Method to deliver a payload to cells, and lipid composition

Abstract

The object of the present invention is to provide a novel method and lipid composition for delivering payload to cells in vitro. The present invention provides a method to deliver a payload to cells, which comprises adding at least one LDLR binding polypeptide to a culture media and contacting the cell with a lipid composition, wherein the lipid composition comprises a payload, an ionizable lipid, a phospholipid, a sterol and a polymer-conjugated lipid, wherein the polymer-conjugated lipid is selected from specified lipids.

Description

METHOD TO DELIVER A PAYLOAD TO CELLS, AND LIPID COMPOSITION

[0001] The present invention relates to a method to deliver a payload to cells, which comprises contacting the cell with a lipid composition. The present invention further relates to a lipid composition which comprises a payload, an ionizable lipid, a phospholipid, a sterol and certain polymer-conjugated lipid.

[0002] Genetically engineered cell therapies have the potential to provide treatments for a wide range of diseases and conditions. These therapies use genetic engineering techniques to introduce or modify genetic information to strengthen or correct their function. Some common examples of genetically engineered cell therapies include Chimeric Antigen Receptor (CAR) T cell therapy, which is a type of immunotherapy approved for cancer treatment.

[0003] For these genetic engineering, transfection methods are used to introduce foreign genetic material, such as plasmid DNA, RNA or proteins, into living cells. These methods can be broadly categorized into three groups: biological, physical and chemical transfection.

[0004] Biological transfection is typically mediated by viruses, utilizing the ability of a virus to inject its DNA inside a host cell. Viral-based transfection have been most widely used in commercial cell therapies to insert genes into genome DNA but has manufacturing and regulatory challenges. Manufacturing of viral vector needs complex manufacturing process that requires specialized equipment and expertise. The process typically involves multiple steps, including cell culture, vector production, purification, and quality control. Each step requires careful monitoring and optimization to ensure a high-quality product, which can drive up the cost of manufacturing. Also, conventional viral vector production methods, such as the use of packaging cell lines or helper viruses, have limited scalability, which can also drive up the manufacturing cost. In addition, because of the infectious and proliferative nature of viruses, the control of residual viral impurities in cell therapies must adhere to strict regulations and standards to ensure the safety of the final product. Furthermore, viral-based transfection also carries the risk that viral genome may be randomly inserted into the human genome, activating oncogene.

[0005] Physical transfection methods such as electroporation and mechanoporation involve the use of mechanical or electrical force to make transient pores to introduce foreign genetic material into cells. Among these methods, electroporation has been most widely used to deliver gene editing enzymes into the cells. However, exposure of cells to high voltage electric pulses usually causes cell death, resulting in extended manufacturing times due additional cell recovery period.

[0006] Chemical transfection utilizes cationic polymer- or lipid-based nanoparticles which encapsulate genetic materials. Since chemical transfection utilizes the cellular intrinsic uptake pathway, it is gentler than the physical transfection method that forcibly perforates the plasma membrane. In addition, these nanoparticles can be chemically manufactured, and thus reducing manufacturing costs and time. Furthermore, since these nanoparticles are not infectious or proliferative like viruses, regulatory challenges can be minimized.

[0007] However, conventional transfection reagents such as PEI (polyethyleneimine), which have been used in protein- or virus-producing HEK and CHO cells, are still too toxic and damaging to use in the cell therapy manufacturing process. In addition, these conventional transfection reagents have low transfection efficiency for primary immune cells and stem cells used in cell therapies.

[0008] Compared to conventional chemical transfection reagents, lipid nanoparticles (LNPs), which use specially designed ionizable cationic lipids, have successfully reduced toxicity, and been approved to be used for liver-targeted siRNA therapeutics and mRNA vaccines in vivo. On the other hand, since the LNPs have been optimized for in vivo applications, their in vitro transfection efficiency against primary immune cells and stem cells remains low.

[0009] Patent Literatures 1 and 2 describe lipid compositions for drug delivery, which comprises ionizable lipid. Patent Literature 3 describes a lipid composition for drug delivery to immune cells. Patent Literature 4 describes a lipid composition for drug delivery to T cells. Patent Literatures 5 and 6 describe in vitro methods of mRNA delivery using lipid nanoparticles, and describe stabilizers such as polyoxyethylene (10) stearyl ether (PEG(10) stearyl ether), polyoxyethylene (20) stearyl ether (PEG(20) stearyl ether), Polysorbate 80 (PEG(20) sorbitan monooleate), and D-alpha-tocopherol polyethylene glycol 1000 succinate. Also, commercial T-cell kits for mRNA are also known (Non-Patent Literature 1).

[0010] As DNA insertion, Patent Literature 7 discloses a method of producing car-t cells, nucleic acid-introducing carrier and kit. Patent Literature 8 discloses composition, lipid particle manufacturing kit, substance delivery method, and detection method.

