍＲＮＡ内包脂質粒子を得た。調製したサンプルは使用時まで－７０℃で凍結して保存した。 GFP mRNA (product name: CleanCap GFP mRNA (5moU); manufactured by TriLink) was diluted with 50 mmol/L citrate buffer at pH 4 so that the weight ratio of the total lipid concentration to the mRNA concentration after mixing of the oil and aqueous phases was as shown in Table 3 to obtain an aqueous phase. The aqueous and oil phases were then mixed using a NanoAssemblr (Precision NanoSystems) so that the volume ratio of the aqueous phase to the oil phase was aqueous phase:oil phase = 3:1, and the mixture was diluted 2-fold with water to obtain a dispersion of mRNA lipid particles. This dispersion was dialyzed against 20 mmol/L Tris buffer pH 7.4 containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to remove the ethanol, yielding mRNA-encapsulated lipid particles. The prepared samples were stored frozen at -70°C until use.

＜ＧＦＰ　ｐＤＮＡ脂質粒子の調製＞
　表４に記載のイオン化可能な脂質、ＤＯＰＥ（Ｌ－α－ジオレオイル　ホスファチジルエタノールアミン　製品名：ＣＯＡＴＳＯＭＥ（Ｒ）ＭＥ－８１８１；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）、ＤＳＰＣ（１，２－ジステアロイル－ｓｎ－グリセロ－３－ホスフォコリン　製品名：ＣＯＡＴＳＯＭＥ（Ｒ）ＭＣ－８０８０；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）、から選択される一つのリン脂質（ヘルパー脂質）、コレステロール、ＤＭＧ－ＰＥＧ２０００（１，２－ジミリストイル－ｒａｃ－グリセロ－３－メトキシポリエチレングリコール－２０００　製品名：ＳＵＮＢＲＩＧＨＴ（Ｒ）ＧＭ－０２０；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）を、表１に記載のモル比で、総脂質濃度が１２．５ｍｍｏｌ／Ｌなるようにエタノールに溶解させ、油相を得た。 <Preparation of GFP pDNA lipid particles>
The ionizable lipids shown in Table 4, DOPE (L-α-dioleoyl phosphatidylethanolamine product name: COATSOME® ME-8181; manufactured by NOF corporation), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine product name: COATSOME® MC-8080; manufactured by NOF corporation), one phospholipid (helper lipid) selected from the group consisting of cholesterol and DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 product name: SUNBRIGHT® GM-020; manufactured by NOF corporation) were dissolved in ethanol in the molar ratios shown in Table 1 so that the total lipid concentration was 12.5 mmol/L to obtain an oil phase.

　ＧＦＰ　ｐＤＮＡ（ＧｅｎＳｃｒｉｐｔ、カスタム合成プラスミドＤＮＡ）を、ｐＨ４の５０ｍｍｏｌ／Ｌクエン酸バッファーで、油相・水相混合後の総脂質濃度のｍＲＮＡ濃度に対する重量比が表４記載のとおりになるように希釈して水相を得た。つづいて水相と油相の体積比が水相：油相＝３：１となるようにＮａｎｏＡｓｓｅｍｂｌｒ（Ｐｒｅｃｉｓｉｏｎ　ＮａｎｏＳｙｓｔｅｍｓ）を使用して混合し、混合液を水で２倍希釈してｍＲＮＡ脂質粒子の分散液を得た。この分散液を透析カセット（Ｓｌｉｄｅ－Ａ－Ｌｙｚｅｒ　Ｇ２、　ＭＷＣＯ：１０ｋＤ、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用いて８％スクロースを含む２０ｍｍｏｌ／Ｌ　Ｔｒｉｓ緩衝液ｐＨ７．４に対して透析することによりエタノールの除去を行い、ＧＦＰ　ｐＤＮＡ内包脂質粒子を得た。調製したサンプルは使用時まで－７０℃で凍結して保存した。 GFP pDNA (GenScript, custom-synthesized plasmid DNA) was diluted with 50 mmol/L citrate buffer at pH 4 so that the weight ratio of the total lipid concentration to the mRNA concentration after mixing of the oil and aqueous phases was as shown in Table 4 to obtain an aqueous phase. The aqueous phase and the oil phase were then mixed using a NanoAssemblr (Precision NanoSystems) so that the volume ratio of the aqueous phase to the oil phase was 3:1, and the mixture was diluted 2-fold with water to obtain a dispersion of mRNA lipid particles. This dispersion was dialyzed against 20 mmol/L Tris buffer pH 7.4 containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to remove ethanol, and GFP pDNA-encapsulated lipid particles were obtained. The prepared samples were frozen and stored at -70°C until use.

＜核酸を含まない脂質粒子（空ＬＮＰ）の調製＞
　表５に記載のイオン化可能な脂質、ＤＯＰＥ（Ｌ－α－ジオレオイル　ホスファチジルエタノールアミン　製品名：ＣＯＡＴＳＯＭＥ（Ｒ）ＭＥ－８１８１；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）、ＤＳＰＣ（１，２－ジステアロイル－ｓｎ－グリセロ－３－ホスフォコリン　製品名：ＣＯＡＴＳＯＭＥ（Ｒ）ＭＣ－８０８０；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）、ＤＯＰＣ（１，２－ジオレオイル－ｓｎ－グリセロ－３－ホスフォコリン　製品名：ＣＯＡＴＳＯＭＥ（Ｒ）ＭＣ－８１８１；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）、ＤＰＰＣ（１，２－ジパルミトイル－ｓｎ－グリセロ－３－ホスフォコリン　製品名：ＣＯＡＴＳＯＭＥ（Ｒ）ＭＣ－６０６０；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）、ＤＭＰＣ（１，２－ジミリストイル－ｓｎ－グリセロ－３－ホスフォコリン　製品名：ＣＯＡＴＳＯＭＥ（Ｒ）ＭＣ－４０４０；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）、から選択される一つのリン脂質（ヘルパー脂質）、コレステロール、ＤＭＧ－ＰＥＧ２０００（１，２－ジミリストイル－ｒａｃ－グリセロ－３－メトキシポリエチレングリコール－２０００　製品名：ＳＵＮＢＲＩＧＨＴ（Ｒ）ＧＭ－０２０；ＮＯＦ　ｃｏｒｐｏｒａｔｉｏｎ社製）を、表５に記載のモル比で、総脂質濃度が１２．５ｍｍｏｌ／Ｌもしくは６２．５ｍｍｏｌ／Ｌとなるようにエタノールに溶解させ、油相を得た。 <Preparation of lipid particles not containing nucleic acid (empty LNPs)>
Ionizable lipids listed in Table 5, DOPE (L-α-dioleoyl phosphatidylethanolamine Product name: COATSOME® ME-8181; manufactured by NOF Corporation), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine Product name: COATSOME® MC-8080; manufactured by NOF Corporation), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine Product name: COATSOME® MC-8181; manufactured by NOF Corporation), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine Product name: COATSOME® MC-6060; manufactured by NOF Corporation), Corporation), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine product name: COATSOME® MC-4040; NOF Corporation), one phospholipid (helper lipid) selected from the group consisting of cholesterol and DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 product name: SUNBRIGHT® GM-020; NOF Corporation) in the molar ratios shown in Table 5. The total lipid concentration was 12.5 mmol/L or 62.5 mmol/L. The oil phase was dissolved in ethanol to obtain an oil phase.

　ｐＨ４の５０ｍｍｏｌ／Ｌクエン酸バッファーを、上記油相と体積比がクエン酸バッファー：油相＝３：１となるようにＮａｎｏＡｓｓｅｍｂｌｒ（Ｐｒｅｃｉｓｉｏｎ　ＮａｎｏＳｙｓｔｅｍｓ）を使用して混合し、混合液を水で２倍希釈して脂質粒子の分散液を得た。この分散液を透析カセット（Ｓｌｉｄｅ－Ａ－Ｌｙｚｅｒ　Ｇ２、　ＭＷＣＯ：１０ｋＤ、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用いて８％スクロースを含む２０ｍｍｏｌ／Ｌ　ＭＥＳ緩衝液ｐＨ６．０に対して透析することによりエタノールの除去を行い、必要に応じて限外ろ過フィルター（Amicon ultra 100kDa, Merck）を用いた濃縮工程を行うことにより、核酸を含まない脂質粒子（空ＬＮＰ）を得た。空ＬＮＰは使用時まで－７０℃にて凍結保存した。 A 50 mmol/L citrate buffer of pH 4 was mixed with the oil phase at a volume ratio of citrate buffer:oil phase = 3:1 using a NanoAssemblr (Precision NanoSystems), and the mixture was diluted 2-fold with water to obtain a lipid particle dispersion. This dispersion was dialyzed against 20 mmol/L MES buffer pH 6.0 containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to remove ethanol, and a concentration step was performed using an ultrafiltration filter (Amicon ultra 100 kDa, Merck) as necessary to obtain lipid particles that do not contain nucleic acids (empty LNPs). The empty LNPs were stored frozen at -70°C until use.

