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

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

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

[0001] The present invention relates to a lipid-containing agent for delivering nucleic acids to immune cells. The present invention further relates to a method for delivering nucleic acids to immune cells using the nucleic acid delivery agent.

[0002] In immune cell therapies, including chimeric antigen receptor (CAR) T-cell therapy, immune cells that have been genetically modified to express a therapeutic foreign gene such as CAR or to alter endogenous gene expression are used. Genetic modification of immune cells generally involves viral vector methods, but these methods pose issues such as safety concerns due to viruses and high costs. Furthermore, electroporation has traditionally been used as a non-viral method, but electroporation can cause cytotoxicity and DNA damage, leading to problems such as growth retardation and chromosomal abnormalities.

[0003] Regarding nucleic acid delivery to immune cells using lipid nanoparticles (LNPs), Patent Document 1 describes encapsulating Cas9 mRNA and gRNA in separate LNPs to perform genome editing at multiple sites, and using these to perform genome editing of T cells. Patent Document 2 describes the delivery of nucleic acid to T cells using LNPs encapsulating nucleic acid (mRNA).

[0004] International Publication No. WO2021 / 222287 International Publication No. WO2020 / 210901

[0005] The lipid nanoparticles described in Patent Documents 1 and 2 require a process to sufficiently activate immune cells, which limits the culturing methods that can be used. Since immune cell activation processes are known to reduce the efficacy of immune cells in treatment, it is desirable to avoid such activation processes.

[0006] In view of the above circumstances, an object of the present invention is to provide an agent for nucleic acid delivery to immune cells that can deliver nucleic acids to both activated and unactivated immune cells. Another object of the present invention is to provide a method for delivering nucleic acids to immune cells using the agent for nucleic acid delivery to immune cells.

[0007] As a result of intensive research to solve the above problems, the present inventors have found that a lipid composition comprising an ionizable lipid that 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, and have completed the present invention.

[0008] <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 represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R １ , R ２ , R ３ and R ４ The substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by the formula (I) are each independently —C(O)O—R １１ , —OC(O)—R １２ , -O-R １３ , -CO-R １４ , -OC(O)OR １５ , or -S-S-R １６ indicates, R １１ , R １２ , R １３ , R １４ , R １５ and R １６ are each independently -S-R １７ represents a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with １７ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by the formula (I) are each independently —OH, —COOH, —NR２１ R ２２ , -OC(O)OR 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 ４２ indicates, R ２１ and R ２２ each independently represents 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 represents 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 , R３４ , R ３５ , R ３６ , R ３７ , R ３８ , R ３９ , R ４０ , R ４１ , and R ４２ The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by is an aryl group having 6 to 20 carbon atoms, a heterocyclic group, —OH, —COOH, or —NR ５１ R ５２ indicates R ５１ and R ５２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; R ７ , R ８ , and R ９ each independently represents a hydrocarbon group having 2 to 8 carbon atoms; R ５ and R ６ , or R ５ and R ７may together form a 4- to 7-membered ring. <2> The agent for nucleic acid delivery to immune cells according to <1>, wherein the ionizable lipid accounts for 20 to 60 mol % in terms of a molar ratio relative to all lipids in the lipid composition. <3> The agent for nucleic acid delivery to immune cells according to <1> or <2>, wherein the non-ionizable lipid comprises a sterol or a derivative thereof, and a phospholipid. <4> The agent for nucleic acid delivery to immune cells according to <3>, wherein the phospholipid is selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. <5> The agent for nucleic acid delivery to immune cells according to <3> or <4>, wherein the sterol or a derivative thereof accounts for 30 to 70 mol % in terms of a molar ratio relative to all lipids in the lipid composition. <6> The agent for nucleic acid delivery to immune cells according to any one of <3> to <5>, wherein the phospholipid accounts for 1 to 30 mol % in terms of a molar ratio relative to all lipids in the lipid composition. <7> The agent for nucleic acid delivery to immune cells 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 nucleic acid delivery to immune cells 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 nucleic acid delivery to immune cells according to any one of <1> to <8>, wherein the lipid having a nonionic polymer accounts for 0.1 to 3 mol % in terms of a molar ratio relative to the total lipids in the lipid composition. <10> The agent for nucleic acid delivery to immune cells according to any one of <1> to <9>, wherein the mass ratio of total lipids to nucleic acids in the lipid composition is 7:1 to 1000:1. <11> The agent for delivering nucleic acids to immune cells according to any one of <1> to <10>, wherein the immune cells are activated cells or non-activated cells. <12> The agent for delivering nucleic acids to immune cells 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 nucleic acid to immune cells, comprising contacting immune cells 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 a 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 method for producing the nucleic acid delivery agent according to any one of <1> to <12>, comprising a step of preparing nucleic acid-free lipid particles using an ionizable lipid that is a compound represented by formula (1) or a salt thereof, a non-ionizable lipid, and a lipid having a nonionic polymer, and a step of mixing the nucleic acid-free lipid particles with nucleic acid. In the formula, R １ , R ２ , R ３ and R ４ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R １ , R ２ , R ３ and R ４ The substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by the formula (I) are each independently —C(O)O—R １１ , —OC(O)—R １２ , -O-R １３ , -CO-R １４ , -OC(O)OR １５ , or -S-S-R １６ indicates, R １１ , R １２ , R １３ , R １４ , R １５ and R １６ are each independently -S-R １７ represents a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with １７represents a hydrocarbon group having 1 to 12 carbon atoms, and R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by the formula (I) are each independently —OH, —COOH, —NR ２１ R ２２ , -OC(O)OR 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 ４２ indicates, R ２１ and R ２２ each independently represents 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 ４２each independently represents 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 ４０ , R ４１ , and R ４２ The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by is an aryl group having 6 to 20 carbon atoms, a heterocyclic group, —OH, —COOH, or —NR ５１ R ５２ indicates R ５１ and R ５２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; R ７ , R ８ , and R ９ each independently represents a hydrocarbon group having 2 to 8 carbon atoms; R ５ and R ６ , or R ５ and R ７ may be joined 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 dioate Bis(2-pentylheptyl) 12-(3-(dimethylamino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(1-ethylpiperidin-4-yl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-isopropylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((4-hydroxybutyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-((4-hydroxybutyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((4-hydroxybutyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-((4-hydroxybutyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-(ethyl(4-hydroxybutyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-(ethyl(4-hydroxybutyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((3-hydroxypropyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-((3-hydroxypropyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((3-hydroxypropyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-((3-hydroxypropyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-(ethyl(3-hydroxypropyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-(ethyl(3-hydroxypropyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-(ethyl(3-hydroxypropyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-(ethyl(3-hydroxypropyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((2-hydroxyethyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-((2-hydroxyethyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-(ethyl(2-hydroxyethyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-(ethyl(2-hydroxyethyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate 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 Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazetidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpyrrolidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazepan-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-((1r,4r)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-((1s,4s)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-(2-hydroxyethyl)piperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(pyrrolidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(piperidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(1-methylpiperidin-4-yl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-(bis(2-hydroxyethyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(2-(diethylamino)ethyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 12-(3-(diethylamino)propyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatricosane dioate Bis(2-pentylheptyl) 7,17-dibutyl-12-(3-(diethylamino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosane dioate

[0009] According to the present invention, it is possible to deliver nucleic acids to both activated and unactivated immune cells.

[0010] Figure 1 shows the results of measuring nucleic acid delivery to activated T cells. Figure 2 shows the results of measuring nucleic acid delivery to non-activated T cells. Figure 3 shows the results of measuring nucleic acid delivery to activated T cells in an activation medium without ApoE3. Figure 4 shows the results of measuring nucleic acid delivery to non-activated T cells in an activation medium without ApoE3. Figure 5 shows the results of measuring TCR KO efficiency in activated T cells. Figure 6 shows the results of measuring nucleic acid delivery to activated T cells under culture conditions with the addition of various proteins. Figure 7 shows the results of measuring nucleic acid delivery to long-term cultured T cells.

[0011] 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.

[0012] [Agent for delivering nucleic acid to immune cells] The agent for delivering nucleic acid to immune cells of the present invention comprises a lipid composition containing 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 represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R １ , R ２ , R ３ and R ４ The substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by the formula (I) are each independently —C(O)O—R １１ , —OC(O)—R １２ , -O-R １３ , -CO-R １４ , -OC(O)OR １５ , or -S-S-R １６ indicates, R １１ , R １２ , R １３ , R １４ , R １５ and R １６ are each independently -S-R １７ represents a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with １７represents a hydrocarbon group having 1 to 12 carbon atoms, and R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by the formula (I) are each independently —OH, —COOH, —NR ２１ R ２２ , -OC(O)OR 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 ４２ indicates, R ２１ and R ２２ each independently represents 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 ４２each independently represents 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 ４０ , R ４１ , and R ４２ The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by is an aryl group having 6 to 20 carbon atoms, a heterocyclic group, —OH, —COOH, or —NR ５１ R ５２ indicates R ５１ and R ５２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; R ７ , R ８ , and R ９ each independently represents a hydrocarbon group having 2 to 8 carbon atoms; R ５ and R ６ , or R ５ and R ７ may be joined together to form a 4- to 7-membered ring.

[0013] The nucleic acid delivery agent for immune cells of the present invention can be used to produce immune cells with modified gene expression, and can deliver target nucleic acids even to inactive immune cells, thereby preventing cell exhaustion and leading to improved cell therapy performance.

[0014] <Compound Represented by Formula (1) or Salts 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.

[0015] The alkyl group may be linear or branched, and may be linear or cyclic. Specific examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, and a 2-butyloctyl group. , 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, and the like.

[0016] The alkenyl group may be linear or branched, linear or cyclic. Specific examples include allyl, prenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably, (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably, (Z)-tridec-8-enyl), tetradecenyl (preferably, tetradec-9-enyl), and pentadecenyl (preferably, (Z)-pentadecen-8-enyl). , a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadeca-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadec-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group), and the like.

[0017] The alkynyl group may be linear or branched, open-chain or cyclic, and specific examples thereof include a propargyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group.

[0018] Preferably, all of the above alkenyl groups have one or two double bonds, and preferably, all of the alkynyl groups have one or two triple bonds.

[0019] R ７ , R ８ , and R ９The hydrocarbon group having 2 to 8 carbon atoms represented by 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 linear 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.

[0020] The aryl group having 6 to 20 carbon atoms is preferably an aryl group having 6 to 18 carbon atoms, more preferably an aryl group having 6 to 10 carbon atoms. Specific examples include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group.

[0021] The heterocyclic group means a heteroaryl group or a heteroaliphatic ring group.