[0011] Non-Patent Literatures 2 to 4 discloses use of transposase and Nanoplasmid DNA for in vitro immune cell transfection. Patent Literature 9 and Non-Patent Literature 5 discloses use of CRISPR / Cas9 and ssDNA template for gene insertion. Patent Literatures 10-12 discloses transfection enhancer.

[0012] WO2019 / 235635WO2022 / 054955WO2019 / 152557WO2020 / 210901WO2019 / 067999WO2021 / 222287WO2021 / 014368WO2021 / 186233WO2020 / 123871WO2019 / 228108WO2021 / 141020WO2022 / 214588

[0013] https: / / www.precisionnanosystems.com / docs / default-source / pni-files / user-guides / t-cell-kit-ignite-user-guide-0322_digital.pdf“TC Buster Transposon engineered CLL-1 car-NK cells efficiently target acute myeloid leukemia” Gurney M, O’Reilly E, Corcoran S, Brophy S, Hardwicke D, Krawczyk J, et al. Blood. 2021;138(Supplement 1):1725. DOI: https: / / doi.org / 10.1182 / blood-2021-147244“Concurrent transposon engineering and CRISPR / Cas9 genome editing of primary CLL-1 chimeric antigen receptor-natural killer cells” Gurney M, O’Reilly E, Corcoran S, Brophy S, Krawczyk J, Otto NM, Hermanson DL, Childs RW, Szegezdi E, O’Dwyer ME. Cytotherapy, Volume 24, Issue 11,2022, Pages 1087-1094, ISSN 1465-3249, DOI: https: / / doi.org / 10.1016 / j.jcyt.2022.07.008“Non-viral engineering of CAR-NK and car-T cells using the TC buster transposon systemTM” Pomeroy EJ, Lahr WS, Chang JW, Krueger J, Wick BJ, Slipek NJ, et al.bioRxiv 2021.08.02.454772; DOI: https: / / doi.org / 10.1101 / 2021.08.02.454772“High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails” Shy BR, Vykunta VS, Ha A, Talbot A, Roth TL, Nguyen DN, Pfeifer WG, Chen YY, Blaeschke F, Shifrut E, Vedova S, Mamedov MR, Chung JJ, Li H, Yu R, Wu D, Wolf J, Martin TG, Castro CE, Ye L, Esensten JH, Eyquem J, Marson A..Nat Biotechnol. 2023 Apr;41(4):521-531. doi: 10.1038 / s41587-022-01418-8.

[0014] The object of the present invention is to provide a novel method and lipid composition for delivering payload to cells in vitro.

[0015] As a result of their diligent study to solve the above object, the inventors have found that when delivering a payload to cells using a lipid composition comprising an ionizable lipid and a polymer-conjugated lipid, the efficiency of introducing the payload into cells in vitro can be greatly improved by using a specified polymer-conjugated lipid as the polymer-conjugate lipid. The present invention was completed based on the above findings. According to the present invention, the following invention is provided.