＜空ＬＮＰへのｇＲＮＡおよびＣａｓ９ｍＲＮＡの内包（後添加法）＞
　ＣｌｅａｎＣａｐ（登録商標）Ｃａｓ９　ｍＲＮＡ（５ｍｏＵ）（ＴｒｉＬｉｎｋ、Ｌ－７２０６）とヒトＴ　ｃｅｌｌ　ｒｅｃｅｐｔｏｒ　ａｌｐｈａ　ｃｏｎｓｔａｎｔ　（ＴＲＡＣ）遺伝子を標的とするｓｇＲＮＡ（配列；Ａ＊Ｇ＊Ａ＊ＧＵＣＵＣＵＣＡＧＣＵＧＧＵＡＣＡ＋ｍｏｄｉｆｉｅｄ　Ｓｃａｆｆｏｌｄ、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ａ３５５１４，カスタム合成）を４：１の重量比で混合し、注射用水を用いて希釈することでＲＮＡ溶液を調製した。－７０℃で保存した空ＬＮＰを４℃で解凍した。本ＬＮＰ液にＲＮＡ溶液を等液量添加し、ピペッティングにて混合した（ＬＮＰ－ＲＮＡ混合液）。室温にて５分間静置後、本ＬＮＰ－ＲＮＡ混合液に、８％スクロースを含む２０ｍｍｏｌ／Ｌトリスバッファーを等液量添加し、ピペッティングにて混合することで、後添加法でＣａｓ９　ｍＲＮＡ／ｇＲＮＡ内包ＬＮＰを調製した。 <Inclusion of gRNA and Cas9 mRNA in empty LNPs (post-addition method)>
CleanCap (registered trademark) Cas9 mRNA (5 moU) (TriLink, L-7206) and sgRNA (sequence; A * G * A * GUCUCUCAGCUGGUACA + modified Scaffold, Thermo Fisher A35514, custom synthesis) targeting the human T cell receptor alpha constant (TRAC) gene were mixed at a weight ratio of 4: 1 and diluted with water for injection to prepare an RNA solution. Empty LNP stored at -70 ° C. was thawed at 4 ° C. An equal amount of RNA solution was added to this LNP solution and mixed by pipetting (LNP-RNA mixture). After standing at room temperature for 5 minutes, an equal volume of 20 mmol/L Tris buffer containing 8% sucrose was added to this LNP-RNA mixture and mixed by pipetting to prepare Cas9 mRNA/gRNA-encapsulated LNPs by the post-addition method.

＜空ＬＮＰへのＧＦＰ　ｐＤＮＡの内包（後添加法）＞
　ＧＦＰ　ｐＤＮＡ（ＧｅｎＳｃｒｉｐｔ、カスタム合成プラスミドDNA）を注射用水を用いて希釈することでＤＮＡ溶液を調製した。－７０℃で保存した空ＬＮＰを４℃で解凍した。本ＬＮＰ液にＤＮＡ溶液を等液量添加し、ピペッティングにて混合した（ＬＮＰ－ＤＮＡ混合液）。室温にて５分間静置後、本ＬＮＰ－ＤＮＡ混合液に、８％スクロースを含む２０ｍｍｏｌ／Ｌトリスバッファー（ｐＨ８．４）を等液量添加し、ピペッティングにて混合することで、後添加法でＧＦＰ　ｐＤＮＡ内包ＬＮＰを調製した。 <Inclusion of GFP pDNA into empty LNPs (post-addition method)>
A DNA solution was prepared by diluting GFP pDNA (GenScript, custom-synthesized plasmid DNA) with water for injection. Empty LNP stored at -70°C was thawed at 4°C. An equal volume of DNA solution was added to the LNP solution and mixed by pipetting (LNP-DNA mixture). After standing at room temperature for 5 minutes, an equal volume of 20 mmol/L Tris buffer (pH 8.4) containing 8% sucrose was added to the LNP-DNA mixture and mixed by pipetting to prepare GFP pDNA-encapsulated LNP by the post-addition method.

＜粒子径の測定＞
　ｍＲＮＡ内包脂質粒子の粒子径は、脂質粒子をリン酸緩衝生理食塩水（ＰＢＳ）で任意に希釈してから、粒径測定システムＮａｎｏＳＡＱＬＡ（大塚電子）を用いて測定した。
粒子径および多分散指数（ＰＤＩ）の測定結果を表７に示す。 <Measurement of particle size>
The particle size of the mRNA-encapsulating lipid particles was measured using a particle size measuring system NanoSAQLA (Otsuka Electronics) after arbitrarily diluting the lipid particles with phosphate buffered saline (PBS).
The particle size and polydispersity index (PDI) measurements are shown in Table 7.

＜核酸の内包率の評価＞
（総核酸濃度定量）
　核酸をＭｉｌｌｉＱ水で希釈して１００μｇ／ｍＬから３．１μｇ／ｍＬまでの２倍希釈系列で希釈サンプルを調製し，検量線溶液を調製した。検量線溶液もしくは脂質粒子５０μＬをメタノール４５０μＬと混合して測定溶液を調製した。ＵＶプレートリーダー（Ｍｕｌｔｉｓｋａｎ　Ｇｏ，　Ｔｈｅｒｍｏ　ｆｉｓｈｅｒ　ｓｃｉｅｎｔｉｆｉｃ）を用いて各測定溶液の２６０ｎｍおよび３３０ｎｍにおける吸光度を測定し，２６０ｎｍの吸光度から３３０ｎｍの吸光度を差し引き，各測定溶液の吸光度とした。各サンプル測定溶液の吸光度を用いて，検量線から総核酸濃度を算出した。 <Evaluation of Nucleic Acid Encapsulation Rate>
(Quantitative determination of total nucleic acid concentration)
The nucleic acid was diluted with MilliQ water to prepare diluted samples in a 2-fold dilution series from 100 μg/mL to 3.1 μg/mL, and a calibration curve solution was prepared. 50 μL of the calibration curve solution or lipid particles were mixed with 450 μL of methanol to prepare a measurement solution. The absorbance of each measurement solution at 260 nm and 330 nm was measured using a UV plate reader (Multiskan Go, Thermo Fisher Scientific), and the absorbance at 330 nm was subtracted from the absorbance at 260 nm to obtain the absorbance of each measurement solution. The total nucleic acid concentration was calculated from the calibration curve using the absorbance of each sample measurement solution.

（外水相における核酸濃度の定量）
　Ｑｕａｎｔ－ｉＴ　ＲｉｂｏＧｒｅｅｎ　ＲＮＡ　Ａｓｓａｙ　Ｋｉｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用い、標準添加法により外水相核酸濃度を定量した。まず、上述のキットに含まれる２０×ＴＥバッファーを水で希釈し、１×ＴＥバッファーとした。なお、ＴＥは、Ｔｒｉｓ／ＥＤＴＡ（エチレンジアミン四酢酸）を示す。核酸を終濃度が０～４００ ｎｇ/ｍＬになるようにＴＥバッファーで希釈し，核酸希釈系列を調製した。脂質粒子１０μＬと核酸希釈系列９０μＬを９６ウェルプレートで混合後、ＴＥバッファーで２００倍に希釈したＲｉｂｏＧｒｅｅｎ試薬１００μLを各ウェルに加え，蛍光プレートリーダー（Ｉｎｆｉｔｉｔｅ　２００　Ｐｒｏ　Ｍ　ｎａｎｏ　＋、ＴＥＣＡＮ）を用いて蛍光 (励起波長：４８５ｎｍ，蛍光波長：５３５ｎｍ) を測定した。得られた結果から標準添加法に則り，各測定溶液の外水相核酸濃度を算出した。 (Quantification of Nucleic Acid Concentration in the External Aqueous Phase)
The nucleic acid concentration in the external aqueous phase was quantified by the standard addition method using Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific). First, the 20x TE buffer included in the above kit was diluted with water to obtain 1x TE buffer. TE stands for Tris/EDTA (ethylenediaminetetraacetic acid). The nucleic acid was diluted with TE buffer to a final concentration of 0 to 400 ng/mL to prepare a nucleic acid dilution series. After mixing 10 μL of lipid particles and 90 μL of the nucleic acid dilution series in a 96-well plate, 100 μL of RiboGreen reagent diluted 200 times with TE buffer was added to each well, and fluorescence (excitation wavelength: 485 nm, fluorescence wavelength: 535 nm) was measured using a fluorescence plate reader (Infitite 200 Pro M nano +, TECAN). From the results obtained, the nucleic acid concentration in the external aqueous phase of each measurement solution was calculated according to the standard addition method.