[0022] The heteroaryl group refers to an aromatic heterocyclic group, and may be an aromatic heterocyclic group fused with an aromatic hydrocarbon ring, an aromatic aliphatic ring, or 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. The five-membered heteroaryl group is a monocyclic heteroaryl group having five atoms constituting the ring.

[0023] Furthermore, the aromatic heterocycle means an aromatic ring having a heteroatom as a ring member, and may be a condensed aromatic heterocycle, an aromatic hydrocarbon ring, a heteroaliphatic ring, or an 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.

[0024] The term "monocyclic nitrogen-containing heteroaryl group" refers to a heteroaryl group having an aromatic ring containing at least one nitrogen atom, such as pyrrolinyl, pyrrolyl, tetrahydropyridyl, pyridyl, imidazolinyl, imidazolyl, pyrazolinyl, pyrazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazolyl, or tetrazolyl (this heteroaryl group may be partially saturated). This heteroaryl group may be further fused with another aromatic ring or an aliphatic ring. The term "monocyclic oxygen-containing heteroaryl group" refers to a heteroaryl group having an aromatic ring containing at least one oxygen atom, such as a furanyl or pyranyl group (this heteroaryl group may be partially saturated). This heteroaryl group may be further fused with another aromatic ring or an aliphatic ring. The term "monocyclic nitrogen-containing oxygen-containing heteroaryl group" refers to an oxazolyl, isoxazolyl, or oxadiazolyl group (this heteroaryl group may be further fused 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.

[0025] The bicyclic nitrogen-containing heteroaryl group includes indolyl, isoindolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, quinolidinyl, 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, means a bicyclic heteroaryl group in which the ring containing at least one nitrogen atom has aromaticity (this heteroaryl group may be partially saturated), such as 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, 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.

[0026] The bicyclic oxygen-containing heteroaryl group means 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).

[0027] The bicyclic nitrogen-containing and oxygen-containing heteroaryl group includes 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, and 7,8-dihydro-5H-pyrano[4,3-c]pyridazinyl. and 5,6-dihydrofuro[2,3-c]pyridazinyl groups, and the like.

[0028] The heteroaliphatic cyclic group refers to a nitrogen-containing heteroaliphatic cyclic group, an oxygen-containing heteroaliphatic cyclic group, a sulfur-containing heteroaliphatic cyclic group, a nitrogen-containing oxygen-containing heteroaliphatic cyclic group, a nitrogen-containing sulfur-containing heteroaliphatic cyclic group, a heterobridged cyclic group, or a heterospirocyclic group. The heteroaliphatic ring refers to an aliphatic ring having a heteroatom as a ring member, and preferred examples 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 heterospirocyclic ring.

[0029] The term "nitrogen-containing heteroaliphatic cyclic group" refers to a heteroaliphatic cyclic group in which the ring containing at least one nitrogen atom is not aromatic, such as azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, octahydroazocinyl, imidazolidinyl, pyrazolidinyl, piperazinyl, and homopiperazinyl groups. This nitrogen-containing heteroaliphatic cyclic group may be further fused with another aromatic ring or an aliphatic ring. The term "oxygen-containing heteroaliphatic cyclic group" refers to a tetrahydrofuranyl, tetrahydropyranyl, oxetanyl, or 1,3-dioxanyl group, etc. This oxygen-containing heteroaliphatic cyclic group may be further fused with another aromatic ring or an aliphatic ring. The term "nitrogen-containing oxygen-containing heteroaliphatic cyclic group" refers to a morpholinyl or 1,4-oxazepanyl group, etc. This nitrogen-containing oxygen-containing heteroaliphatic cyclic group may be further fused with another aromatic ring or an aliphatic ring.

[0030] Heteroaliphatic ring C １－８ The alkyl group is a straight-chain, branched-chain or cyclic C alkyl group to which 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 is bonded. １－８ It means an alkyl group.

[0031] In formula (1), preferably, R １ Ga-R １ａ -L １ -R １ｂ indicates 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 Ga-R ３ａ -L ３ -R ３ｂ indicates R ３ａ represents a hydrocarbon group having 1 to 18 carbon atoms; L ３ 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 ４each independently represents an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; R ２ and R ４ The substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by the formula (I) are each independently —C(O)O—R １１ , —OC(O)—R １２ , -O-R １３ , -CO-R １４ , -OC(O)OR １５ , or -S-S-R １６ indicates, R １１ , R １２ , R １３ , R １４ , R １５ and R １６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 12 carbon atoms; R ５ and R ６ The substituents on the hydrocarbon group having 1 to 12 carbon atoms which may be substituted are each independently —OH, —O—R ２６ , —C(O)NR ２７ R ２８ , or -NR ２９ C(O)R ３０ indicates, R ２６ , R ２７ , R ２８ , R ２９ , and R ３０ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 12 carbon atoms; R ２６ , R ２７ , R ２８ , R ２９ , and R ３０ The substituent on the optionally substituted hydrocarbon group having 1 to 12 carbon atoms represented by R is an aryl group or heterocyclic group having 6 to 10 carbon atoms, ７ , R ８ and R ９ are each independently -(CH ２ ) ｎ -, and n is an integer of 2 to 8. ５ and R ６ , or R５ and R ７ may be joined together to form a 4- to 7-membered ring.

[0032] In formula (1), R is more preferably １ Ga-R １ａ -L １ -R １ｂ indicates R １ａ represents a hydrocarbon group having 1 to 18 carbon atoms; L １ represents —C(O)O— or —OC(O)—, and R １ｂ represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ Ga-R ３ａ -L ３ -R ３ｂ indicates R ３ａ represents a hydrocarbon group having 1 to 18 carbon atoms; L ３ represents —C(O)O— or —OC(O)—, and R ３ｂ represents a hydrocarbon group having 1 to 18 carbon atoms; R ２ and R ４ each independently represents a hydrocarbon group having 1 to 10 carbon atoms; R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 6 carbon atoms; R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 6 carbon atoms represented by the formula (I) are each independently —OH, —O—R ２６ , —C(O)NR ２７ R ２８ , or -NR ２９ C(O)R ３０ indicates, R ２６ , R ２７ , R ２８ , R ２９ , and R ３０ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 12 carbon atoms; R ２６ , R ２７ , R ２８ , R ２９ , and R ３０ The substituent on the optionally substituted hydrocarbon group having 1 to 12 carbon atoms represented by R represents an aryl group having 6 to 10 carbon atoms, ７ , R８ and R ９ are each independently -(CH ２ ) ｎ -, and n is an integer of 2 to 8.

[0033] In formula (1), R is most preferably １ Ga-R １ａ -L １ -R １ｂ indicates 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 Ga-R ３ａ -L ３ -R ３ｂ indicates 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 represents a hydrocarbon group having 3 to 8 carbon atoms; R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 4 carbon atoms, and R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 4 carbon atoms represented by R are each independently preferably an —OH group. ７ , R ８ and R ９ are each independently -(CH ２ ) ｎ -, and n is an integer of 2 to 4. ５ and R ６ , or R ５ and R ７ may be joined together to form a 5- to 7-membered ring.

[0034] The compound represented by formula (1) may form a salt. Examples of the salt of a basic group 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. Salts of acidic groups include, for example, 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. Of the above-mentioned salts, preferred salts include pharmacologically acceptable salts.

[0035] Preferably, the ionizable lipid is one or more of the following compounds, although the invention is not intended to be limited thereto:

[0036] 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.

[0037] In the lipid composition, the amount of the compound represented by formula (1) or a salt thereof 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%.

[0038] <Method for producing the compound represented by formula (1)> A method for producing the compound represented by formula (1) will be described. The compound represented by formula (1) can be produced by combining known methods, and can be produced, for example, according to the production method shown below.

[0039] [Production Method 1] A method for producing a compound of formula [1] from a compound of formula [2].

[0040] In the formula, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ and R ９ has the same meaning as above; R ８ａ , R ９ａ and R Ａ means a hydrocarbon group having 1 to 7 carbon atoms.

[0041] (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, with or without a solvent. The acid used in this reaction can be an inorganic or organic acid. Organic acids are preferred, and specific examples include formic acid, acetic acid, trifluoroacetic acid, 4-toluenesulfonic acid, and methanesulfonic acid, with formic acid being more preferred. The amount of acid used can be 1 to 100 times (v / w), preferably 1 to 10 times (v / w), relative to the compound of formula [2]. The amount of water used can be 0.1 to 100 times (v / w), preferably 0.1 to 10 times (v / w), relative to the compound of formula [2]. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples 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 formula [2]. This reaction may be carried out at −30 to 150° C., preferably 0 to 100° C., for 5 minutes to 48 hours.

[0042] (1-2) The compound of formula [1] can be produced by reacting a compound of formula [3A] with a compound of formula [4] in the presence of a reducing agent. Known examples of compounds of 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, and examples 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 solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of formula [3A]. Examples of reducing agents used in this reaction include 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 formula [3A]. The amount of the compound of formula [4] used may be 0.1 to 1 times the molar amount of the compound of formula [3A]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0043] (1-3) The compound of formula [5] can be produced by reacting the compound of formula [3A] with the compound of formula [4] in the presence of a reducing agent. This reaction can be carried out in accordance with the production method (1-2), and the compound of formula [4] can be used in an amount of 1 to 10 times the molar amount of the compound of formula [3A].

[0044] (1-4) The compound of formula [1] can be produced by reacting the compound of formula [3B] with the compound of formula [5] in the presence of a reducing agent. This reaction can be carried out in accordance with the production method (1-2), and the compound of formula [3B] can be used in an amount of 1 to 10 times the molar amount of the compound of formula [5].

[0045] [Production Method 2] A method for producing a compound of formula [2] from compounds of formula [6] and formula [7].

[0046] In the formula, R １ , R ２ , R ９ａ and R Ａ has the same meaning as above; X １ and X ２ means a leaving group. Examples of the leaving group 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.

[0047] (2-1) A compound of formula [9] can be produced by reacting a compound of formula [7] with a compound of formula [8] in the presence or absence of a base. Known examples of compounds of 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, and examples 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 solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of formula [7]. The base used in this reaction includes inorganic and organic bases. The base is preferably an organic base, and specific examples include triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, and 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 formula [7]. The amount of the compound of formula [8] used is not particularly limited, but may be 1 to 10 times the molar amount of the compound of formula [7]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0048] (2-2) The compound of formula [2] can be produced by reacting a compound of formula [6] with a compound of formula [9] in the presence of a base. Dioctylamine, for example, is known as a compound of formula [6]. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Nitriles are preferred, with acetonitrile being more preferred. The amount of solvent used is not particularly limited, and may be 1 to 500 times (v / w) the amount of the compound of formula [6]. The base used in this reaction can be an inorganic base or an organic base. Specific examples of the base include 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, and 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 formula [6]. The amount of the compound of formula [9] used is not particularly limited, but may be 0.1 to 10 times the molar amount of the compound of formula [6]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0049] [Production Method 3] A method for producing a compound of formula [6A].