[0016] <1> A method to deliver a payload to cells, which comprises adding at least one LDLR binding polypeptide to a culture media and contacting the cell with a lipid composition, wherein the lipid composition comprises a payload, an ionizable lipid, a phospholipid, a sterol and a polymer-conjugated lipid, wherein the polymer-conjugated lipid is selected from; (a) aceramide-polyethylene glycol, a phospholipid-polyethylene glycol, or a glyceride- polyethylene glycol with average polyethylene glycol molecular weight of 550-1100; (b) acholesterol-polyethylene glycol with average polyethylene glycol molecular weight of 350-5000 or 550-2000; (c) a polyethylene glycol fatty acid ester with average polyethylene glycol molecular weight of 350-2000, or 550-2000, or 1000-2000; (d) a polyoxyethylene alkyl ether with the number of ethylene oxide units of 10-25; (e) a polyethylene glycol hydrogenated castor oil or a polyethylene glycol castor oil with an average ethoxylation degree of 30-50; or (f) a polyethylene glycol(20) sorbitan fatty acid monoester. <2> The method of <1>,wherein the cell is an immune cell, a stem cell, or a progenitor cell. <3> The method of <2>, wherein the imune cell is selected from lymphocytes, monocytes, macrophages, mast cells, dendritic cells, granulocytes, primary immune cells, CD3+ cells, CD4+ cells, CD8+ T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DC). <4> The method of <2>, wherein the stem cell is selected from mesenchymal stem cells (MSCs); hematopoietic stem cells (HSCs); endothelial progenitor cell (EPCs); neural stem cell (NSCs); limbal stem cell (LSCs); induced pluripotent stem cell (iPSCs); ocular stem cells; pluripotent stem cell (PSCs); or embryonic stem cell (ESCs). <5> The method of <1>,wherein the cell is used for transplantation <6> The method of <1>, which further comprises inducing expression of low density lipoprotein receptor (LDLR). <7> The method of <6>, wherein the LDLR binding polypeptide is an apolipoprotein. <8> The method of <7>, wherein the apolipoprotein is ApoE1, ApoE2, ApoE3 or ApoE4, or their fragment. <9> The method of <1>, wherein the payload is delivered to the cells in vitro. <10> A lipid composition which comprises an ionizable lipid, a phospholipid, a sterol and a polymer-conjugated lipid, wherein the polymer-conjugated lipid is selected from; (a) aceramide-polyethylene glycol, a phospholipid-polyethylene glycol, or a glyceride- polyethylene glycol with average polyethylene glycol molecular weight of 550-1100; (b) acholesterol-polyethylene glycol with average polyethylene glycol molecular weight of 350-5000 or 550-2000; (c) a polyethylene glycol fatty acid ester with average polyethylene glycol molecular weight of 350-2000, or 550-2000, or 1000-2000; (d) a polyoxyethylene alkyl ether with the number of ethylene oxide units of 10-25; (e) a polyethylene glycol hydrogenated castor oil or a polyethylene glycol castor oil with an average ethoxylation degree of 30-50; or (f) a polyethylene glycol(20) sorbitan fatty acid monoester. <11> The lipid composition of <10> which further comprises a payload. The lipid composition of <10> wherein the polymer-conjugated lipid is selected from; (b) acholesterol-polyethylene glycol with average polyethylene glycol molecular weight of 350-5000 or 550-2000 or 1000-2000; or (d) a polyoxyethylene alkyl ether with the number of ethylene oxide units of 10-25; The lipid composition of <10>, wherein the polymer-conjugated lipid is selected from; (d) polyoxyethylene (21) lauryl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (25) lauryl ether, polyoxyethylene (10) cethyl ether, polyoxyethylene (15) cethyl ether, polyoxyethylene (20) cethyl ether, polyoxyethylene (23) cethyl ether, polyoxyethylene (25) cethyl ether, polyoxyethylene (12) cetostearyyl ether, polyoxyethylene (20) cetostearyyl (25) ether, polyoxyethylene (10) stearyl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (10) oleyl ether, or polyoxyethylene (20) oleyl ether. <C> The lipid composition of <10>, wherein the polymer-conjugated lipid is present in the lipid composition in a range of 0.25-10 mol %, or 0.5-10 mol %, or 1-10 mol %, or 1-5 mol %, or 2-4 mol % at a mol ratio to total lipids. <12> The lipid composition of <10>, wherein the composition comprises 10-55 mol % ionizable lipid, 5-25 mol % phospholipid, 30 to 70 mol % sterol, and 0.25-10 mol % polymer-conjugated lipid at a mol ratio to total lipids. <D> The lipid composition of <10>, wherein the composition comprises 15-50 mol % ionizable lipid, 10-20 mol % phospholipid, 40 to 65 mol % sterol, and 0.5-10 mol % polymer-conjugated lipid at a mol ratio to total lipids. <E> The lipid composition of <10>, wherein the composition comprises 20-40 mol % ionizable lipid, 10-20 mol % phospholipid, 40 to 60 mol % sterol, and 1-5 mol % polymer-conjugated lipid at a mol ratio to total lipids. <F> The lipid composition of <10>, wherein the composition comprises 20-40 mol % ionizable lipid, 15-20 mol % phospholipid, 40 to 55 mol % sterol, and 2-4 mol % polymer-conjugated lipid at a mol ratio to total lipids. <13> The lipid composition of <10>, wherein the ionizable lipid is a compound represented by the formula (2), the formula (3) or the formula (4): In the formula, X represents -NR1- or -O-, R1represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R21-L1-R22-, where R21represents a hydrocarbon group having 1 to 24 carbon atoms, L1represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R22represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R2and R3each independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R31-L2-R32-, where R31represents a hydrocarbon group having 1 to 24 carbon atoms, L2represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R32represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R4, R5, R6, R7, R8, R9, R10, R11, and R12each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted, groups in any one or more pairs among R4and R5, R10and R5, R5and R12, R4and R6, R5and R6, R6and R7, R6and R10, R12and R7, and R7and R8may be linked to each other to form a 4- to 7-membered ring which may contain an O atom, a substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, the substituent on the substituted or unsubstituted aryl group and on the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and a, b, c, and d each independently represent an integer of 0 to 3, a + b is 1 or more, and c + d is 1 or more. wherein R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with one or more substituents selected from -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, and -O-R156, R104represents a hydrocarbon group having 1 to 8 carbon atoms, R105and R106each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R108-L101-R109, excluding a case that both R105and R106are hydrocarbon groups having 1 to 8 carbon atoms, R107represents -R110-L102-R111-L103-R112, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R153, R154, R155, and R156may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, R158represents a hydrocarbon group having 1 to 12 carbon atoms, and R157represents -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, or -O-R166. R161and R162each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R163, R164, R165, and R166each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R163, R164, R165, and R166may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R168, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, -O-R166, or -(hydrocarbon group having 1 to 12 carbon atoms), R168represents a hydrocarbon group having 1 to 12 carbon atoms, and L101, L102, and L103each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-. R108represents a hydrocarbon group having 1 to 12 carbon atoms, R109represents a hydrocarbon group having 1 to 24 carbon atoms, R110represents a hydrocarbon group having 1 to 8 carbon atoms, R111represents a hydrocarbon group having 1 to 24 carbon atoms, R112represents a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R109and R112may be substituted with an aryl group, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, where definitions of R153, R154, R155, and R158are as described above, and the hydrocarbon group represented by R111may be substituted with -OC(O)O-R153, -C(O)O-R154, or -OC(O)-R155, where the definitions of R153, R154, and R155are as described above. wherein R201, R202, R203, and R204each independently represent a hydrogen, a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, the substituted or unsubstituted hydrocarbon groups represented by R201, R202, R203, and R204each independently represent -C(O)O-R2011, -OC(O)-R2012, -O-R2013, -CO-R2014-, -OC(O)O-R2015, or -S-S-R2016, R2011, R2012, R2013, R2014-, R2015, and