（内包率の算出）
　上述の工程で得られた総ＲＮＡ濃度および外水相での核酸濃度の定量結果を用いて、下記式に従って、核酸脂質ナノ粒子の核酸内包率を算出した。
核酸内包率（％）＝（総核酸濃度－外水相における核酸濃度）÷総核酸濃度×
１００
　結果を表７に示す。 (Calculation of Inclusion Rate)
Using the quantitative results of the total RNA concentration and the nucleic acid concentration in the external aqueous phase obtained in the above steps, the nucleic acid encapsulation rate of the nucleic acid-lipid nanoparticles was calculated according to the following formula.
Nucleic acid encapsulation rate (%) = (total nucleic acid concentration - nucleic acid concentration in external aqueous phase) ÷ total nucleic acid concentration x
100
The results are shown in Table 7.

























［試験例２］
［材料及び方法］
＜活性化用培地の調製＞
　Ｔ細胞を活性化培養条件で培養する際に用いる培地は、ＴｅｘＭＡＣＳｔｍ　Ｍｅｄｉｕｍ（Ｍｉｌｔｅｎｙｉ　ｂｉｏｔｅｃｈ，１３０－０９７－１９６）、５ｎｇ／ｍｌｈｕｍａｎ　ｉｎｔｅｒｌｅｕｋｉｎ－２（ＩＬ－２，Ｒｏｃｈｅ，１１１４７５２８００１）からなる（以下、活性化用培地）。 [Test Example 2]
Materials and Methods
<Preparation of activation medium>
The medium used for culturing T cells under activation culture conditions consists of TexMACStm Medium (Miltenyi biotech, 130-097-196) and 5 ng/ml human interleukin-2 (IL-2, Roche, 11147528001) (hereinafter, activation medium).

＜Ｔ細胞の調製および活性化培養＞
　健康なヒトドナーの末梢血に由来する凍結Ｔ細胞（Ｈｕｍａｎ　ＰＢ　Ｐａｎ－Ｔ，　Ｃｒｙｏ、ＳＴＥＭＣＥＬＬ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，ＳＴ－７００２４）を、３７℃にて数分間湯浴させることで解凍した。解凍したＴ細胞は、１％ＢＳＡ（牛血清アルブミン）（ＳＩＧＭＡ，Ａ９５７６），２０Ｕ／ｍｌ　ＤＮａｓｅＩ（Ｗｏｒｔｈｉｎｇｔｏｎ　Ｂｉｏｃｈｅｍｉｃａｌ，ＬＳ００２１３９）を含有させたＴｅｘＭＡＣＳ　Ｍｅｄｉｕｍに再懸濁し、さらに遠心により洗浄し、活性化用培地にて再懸濁した。Ｔ細胞は、本培地を用いて１．０×１０６ｃｅｌｌｓ／ｍｌの細胞濃度に調整し、Ｄｙｎａｂｅａｄｓ　Ｈｕｍａｎ　Ｔ－Ａｃｔｉｖａｔｏｒ　ＣＤ３／ＣＤ２８（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　ＤＢ１１１３１）を１．０×１０６ｂｅａｄｓ／ｍｌとなるように添加した。細胞培養用の２４ウェルプレートに播種し、３７℃５％ＣＯ２インキュベーターにて３日間培養することで、活性化させた。活性化３日目にＴ細胞培養液からＤｙｎａｂｅａｄｓを除去した。このように前処理したＴ細胞を活性化Ｔ細胞として使用した。 <Preparation and activation culture of T cells>
Frozen T cells derived from peripheral blood of healthy human donors (Human PB Pan-T, Cryo, STEMCELL Technologies, ST-70024) were thawed by placing in a water bath at 37° C. for several minutes. The thawed T cells were resuspended in TexMACS Medium containing 1% BSA (bovine serum albumin) (SIGMA, A9576) and 20 U/ml DNase I (Worthington Biochemical, LS002139), further washed by centrifugation, and resuspended in activation medium. The T cells were adjusted to a cell concentration of 1.0 x 10 6 cells/ml using this medium, and Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher DB11131) was added to a concentration of 1.0 x 10 6 beads/ml. The cells were seeded in a 24-well cell culture plate and activated by culturing for 3 days in a 37°C 5% CO 2 incubator. On the third day of activation, the Dynabeads were removed from the T cell culture medium. The T cells thus pretreated were used as activated T cells.

＜非活性化用培地の調製＞
　Ｔ細胞を非活性化培養条件で培養する際に用いる培地は、ＴｅｘＭＡＣＳｔｍ　Ｍｅｄｉｕｍ（Ｍｉｌｔｅｎｙｉ　ｂｉｏｔｅｃｈ，１３０－０９７－１９６）、５ｎｇ／ｍｌ　ｈｕｍａｎ　ｉｎｔｅｒｌｅｕｋｉｎ－２（ＩＬ－２，Ｒｏｃｈｅ，１１１４７５２８００１）、５ｎｇ／ｍｌ　Ｈｕｍａｎ　ＩＬ－７（Ｍｉｌｔｅｎｙｉ　ｂｉｏｔｅｃｈ　１３０－０９５－３６７）、５ｎｇ／ｍｌ　Ｈｕｍａｎ　ＩＬ－１５（Ｍｉｌｔｅｎｙｉ　ｂｉｏｔｅｃｈ、１３０－０９５－７６０）からなる（以下、非活性化用培地）。 <Preparation of non-activation medium>
The medium used for culturing T cells under non-activation culture conditions consists of TexMACStm Medium (Miltenyi biotech, 130-097-196), 5 ng/ml human interleukin-2 (IL-2, Roche, 11147528001), 5 ng/ml human IL-7 (Miltenyi biotech 130-095-367), and 5 ng/ml human IL-15 (Miltenyi biotech, 130-095-760) (hereinafter, non-activation medium).

＜Ｔ細胞の調製および非活性化培養＞
　健康なヒトドナーの末梢血に由来する凍結Ｔ細胞（Ｈｕｍａｎ　ＰＢ　Ｐａｎ－Ｔ，　Ｃｒｙｏ、ＳＴＥＭＣＥＬＬ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，ＳＴ－７００２４）を、３７℃にて数分間湯浴させることで解凍した。解凍したＴ細胞は、１％ＢＳＡ、２０Ｕ／ｍｌ　ＤＮａｓｅＩを含有させたＴｅｘＭＡＣＳ　Ｍｅｄｉｕｍに再懸濁し、さらに遠心により洗浄し、非活性化用培地にて再懸濁した。Ｔ細胞は、本培地を用いて１．０×１０６ｃｅｌｌｓ／ｍｌの細胞濃度に調整し、細胞培養用の２４ウェルプレートに播種し、３７℃５％ＣＯ２インキュベーターにて３日間培養した。このように前処理したＴ細胞を非活性
化Ｔ細胞として使用した。 <Preparation of T cells and non-activation culture>
Frozen T cells (Human PB Pan-T, Cryo, STEMCELL Technologies, ST-70024) derived from peripheral blood of healthy human donors were thawed by placing them in a water bath at 37°C for several minutes. The thawed T cells were resuspended in TexMACS Medium containing 1% BSA and 20 U/ml DNaseI, washed by centrifugation, and resuspended in non-activation medium. The T cells were adjusted to a cell concentration of 1.0 x 10 6 cells/ml using this medium, seeded on a 24-well plate for cell culture, and cultured for 3 days in a 37°C, 5% CO 2 incubator. The T cells thus pretreated were used as non-activated T cells.

＜フローサイトメトリー＞
　培養開始４日目に、各手法で核酸送達したＴ細胞をフローサイトメトリーによって、ＧＦＰ陽性率を評価した。
　ＧＦＰ陽性率の評価は、Ｔ細胞を、ＢＤ　ＨｏｒｉｚｏｎTM　Ｆｉｘａｂｌｅ　Ｖｉａｂｉｌｉｔｙ　Ｓｔａｉｎ　（ＦＶＳ）Ｒｅａｇｅｎｔｓ（ＢＤ、５６５３８８）で死細胞を染色した。染色操作の後、細胞を固定、洗浄し、Ａｔｔｕｎｅ装置（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ）にて細胞状態を解析した。解析データは、Ｆｌｏｗｊｏソフトウェアを使用して分析した。Ｔ細胞を、サイズ、シングルセル、生細胞でゲーティングした後、ＧＦＰ陽性細胞の比率および蛍光強度中央値（Ｍｅｄｉａｎ　Ｆｌｕｏｒｅｓｃｅｎｃｅ　Ｉｎｔｅｎｓｉｔｙ、ＭＦＩ）を分析した。 <Flow cytometry>
On day 4 after the start of culture, the GFP positivity rate of T cells to which nucleic acid had been delivered by each method was evaluated by flow cytometry.
To evaluate the GFP positivity rate, T cells were stained for dead cells with BD Horizon ™ Fixable Viability Stain (FVS) Reagents (BD, 565388). After staining, the cells were fixed and washed, and the cell status was analyzed using an Attune device (Thermo Fisher). The analysis data was analyzed using Flowjo software. After gating T cells by size, single cells, and live cells, the ratio of GFP-positive cells and median fluorescence intensity (MFI) were analyzed.