[0050] In the formula, R ２ , R １a and R １b has the same meaning as above; X ３ represents a hydroxyl group and a leaving group; X ４ means a leaving group; leaving group has the same meaning as above.

[0051] (3-1) A 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 compounds of formula [10A] include 5-bromovaleric acid and chloroacetyl chloride. Known examples of compounds of 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, and 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 formula [10A]. The acid used in this reaction may be an inorganic acid or an organic acid. The acid is preferably a sulfonic acid, and specific examples include sulfuric acid, 4-toluenesulfonic acid, and methanesulfonic acid. Examples of the condensing agent used in this reaction include 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; and 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.Examples of acid halides used in this reaction include carboxylic acid halides such as acetyl chloride and trifluoroacetyl chloride; sulfonic acid halides such as methanesulfonyl chloride and tosyl chloride; and chloroformates such as ethyl chloroformate and isobutyl chloroformate. Examples of bases used in this reaction include inorganic and organic bases. Organic bases are preferred, and specific examples include triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, and N,N-dimethylaminopyridine. The amount of base used may be 1 to 50 times, preferably 1 to 10 times, the molar ratio of the compound of formula [10A]. The amount of compound of formula [11A] used is not particularly limited, but may be 0.8 to 10 times (v / w) the amount of compound of formula [10A]. This reaction may be carried out at a temperature of from -30 to 150°C, preferably from 0 to 100°C, for 5 minutes to 48 hours.

[0052] (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. Examples of compounds of 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, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include nitriles and ethers, with acetonitrile and tetrahydrofuran being more preferred. The amount of solvent used is not particularly limited, and may be 1 to 500 times (v / w) the amount of the compound of formula [12A]. The base used in this reaction may be an inorganic base or an organic base. Specific examples of the base include 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, and N,N-dimethylaminopyridine, with potassium carbonate being more preferred. The amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar ratio of the compound of Formula [12A]. The amount of the compound of Formula

[13] used is not particularly limited, but may be 1 to 10 times the molar ratio 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 ratio of the compound of Formula [12A]. This reaction may be carried out at a temperature of from -30 to 150°C, preferably from 0 to 100°C, for 5 minutes to 48 hours.

[0053] [Production Method 4] A method for producing a compound of formula [6B].

[0054] In the formula, R２ , R １ａ , R １ｂ , X ３ and X ４ has the same meaning as above.

[0055] (4-1) The compound of formula [12B] can be produced by the same method as in Production Method (3-1), except that the compound of formula [10B] is used instead of the compound of formula [10A] and the compound of formula [11B] is used instead of the compound of formula [11A].

[0056] (4-2) The compound of formula [6B] can be produced in the same manner as in production method (3-2) by using the compound of formula [12B] instead of the compound of formula [12A].

[0057] [Production Method 5] A method for producing a compound of formula [6C].

[0058] In the formula, R １ａ , R １ｂ and X ３ has the same meaning as above; R Ｂ means an amino protecting group.

[0059] (5-1) The compound of formula

[15] can be produced by reacting the compound of formula [10B] with the 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. Examples of known compounds of formula [10B] include decanoic acid and decanoic acid chloride. Examples of known compounds of formula

[15] include tert-butyl bis(2-hydroxyethyl)carbamate. This reaction may be carried out in accordance with Production Method (3-1).

[0060] (5-2) The compound of formula [6C] can be produced 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.

[0061] [Production Method 6] A method for producing a compound of formula [6D].

[0062] In the formula, R １ａ , R １ｂ and R Ｂ has the same meaning as above.

[0063] (6-1) The compound of formula [6D] can be produced by reacting the compound of formula

[11] with the 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. Known examples of the compound of formula

[11] include 1-decanol. Known examples of the compound of formula

[16] include N-(tert-butoxycarbonyl)iminodiacetic acid. This reaction may be carried out in accordance with Production Method (3-1).

[0064] (6-2) The compound of formula [6D] can be prepared by deprotecting the compound of formula

[17] . This reaction can be carried out in accordance with the preparation method (3-2).

[0065] In the compounds used in the above-mentioned production methods, when isomers (e.g., 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.

[0066]

[0033] In the compounds used in the above-mentioned production methods, for example, compounds having an amino group, a hydroxyl group, or a carboxyl group 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 a reaction known per se, such as condensation, addition, oxidation, reduction, rearrangement, substitution, halogenation, dehydration, or hydrolysis, or by an appropriate combination of these reactions.

[0067] <Sterol> The lipid composition of the present invention preferably contains a sterol or a derivative thereof as a non-ionized lipid. By including a sterol in the lipid composition, 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, and cholesteryl-4'-hydroxybutyl ether. Among these, cholesterol is preferred.

[0068] 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 molar ratio to the total lipids in the lipid composition.

[0069] <Phospholipid> The lipid composition of the present invention preferably contains a phospholipid as a 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 phospholipid or a combination of multiple different neutral lipids.

[0070] Examples of phosphatidylcholines include, but are not limited to, soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated egg yolk lecithin (HEPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC).

[0071] The phosphatidylethanolamine is not particularly limited, and examples thereof include dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, dioctadecylphosphatidylethanolamine, and diphytanylphosphatidylethanolamine.

[0072] Examples of sphingomyelin include, but are not limited to, egg yolk-derived sphingomyelin, milk-derived sphingomyelin, etc. Examples of ceramide include, but are not limited to, egg yolk-derived ceramide, milk-derived ceramide, etc.

[0073] The phospholipid is preferably selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine.

[0074] In the lipid composition, the blending amount of the phospholipid is preferably 1 to 30 mol %, more preferably 1 to 20 mol %, in terms of molar ratio to the total lipids in the lipid composition.

[0075] <Lipids Having Nonionic Hydrophilic Polymer Chains> The lipid composition of the present invention may contain a lipid having a nonionic hydrophilic polymer. In the present invention, the inclusion of a lipid having a nonionic hydrophilic polymer can achieve a dispersion stabilization effect of the lipid composition. 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 building blocks. Among these nonionic hydrophilic polymers, nonionic polyethers, nonionic polyesters, nonionic polyamino acids, or nonionic synthetic polypeptides are preferred, nonionic polyethers or nonionic polyesters are more preferred, nonionic polyethers or nonionic monoalkoxy polyethers are even more preferred, and polyethylene glycol (polyethylene glycol will also be referred to as PEG hereinafter) is particularly preferred. That is, the lipid having a nonionic polymer is preferably a lipid having a polyethylene glycol chain.

[0076] The lipid having a nonionic hydrophilic polymer is not particularly limited, but examples thereof include PEG-modified phosphoethanolamine, diacylglycerol PEG derivatives, monoacylglycerol PEG derivatives, dialkylglycerol PEG derivatives, cholesterol PEG derivatives, ceramide PEG derivatives, etc. Among these, monoacylglycerol PEG or diacylglycerol PEG is preferred.

[0077] 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.

[0078] The weight-average molecular weight of the PEG chain of the nonionic hydrophilic polymer derivative is preferably 500 to 5000, more preferably 750 to 3000. The nonionic hydrophilic polymer chain may be branched and may have a substituent such as a hydroxymethyl group.

[0079] 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.

[0080] <Nucleic Acid> The lipid composition contains a nucleic acid. Examples of nucleic acids include circular double-stranded DNA (plasmid DNA, small circular double-stranded DNA without a drug resistance gene, etc.), single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (micro RNA), mRNA, antisense oligonucleotides (also known as ASO), ribozymes, aptamers, saRNA, sgRNA, etc., and any of these may be included. Two or more types of nucleic acids may be used. Modified nucleic acids may also 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.

[0081] The nucleic acid may be a sequence for gene editing, such as mRNA encoding a DNA nuclease. The nucleic acid may be a sequence for gene editing, such as guide RNA. The nucleic acid may be a sequence for gene editing, such as a nucleic acid mixture containing mRNA encoding a Cas nuclease and guide RNA. That is, the nucleic acid may be a nucleic acid for gene editing that contains mRNA encoding a Cas nuclease and guide RNA. The mixture may further contain any donor DNA. The nucleic acid may be a sequence for single base editing, such as a nucleic acid mixture containing mRNA encoding a deaminase and a mutant Cas nuclease and guide RNA. The nucleic acid may be a sequence for replacing a target DNA nucleotide, such as a nucleic acid mixture containing mRNA encoding a fusion protein of an artificial reverse transcriptase and a Cas9 endonuclease and a prime editing guide RNA.

[0082] The nucleic acid may be a nucleic acid mixture containing a sequence for guide RNA-dependent gene transcription activation (such as the CRISPR activator system), mRNA encoding a fusion protein of an activator protein (VP64, p65, Rta) and a mutant Cas nuclease, and guide RNA. Alternatively, it may be a nucleic acid mixture containing an mRNA encoding an activator protein (MS2, p65, HSF1), mRNA encoding a fusion protein of VP64 and a mutant Cas nuclease, and guide RNA. The nucleic acid may be a sequence for guide RNA-dependent gene transcription suppression (such as the CRISPR interference system), mRNA encoding a fusion protein of a transcription suppressor (such as KRAB) and a mutant Cas nuclease, and guide RNA. The nucleic acid may be mRNA or DNA encoding a DNA recombinase, and optionally a nucleic acid mixture containing donor DNA. The nucleic acid may be mRNA or DNA encoding a DNA recombinase, and may also be a nucleic acid mixture optionally containing donor DNA. Examples of DNA recombinase include, but are not limited to, transposases (e.g., Sleeping Beauty transposase, piggyBac transposase, Tol2, etc.), Cre recombinase, and serine integrase. The nucleic acid may be mRNA encoding a reverse transcriptase, which is a sequence for inserting a foreign gene into the host cell genome, and may also be a nucleic acid mixture optionally containing donor RNA. The nucleic acid may be mRNA encoding a sequence for expressing the foreign gene, or DNA containing the foreign gene, a promoter sequence, and a terminal sequence.