R2016each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R2017, and R2017represents a hydrocarbon group having 1 to 12 carbon atoms, R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 18 carbon atoms, the substituted groups on the substituted hydrocarbon groups having 1 to 18 carbon atoms represented by R205and R206each represent -OH, -COOH, -NR2021R2022, -OC(O)O-R2023, -C(O)O-R2024, -OC(O)-R2025, -O-R2026, -C(O)NR2027R2028, -NR2029C(O)R2030, -N(R2031)S(O)2R2032, -N(R2033)C(O)N(R2034)R2035, -N(R2036)C(S)N(R2037)R2038, -OC(O)N(R2039)R2040, or -N(R2041)C(O)OR2042, R2021and R2022each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030, R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042each independently represent a hyrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, where the substituted groups on the substituted hydrocarbon group having 1 to 24 carbon atoms represented by R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030,R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042represent an aryl group having 6 to 20 carbon atoms, a heterocyclic group, -OH, -COOH, or NR2051R2052, and R2051and R2052each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R207, R208, and R209each independently represent a hydrocarbon group having 2 to 8 carbon atoms, R205and R206, or R205and R207may form a 4 to 7-membered ring together. <G>. The lipid composition of <10>, wherein the ionizable lipid is FL-A or FL-B: FL-A FL-B FL-E FL-F FL-H FL-I FL-J FL-K FL-L FL-M FL-N <14> The lipid composition of <10>, wherein the ionizable lipid is a compound represented by the following formula (1): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, the substituent A represents a hydroxyl group, or a group represneted by -G20-CH(R55)(R56), -N(R58)(R59) or -G20-R60, G20represents -O(CO)-, or-(CO)O-, R55and R56each independently represent a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R58and R59each independently represent a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, the substituent B is-N(R61)(R62), R61and R62each independently represent a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R60represents a hydrocarbon group having 1 to 18 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G30indicates-S-(CO)-NR64, R64represents a group represented by-L30-G20-CH(R55)(R56), a represents 0 or 1, L30represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G10represents -O(CO)-, -(CO)O-, -O(CO)O-, or -N(C(O)R63)-, R63represents a hydrocarbon group having 1 to 18 carbon atoms, L20represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, the substituent C represents a group represented by-(CO)O R65or-O(CO)-R65, R65represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by-L40-CH(R66)(R67), L40represents a hydrocarbon group having 1 to 6 carbon atoms, R66and R67represent a hydrocarbon group having 1 to 10 carbon atoms or an alkoxy group. <H>. The lipid composition of <10>, wherein the ionizable lipid is selectected from: MC3 ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate) L-319 (bis[(Z)-non-2-enyl] 9-[4-(dimethylamino)butanoyloxy]heptadecanedioate) ALC-0315 (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate) SM-102 (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) Lipid 5 (nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate) Lipid 29 (undecan-3-yl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-[3-[[2-(methylamino)-3,4-dioxocyclobuten-1-yl]amino]propyl]amino]octanoate) ATX-100 (pentadecan-8-yl 4-[3-(dimethylamino)propylsulfanylcarbonyl-(4-oxo-4-pentadecan-8-yloxybutyl)amino]butanoate) Lipid A9 (bis(2-butyloctyl) 10-[3-(dimethylamino)propyl-nonanoylamino]nonadecanedioate) Lp01 ([2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate) GCL1 ([(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate) The lipid composition of <10>, wherein the ionizable lipid is selectected from: ATX-100 Lipid A9 GCL1 <15> The lipid composition of <10>, wherein the phospholipid is a phosphatidylcholine with fatty acid tails having 12 to 20 carbon atoms, a phosphatidylcholine with saturated fatty acid tails having 14 to 18 carbon atoms, or a phosphatidylcholine with saturated fatty acid tails having 18 carbon atoms. <16> The lipid composition of <10>, wherein the sterol is selected from cholesterol, or phytosterol. <17> The lipid composition of <10>, wherein the payload is a nucleic acid molecule. <J>. The lipid composition of <17>, wherein the nucleic acid molecule is an RNA. <K> The lipid composition of <17>, wherein the nucleic acid molecule is an mRNA. <L> The lipid composition of <17>, wherein the nucleic acid molecule is a guide RNA. <M> The lipid composition of <K>, wherein the mRNA encodes a DNA binding protein. <N> The lipid composition of <M>, wherein the DNA binding protein is TALEN, ZFN, CRIPSR-Cas system, transposase, or their derivatives. <O> The lipid composition of <17>, wherein the nucleic acid molecule is a DNA. The lipid composition of <O>, wherein the DNA is a single strand DNA. <Q> The lipid composition of , wherein the single strand DNA is a linear or circular single strand DNA. <R> The lipid composition of <Q>, wherein the linear single strand DNA is hybridized with a short single strand DNA to form guide RNA-Cas enzyme complex binding domain. <S> The lipid composition of <O>, wherein the DNA is a double strand DNA. <T> The lipid composition of <S>, wherein the double strand DNA is a circular or linear double strand DNA. <18> A method for generating one or more genetically modified cells, comprising: (a) contacting the cells with a lipid nanoparticle (LNP) composition comprising a first nucleic acid encoding a DNA binding protein with at least one nuclear localization signal sequence, (b) contacting the cells with an LNP composition comprising a second nucleic acid encoding a sequence for insertion, and the second nucleic acid comprises a sequence which bind to the DNA binding protein, wherein the LNP composition is a lipid composition of <10> <19> The method of <18>, wherein (a) the first nucleic acid is mRNA, and (b) the second nucleic acid is DNA or RNA. <20> The method of <18>, further comprising (c) loading the nucleic acid into pre-formed empty LNPs. <18-1> The method of <18>, wherein (a) the DNA binding protein is a transposase. <18-2> The method of <18>, , wherein (b) the DNA is a circular double strand DNA, or a linear double strand DNA. <18-3> The method of <18>, wherein (b) the DNA comprises: Terminal repeat, a promoter region, therapeutic gene-coding region, poly A signal region, terminal repeat, wherein the terminal repeat can bind to the transposase. <18-4> The method of <18>, which further comprises (d) treating the cells with an immune modulating agent. <18-5> The method of <18-4>,, wherein (d) the immune modulating agent is selected from cGASinhibitor, STING inhibitor, TBK1 inhibitor, IKKε inhibitor, JAK inhibitor, TYK2 inhibitor, interferon inhibitor. <18-6> The method of <18-4>, wherein (d) the immune modulating agent is selected from BX-795, MRT67307, GSK8612,Peficitinib, JAK Inhibitor I, or Baricitinib. <18-7> The method of <18>, wherein (a) the DNA binding protein is an RNA-guided DNA binding protein, zinc-finger nuclease, meganuclease, or TALEN nuclease. <18-8> The method of <18-7>, wherein (a) the RNA-guided DNA binding protein is a Cas nuclease or its derivative. <18-9> The method of <18>, wherein (b) the DNA is a single strand DNA. <18-10> The method of <18>, wherein (b) the single strand DNA is a linear or circular single strand DNA. <18-11> The method of <18-10>, wherein (b) the linear single strand DNA is hybridized with a short single strand DNA to form guide RNA-Cas enzyme complex binding domain. <18-12> The method of <18>, wherein (b) the DNA comprises: a 5′ homology arm, a therapeutic gene-coding region, and a 3′ homology arm to a cell, wherein the 5′ homology arm and the 3′ homology arm are complementary to the polynucleotides in a target region of a genomic DNA in the cell. <18-13> The method of <18>, wherein (a) the DNA binding protein further comprise single strand DNA binding domain, and (b) the DNA is a circular single strand DNA. <18-14> The method of <18>, which further comprises (e) treating the cells with a DNA damage repair inhibitor. <18-15> The method of <18-14>, wherein the DNA damage repair inhibitor is selected from NHEJ inhibitors. <18-16> The method of <18-15>, wherein (e) the NHEJ inhibitor is DNA-dependent protein kinase (DNA-PK) inhibitor or DNA polymerase theta (Polθ) inhibitor. <18-17> The method of <18-15>, wherein (e) the NHEJ inhibitor is selected from Nu-7441,AZD7648, LTURM 34, Ku-0060648, Compound 401, LY294002, KU55933, Wortmannin, Nu7206, MSC2490484, VX-984, CC-115, STL127705, or SCR-7