＜試験例２－１＞活性化Ｔ細胞への核酸送達
　培養３日目の活性化Ｔ細胞を必要細胞数分取し、遠心および上清を除去した。予め用時調製した最終濃度１μｇ／ｍｌで組換えヒトアポリポプロテインＥ３（ＡｐｏＥ３）（富士フイルム和光、０１０－２０２６１）を含有させた活性化用培地で、活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。 Test Example 2-1: Nucleic acid delivery to activated T cells On the third day of culture, the required number of activated T cells were collected, centrifuged, and the supernatant was removed. The activated T cells were adjusted to a concentration of 1.0 x 106 cells/ml in an activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg/ml, which had been prepared just before use, and seeded on a 96-well plate.

　実施例１～５で調製したＧＦＰ　ｍＲＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり１．０μｇ（総ＲＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。ＬＮＰ添加から２４時間後に細胞を回収し、フローサイトメトリーにて各ＬＮＰで処理したＴ細胞のＧＦＰ陽性細胞比率およびＭＦＩを測定し、ＧＦＰ　ｍＲＮＡ導入効率を評価した。本結果を図１に示す。
　実施例１～５のＬＮＰはいずれも活性化Ｔ細胞へ高効率にｍＲＮＡ送達が可能であった。 The GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5 were added at 1.0 μg (total RNA amount) per 1.0 × 10 6 cells, and cultured in a 37°C, 5% CO 2 incubator. 24 hours after the addition of LNP, the cells were collected, and the GFP-positive cell ratio and MFI of T cells treated with each LNP were measured by flow cytometry to evaluate the GFP mRNA introduction efficiency. The results are shown in Figure 1.
All of the LNPs of Examples 1 to 5 were capable of highly efficient delivery of mRNA to activated T cells.

＜試験例２―２＞非活性化Ｔ細胞への核酸送達
　培養３日目の非活性化Ｔ細胞を必要細胞数分取し、遠心および上清を除去した。予め用時調製した最終濃度１μｇ／ｍｌ　ＡｐｏＥ３を含有させた活性化用培地で、非活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。　実施例１～５で調製したＧＦＰ　ｍＲＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり１．０μｇ（総ＲＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。
　ＬＮＰ添加から２４時間後に細胞を回収し、フローサイトメトリーにて各ＬＮＰで処理したＴ細胞のＧＦＰ陽性細胞比率およびＭＦＩを測定し、ＧＦＰ　ｍＲＮＡ導入効率を評価した。本結果を図２に示す。
　実施例１～５のＬＮＰはいずれも非活性化Ｔ細胞へｍＲＮＡ送達が可能であり、特に、実施例１、３、４のＬＮＰは効率が高いことが示された。 <Test Example 2-2> Nucleic acid delivery to non-activated T cells On the third day of culture, the required number of non-activated T cells were collected, centrifuged, and the supernatant was removed. The non-activated T cells were adjusted to a concentration of 1.0 x 10 6 cells/ml using an activation medium containing ApoE3 at a final concentration of 1 μg/ml, which had been prepared just before use, and seeded on a 96-well plate. Using the GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5, 1.0 μg (total RNA amount) was added per 1.0 x 10 6 cells, and the cells were cultured in a 37°C, 5% CO 2 incubator.
The cells were harvested 24 hours after the addition of LNPs, and the GFP-positive cell ratio and MFI of T cells treated with each LNP were measured by flow cytometry to evaluate the efficiency of GFP mRNA transfer. The results are shown in FIG.
It was demonstrated that all of the LNPs of Examples 1 to 5 were capable of delivering mRNA to non-activated T cells, and that the LNPs of Examples 1, 3, and 4 in particular were highly efficient.

＜試験例３－１＞活性化Ｔ細胞への核酸送達
　培養３日目の活性化Ｔ細胞を必要細胞数分取し、遠心および上清を除去した。ＡｐｏＥ３を添加しない活性化用培地で、活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。 Test Example 3-1: Nucleic acid delivery to activated T cells The activated T cells on day 3 of culture were collected in the required number of cells, centrifuged, and the supernatant was removed. The activated T cells were adjusted to a concentration of 1.0 x 10 6 cells/ml in an activation medium containing no ApoE3, and seeded on a 96-well plate.

　実施例１～５で調製したＧＦＰ　ｍＲＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり１．０μｇ（総ＲＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。ＬＮＰ添加から２４時間後に細胞を回収し、フローサイトメトリーにて各ＬＮＰで処理したＴ細胞のＧＦＰ陽性細胞比率およびＭＦＩを測定し、ＧＦＰ　ｍＲＮＡ導入効率を評価した。本結果を図３に示す。（参考例として図１で示したＡｐｏＥ３存在下でのデータを併記した）
　実施例１、３、４のＬＮＰは、ＡｐｏＥ３活性化が存在しない環境においても活性化Ｔ細胞へ高効率にｍＲＮＡ送達が可能であった。 The GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5 were added at 1.0 μg (total RNA amount) per 1.0×10 6 cells, and cultured in a 37° C., 5% CO 2 incubator. 24 hours after the addition of LNP, the cells were collected, and the GFP-positive cell ratio and MFI of T cells treated with each LNP were measured by flow cytometry to evaluate the GFP mRNA introduction efficiency. The results are shown in FIG. 3. (As a reference example, data in the presence of ApoE3 shown in FIG. 1 are also shown.)
The LNPs of Examples 1, 3, and 4 were capable of highly efficient delivery of mRNA to activated T cells even in an environment in which ApoE3 activation was not present.

＜試験例３―２＞非活性化Ｔ細胞への核酸送達
　培養３日目の非活性化Ｔ細胞を必要細胞数分取し、遠心および上清を除去した。ＡｐｏＥ３を含まない活性化用培地で、非活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。　実施例１～５で調製したＧＦＰ　ｍＲＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり１．０μｇ（総ＲＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。
　ＬＮＰ添加から２４時間後に細胞を回収し、フローサイトメトリーにて各ＬＮＰで処理したＴ細胞のＧＦＰ陽性細胞比率およびＭＦＩを測定し、ＧＦＰ　ｍＲＮＡ導入効率を評価した。本結果を図４に示す。（参考例として図２で示したＡｐｏＥ３存在下でのデータを併記した）
　実施例１、３、４のＬＮＰは、ＡｐｏＥ３活性化が存在しない環境においても非活性化Ｔ細胞へ高効率にｍＲＮＡ送達が可能であった。 <Test Example 3-2> Nucleic acid delivery to non-activated T cells On the third day of culture, the required number of non-activated T cells were collected, centrifuged, and the supernatant was removed. The non-activated T cells were adjusted to a concentration of 1.0 x 106 cells/ml using an activation medium that does not contain ApoE3, and seeded on a 96-well plate. Using the GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5, 1.0 μg (total RNA amount) was added per 1.0 x 106 cells, and the cells were cultured in a 37°C, 5% CO2 incubator.
The cells were harvested 24 hours after the addition of LNP, and the GFP-positive cell ratio and MFI of T cells treated with each LNP were measured by flow cytometry to evaluate the efficiency of GFP mRNA transfer. The results are shown in Figure 4. (The data in the presence of ApoE3 shown in Figure 2 are also shown as a reference example.)
The LNPs of Examples 1, 3, and 4 were capable of highly efficient delivery of mRNA to non-activated T cells even in an environment in which ApoE3 activation was not present.

＜試験例４＞従来法ＬＮＰを用いた活性化Ｔ細胞へのプラスミドＤＮＡ送達
＜１＞活性化Ｔ細胞のＬＮＰ処理
　培養３日目の活性化Ｔ細胞を必要細胞数分取し、遠心および上清を除去した。予め用時調製した最終濃度１μｇ／ｍｌで組換えヒトアポリポプロテインＥ３（ＡｐｏＥ３）（富士フイルム和光、０１０－２０２６１）を含有させた活性化用培地または活性化用CDM培地で、活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。
　実施例１３０～１４７のＧＦＰ　ｐＤＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり０．４～１０．０μｇ（総ＤＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。
＜２＞活性化Ｔ細胞におけるＧＦＰ陽性細胞比率
　培養開始４日目に、フローサイトメトリーにて各手法で処理したＴ細胞のＧＦＰ陽性細胞比率を測定し、プラスミドＤＮＡ導入効率を評価した。その結果を表８に示した。 <Test Example 4> Delivery of plasmid DNA to activated T cells using conventional LNP <1> LNP treatment of activated T cells The required number of activated T cells on the third day of culture were collected, centrifuged, and the supernatant was removed. The activated T cells were adjusted to a concentration of 1.0 x 10 6 cells/ml in activation medium or activation CDM medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg/ml, which had been prepared just before use, and seeded on a 96-well plate.
The GFP pDNA-encapsulated LNPs of Examples 130 to 147 were added at 0.4 to 10.0 μg (total DNA amount) per 1.0×10 6 cells, and cultured in a 37° C., 5% CO 2 incubator.
<2> Ratio of GFP-positive cells in activated T cells On day 4 of the culture, the ratio of GFP-positive cells in the T cells treated by each method was measured by flow cytometry to evaluate the efficiency of plasmid DNA transfer. The results are shown in Table 8.