[0083] The nucleic acid may be a sequence for gene editing or gene transcription suppression of a gene (target gene) present in an immune cell. The target gene is not particularly limited, but includes T cell receptor genes (TRAC, TRBC), MHC-I (or HLA-I), MHC-II, B2M, genes related to autoantigens, 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 involved in inflammatory responses (CGAS, STING, TBK1, etc.), methylation genes (TET1, TET2, DNMT3A), genes related to immune evasion Involved genes (CD47, NKG2A), others (DGK, EZH2, CSF2, PAX5, LDLR, MAP4K1, CISH, CD5, CD52, ADORA2A, CD39, CD73, CD5, MCM3AP, EIF3D, CAD, HGS, RPL19, MAK16, PDGFRA, NRF1, EP400, CBL B, RPS7, CPSF4, IL2RG, RPL38, IL2RB, JAK3, MCM2, SNRPC, PSMD4, MAP4K1, BRD9, RNF20, RNF40, NFKB2, NMT1, MYB, TSC1, EIF3K, RPL19, TBX21, PRDM1, SUV39H1, ARID1A), and the like.

[0084] 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 forms of 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 Examples of nucleic acids include sequences encoding secreted proteins that induce inflammation (e.g., HX6, FOXO4, IRX4, FOXQ1, OTUD7B, LCP2, FOSB, RAC2, FOSL1, APOBEC3D, RIPK3, EMP1, ANXA2R, CDKN2C, OTUD7A, CD2, LAT, LCP2, TBX21, and EOMES). Nucleic acids may also be sequences for expressing chimeric antigen receptor genes, T cell receptor genes, antibodies, bispecific antibodies, multispecific antibodies, single chain Fvs (scFvs), nanobodies, and bispecific T cell-inducing antibodies (BiTEs). Nucleic acids may also be sequences for expressing exogenous genes involved in cell reprogramming. Examples of the foreign gene include, but are not limited to, Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and Lin28.

[0085] In the lipid composition, the mass ratio of total lipids to 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.

[0086] <Method for producing lipid composition> A method for producing a lipid composition will be described. The method for producing a lipid composition is not limited, but it can be produced by dissolving all or some 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 emulsification may be performed using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure injection emulsifier, or the like. Alternatively, a solution containing lipids may be dried under reduced pressure using an evaporator or the like, or spray-dried using a spray dryer or the like to prepare a dried mixture containing lipids, and this mixture is added to an aqueous solvent and further emulsified using the above-mentioned emulsifier or the like.

[0087] An example of a method for producing a lipid composition containing nucleic acid includes the following steps: 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.

[0088] 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 acids (e.g., mRNA, etc.) in water or a buffer solution. The nucleic acid concentration is not particularly limited, but is preferably 1 to 1000 μg / mL, more preferably 10 to 500 μg / mL. If necessary, components such as buffer components and antioxidants for pH adjustment 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, 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 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.

[0089] 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 channel device. For the in-line method, a micro-flow channel device is preferably used, and examples of the micro-flow channel device that can be used include a Y-mixer, a T-mixer, a herringbone mixer, a ring micromixer, 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.

[0090] 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 may be water, but may also include adjusting the pH or salt strength. The components contained in the diluent 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 to adjust the pH. In addition, sodium chloride, potassium chloride, sucrose, trehalose, fructose, mannitol, etc. may be included to adjust the salt strength or osmotic pressure, and the above buffer solutions may also be used with the addition of these additives.

[0091] The lipid particle dispersion and the diluted solution may be mixed by any method, including a batch method or 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 the dilution is preferably carried out 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 diluted solution is preferably 1:0.5 to 1:10, and more preferably 1:1 to 1:5.

[0092] 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.

[0093] Furthermore, multiple dilution steps may be carried out consecutively, and the interval between one dilution step and the next dilution step may be set arbitrarily, for example, 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.

[0094] The pH of the lipid particle dispersion after step (c) is preferably from pH 3.0 to 10.0, more preferably from pH 3.5 to pH 9.0, and particularly preferably from pH 4.0 to pH 8.5.

[0095] The lipid composition can be subjected to sizing as needed. The sizing method is not particularly limited, but the particle size can 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.

[0096] In step (d), the method for removing the organic solvent from the lipid particle dispersion is not particularly limited, and a common method can be used. For example, a pH buffer solution such as phosphate buffered saline or Tris buffer can be used as the dialysis solution, and additives such as any salt or sugar can be added as needed to adjust the osmotic pressure or protect from freezing.

[0097] In step (e), the concentration of the lipid particle dispersion obtained in step (d) can be adjusted.When diluting, it can be diluted to an appropriate concentration using a solution such as phosphate buffered saline, physiological saline, Tris buffer, or sucrose-containing Tris buffer as a diluent.When concentrating, it can be concentrated by ultrafiltration using an ultrafiltration membrane, etc., with the dispersion obtained in step (d).It is preferable to use the concentrated dispersion as it is, or it is also preferable to use the above-mentioned diluent after concentrating to adjust to a desired concentration.

[0098] 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.

[0099] The solution that can be used for dialysis in step (d) or dilution in step (e) may contain an excipient, cryoprotectant, buffer, or antioxidant. 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.

[0100] The lipid particle dispersion may be subjected to sterile filtration. As a filtration method, insoluble materials can be removed from the lipid particle dispersion using a hollow fiber membrane, a reverse osmosis membrane, a membrane filter, or the like. In the present invention, although not particularly limited, filtration is preferably performed using a filter having a sterilizable pore size (preferably a 0.2 μm filtration sterilization filter). In addition, sterile filtration is preferably performed after step (d) or step (e). Furthermore, if necessary, the lipid particle dispersion can be subjected to freezing or lyophilization. The lipid particle dispersion can be subjected to freezing or lyophilization by a general method, and the method is not particularly limited.

[0101] In addition, the method for producing a nucleic acid delivery agent is not particularly limited, and examples include a method comprising the steps of preparing nucleic acid-free lipid particles using an ionizable lipid that is a compound represented by formula (1) or its salt, a non-ionizable lipid, and a lipid having a nonionic polymer, and mixing the nucleic acid-free lipid particles with the nucleic acid. The nucleic acid-free lipid particles can be produced by dissolving all or some of the oil-soluble components of the nucleic acid-free lipid particles 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 mixture may be emulsified using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. Alternatively, a lipid-containing solution may be dried under reduced pressure using an evaporator or the like, or spray-dried using a spray dryer or the like to prepare a dried mixture containing lipids, and this mixture may be added to an aqueous solvent and further emulsified using the above-mentioned emulsifier or the like.

[0102] An example of a method for producing nucleic acid-free lipid particles includes the following steps: Step (A): dissolving components of nucleic acid-free lipid particles 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; and Step (E): adjusting the concentration of the lipid particle dispersion.

[0103] 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, 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.

[0104] 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 channel device. For the in-line method, a micro-flow channel device is preferably used, and examples of the micro-flow channel device that can be used include a Y-mixer, a T-mixer, a herringbone mixer, a ring micromixer, 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.

[0105] Components such as buffer components for pH adjustment and antioxidants can be added to the aqueous phase as needed. 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, 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 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.

[0106] 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 may be water, but may also include adjusting the pH or salt strength. The components contained in the diluent 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 to adjust the pH. In addition, sodium chloride, potassium chloride, sucrose, trehalose, fructose, mannitol, etc. may be included to adjust the salt strength or osmotic pressure, and the above buffer solutions may also be used with these additives added.

[0107] The lipid particle dispersion and the diluted solution may be mixed by any method, including a batch method or 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 the dilution is preferably carried out 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 diluted solution is preferably 1:0.5 to 1:10, and more preferably 1:1 to 1:5.

[0108] 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.

[0109] Furthermore, multiple dilution steps may be carried out consecutively, and the interval between one dilution step and the next dilution step may be set arbitrarily, for example, 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.

[0110] The pH of the lipid particle dispersion after step (C) is preferably from pH 3.0 to 10.0, more preferably from pH 3.5 to pH 9.0, and particularly preferably from pH 4.0 to pH 8.5.

[0111] The lipid particles can be sized as needed. The sizing method is not particularly limited, but the particle size can be reduced using an extruder or the like. In addition, the dispersion containing the lipid composition can be frozen or freeze-dried by a general method.

[0112] In step (D), the method for removing the organic solvent from the lipid particle dispersion is not particularly limited, and a common method can be used. For example, a pH buffer solution such as phosphate buffered saline or Tris buffer can be used as the dialysis solution, and additives such as any salt or sugar can be added as needed for the purpose of adjusting the osmotic pressure or protecting the lipid particle from freezing.

[0113] In step (E), the concentration of the lipid particle dispersion obtained in step (D) can be adjusted.When diluting, it can be diluted to an appropriate concentration using a solution such as phosphate buffered saline, physiological saline, Tris buffer, or sucrose-containing Tris buffer as a diluent.When concentrating, it can be concentrated by ultrafiltration using an ultrafiltration membrane, etc., of the dispersion obtained in step (D).It is preferable to use the concentrated dispersion as it is, or it is also preferable to adjust to a desired concentration using the above-mentioned diluent after concentrating.

[0114] 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 step, 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.

[0115] The solution that can be used for dialysis in step (D) or dilution in step (E) may contain an excipient, cryoprotectant, buffer, or antioxidant. 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.

[0116] The lipid particle dispersion may be subjected to sterile 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 preferred to use a filter with a sterilizable pore size (preferably a 0.2 μm filter sterilization filter).In addition, it is preferred to carry out sterile filtration after step (D) or step (E).

[0117] Furthermore, if necessary, the dispersion of lipid particles not containing nucleic acid can be subjected to freezing or lyophilization. The dispersion of lipid particles can be subjected to freezing or lyophilization by a general method, and the method is not particularly limited.

[0118] Preferably, in the present invention, the nucleic acid-free lipid particles are cryopreserved, and the cryopreserved nucleic acid-free lipid particles can be thawed before mixing with nucleic acid.

[0119] In the present invention, a step of mixing the nucleic acid-free lipid particles prepared as described above with nucleic acid is carried out. The step of mixing the nucleic acid-free lipid particles with nucleic acid is not particularly limited, and can be performed by any of the following methods: mixing the liquid using a flow channel, mixing by moving the liquid back and forth in a container, pipette mixing, stirrer mixing in a batch container, mixing by rotating the container to agitate the contents, or flask stirring. The step of mixing the nucleic acid-free lipid particles with nucleic acid preferably includes a step of incubating the nucleic acid-free lipid particles with an aqueous solution containing nucleic acid at 0°C to 30°C for 0.1 to 120 minutes, and a step of adjusting the pH of the resulting mixture to 6.5 to 8.5. The aqueous solution containing nucleic acid can be obtained by dissolving the nucleic acid in water or a buffer solution. The concentration of the nucleic acid is not particularly limited, but is preferably 1 to 2000 μg / mL, and more preferably 10 to 1000 μg / mL. If necessary, components such as a buffer component for pH adjustment or an antioxidant can be added.