[0017] The lipid composition and method of the invention allow for efficient delivery of payloads to cells.

[0018] Figure 1 shows that LDLR expression level was upregulated by CD3 / CD28 agonistic antibody activation or IL-2 / IL-7 / IL-15 cytokine stimulation on human primary T Cells.Figure 2 shows the mCherry expression in human primary T cells.Figure 3 shows the luciferase expression after treating with the LNP library and commercial transfection reagents (TransITRand LipofectamineTMMessengerMaxTM).Figure 4 shows the luciferase expression after treating with the LNP library.Figure 5 shows luciferase expression in human primary T cells after treatment with LNPs for 24 h at a dose of 5 μg / 106cells.Figure 6 shows the luciferase expression in human primary T cells after treatment with LNPs for 24 h at a dose of 5 μg / 106cells.Figure 7 shows the luciferase expression in human primary T cells after treatment with LNPs for 24 h at a dose of 5 μg / 106cells.Figure 8 shows the quantification of gene editing efficiency and cell viability after transfecting with different LNP formulations in human primary Tcells.Figure 9 shows the gene editing efficiency in different T cell subsets.Figure 10 shows the comparison of gene editing efficiency and cell viability after transfecting with LNPs encapsulating 5moU and unmodified Cas9 mRNA in human primary Tcells.Figure 11 shows B2M knockout efficiency in DOE-based lipid ratio optimization (Library A).Figure 12 shows B2M knockout efficiency in DOE-based lipid ratio optimization (Library B).Figure 13 shows polymer-conjugated lipid screening in FL-B LNPs using firefly luciferase mRNA.Figure 14 shows dose dependent B2M knockout using optimized and unoptimized LNP formulations.Figure 15 shows B2M knockout efficiency using different LNP formulations (12 μg / 106cells).Figure 16 shows B2M knockout efficiency using different LDLR-binding proteins (2.2 μg / 106cells).Figure 17 shows effect of Cas9 mRNA: sgRNA mass ratio.Figure 18 shows the gene editing efficiency after transfection with different LNP formulations in primary human hematopoietic stem cells (HSCs).Figure 19 shows the results of comparison of T cell activation method.Figure 20 shows the comparison of gene editing efficiency and cell viability after transfecting with LNPs encapsulating 5moU and unmodified Cas9 mRNA in human primary T cells.Figure 21 shows the results of ionizable lipid generalization.Figure 22 shows the results of transgene expression using nanoplasmid DNA.Figure 23 shows schematic of gene insertion using transposase mRNA and transposon DNA.Figure 24 shows the results of in vitro gene insertion using Sleeping Beautytransposase.Figure 25 shows the results of LNP formulation optimization.Figure 26 shows the results of using different polymer-conjugated lipids.Figure 27 shows the results of using different polymer-conjugated lipids.Figure 28 shows the results of optimization of Sleeping Beautytransposase mRNA and EGFP npDNA treatment.Figure 29 shows the results of enhancement of transgene integration using immune modulating agents.Figure 30 shows HDR insertion template DNA design.Figure 31 shows CTS design.Figure 32 shows schematic of gene insertion using Cas9 mRNA and ssDNA with CTS.Figure 33 shows the results of in vitro gene insertion using CRISPR / Cas9.Figure 34 shows B2M knockout efficiency using various polymer-conjugated lipids.