＜試験例５＞後添加ＬＮＰを用いた活性化Ｔ細胞への核酸送達（ＴＣＲ　ＫＯ）
＜１＞活性化Ｔ細胞のＬＮＰ処理
　培養３日目の活性化Ｔ細胞を必要細胞数分取し、遠心および上清を除去した。予め用時調製した最終濃度１μｇ／ｍｌで組換えヒトアポリポプロテインＥ３（ＡｐｏＥ３）（富士フイルム和光、０１０－２０２６１）を含有させた活性化用培地で、活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。
　実施例８～１２９および実施例４０５の後添加法で調製したＲＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり１．８～４．０μｇ（総ＲＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。 <Test Example 5> Nucleic acid delivery to activated T cells using post-added LNPs (TCR KO)
<1> LNP treatment of activated T cells On day 3 of culture, the required number of activated T cells were collected, centrifuged, and the supernatant was removed. The activated T cells were adjusted to a concentration of 1.0 x 10 6 cells/ml in an activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg/ml, which had been prepared just before use, and seeded onto a 96-well plate.
RNA-encapsulating LNPs prepared by the post-addition method of Examples 8 to 129 and 405 were added at 1.8 to 4.0 μg (total RNA amount) per 1.0×10 6 cells, and cultured in a 37° C., 5% CO 2 incubator.

　ＬＮＰ添加から２４時間後に、Ｔ細胞懸濁液に活性化用培地を体積比１：３で添加して拡大培養し、さらに３日間培養した。 24 hours after the addition of LNP, the T cell suspension was expanded by adding activation medium at a volume ratio of 1:3, and then cultured for an additional 3 days.

＜２＞活性化Ｔ細胞におけるＴＣＲ　ＫＯ効率
　培養開始７日目に、フローサイトメトリーにて各手法で処理したＴ細胞のＴＣＲ　陰性細胞比率を測定し、ＴＣＲ　ＫＯ効率を評価した。その結果を表９に示した。 <2> TCR KO efficiency in activated T cells On day 7 after the start of culture, the ratio of TCR negative cells in the T cells treated by each method was measured by flow cytometry to evaluate the TCR KO efficiency. The results are shown in Table 9.

＜試験例６＞後添加ＬＮＰを用いた活性化Ｔ細胞へのプラスミドＤＮＡ送達
＜１＞活性化Ｔ細胞のＬＮＰ処理
　培養３日目の活性化Ｔ細胞を必要細胞数分取し、遠心および上清を除去した。予め用時調製した最終濃度１μｇ／ｍｌで組換えヒトアポリポプロテインＥ３（ＡｐｏＥ３）（富士フイルム和光、０１０－２０２６１）を含有させた活性化用培地または活性化用CDM培地で、活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。
　実施例１４８～４０４の後添加法で調製したプラスミドＤＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり０．４～１０．０μｇ（総ＤＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。 <Test Example 6> Delivery of plasmid DNA to activated T cells using post-added LNPs <1> LNP treatment of activated T cells The activated T cells on day 3 of culture were collected in the required number of cells, centrifuged, and the supernatant was removed. The activated T cells were adjusted to a concentration of 1.0 x 10 6 cells/ml in activation medium or activation CDM medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg/ml, which had been prepared just before use, and seeded on a 96-well plate.
Plasmid DNA-encapsulating LNPs prepared by the post-addition method of Examples 148 to 404 were added at 0.4 to 10.0 μg (total DNA amount) per 1.0×10 6 cells, and cultured in a 37° C., 5% CO 2 incubator.

＜２＞活性化Ｔ細胞におけるＧＦＰ陽性細胞比率
　培養開始４日目に、フローサイトメトリーにて各手法で処理したＴ細胞のＧＦＰ陽性細胞比率を測定し、プラスミドＤＮＡ導入効率を評価した。その結果を表１０に示した。 <2> Ratio of GFP-positive cells in activated T cells On day 4 of the culture, the ratio of GFP-positive cells in the T cells treated by each method was measured by flow cytometry to evaluate the efficiency of plasmid DNA introduction. The results are shown in Table 10.

＜試験例７＞後添加ＬＮＰを用いた活性化Ｔ細胞への核酸送達（ＴＣＲ　ＫＯ）
＜１＞活性化Ｔ細胞のＬＮＰ処理
　培養３日目の活性化Ｔ細胞を必要細胞数分取し、遠心および上清を除去した。予め用時調製した最終濃度１μｇ／ｍｌで組換えヒトアポリポプロテインＥ３（ＡｐｏＥ３）（富士フイルム和光、０１０－２０２６１）を含有させた活性化用培地で、活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。
　実施例８８～９０の後添加法で調製したＲＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり０．４～４．０μｇ（総ＲＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。 <Test Example 7> Nucleic acid delivery to activated T cells using post-added LNPs (TCR KO)
<1> LNP treatment of activated T cells On day 3 of culture, the required number of activated T cells were collected, centrifuged, and the supernatant was removed. The activated T cells were adjusted to a concentration of 1.0 x 10 6 cells/ml in an activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg/ml, which had been prepared just before use, and seeded onto a 96-well plate.
The RNA-encapsulating LNPs prepared by the post-addition method of Examples 88 to 90 were added at 0.4 to 4.0 μg (total RNA amount) per 1.0×10 6 cells, and the cells were cultured in a 37° C., 5% CO 2 incubator.

　ＬＮＰ添加から２４時間後に、Ｔ細胞懸濁液に活性化用培地を体積比１：３で添加して拡大培養し、さらに３日間培養した。 24 hours after the addition of LNP, the T cell suspension was expanded by adding activation medium at a volume ratio of 1:3, and then cultured for an additional 3 days.

＜２＞活性化Ｔ細胞におけるＴＣＲ　ＫＯ効率
　培養開始７日目に、フローサイトメトリーにて各手法で処理したＴ細胞のＴＣＲ　陰性細胞比率を測定し、ＴＣＲ　ＫＯ効率を評価した。その結果を図５に示した。 <2> TCR KO efficiency in activated T cells On day 7 after the start of culture, the ratio of TCR-negative cells in the T cells treated by each method was measured by flow cytometry to evaluate the TCR KO efficiency. The results are shown in FIG.

＜試験例８＞添加剤を用いた活性化Ｔ細胞への核酸送達
　培養３日目の活性化Ｔ細胞を必要細胞数分取し、遠心および上清を除去した。予め用時調製した最終濃度１μｇ／ｍｌで組換えヒトアポリポプロテインＥ３（ＡｐｏＥ３）（富士フイルム和光、０１０－２０２６１）、もしくはレトロネクチン（タカラバイオ、）を含有もしくは非含有の活性化用培地で、活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。 Test Example 8: Nucleic acid delivery to activated T cells using additives The activated T cells on day 3 of culture were collected in the required number of cells, centrifuged, and the supernatant was removed. The activated T cells were adjusted to a concentration of 1.0 x 10 6 cells/ml in an activation medium containing or not containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) or Retronectin (Takara Bio) at a final concentration of 1 μg/ml, which had been prepared just before use, and seeded on a 96-well plate.

　実施例１、４で調製したＧＦＰ　ｍＲＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり４．０μｇ（総ＲＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。ＬＮＰ添加から２４時間後に細胞を回収し、フローサイトメトリーにて各ＬＮＰで処理したＴ細胞のＧＦＰ陽性細胞比率を測定し、ＧＦＰ　ｍＲＮＡ導入効率を評価した。本結果を図６に示す。
　実施例１、４のＬＮＰは、レトロネクチン単独添加により活性化Ｔ細胞へのｍＲＮＡ送達効率を向上させ、さらにＡｐｏＥ３とレトロネクチンの併用によりさらに効率が向上した。 The GFP mRNA-encapsulated LNPs prepared in Examples 1 and 4 were added to 1.0 x 106 cells at 4.0 μg (total RNA amount) and cultured in a 37°C, 5% CO2 incubator. 24 hours after the addition of LNP, the cells were collected, and the GFP-positive cell ratio of T cells treated with each LNP was measured by flow cytometry to evaluate the GFP mRNA introduction efficiency. The results are shown in Figure 6.
The LNPs of Examples 1 and 4 improved the efficiency of mRNA delivery to activated T cells by adding RetroNectin alone, and the efficiency was further improved by using ApoE3 in combination with RetroNectin.

＜試験例９＞長期培養後のＴ細胞への核酸送達
　培養１０日目の（活性化後７日目）のＴ細胞を必要細胞数分取し、遠心および上清を除去した。予め用時調製した最終濃度１μｇ／ｍｌで組換えヒトアポリポプロテインＥ３（ＡｐｏＥ３）（富士フイルム和光、０１０－２０２６１）を含有させた活性化用培地で、活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。 Test Example 9: Nucleic acid delivery to T cells after long-term culture The required number of T cells on day 10 of culture (day 7 after activation) were collected, centrifuged, and the supernatant was removed. The activated T cells were adjusted to a concentration of 1.0 x 106 cells/ml in an activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg/ml, which had been prepared just before use, and seeded on a 96-well plate.