[0120] In the step of mixing nucleic acid-free lipid particles with nucleic acid, the mass ratio of lipid concentration to 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.

[0121] <Lipid Composition> The lipid composition may be a lipid particle. Lipid particles refer to particles composed of lipids, and include compositions having any structure selected from lipid aggregates in which lipids are aggregated, micelles, liposomes, lipid nanoparticles (LNPs), and lipoplexes. Liposomes have a lipid bilayer structure, an internal aqueous phase, and include liposomes with a single bilayer membrane and multilayer liposomes with multiple layers stacked on top of each other. Either type of liposome may be included in the present invention. Lipid particles are preferably lipid nanoparticles (LNPs).

[0122] The morphology of lipid particles can be confirmed by electron microscope observation or X-ray structural analysis.For example, by using a cryo-transmission electron microscope (cryo-TEM) method, 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 a structure packed with lipids and other components.Small-angle X-ray scattering (SAXS) measurement can also be used to confirm whether the lipid particles have a lipid bilayer structure (lamellar structure).

[0123] 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.).

[0124] <Method for delivering nucleic acid to immune cells> The present invention provides a method for delivering nucleic acid to immune cells, which comprises contacting immune cells with the above-described nucleic acid delivery agent for immune cells of the present invention. However, in vivo delivery methods may be excluded. That is, nucleic acids can be introduced into immune cells by mixing a lipid composition with nucleic acid and transfecting the immune cells ex vivo, in vitro, or in vivo.

[0125] <Medical Use> According to the present invention, the nucleic acid delivery agent for immune cells of the present invention described above 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 a mixture with a pharmaceutically acceptable carrier. Furthermore, 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.

[0126] The nucleic acid delivery agent of the present invention may be bound to the surface of the lipid composition with a molecule that targets immune cells (hereinafter also referred to as a target molecule) to more efficiently deliver the nucleic acid to immune cells. The target molecule is not particularly limited, but small molecules, peptides, nucleic acids, and antibodies can be used. Furthermore, 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 bonding the lipid composition to the target molecule. Examples of lipids having such a modifying group include lipids having a maleimide group and a polyethylene glycol chain.

[0127] Immune cells may be either activated or non-activated cells, and can be selected as desired depending on the cell production method. For example, in the case of T cells, activation treatment refers to stimulation via the TCR / CD3 complex using an anti-CD3 antibody and an anti-CD28 antibody, or via a 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. Furthermore, non-activated cells refer to T cells that have been cultured in the presence of cytokine signals such as IL-2, IL-7, and IL-15 without undergoing the above-mentioned activation treatment. However, the activation methods are not limited to those listed here.

[0128] The immune cells are preferably mammalian-derived cells, more preferably human-derived cells. Examples of immune cells include, but are not limited to, lymphocytes (e.g., T cells, B cells, natural killer cells (NK cells), NKT cells, and iNKT cells), monocytes, macrophages, mast cells, dendritic cells, granulocytes (e.g., neutrophils, eosinophils, and basophils), hematopoietic stem / progenitor cells, primary immune cells, and CD3 ＋ cells, CD4 ＋ cells, CD8 ＋The immune cells can be selected from T cells, regulatory T cells (Treg), B cells, NK cells, innate lymphocytes, or dendritic cells (DC). The immune cells are preferably peripheral blood mononuclear cells (PBMC), lymphocytes, T cells, CD4 ＋ cells, CD8 ＋ The immune cells may be selected from primary T 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.

[0129] The method may include a 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.

[0130] 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, apolipoproteins A to E, based on their structure and function, and some of these are divided into subclasses, such as apolipoproteins 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 can be used.

[0131] 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 a recombinant protein containing only a cell-binding domain and a heparin-binding domain, more preferably retronectin.

[0132] The present invention will now be described with reference to examples, but the present invention is not limited to these examples.

[0133] Unless otherwise specified, purification by column chromatography was performed using an automatic purification system ISOLERA (Biotage), a medium-pressure fractionation and purification system Purif-espoir-2 (Shoko Science Co., Ltd.), or a medium-pressure liquid chromatograph YFLC W-prep 2XY (Yamazen Corporation).

[0134] Unless otherwise specified, the carriers used in silica gel column chromatography were Chromatorex Q-Pack SI 50 (Fuji Silysia Chemical Ltd.) and Hi-Flash Columns W001, W002, W003, W004, or W005 (Yamazen Corporation). NH silica gel was Chromatorex Q-Pack NH 60 (Fuji Silysia Chemical Ltd.).

[0135] NMR spectra were measured using a Bruker AVNEO400 (manufactured by Bruker) with tetramethylsilane as an internal standard, and all δ values ​​were expressed in ppm. MS spectra were measured using an ACQUITY SQD LC / MS System (manufactured by Waters).

[0136] [Synthesis Example 1] (1)

[0137] To a mixture of 2,2-diethoxyethanol (5.0 g), tetrahydrofuran (25 mL), and triethylamine (15.6 mL) was added 4-nitrophenyl chloroformate (11.3 g) 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 resulting organic layer was washed with water (25 mL) and saturated brine, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to yield 2,2-diethoxyethyl(4-nitrophenyl)carbonate (12.2 g) as a pale yellow oil. 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).

[0138] (2)

[0139] To a mixture of 2-hexyl-1-octanol (5.0 g), 5-bromovaleric acid (4.6 g) and toluene (25 mL) was added sulfuric acid (0.5 mL), and the mixture was stirred for 5 hours at 110° C. After cooling to room temperature, the reaction mixture was 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(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).

[0140] (3)

[0141] To a mixture of 2-hexyloctyl 5-bromopentanoate (1.2 g), n-octylamine (1.2 g), and 1-methyl-2-pyrrolidone (6 mL), potassium carbonate (1.3 g) was added and 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 brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane-ethyl acetate-methanol) to yield 2-hexyloctyl 5-(octylamino)pentanoate (1.25 g) as a pale yellow oil. 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).

[0142] (4)

[0143] A mixture of 2-hexyloctyl 5-(octylamino)pentanoate (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, which was cooled to room temperature, and the organic layer was separated. The resulting organic layer was washed with water and saturated brine, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to yield 2-hexyloctyl 5-(((2,2-diethoxyethoxy)carbonyl)(octyl)amino)pentanoate (0.83 g) as a pale yellow oil. 1 H-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).

[0144] (5)

[0145] 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 solvent was evaporated under reduced pressure. This procedure of adding toluene again and evaporating 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(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).

[0146] (6)

[0147] To a solution of 2-hexyloctyl 5-(octyl((2-oxoethoxy)carbonyl)amino)pentanoate (0.73 g) in ethyl acetate (8 mL) were added N,N-diethylethylenediamine (0.083 g), acetic acid (43 mg), and sodium triacetoxyborohydride (0.91 g) at room temperature, followed by stirring at room temperature for 5 hours. 20% aqueous potassium carbonate solution (10 mL) was added to the reaction mixture, and the organic layer was separated and washed with water and saturated brine. Anhydrous sodium sulfate was then added for drying, and the solvent was then distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (methanol-ethyl acetate-hexane) to obtain 0.39 g of 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 (referred to as Compound 1). 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.

[0148] [Synthesis Example 2] (1)

[0149] To a solution of 2,2-diethoxyethanol (10.0 g) in tetrahydrofuran (100 mL), 1,1'-carbonyldi(1,2,4-triazole) (18.3 g) was added, heated to 30°C, and stirred for 1 hour. After the reaction mixture was cooled to room temperature, hexane (100 mL) and saturated aqueous sodium bicarbonate (100 mL) were added, and the organic layer was separated. The resulting organic layer was washed with water (50 mL) and saturated brine, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to yield 2,2-diethoxyethyl 1H-1,2,4-triazole-1-carboxylate (10.4 g) as a colorless oil. 1 H-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).

[0150] (2)

[0151] To a solution of tert-butyl N,N-bis(2-hydroxyethyl)carbamate (5.00 g) and triethylamine (8.15 mL) in tetrahydrofuran (50 mL), decanoic acid chloride (11 mL) was added dropwise under ice cooling, followed by stirring 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 resulting organic layer was washed with water (50 mL) and saturated brine, then dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give crude ((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) (13.1 g) as a yellow oil. 1 H-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).

[0152] (3)

[0153] To a mixture of ((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) (13.0 g) and water (1 mL) was added trifluoroacetic acid (20 mL) at room temperature and stirred overnight. The solvent was evaporated under reduced pressure, and hexane (60 mL), ethyl acetate (30 mL), and 20% aqueous potassium carbonate (40 mL) were added to the residue, and the organic layer was separated. Anhydrous sodium sulfate was added to the resulting organic layer for drying, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate-hexane) to yield azanediylbis(ethane-2,1-diyl)bis(decanoic acid) (7.39 g) as a pale yellow oil. MS m / z (M+H): 415.

[0154] (4)

[0155] 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, which was cooled to room temperature, and the organic layer was separated. Anhydrous sodium sulfate was added to the resulting organic layer for drying, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to yield (((2,2-diethoxyethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid) (0.27 g) as a colorless oil. 1 H-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).

[0156] (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 evaporated under reduced pressure. To the obtained crude (((2-oxoethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)bis(decanoic acid), ethyl acetate (2.4 mL), N,N-diethylethylenediamine (27.6 mg), and sodium triacetoxyborohydride (303 mg) were added 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 brine. After drying over anhydrous sodium sulfate, the solvent was evaporated 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(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, MS m / z(M+H):1084.

[0157] [Synthesis Example 3] (1)

[0158] 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 under reflux while removing water using a Dean-Stark apparatus. After cooling the reaction mixture to room temperature, it was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2-pentylheptyl 5-bromopentanoate (10.8 g) as a colorless oil. 1 H-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).

[0159] (2)

[0160] To a mixture of 2-pentylheptyl 5-bromopentanoate (1.2 g), isopropylamine (0.65 g), and acetonitrile (6 mL), potassium carbonate (1.45 g) was added and 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 brine, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane-ethyl acetate-methanol) to give 2-pentylheptyl 5-(isopropylamino)pentanoate (0.69 g) as a pale yellow oil. 1 H-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.

[0161] (3)

[0162] A mixture of 2-pentylheptyl 5-(isopropylamino)pentanoate (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, which was cooled to room temperature, and the organic layer was separated. The resulting organic layer was washed with saturated aqueous ammonium chloride and saturated brine, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to yield 2-pentylheptyl 5-(((2,2-diethoxyethoxy)carbonyl)(isopropyl)amino)pentanoate (0.55 g) as a colorless oil.