[0019] Hereinafter, the present invention will be described in detail. In this specification, "~" denotes a range including a numerical value described before and after it as a minimum value and a maximum value, respectively.

[0020] The present invention relates to a lipid composition which comprises a payload, an ionizable lipid, a phospholipid, a sterol and a polymer-conjugated lipid, wherein the polymer-conjugated lipid is selected from; (a) aceramide-polyethylene glycol, a phospholipid-polyethylene glycol, or a glyceride- polyethylene glycol with average polyethylene glycol molecular weight of 550-1100; (b) acholesterol-polyethylene glycol with average polyethylene glycol molecular weight of 350-5000 or 550-2000; (c) a polyethylene glycol fatty acid ester with average polyethylene glycol molecular weight of 350-2000, or 550-2000, or 1000-2000; (d) a polyoxyethylene alkyl ether with the number of ethylene oxide units of 10-25; (e) a polyethylene glycol hydrogenated castor oil or a polyethylene glycol castor oil with an average ethoxylation degree of 30-50; or (f) a polyethylene glycol(20) sorbitan fatty acid monoester.

[0021] The present invention further relates to a method to deliver a payload to cells, which comprises adding at least one LDLR binding polypeptide to a culture media and contacting the cell with the aforementioned lipid composition of the present invention.

[0022] The present invention comprises using lipid composition which comprises lipids and payload and can more safely deliver genome editing tools and can be used for multiplex genome editing applications, providing substantial advantages over traditional methods such as viral-based methods and physical methods (e.g. electroporation).

[0023] In some embodiments of the present invention, the present invention produces cells with a fewer DNA damage, less random insertion, fewer chromosomal translocations, and greater survival and expansion, thereby shortening the time required for manufacturing and increasing yield. In some embodiments of the present invention, the present invention provides for highly efficient multiplex gene editing in immune cells and stem cells, resulting in favorable early-stern cell memory phenotype and continued proliferation.