　実施例１、３、４で調製したＧＦＰ　ｍＲＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり４．０μｇ（総ＲＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。ＬＮＰ添加から２４時間後に細胞を回収し、フローサイトメトリーにて各ＬＮＰで処理したＴ細胞のＧＦＰ陽性細胞比率を測定し、ＧＦＰ　ｍＲＮＡ導入効率を評価した。本結果を図７(Day10 TF条件)に示す。 The GFP mRNA-encapsulated LNPs prepared in Examples 1, 3, and 4 were added to 1.0 x 10 6 cells at 4.0 μg (total RNA amount) and cultured in a 37°C, 5% CO 2 incubator. 24 hours after the addition of LNP, the cells were collected, and the GFP-positive cell ratio of T cells treated with each LNP was measured by flow cytometry to evaluate the GFP mRNA introduction efficiency. The results are shown in Figure 7 (Day 10 TF condition).

＜試験例１０＞長期培養後のＴ細胞への核酸送達
　活性化培地で培養９日目の（活性化後６日目）のＴ細胞を回収し、新たに調整した活性化培地、もしくは５ｎｇ／ｍｌ　ＩＬ－２含有Ｏｐｔｉ－ＭＥＭ（ＴＭ）　Ｉ　Ｒｅｄｕｃｅｄ　Ｓｅｒｕｍ　Ｍｅｄｉｕｍ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ，３１９８５０６２）に再懸濁し、１日間培養した。これら培養１０日目のＴ細胞を必要細胞数分取し、遠心および上清を除去した。予め用時調製した最終濃度１μｇ／ｍｌで組換えヒトアポリポプロテインＥ３（ＡｐｏＥ３）（富士フイルム和光、０１０－２０２６１）を含有させた活性化用培地で、活性化Ｔ細胞を１．０×１０６ｃｅｌｌｓ／ｍｌ濃度に調整し、９６ウェルプレートに播種した。 <Test Example 10> Nucleic acid delivery to T cells after long-term culture T cells were collected on the 9th day of culture in activation medium (6th day after activation), resuspended in a freshly prepared activation medium or Opti-MEM™ I Reduced Serum Medium (Thermo Fisher, 31985062) containing 5 ng/ml IL-2, and cultured for 1 day. The required number of T cells on the 10th day of culture were collected, centrifuged, and the supernatant was removed. The activated T cells were adjusted to a concentration of 1.0 x 10 6 cells/ml in activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg/ml prepared in advance, and seeded on a 96-well plate.

　実施例１、３，４で調製したＧＦＰ　ｍＲＮＡ内包ＬＮＰを使用して、１．０×１０６ｃｅｌｌｓあたり４．０μｇ（総ＲＮＡ量）となるように添加し、３７℃５％ＣＯ２インキュベーターにて培養した。ＬＮＰ添加から２４時間後に細胞を回収し、フローサイトメトリーにて各ＬＮＰで処理したＴ細胞のＧＦＰ陽性細胞比率を測定し、ＧＦＰ　ｍＲＮＡ導入効率を評価した。本結果を図７（Ｄａｙ９　ＭＣ＿Ｄａｙ１０　ＴＦ条件、Ｄａｙ９　ＯｐｔｉＭＥＭ＿Ｄａｙ１０　ＴＦ条件）に示す。 The GFP mRNA-encapsulated LNP prepared in Examples 1, 3, and 4 was added to 1.0 x 10 6 cells at 4.0 μg (total RNA amount) and cultured in a 37°C 5% CO 2 incubator. 24 hours after the addition of LNP, the cells were collected, and the GFP positive cell ratio of T cells treated with each LNP was measured by flow cytometry to evaluate the GFP mRNA introduction efficiency. The results are shown in Figure 7 (Day 9 MC_Day 10 TF condition, Day 9 OptiMEM_Day 10 TF condition).

Claims (17)

式（１）で表される化合物またはその塩であるイオン化可能な脂質と、非イオン化脂質と、非イオン性高分子を有する脂質と、核酸とを含む脂質組成物を含む、免疫細胞への核酸送達剤。
式中、Ｒ１、Ｒ２、Ｒ３およびＲ４はそれぞれ独立に、水素原子、置換されていてもよい炭素数１～２４の炭化水素基を示し、
　Ｒ１、Ｒ２、Ｒ３およびＲ４が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基はそれぞれ独立に、－Ｃ（Ｏ）Ｏ－Ｒ１１、－ＯＣ（Ｏ）－Ｒ１２、－Ｏ－Ｒ１３、－ＣＯ－Ｒ１４、－ＯＣ（Ｏ）Ｏ－Ｒ１５、または－Ｓ－Ｓ－Ｒ１６を示し、
　Ｒ１１、Ｒ１２、Ｒ１３、Ｒ１４、Ｒ１５およびＲ１６はそれぞれ独立に、－Ｓ－Ｒ１７で置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ１７は、炭素数１～１２の炭化水素基を示し、
　Ｒ５およびＲ６はそれぞれ独立に、置換されていてもよい炭素数１～１８の炭化水素基を示し、
　Ｒ５およびＲ６が示す置換されていてもよい炭素数１～１８の炭化水素基上の置換基はそれぞれ独立に、－ＯＨ、－ＣＯＯＨ、－ＮＲ２１Ｒ２２、－ＯＣ（Ｏ）Ｏ－Ｒ２３、－Ｃ（Ｏ）Ｏ－Ｒ２４、－ＯＣ（Ｏ）－Ｒ２５、－Ｏ－Ｒ２６、－Ｃ（Ｏ）ＮＲ２７Ｒ２８、－ＮＲ２９Ｃ（Ｏ）Ｒ３０、－Ｎ（Ｒ３１）Ｓ（Ｏ）２Ｒ３２、－Ｎ（Ｒ３３）Ｃ（Ｏ）Ｎ（Ｒ３４）Ｒ３５、－Ｎ（Ｒ３６）Ｃ（Ｓ）Ｎ（Ｒ３７）Ｒ３８、－ＯＣ（Ｏ）Ｎ（Ｒ３９）Ｒ４０、または－Ｎ（Ｒ４１）Ｃ（Ｏ）ＯＲ４２を示し、
　Ｒ２１およびＲ２２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２はそれぞれ独立に、水素原子、または置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基は、炭素数６～２０のアリール基、ヘテロ環基、－ＯＨ、－ＣＯＯＨ、または－ＮＲ５１Ｒ５２を示し、Ｒ５１およびＲ５２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ７、Ｒ８、およびＲ９はそれぞれ独立に、炭素数２～８の炭化水素基を示し、
　Ｒ５とＲ６、またはＲ５とＲ７は一緒になって４～７員環を形成してもよい。 A nucleic acid delivery agent for immune cells, comprising a lipid composition comprising an ionizable lipid which is a compound represented by formula (1) or a salt thereof, a nonionizable lipid, a lipid having a nonionic polymer, and a nucleic acid.
In the formula, R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms;
Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ;
R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R 17 , R 17 represents a hydrocarbon group having 1 to 12 carbon atoms;
R 5 and R 6 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted;
Substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by R 5 and R 6 are each independently -OH, -COOH, -NR 21 R 22 , -OC(O)O-R 23 , -C(O)O-R 24 , -OC(O)-R 25 , -O-R 26 , -C(O)NR 27 R 28 , -NR 29 C(O)R 30 , -N(R 31 )S(O) 2 R 32 , -N(R 33 )C(O)N(R 34 ) R 35 , -N(R 36 )C(S)N(R 37 )R 38 , -OC(O)N(R 39 )R 40 , or -N(R 41 )C(O)OR Showing 42 ,
R 21 and R 22 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , and R 42 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 40 , R 41 , and R 42 represents an aryl group, heterocyclic group, -OH, -COOH, or -NR 51 R 52 having 6 to 20 carbon atoms, and R 51 and R 52 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 7 , R 8 , and R 9 each independently represent a hydrocarbon group having 2 to 8 carbon atoms;
R 5 and R 6 , or R 5 and R 7 may join together to form a 4- to 7-membered ring. 前記イオン化可能な脂質が、脂質組成物中の全脂質に対するモル比で、２０～６０モル％である、請求項１に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to claim 1, wherein the ionizable lipid is present in an amount of 20 to 60 mol % in terms of a molar ratio relative to the total lipids in the lipid composition. 前記非イオン化脂質が、ステロールまたはその誘導体、およびリン脂質を含む、請求項１に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to claim 1, wherein the non-ionized lipid comprises a sterol or a derivative thereof, and a phospholipid. 前記リン脂質が、ジステアロイルホスファチジルコリン、ジオレオイルホスファチジルコリン、およびジオレオイルホスファチジルエタノールアミンからなる群から選択される、請求項３に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to claim 3, wherein the phospholipid is selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. 前記ステロールまたはその誘導体が、脂質組成物中の全脂質に対するモル比で、３０～７０モル％である、請求項３に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to claim 3, wherein the sterol or its derivative is present in an amount of 30 to 70 mol % in terms of a molar ratio relative to the total lipids in the lipid composition. 前記リン脂質が、脂質組成物中の全脂質に対するモル比で、１～３０モル％である、請求項３に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to claim 3, wherein the phospholipid is present in an amount of 1 to 30 mol % relative to the total lipids in the lipid composition. 前記非イオン性高分子を有する脂質が、ポリエチレングリコール鎖を有する脂質である、請求項１から６の何れか一項に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to any one of claims 1 to 6, wherein the lipid having a nonionic polymer is a lipid having a polyethylene glycol chain. 前記ポリエチレングリコール鎖を有する脂質が、ジミリストイル－ｒａｃ－グリセロールポリエチレングリコール、ジステアロイル－ｒａｃ－グリセロールポリエチレングリコール、およびジステアロイルホスファチジルエタノールアミンポリエチレングリコールから選択される、請求項７に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to claim 7, wherein the lipid having a polyethylene glycol chain is selected from dimyristoyl-rac-glycerol polyethylene glycol, distearoyl-rac-glycerol polyethylene glycol, and distearoylphosphatidylethanolamine polyethylene glycol. 前記非イオン性高分子を有する脂質が、脂質組成物中の全脂質に対するモル比で、０．１～３モル％である、請求項１から６の何れか一項に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to any one of claims 1 to 6, wherein the lipid having the nonionic polymer is present in an amount of 0.1 to 3 mol% in terms of a molar ratio relative to the total lipid in the lipid composition. 前記脂質組成物の全脂質と核酸の質量比が７：１～１０００：１である、請求項１から６の何れか一項に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to any one of claims 1 to 6, wherein the mass ratio of the total lipids to the nucleic acid in the lipid composition is 7:1 to 1000:1. 前記免疫細胞が、活性化細胞であるか、または非活性化細胞である、請求項１から６の何れか一項に記載の免疫細胞への核酸送達剤。 The nucleic acid delivery agent for immune cells according to any one of claims 1 to 6, wherein the immune cells are activated cells or non-activated cells. 前記イオン化可能な脂質が以下の化合物のうちの１つ以上である、請求項１から６の何れか一項に記載の免疫細胞への核酸送達剤。
