[0163] (4)

[0164] A mixture of 2-pentylheptyl 5-(((2,2-diethoxyethoxy)carbonyl)(isopropyl)amino)pentanoate (0.55 g), formic acid (2.2 mL), and water (0.55 mL) was stirred at 40°C for 2 hours. To the reaction mixture cooled to room temperature, ethyl acetate and water were added, and the organic layer was washed twice with saturated aqueous sodium bicarbonate. Anhydrous sodium sulfate was added to the resulting organic layer for drying, 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 resulting crude 2-pentylheptyl 5-(isopropyl((2-oxoethoxy)carbonyl)amino)pentanoate (0.15 g) at room temperature, and the mixture was stirred at room temperature for 1 hour. After adding 10% aqueous potassium carbonate to the reaction mixture, the organic layer was separated and washed with water and saturated brine, then dried over anhydrous sodium sulfate, 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 give 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) as a colorless oil. 1 H-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.

[0165] [Synthesis Example 4]

[0166] 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(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.

[0167] [Synthesis Example 5]

[0168] 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 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(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, MS m / z(M+H):1010.

[0169] [Synthesis Example 6] (1)

[0170] 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 the mixture was 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, and the organic layer was washed with saturated brine. The organic layer was combined with the previously obtained organic layer, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain a colorless oily substance, ditridecyl 2,2'-((tert-butoxycarbonyl)azanediyl)diacetate (4.26 g). 1 H-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).

[0171] (2)

[0172] 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 evaporated under reduced pressure. To the residue, toluene (20 mL) was added, and the mixture was evaporated under reduced pressure. This operation was repeated three times, and 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). 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).

[0173] (3)

[0174] 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 resulting organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to yield ditridecyl 2,2'-(((2,2-diethoxyethoxy)carbonyl)azanediyl)diacetate (1.06 g) as a colorless oil. 1 H-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).

[0175] (4)

[0176] 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(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).

[0177] [Synthesis Example 7]

[0178] 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(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, MS m / z(M+H):1123.

[0179] [Synthesis Example 8]

[0180] 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 example described in WO 2024 / 158042.

[0181] [Synthesis Example 9]

[0182] 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 example described in WO 2024 / 158042.

[0183] [Synthesis Example 10]

[0184] 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 example described in WO 2024 / 158042.

[0185] [Synthesis Example 11]

[0186] 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 example described in WO 2024 / 158042.

[0187] [Synthesis Example 12]

[0188] 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 example described in WO 2024 / 158042.

[0189] [Synthesis Example 13]

[0190] 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 example described in WO 2024 / 158042.

[0191] [Synthesis Example 14]

[0192] 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 example described in WO 2024 / 158042.

[0193] [Synthesis Example 15]

[0194] 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 example described in WO 2024 / 158042.

[0195] [Synthesis Example 16]

[0196] 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 example described in WO 2024 / 158042.

[0197] [Synthesis Example 17]

[0198] 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 example described in WO 2024 / 158042.

[0199] [Synthesis Example 18]

[0200] 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 example described in WO 2024 / 158042.

[0201] [Synthesis Example 19]

[0202] 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 example described in WO 2024 / 158042.

[0203] [Synthesis Example 20]

[0204] 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 example described in WO 2024 / 158042.

[0205] [Synthesis Example 21]

[0206] 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 example described in WO 2024 / 158042.

[0207] [Synthesis Example 22]

[0208] 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 example described in WO 2024 / 158042.

[0209] [Synthesis Example 23]

[0210] 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 example described in WO 2024 / 158042.

[0211] [Synthesis Example 24]

[0212] 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 example described in WO 2024 / 158042.

[0213] [Synthesis Example 25]

[0214] 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 example described in WO 2024 / 158042.

[0215] [Synthesis Example 26]

[0216] 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 example described in WO 2024 / 158042.

[0217] [Synthesis Example 27]

[0218] 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 WO 2024 / 158042.

[0219] [Synthesis Example 28]

[0220] 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 example described in WO 2024 / 158042.

[0221] [Synthesis Example 29]

[0222] 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 example described in WO 2024 / 158042.

[0223] [Synthesis Example 30]

[0224] 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 example described in WO 2024 / 158042.

[0225] [Synthesis Example 31]

[0226] A solution of 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate (1.0 g), synthesized according to the example described in WO 2024 / 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 sequentially. The cooling medium was removed, and the mixture was stirred at room temperature for 1 hour. After confirming completion of the reaction, the reaction was quenched by adding 10% aqueous sodium bicarbonate solution. The organic layer was washed with 10% aqueous sodium bicarbonate solution and then with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The resulting residue was purified by silica gel column chromatography (ethyl acetate / methanol) followed by NH silica gel column chromatography (hexane / ethyl acetate) to give 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] +

[0227] [Synthesis Example 32]

[0228] 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] +

[0229] [Synthesis Example 33]

[0230] 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] +

[0231] [Synthesis Example 34]

[0232] 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] +

[0233] [Synthesis Example 35]

[0234] 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] +

[0235] [Synthesis Example 36]

[0236] (1) To a solution of tert-butyl (2-bromoethyl)carbamate (A) (1.12 g) and potassium carbonate (2.0 g) in dimethylformamide (10 mL), 4-(ethylamino)butan-1-ol (II) (1.3 mL) was added and stirred at 80 °C for 30 minutes. The reaction mixture 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 evaporated to give 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] +

[0237] (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 distilled off under reduced pressure, and the resulting residue was desalted using an ion exchange resin (Diaion SA10A (Mitsubishi Chemical), regenerated to OH form) and azeotropically dehydrated with ethanol to obtain 4-((2-aminoethyl)(ethyl)amino)butan-1-ol (IIA-NH). LC / MS rt (min): 0.19 MS (ESI, m / z): 161.2 [M+H] +

[0238] (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) (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] +

[0239] Synthesis Examples 37 to 59 In the same manner as in Synthesis Example 36(1) to (3), tert-butyl (2-((4-hydroxybutyl)(methyl)amino)ethyl)carbamate (IA), tert-butyl (3-((4-hydroxybutyl)(methyl)amino)propyl)carbamate (IB), and tert-butyl (2-((4-hydroxybutyl)(methyl)amino)ethyl)carbamate (IA) were obtained using 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) and tert-butyl (3-bromopropyl)carbamate (B). (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) were synthesized, and then the BOC group was 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-NH2), 4-((3-aminopropyl)(ethyl)amino)butan-1-ol (IIB-NH2), 3-((2-aminoethyl)(methyl)amino)propan-1-ol (IIIA-NH2), 3-((3-aminopropyl)(methyl)amino)propan-1-ol (IIIB-NH2), 3-((2-aminoethyl)(ethyl)amino)propan-1-ol (IVA-NH2), 3-((3-aminopropyl)(ethyl)amino)propan-1-ol (IVB-NH2), 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) were synthesized (Table 1). Using these obtained amines and 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate or 2-pentyl 6-(octyl((2-oxoethoxy)carbonyl)amino)hexanoate, the compounds (Compounds 37 to 59) were synthesized in the same manner as in Synthesis Example 31 (Table 2).

[0240]

[0241]

[0242] [Synthesis Example 60]

[0243] 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] +

[0244] [Synthesis Example 61]

[0245] 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] +

[0246] [Synthesis Example 62]

[0247] 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] +

[0248] [Synthesis Example 63]

[0249] 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] +

[0250] [Synthesis Example 64]

[0251] 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] +

[0252] [Synthesis Examples 65 and 66]

[0253] A condensate was synthesized in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(heptyl((2-oxoethoxy)carbonyl)amino)hexanoate and rac-N1,N1-dimethylcyclohexane-1,4-diamine, and two compounds with different polarities (low-polarity compound: Compound 65, high-polarity compound: Compound 66) were obtained by silica gel column chromatography.

[0254] 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] +

[0255] 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] +

[0256] [Synthesis Example 67]

[0257] 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] +

[0258] [Synthesis Example 68]

[0259] 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] +

[0260] [Synthesis Example 69]

[0261] 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] +

[0262] [Synthesis Example 70]

[0263] 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] +

[0264] [Synthesis Example 71]

[0265] 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] +

[0266] [Synthesis Example 72]

[0267] 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 in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(((2-oxoethoxy)carbonyl)(propyl)amino)hexanoate and 2-(diethylamino)ethylamine. LC / MS rt (min): 0.91 MS (ESI, m / z): 940.2 [M+H] +

[0268] [Synthesis Example 73]

[0269] 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] +

[0270] [Synthesis Example 74]

[0271] 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 in the same manner as in Synthesis Example 31 using 2-pentylheptyl 6-(butyl((2-oxoethoxy)carbonyl)amino)hexanoate and 3-diethylaminopropylamine. LC / MS rt (min): 0.99 MS (ESI, m / z): 982.3 [M+H] +

[0272] Test Example 1 Preparation of GFP mRNA Lipid Particles The ionizable lipids listed 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 at the molar ratios listed in Table 3 to a total lipid concentration of 12.5 mmol / L to obtain an oil phase.

[0273] 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 total lipid concentration to 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 aqueous phase to 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 mRNA-encapsulated lipid particles were obtained. The prepared samples were frozen and stored at −70° C. until use.

[0274]

[0275] <Preparation of GFP pDNA Lipid Particles> An ionizable lipid listed in Table 4, one phospholipid (helper lipid) selected from DOPE (L-α-dioleoyl phosphatidylethanolamine, product name: COATSOME® ME-8181; manufactured by NOF Corporation), and 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 at the molar ratio listed in Table 1 to a total lipid concentration of 12.5 mmol / L to obtain an oil phase.

[0276] GFP pDNA (GenScript, custom-synthesized plasmid DNA) was diluted with 50 mmol / L citrate buffer at pH 4 so that the weight ratio of total lipid concentration to mRNA concentration after mixing of the oil and aqueous phases was as shown in Table 4 to obtain an aqueous phase. The aqueous and oil phases were then mixed using a NanoAssemblr (Precision NanoSystems) so that the volume ratio of aqueous phase to 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, yielding GFP pDNA-encapsulated lipid particles. The prepared samples were frozen and stored at −70° C. until use.

[0277]

[0278] <Preparation of Nucleic Acid-Free Lipid Particles (Empty LNPs)> The 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), and DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, product name: COATSOME® MC-6060; manufactured by NOF Corporation) were used. One phospholipid (helper lipid) selected from the group consisting of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine, product name: COATSOME® MC-4040; 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) was dissolved in ethanol at the molar ratios shown in Table 5 so that the total lipid concentration was 12.5 mmol / L or 62.5 mmol / L to obtain an oil phase.