[0024] <Sterols> The lipid composition contains a sterol. In the present invention, since a sterol is contained, the membrane fluidity can be reduced and the effect to stabilize the lipid particles can be obtained. The sterols are not particularly limited, and examples thereof include cholesterol, phytosterol (for example, fucosterol, campesterol, beta-sitosterol, stigmasterol, spinasterol, brassicasterol, and the like), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2’-hydroxyethyl ether, cholesteryl-4’-hydroxybutyl ether, and the like.

[0025] Cholesterol

[0026] The content of the sterols with respect to the total lipids is preferably 5 mol% to 80 mol%, more preferably 10 mol% to 80 mol%, still more preferably 10 mol% to 60 mol%, and further preferably 30 mol% to 50 mol%.

[0027] <Ionizable Lipids> In the present invention, an ionizable lipid is used. The ionizable lipid may be a lipid having at least one biodegradable group. The ionizable lipid may be a lipid having at least one ionizable amino group and at least one biodegradable group. Examples of the above-mentioned biodegradable group include groups represented by-O (CO) O-, -O (CO)-, or -(CO) O-.

[0028] For example, the ionizable lipid may be a compound represented by the formula (2).

[0029] In the formula, X represents -NR1- or -O-, R1represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R21-L1-R22-, where R21represents a hydrocarbon group having 1 to 24 carbon atoms, L1represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R22represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R2and R3each independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R31-L2-R32-, where R31represents a hydrocarbon group having 1 to 24 carbon atoms, L2represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R32represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R4, R5, R6, R7, R8, R9, R10, R11, and R12each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted, groups in any one or more pairs among R4and R5, R10and R5, R5and R12, R4and R6, R5and R6, R6and R7, R6and R10, R12and R7, and R7and R8may be linked to each other to form a 4- to 7-membered ring which may contain an O atom, a substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, the substituent on the substituted or unsubstituted aryl group and on the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and a, b, c, and d each independently represent an integer of 0 to 3, a + b is 1 or more, and c + d is 1 or more.

[0030] As the hydrocarbon group having 6 to 24 carbon atoms that is represented by R1and the hydrocarbon group having 3 to 24 carbon atoms that is represented by R2and R3, an alkyl group, an alkenyl group, or an alkynyl group is preferable, and an alkyl group or an alkenyl group is more preferable. The alkyl group having 6 to 24 carbon atoms and the alkyl group having 3 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. The alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 6 to 20 carbon atoms, and the alkyl group having 3 to 24 carbon atoms is more preferably an alkyl group having 6 to 20 carbon atoms. Specifically, examples thereof include a hexyl 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 nonadecyl group, an icosyl group, and the like. The alkenyl group having 6 to 24 carbon atoms and the alkenyl group having 3 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. The alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 6 to 20 carbon atoms, and the alkenyl group having 3 to 24 carbon atoms is more preferably an alkenyl group having 6 to 20 carbon atoms. Specifically, examples thereof include a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-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), a nonadecenyl group, an icosenyl group (preferably a (Z)-icos-11-enyl group), an icosadienyl group (preferably a (11Z,14Z)-icosa-11,14-dienyl group), and the like. The alkynyl group having 6 to 24 carbon atoms is preferably an alkynyl group having 6 to 20 carbon atoms, and the alkynyl group having 3 to 24 carbon atoms is more preferably an alkynyl group having 6 to 20 carbon atoms. Specifically, examples thereof include 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, an octadecynyl group, and the like. All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0031] The hydrocarbon group having 1 to 24 carbon atoms that is represented by R21and R31is preferably an alkyl group having 10 to 24 carbon atoms, an alkenyl group having 10 to 24 carbon atoms, or an alkynyl group having 10 to 24 carbon atoms. The alkyl group having 10 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. The alkyl group having 10 to 24 carbon atoms is preferably an alkyl group having 12 to 24 carbon atoms. Specifically, examples thereof include 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, a 2-butyloctyl group, a 1-pentylhexyl group, a 2-pentylheptyl group, a 3-pentyloctyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 2-hexyloctyl group, a 2-hexyldecyl group, a 3-hexylnonyl group, a 1-heptyloctyl group, a 2-heptylnonyl group, a 2-heptylundecyl group, a 3-heptyldecyl group, a 1-octylnonyl group, a 2-octyldecyl group, a 2-octyldodecyl group, a 3-octylundecyl group, a 2-nonylundecyl group, a 3-nonyldodecyl group, a 2-decyldodecyl group, a 2-decyltetradecyl group, a 3-decyltridecyl group, a 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like. The alkenyl group having 10 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. Specifically, examples thereof include a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, tridecenyl group (preferably a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably a tetradec-9-enyl group), a pentadecenyl group (preferably a (Z)-pentadec-8-enyl group), a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-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. The alkynyl group having 10 to 24 carbon atoms may be linear or branched or may be chainlike or cyclic. Specifically, examples thereof include a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, an octadecynyl group, and the like. All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0032] The divalent hydrocarbon linking group having 1 to 18 carbon atoms that is represented by R22and R32is preferably an alkylene group having 1 to 18 carbon atoms or an alkenylene group having 2 to 18 carbon atoms. The alkylene group having 1 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkylene group is preferably 1 to 12, more preferably 1 to 10, and still more preferably 2 to 10. Specifically, examples thereof include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a decamethylene group, an undecamethylene group, a dodecamethylene group, and the like. The alkenylene group having 2 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkenylene group is preferably 1 to 12, and more preferably 2 to 10.