 The nucleic acid delivery agent for immune cells according to any one of claims 1 to 6, wherein the ionizable lipid is one or more of the following compounds:
























請求項１から６の何れか一項に記載の免疫細胞への核酸送達剤を、免疫細胞と接触させることを含む、免疫細胞に核酸を送達する方法（ただし、インビボでの送達方法を除く）。 A method for delivering nucleic acid to an immune cell (excluding in vivo delivery methods), comprising contacting the immune cell with a nucleic acid delivery agent for immune cells according to any one of claims 1 to 6. 前記免疫細胞が、活性化細胞または非活性化細胞である、請求項１３に記載の方法。 The method of claim 13, wherein the immune cells are activated or non-activated cells. 前記核酸送達剤を免疫細胞に接触させる前に、核酸送達剤もしくは免疫細胞に対して（ｉ）アポリポタンパク、及び／又は（ｉｉ）細胞結合ドメインとヘパリン結合ドメインとを含むタンパク質を添加する工程を含む、請求項１３に記載の方法。 The method according to claim 13, comprising the step of adding (i) an apolipoprotein and/or (ii) a protein including a cell-binding domain and a heparin-binding domain to the nucleic acid delivery agent or the immune cells before contacting the nucleic acid delivery agent with the immune cells. 式（１）で表される化合物またはその塩であるイオン化可能な脂質と、非イオン化脂質と、非イオン性高分子を有する脂質とを用いて、核酸を含まない脂質粒子を調製する工程と、
前記の核酸を含まない脂質粒子と核酸とを混合する工程とを含む、
請求項１に記載の核酸送達剤の製造方法。
式中、Ｒ１、Ｒ２、Ｒ３およびＲ４はそれぞれ独立に、水素原子、置換されていてもよい炭素数１～２４の炭化水素基を示し、
　Ｒ１、Ｒ２、Ｒ３およびＲ４が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基はそれぞれ独立に、－Ｃ（Ｏ）Ｏ－Ｒ１１、－ＯＣ（Ｏ）－Ｒ１２、－Ｏ－Ｒ１３、－ＣＯ－Ｒ１４、－ＯＣ（Ｏ）Ｏ－Ｒ１５、または－Ｓ－Ｓ－Ｒ１６を示し、
　Ｒ１１、Ｒ１２、Ｒ１３、Ｒ１４、Ｒ１５およびＲ１６はそれぞれ独立に、－Ｓ－Ｒ１７で置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ１７は、炭素数１～１２の炭化水素基を示し、
　Ｒ５およびＲ６はそれぞれ独立に、置換されていてもよい炭素数１～１８の炭化水素基を示し、
　Ｒ５およびＲ６が示す置換されていてもよい炭素数１～１８の炭化水素基上の置換基はそれぞれ独立に、－ＯＨ、－ＣＯＯＨ、－ＮＲ２１Ｒ２２、－ＯＣ（Ｏ）Ｏ－Ｒ２３、－Ｃ（Ｏ）Ｏ－Ｒ２４、－ＯＣ（Ｏ）－Ｒ２５、－Ｏ－Ｒ２６、－Ｃ（Ｏ）ＮＲ２７Ｒ２８、－ＮＲ２９Ｃ（Ｏ）Ｒ３０、－Ｎ（Ｒ３１）Ｓ（Ｏ）２Ｒ３２、－Ｎ（Ｒ３３）Ｃ（Ｏ）Ｎ（Ｒ３４）Ｒ３５、－Ｎ（Ｒ３６）Ｃ（Ｓ）Ｎ（Ｒ３７）Ｒ３８、－ＯＣ（Ｏ）Ｎ（Ｒ３９）Ｒ４０、または－Ｎ（Ｒ４１）Ｃ（Ｏ）ＯＲ４２を示し、
　Ｒ２１およびＲ２２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２はそれぞれ独立に、水素原子、または置換されていてもよい炭素数１～２４の炭化水素基を示し、Ｒ２３、Ｒ２４、Ｒ２５、Ｒ２６、Ｒ２７、Ｒ２８、Ｒ２９、Ｒ３０、Ｒ３１、Ｒ３２、Ｒ３３、Ｒ３４、Ｒ３５、Ｒ３６、Ｒ３７、Ｒ３８、Ｒ３９、Ｒ４０、Ｒ４１、およびＲ４２が示す置換されていてもよい炭素数１～２４の炭化水素基上の置換基は、炭素数６～２０のアリール基、ヘテロ環基、－ＯＨ、－ＣＯＯＨ、または－ＮＲ５１Ｒ５２を示し、Ｒ５１およびＲ５２はそれぞれ独立に、水素原子、または炭素数１～８の炭化水素基を示し、
　Ｒ７、Ｒ８、およびＲ９はそれぞれ独立に、炭素数２～８の炭化水素基を示し、
　Ｒ５とＲ６、またはＲ５とＲ７は一緒になって４～７員環を形成してもよい。 A step of preparing a lipid particle that does not contain nucleic acid using an ionizable lipid that is a compound represented by formula (1) or a salt thereof, a nonionizable lipid, and a lipid having a nonionic polymer;
mixing the nucleic acid-free lipid particles with nucleic acid;
A method for producing the nucleic acid delivery agent according to claim 1.
In the formula, R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms;
Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ;
R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R 17 , R 17 represents a hydrocarbon group having 1 to 12 carbon atoms;
R 5 and R 6 each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted;
Substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by R 5 and R 6 are each independently -OH, -COOH, -NR 21 R 22 , -OC(O)O-R 23 , -C(O)O-R 24 , -OC(O)-R 25 , -O-R 26 , -C(O)NR 27 R 28 , -NR 29 C(O)R 30 , -N(R 31 )S(O) 2 R 32 , -N(R 33 )C(O)N(R 34 ) R 35 , -N(R 36 )C(S)N(R 37 )R 38 , -OC(O)N(R 39 )R 40 , or -N(R 41 )C(O)OR Showing 42 ,
R 21 and R 22 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , and R 42 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 40 , R 41 , and R 42 represents an aryl group, heterocyclic group, -OH, -COOH, or -NR 51 R 52 having 6 to 20 carbon atoms, and R 51 and R 52 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms;
R 7 , R 8 , and R 9 each independently represent a hydrocarbon group having 2 to 8 carbon atoms;
R 5 and R 6 , or R 5 and R 7 may join together to form a 4- to 7-membered ring. 下記の化合物からなる群から選択される少なくとも１つの化合物またはその塩。