[0279] A 50 mmol / L citrate buffer solution at pH 4 was mixed with the oil phase at a volume ratio of 3:1 citrate buffer to oil phase using a NanoAssemblr (Precision NanoSystems). The mixture was then diluted 2-fold with water to obtain a lipid particle dispersion. This dispersion was dialyzed against 20 mmol / L MES buffer solution at pH 6.0 containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to remove ethanol. Nucleic acid-free lipid particles (empty LNP) were obtained by a concentration step using an ultrafiltration filter (Amicon ultra 100 kDa, Merck) as needed. The empty LNP was stored frozen at -70°C until use.

[0280] <Inclusion of gRNA and Cas9 mRNA into empty LNP (post-addition method)> CleanCap (registered trademark) Cas9 mRNA (5 moU) (TriLink, L-7206) and sgRNA targeting the human T cell receptor alpha constant (TRAC) gene (sequence; A * G * A * GUCUCUCAGCUGGUACA + modified Scaffold, Thermo Fisher A35514, custom synthesis) 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 volume of RNA solution was added to this LNP solution and mixed by pipetting (LNP-RNA mixture). After allowing to stand at room temperature for 5 minutes, an equal volume of 20 mmol / L Tris buffer containing 8% sucrose was added to the LNP-RNA mixture and mixed by pipetting to prepare Cas9 mRNA / gRNA-encapsulated LNPs using the post-addition method.

[0281]

[0282] <Encapsulation of GFP pDNA into empty LNP (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 the 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 using the post-addition method.

[0283]

[0284] <Particle size measurement> The particle size of the mRNA-encapsulated lipid particles was measured using a particle size measurement system NanoSAQLA (Otsuka Electronics) after arbitrarily diluting the lipid particles with phosphate-buffered saline (PBS). The measurement results of particle size and polydispersity index (PDI) are shown in Table 7.

[0285] <Evaluation of Nucleic Acid Encapsulation Rate> (Quantification of Total Nucleic Acid Concentration) 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 was 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.

[0286] (Quantification of nucleic acid concentration in the external aqueous phase) Using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific), the nucleic acid concentration in the external aqueous phase was quantified by the standard addition method. First, the 20x TE buffer included in the kit was diluted with water to prepare 1x TE buffer. TE stands for Tris / EDTA (ethylenediaminetetraacetic acid). A nucleic acid dilution series was prepared by diluting the nucleic acid with TE buffer to a final concentration of 0 to 400 ng / mL. 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-fold with TE buffer was added to each well, and fluorescence (excitation wavelength: 485 nm, fluorescence wavelength: 535 nm) was measured using a fluorescent plate reader (Infitite 200 Pro M nano +, TECAN). The nucleic acid concentration in the external aqueous phase of each measurement solution was calculated from the obtained results according to the standard addition method.

[0287] (Calculation of Encapsulation 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 the external aqueous phase) ÷ total nucleic acid concentration × 100 The results are shown in Table 7.

[0288]

[0289] Test Example 2 Materials and Methods Preparation of Activation Medium The medium used for culturing T cells under activation culture conditions consisted of TexMACStm Medium (Miltenyi biotech, 130-097-196) and 5 ng / ml human interleukin-2 (IL-2, Roche, 11147528001) (hereinafter referred to as the activation medium).

[0290] <Preparation of T Cells and Activation Culture> Frozen T cells (Human PB Pan-T, Cryo, STEMCELL Technologies, ST-70024) derived from peripheral blood of a healthy human donor 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), washed by centrifugation, and resuspended in activation medium. T cells were cultured at a concentration of 1.0 x 10 ６ The cell concentration was adjusted to 1.0 × 10 cells / ml, and Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher DB11131) was added at 1.0 × 10 ６ The cells were seeded in a 24-well cell culture plate and incubated at 37°C and 5% CO ２ The cells were cultured in an incubator for 3 days for activation. On day 3 of activation, the Dynabeads were removed from the T cell culture medium. The T cells thus pretreated were used as activated T cells.

[0291] <Preparation of Non-activation Medium> The medium used when 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 referred to as non-activation medium).

[0292] <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 a healthy human donor 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 and 20 U / ml DNase I, washed by centrifugation, and resuspended in non-activation medium. T cells were cultured at a concentration of 1.0 x 10 ６ The cell concentration was adjusted to 1000 cells / ml, and the cells were seeded in a 24-well cell culture plate. The cells were incubated at 37°C in 5% CO ２ The cells were cultured in an incubator for 3 days, and the pretreated T cells were used as non-activated T cells.

[0293] <Flow cytometry> On day 4 of the culture, the GFP-positive rate of T cells to which nucleic acid had been delivered by each method was evaluated by flow cytometry. The GFP-positive rate was evaluated by measuring the T cells with BD Horizon TM Dead cells were stained with Fixable Viability Stain (FVS) Reagents (BD, 565388). After staining, the cells were fixed and washed, and the cell status was analyzed using an Attune instrument (Thermo Fisher). Data were analyzed using Flowjo software. T cells were gated by size, single cells, and live cells, and the percentage of GFP-positive cells and median fluorescence intensity (MFI) were analyzed.

[0294] Test Example 2-1: Nucleic acid delivery to activated T cells The required number of activated T cells were collected on day 3 of culture, centrifuged, and the supernatant was removed. The activated T cells were cultured at a concentration of 1.0 x 10 in an activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg / ml. ６ The cells were adjusted to a concentration of 1000 cells / ml and seeded onto a 96-well plate.

[0295] 1.0 × 10 GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5 were used. ６ The total RNA was added to each cell at 1.0 μg per cell, and incubated at 37°C in 5% CO ２ The cells were cultured in an incubator. 24 hours after 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 efficiency of GFP mRNA transfer. The results are shown in Figure 1. All of the LNPs of Examples 1 to 5 were capable of highly efficient mRNA delivery to activated T cells.

[0296] Test Example 2-2: Nucleic acid delivery to non-activated T cells On day 3 of culture, the required number of non-activated T cells were collected, centrifuged, and the supernatant was removed. 1.0 x 10 non-activated T cells were cultured in an activation medium containing ApoE3 at a final concentration of 1 μg / ml, which had been prepared just before use. ６ The GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5 were used to prepare 1.0 × 10 cells / ml of LNPs, which were then seeded onto a 96-well plate. ６ The total RNA was added to each cell at 1.0 μg per cell, and incubated at 37°C in 5% CO ２ The cells were cultured in an incubator. 24 hours after 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 efficiency of GFP mRNA transfer. The results are shown in Figure 2. All of the LNPs of Examples 1 to 5 were capable of delivering mRNA to non-activated T cells, and the LNPs of Examples 1, 3, and 4 were shown to be particularly efficient.

[0297] Test Example 3-1: Nucleic acid delivery to activated T cells The required number of activated T cells were collected on day 3 of culture, centrifuged, and the supernatant was removed. 1.0 x 10 activated T cells were cultured in an activation medium without ApoE3. ６ The cells were adjusted to a concentration of 1000 cells / ml and seeded onto a 96-well plate.

[0298] 1.0 × 10 GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5 were used. ６ The total RNA was added to each cell at 1.0 μg per cell, and incubated at 37°C in 5% CO ２ The cells were cultured in an incubator. 24 hours after 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 efficiency of GFP mRNA transfer. The results are shown in Figure 3. (Data in the presence of ApoE3 shown in Figure 1 are also shown as a reference example.) The LNPs of Examples 1, 3, and 4 were capable of highly efficient mRNA delivery to activated T cells even in an environment without ApoE3 activation.

[0299] Test Example 3-2: Nucleic acid delivery to non-activated T cells On day 3 of culture, the required number of non-activated T cells were collected, centrifuged, and the supernatant was removed. 1.0 x 10 non-activated T cells were cultured in an activation medium containing no ApoE3. ６ The GFP mRNA-encapsulated LNPs prepared in Examples 1 to 5 were used to prepare 1.0 × 10 cells / ml of LNPs, which were then seeded onto a 96-well plate. ６ The total RNA was added to each cell at 1.0 μg per cell, and incubated at 37°C in 5% CO ２ The cells were cultured in an incubator. 24 hours after 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 efficiency of GFP mRNA transfer. The results are shown in Figure 4. (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 mRNA delivery to non-activated T cells even in an environment without ApoE3 activation.

[0300] Test Example 4: Delivery of plasmid DNA to activated T cells using conventional LNPs. <1> LNP treatment of activated T cells. The required number of activated T cells were collected on day 3 of culture, centrifuged, and the supernatant was removed. 1.0 x 10 activated T cells were cultured 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. ６ The GFP pDNA-encapsulated LNPs of Examples 130 to 147 were used to prepare 1.0 × 10 ６ The total DNA was added at 0.4 to 10.0 μg per cell, and the cells were cultured in a 37°C, 5% CO2 incubator. <2> Ratio of GFP-positive cells in activated T cells On day 4 of culture, the ratio of GFP-positive cells in T cells treated with each method was measured by flow cytometry to evaluate the efficiency of plasmid DNA introduction. The results are shown in Table 8.

[0301]

[0302] <Test Example 5> Nucleic acid delivery to activated T cells using post-added LNPs (TCR KO) <1> LNP treatment of activated T cells The required number of activated T cells on day 3 of culture were collected, centrifuged, and the supernatant was removed. The activated T cells were cultured at a concentration of 1.0 × 10 in an activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg / ml. ６ The RNA-encapsulated LNPs prepared by the post-addition method in Examples 8 to 129 and Example 405 were used to prepare 1.0 × 10 ６ The total amount of RNA was added to each cell at 1.8 to 4.0 μg (total RNA amount). The mixture was incubated at 37°C in 5% CO ２ The cells were cultured in an incubator.

[0303] 24 hours after the addition of LNP, activation medium was added to the T cell suspension at a volume ratio of 1:3 for expansion, and the cells were further cultured for 3 days.

[0304] <2> TCR KO efficiency in activated T cells On day 7 after the start of culture, the ratio of TCR-negative cells in T cells treated by each method was measured by flow cytometry to evaluate the TCR KO efficiency. The results are shown in Table 9.

[0305]

[0306] Test Example 6: Delivery of plasmid DNA to activated T cells using post-added LNPs <1> LNP treatment of activated T cells The required number of activated T cells were collected on day 3 of culture, centrifuged, and the supernatant was removed. The activated T cells were cultured in an 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. ６ The plasmid DNA-encapsulated LNPs prepared by the post-addition method in Examples 148 to 404 were used to prepare 1.0 x 10 cells / ml of LNPs and seeded in a 96-well plate. ６ The cells were cultured in a 37°C, 5% CO 2 incubator, with the total amount of DNA added being 0.4 to 10.0 μg per cell (total DNA amount).