ビス（２－ヘキシロクチル）　１１－（２－（ジエチルアミノ）エチル）－６，１６－ジオクチル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
２－（２－（２－（ビス（２－デカノイルオキシエチル）カルバモイルオキシ）エチル－（２－（ジエチルアミノ）エチル）アミノ）エトキシカルボニル－（２－デカノイルオキシエチル）アミノ）エチル　デカノエート
ビス（２－ペンチルヘプチル）　１１－（３－（ジエチルアミノ）プロピル）－６，１６－ジイソプロピル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
ビス（２－ペンチルヘプチル）　１１－（３－（ジエチルアミノ）プロピル）－７，１５－ジオキソ－６，１６－ジプロピル－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
ビス（２－ヘキシルオクチル）　６，１６－ジブチル－１１－（３－（ジエチルアミノ）プロピル）－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
ジトリデシル　８－（２－（ジエチルアミノ）エチル）－４，１２－ジオキソ－３，１３－ビス（２－オキソ－２－（トリデシルオキシ）エチル）－５，１１－ジオキサ－３，８，１３－トリアザペンタデカンジオエート
ビス（２－ヘキシルオクチル）　１１－（３－（ジエチルアミノ）プロピル）－６，１６－ジオクチル－７，１５－ジオキソ－８，１４－ジオキサ－６，１１，１６－トリアザヘニコサンジオエート
ビス（２－ペンチルヘプチル）　１２－（４－（ジエチルアミノ）ブチル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（ジメチルアミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－メチルピペリジン－４－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（１－エチルピペリジン－４－イル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－イソプロピルピペリジン－４－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（４－ヒドロキシブチル）アミノ）エチル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（２－（（４－ヒドロキシブチル）（メチル）アミノ）エチル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（（４－ヒドロキシブチル）（メチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（３－（（４－ヒドロキシブチル）（メチル）アミノ）プロピル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（（４－ヒドロキシブチル）（メチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（４－ヒドロキシブチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（４－ヒドロキシブチル）アミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（４－ヒドロキシブチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（２－（（３－ヒドロキシプロピル）（メチル）アミノ）エチル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（（３－ヒドロキシプロピル）（メチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（３－（（３－ヒドロキシプロピル）（メチル）アミノ）プロピル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（（３－ヒドロキシプロピル）（メチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（３－ヒドロキシプロピル）アミノ）エチル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（３－ヒドロキシプロピル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（３－ヒドロキシプロピル）アミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（３－ヒドロキシプロピル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（２－（（２－ヒドロキシエチル）（メチル）アミノ）エチル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（（２－ヒドロキシエチル）（メチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（３－（（２－ヒドロキシエチル）（メチル）アミノ）プロピル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（（２－ヒドロキシエチル）（メチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（２－ヒドロキシエチル）アミノ）エチル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（エチル（２－ヒドロキシエチル）アミノ）エチル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（２－ヒドロキシエチル）アミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（エチル（２－ヒドロキシエチル）アミノ）プロピル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（８－メチル－８－アザビシクロ［３．２．１］オクタン－３－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２、　１７－トリアザトリコサン二酸
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－８，１６－ジオキソ－１２－（１，２，２，６，６－ペンタメチルピペリジン－４－イル）－９，１５－ジオキサ－７，１２，１７－トリアザトリコサン二酸塩
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－メチルアゼチジン－３－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－メチルピロリジン－３－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－メチルアゼパン－４－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（（１ｒ，４ｒ）－４－（ジメチルアミノ）シクロヘキシル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（（１ｓ，４ｓ）－４－（ジメチルアミノ）シクロヘキシル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－１２－（１－（２－ヒドロキシエチル）ピペリジン－４－イル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－８，１６－ジオキソ－１２－（２－（ピロリジン－１－イル）エチル）－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジヘプチル－８，１６－ジオキソ－１２－（２－（ピペリジン－１－イル）エチル）－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（１－メチルピペリジン－４－イル）－７，１７－ジオクチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（ビス（２－ヒドロキシエチル）アミノ）プロピル）－７，１７－ジヘプチル－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（２－（ジエチルアミノ）エチル）－８，１６－ジオキソ－７，１７－ジプロピル－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　１２－（３－（ジエチルアミノ）プロピル）－８，１６－ジオキソ－７，１７－ジプロピル－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート
ビス（２－ペンチルヘプチル）　７，１７－ジブチル－１２－（３－（ジエチルアミノ）プロピル）－８，１６－ジオキソ－９，１５－ジオキサ－７，１２，１７－トリアザトリコサンジオアート At least one compound selected from the group consisting of the following compounds or a salt thereof:


Bis(2-hexyloctyl) 11-(2-(diethylamino)ethyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate
2-(2-(2-(bis(2-decanoyloxyethyl)carbamoyloxy)ethyl-(2-(diethylamino)ethyl)amino)ethoxycarbonyl-(2-decanoyloxyethyl)amino)ethyl decanoate
Bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-6,16-diisopropyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate
Bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-7,15-dioxo-6,16-dipropyl-8,14-dioxa-6,11,16-triazahenicosanedioate
Bis(2-hexyloctyl) 6,16-dibutyl-11-(3-(diethylamino)propyl)-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate
Ditridecyl 8-(2-(diethylamino)ethyl)-4,12-dioxo-3,13-bis(2-oxo-2-(tridecyloxy)ethyl)-5,11-dioxa-3,8,13-triazapentadecanedioate
Bis(2-hexyloctyl) 11-(3-(diethylamino)propyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate
Bis(2-pentylheptyl) 12-(4-(diethylamino)butyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(dimethylamino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(1-ethylpiperidin-4-yl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-isopropylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((4-hydroxybutyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-((4-hydroxybutyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((4-hydroxybutyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-((4-hydroxybutyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(4-hydroxybutyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(4-hydroxybutyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((3-hydroxypropyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-((3-hydroxypropyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((3-hydroxypropyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-((3-hydroxypropyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(3-hydroxypropyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(3-hydroxypropyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(3-hydroxypropyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(3-hydroxypropyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((2-hydroxyethyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-((2-hydroxyethyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(2-hydroxyethyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(ethyl(2-hydroxyethyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)-8,16-dioxo-9,15-dioxa-7,12, 17-triazatricosane diacid
Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(1,2,2,6,6-pentamethylpiperidin-4-yl)-9,15-dioxa-7,12,17-triazatricosane diacid salt
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazetidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpyrrolidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazepan-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-((1r,4r)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-((1s,4s)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-(2-hydroxyethyl)piperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(pyrrolidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(piperidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(1-methylpiperidin-4-yl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(bis(2-hydroxyethyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(2-(diethylamino)ethyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 12-(3-(diethylamino)propyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatricosane diate
Bis(2-pentylheptyl) 7,17-dibutyl-12-(3-(diethylamino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane diate
PCT/JP2024/046426 2023-12-28 2024-12-27 Agent for delivering nucleic acid to immune cells and method for delivering nucleic acid to immune cells Pending WO2025143232A1 (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3872171A (en) * 1971-05-24 1975-03-18 Pfizer Polyamines as antiviral agents in animals
JPH0222259A (en) * 1987-02-06 1990-01-25 Takeda Chem Ind Ltd Substituted amine derivative
WO2021095876A1 (en) * 2019-11-15 2021-05-20 富士フイルム株式会社 Lipid composition
WO2022170833A1 (en) * 2021-02-09 2022-08-18 中山大学 Ionizable cationic lipid analog material and use thereof as drug delivery carrier
WO2022251665A1 (en) * 2021-05-28 2022-12-01 Renagade Therapeutics Management Inc. Lipid nanoparticles and methods of use thereof
WO2022270941A1 (en) * 2021-06-24 2022-12-29 주식회사 테르나테라퓨틱스 Lipid nanoparticles and method for preparing same
WO2024158042A1 (en) * 2023-01-27 2024-08-02 富士フイルム株式会社 Compound or salt thereof, lipid composition, pharmaceutical composition, and delivery carrier

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3872171A (en) * 1971-05-24 1975-03-18 Pfizer Polyamines as antiviral agents in animals
JPH0222259A (en) * 1987-02-06 1990-01-25 Takeda Chem Ind Ltd Substituted amine derivative
WO2021095876A1 (en) * 2019-11-15 2021-05-20 富士フイルム株式会社 Lipid composition
WO2022170833A1 (en) * 2021-02-09 2022-08-18 中山大学 Ionizable cationic lipid analog material and use thereof as drug delivery carrier
WO2022251665A1 (en) * 2021-05-28 2022-12-01 Renagade Therapeutics Management Inc. Lipid nanoparticles and methods of use thereof
WO2022270941A1 (en) * 2021-06-24 2022-12-29 주식회사 테르나테라퓨틱스 Lipid nanoparticles and method for preparing same
WO2024158042A1 (en) * 2023-01-27 2024-08-02 富士フイルム株式会社 Compound or salt thereof, lipid composition, pharmaceutical composition, and delivery carrier

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