[0307] <2> Ratio of GFP-positive cells in activated T cells On day 4 of culture, the ratio of GFP-positive cells in 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.

[0308]

[0309] <Test Example 7> Nucleic acid delivery to activated T cells using post-added LNPs (TCR KO) <1> LNP treatment of activated T cells The required number of activated T cells on day 3 of culture were collected, centrifuged, and the supernatant was removed. The activated T cells were cultured at a concentration of 1.0 × 10 in an activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg / ml. ６The RNA-encapsulated LNPs prepared by the post-addition method of Examples 88 to 90 were used to prepare 1.0 × 10 ６ The total RNA was added at 0.4 to 4.0 μg per cell, and the cells were cultured in a 37° C., 5% CO 2 incubator.

[0310] 24 hours after the addition of LNP, activation medium was added to the T cell suspension at a volume ratio of 1:3 for expansion, and the cells were further cultured for 3 days.

[0311] <2> TCR KO efficiency in activated T cells On day 7 after the start of culture, the ratio of TCR-negative cells in T cells treated by each method was measured by flow cytometry to evaluate the TCR KO efficiency. The results are shown in Figure 5.

[0312] Test Example 8: Nucleic acid delivery to activated T cells using additives The required number of activated T cells were collected on day 3 of culture, centrifuged, and the supernatant was removed. The activated T cells were cultured at a concentration of 1.0 x 10 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. ６ The cells were adjusted to a concentration of 1000 cells / ml and seeded onto a 96-well plate.

[0313] Using the GFP mRNA-encapsulated LNPs prepared in Examples 1 and 4, 1.0 × 10 ６ The total amount of RNA was added to each cell at 4.0 μg (total RNA amount). ２ The cells were cultured in an incubator. 24 hours after 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 efficiency of GFP mRNA transfer. 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 and RetroNectin in combination.

[0314] Test Example 9: Nucleic acid delivery to T cells after long-term culture The required number of T cells were collected on day 10 of culture (day 7 after activation), centrifuged, and the supernatant was removed. 1.0 x 10 activated T cells were cultured in an activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg / ml. ６ The cells were adjusted to a concentration of 1000 cells / ml and seeded onto a 96-well plate.

[0315] 1.0 × 10 GFP mRNA-encapsulated LNPs prepared in Examples 1, 3, and 4 were used. ６ The total amount of RNA was added to each cell at 4.0 μg (total RNA amount). ２ The cells were cultured in an incubator. 24 hours after addition of LNP, the cells were collected, and the percentage of GFP-positive T cells treated with each LNP was measured by flow cytometry to evaluate the efficiency of GFP mRNA transduction. The results are shown in Figure 7 (Day 10 TF condition).

[0316] Test Example 10: Nucleic acid delivery to T cells after long-term culture T cells were collected on day 9 of culture in activation medium (day 6 after activation) and resuspended in 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 day 10 of culture were collected, centrifuged, and the supernatant was removed. The activated T cells were then cultured at a concentration of 1.0 x 10 in activation medium containing recombinant human apolipoprotein E3 (ApoE3) (Fujifilm Wako, 010-20261) at a final concentration of 1 μg / ml, prepared just before use. ６ The cells were adjusted to a concentration of 1000 cells / ml and seeded onto a 96-well plate.

[0317] Using the GFP mRNA-encapsulated LNPs prepared in Examples 1, 3, and 4, 1.0 × 10 ６ The total amount of RNA was added to each cell at 4.0 μg (total RNA amount). ２The cells were cultured in an incubator. 24 hours after 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 transduction efficiency. The results are shown in Figure 7 (Day 9 MC_Day 10 TF condition, Day 9 OptiMEM_Day 10 TF condition).

Claims

A nucleic acid delivery agent for immune cells, comprising a lipid composition containing an ionizable lipid which 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, and R １ , R ２ , R ３ and R ４ The substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by are each independently -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 represents, and 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, and R ５ and R ６ each independently represent an optionally substituted hydrocarbon group having 1 to 18 carbon atoms, and R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by 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 ４２ , where R ２１ and R ２２ each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms, and 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, and the substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by 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 are an aryl group having 6 to 20 carbon atoms, a heterocyclic group, -OH, -COOH, or -NR ５１ R ５２ is shown, and R ５１ and R ５２ each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms, and R ７ , R ８ , and R ９ each independently represent a hydrocarbon group having 2 to 8 carbon atoms, and R ５ and R ６ , or R ５ and R ７ may combine together to form a 4- to 7-membered ring.

2. The nucleic acid delivery agent to immune cells according to claim 1, wherein the ionizable lipid is 20 to 60 mol% in terms of the molar ratio to the total lipids in the lipid composition.

3. The nucleic acid delivery agent to immune cells according to claim 1, wherein the non-ionizable lipid contains a sterol or its derivative, and a phospholipid.

4. The nucleic acid delivery agent to immune cells according to claim 3, wherein the phospholipid is selected from the group consisting of distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, and dioleoyl phosphatidylethanolamine.

5. The nucleic acid delivery agent to immune cells according to claim 3, wherein the sterol or its derivative is 30 to 70 mol% in terms of the molar ratio to the total lipids in the lipid composition.

6. The nucleic acid delivery agent to immune cells according to claim 3, wherein the phospholipid is 1 to 30 mol% in terms of the molar ratio to the total lipids in the lipid composition.

7. The nucleic acid delivery agent to immune cells according to any one of claims 1 to 6, wherein the lipid having the non-ionic polymer is a lipid having a polyethylene glycol chain.

8. The nucleic acid delivery agent to 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 distearoyl phosphatidylethanolamine polyethylene glycol.

9. The nucleic acid delivery agent to immune cells according to any one of claims 1 to 6, wherein the lipid having the non-ionic polymer is 0.1 to 3 mol% in terms of the molar ratio to the total lipids in the lipid composition.

10. The nucleic acid delivery agent to immune cells according to any one of claims 1 to 6, wherein the mass ratio of the total lipids of the lipid composition to the nucleic acid is 7:1 to 1000:

1.

11. The nucleic acid delivery agent to immune cells according to any one of claims 1 to 6, wherein the immune cells are activated cells or non-activated cells.

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

13. A method for delivering nucleic acid to immune cells (excluding the delivery method in vivo), comprising contacting the nucleic acid delivery agent to immune cells according to any one of claims 1 to 6 with immune cells.

14. The method according to claim 13, wherein the immune cells are activated cells or non-activated cells.

15. The method according to claim 13, comprising the step of adding (i) apolipoprotein and / or (ii) a protein comprising a cell-binding domain and a heparin-binding domain to the nucleic acid delivery agent or immune cells before contacting the nucleic acid delivery agent with the immune cells.

16. A step of preparing lipid particles not containing nucleic acid using an ionizable lipid which is a compound represented by formula (1) or a salt thereof, a non-ionizable lipid, and a lipid having a non-ionic polymer, and a step of mixing the lipid particles not containing nucleic acid and nucleic acid, the method for producing a 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 a hydrocarbon group having 1 to 24 carbon atoms which may be substituted, and R １ , R ２ , R ３ and R ４ The substituents on the hydrocarbon group having 1 to 24 carbon atoms which may be substituted represented by are each independently -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 represents, and 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, and R ５ and R ６ each independently represent a hydrocarbon group having 1 to 18 carbon atoms which may be substituted, and R ５ and R ６ The substituents on the hydrocarbon group having 1 to 18 carbon atoms which may be substituted represented by 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 ４２ represents, and R ２１ and R ２２ each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms, and 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, and 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 The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms shown is an aryl group having 6 to 20 carbon atoms, a heterocyclic group, -OH, -COOH, or -NR ５１ R ５２ represents, and R ５１ and R ５２ each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms, and R ７ , R ８ , and R ９ each independently represent a hydrocarbon group having 2 to 8 carbon atoms, and R ５ and R ６ , or R ５ and R ７ may together 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-triazapentacosanedioate 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-triazapentacosanedioate Bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-7,15-dioxo-6,16-dipropyl-8,14-dioxa-6,11,16-triazapentacosanedioate Bis(2-hexyloctyl) 6,16-dibutyl-11-(3-(diethylamino)propyl)-7,15-dioxo-8,14-dioxa-6,11,16-triazapentacosanedioate 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-triazapentacosanedioate Bis(2-pentylheptyl) 12-(4-(diethylamino)butyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-(dimethylamino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(1-ethylpiperidin-4-yl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-isopropylpiperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((4-hydroxybutyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(2-((4-hydroxybutyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((4-hydroxybutyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-((4-hydroxybutyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-(ethyl(4-hydroxybutyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-(ethyl(4-hydroxybutyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((3-hydroxypropyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(2-((3-hydroxypropyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((3-hydroxypropyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-((3-hydroxypropyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(2-(ethyl(3-hydroxypropyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(2-(ethyl(3-hydroxypropyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-(ethyl(3-hydroxypropyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-(ethyl(3-hydroxypropyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(3-((2-hydroxyethyl)(methyl)amino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-((2-hydroxyethyl)(methyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-(ethyl(2-hydroxyethyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-(3-(ethyl(2-hydroxyethyl)amino)propyl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate 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-triazatricosanedioic acid Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(1,2,2,6,6-pentamethylpiperidin-4-yl)-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazetidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylpyrrolidin-3-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-methylazepan-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-((1r,4r)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 12-((1s,4s)-4-(dimethylamino)cyclohexyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatricosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-12-(1-(2-hydroxyethyl)piperidin-4-yl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatriacosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(pyrrolidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatriacosanedioate Bis(2-pentylheptyl) 7,17-diheptyl-8,16-dioxo-12-(2-(piperidin-1-yl)ethyl)-9,15-dioxa-7,12,17-triazatriacosanedioate Bis(2-pentylheptyl) 12-(1-methylpiperidin-4-yl)-7,17-dioctyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatriacosanedioate Bis(2-pentylheptyl) 12-(3-(bis(2-hydroxyethyl)amino)propyl)-7,17-diheptyl-8,16-dioxo-9,15-dioxa-7,12,17-triazatriacosanedioate Bis(2-pentylheptyl) 12-(2-(diethylamino)ethyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatriacosanedioate Bis(2-pentylheptyl) 12-(3-(diethylamino)propyl)-8,16-dioxo-7,17-dipropyl-9,15-dioxa-7,12,17-triazatriacosanedioate Bis(2-pentylheptyl) 7,17-dibutyl-12-(3-(diethylamino)propyl)-8,16-dioxo-9,15-dioxa-7,12,17-triazatriacosanedioate

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