[0033] -O(CO)O-, -O(CO)-, and -(CO)O- are in a preferred range of L1, and -O(CO)- and -(CO)O- are in a more preferred range of L1. -O(CO)O-, -O(CO)-, and -(CO)O- are in a preferred range of L2, and -O(CO)- and -(CO)O- are in a more preferred range of L2.

[0034] The alkyl group having 1 to 18 carbon atoms which may be substituted and which represented by R4, R6, R9, R10, R11, and R12may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 12. Specifically, examples thereof 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, and the like. In a case where the alkyl group has a substituent, as the substituent, a hydroxyl group, a carboxyl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44is preferable, and a group represented by -O(CO)-R42or -(CO)O-R43is more preferable.

[0035] The alkyl group having 1 to 18 carbon atoms which may be substituted and which represented by R5, R7, and R8may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 12, and more preferably 1 to 8. Specifically, examples thereof 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, and the like. In a case where the alkyl group has a substituent, as the substituent, a hydroxyl group, a carboxyl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44is preferable, and a group represented by -O(CO)-R42, or -(CO)O-R43is more preferable.

[0036] Examples of the 4- to 7-membered ring which may contain an O atom include an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring, and an azepane ring. The 4- to 7-membered ring is preferably a 6-membered ring and is preferably a piperidine ring or a morpholine ring.

[0037] In a case where the alkyl group having 1 to 18 carbon atoms which is represented by R4, R5, R6, R7, R8, R9, R10, R11, and R12and which may be substituted has a substituted or unsubstituted aryl group as a substituent, the number of carbon atoms in the aryl group is preferably 6 to 22, more preferably 6 to 18, and still more preferably 6 to 10. Specifically, examples of the aryl group include a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, and the like. As the substituent on the aryl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44is preferable, and a hydroxyl group or a carboxyl group is more preferable. Specifically, examples of the substituted aryl group include a hydroxyphenyl group, a carboxyphenyl group, and the like.

[0038] In a case where the alkyl group having 1 to 18 carbon atoms which is represented by R4, R5, R6, R7, R8, R9, R10, R11, and R12and which may be substituted has a substituted or unsubstituted heteroaryl group as a substituent, the number of carbon atoms in the heteroaryl group is preferably 1 to 12, and more preferably 1 to 6. Specifically, examples of the heteroaryl group include a pyridyl group, a pyrazolyl group, an imidazolyl group, a benzimidazolyl group, a thiazolyl group, an oxazolyl group, and the like. As the substituent on the heteroaryl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44is preferable, and a hydroxyl group or a carboxyl group is more preferable. Specifically, examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxypyridyl group, a pyridonyl group, and the like.

[0039] As hydrocarbon group having 1 to 18 carbon atoms that is represented by R41, R42, R43, R44, R45, and R46, an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 2 to 18 carbon atoms, or an alkynyl group having 2 to 18 carbon atoms is preferable, and an alkyl group having 1 to 18 carbon atoms or an alkenyl group having 2 to 18 carbon atoms is more preferable. The alkyl group having 1 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkyl group is preferably 3 to 18, and more preferably 5 to 18. Specifically, examples thereof include 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 heptadecyl group, an octadecyl group, and the like. The alkenyl group having 2 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkenyl group is preferably 3 to 18, and more preferably 5 to 18. Specifically, examples thereof include an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably a tetradec-9-enyl group), a pentadecenyl group (preferably a (Z)-pentadec-8-enyl group), a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-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. The alkynyl group having 2 to 18 carbon atoms may be linear or branched or may be chainlike or cyclic. The number of carbon atoms in the alkynyl group is preferably 3 to 18, and more preferably 5 to 18. Specifically, 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, an octadecynyl group, and the like.

[0040] In a case where X represents -NR1-, R1preferably represents a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R21-L1-R22-. In this case, it is preferable that one of R2and R3represent a hydrogen atom and the other represent a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R31-L2-R32-.

[0041] In a case where X represents -O-, it is preferable that R2and R3each independently represent a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R31-L2-R32-.

[0042] It is preferable that R4, R6, R9, R10, R11, and R12each represent a hydrogen atom.

[0043] R5is preferably a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43, an alkyl group having 1 to 18 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 18 carbon atoms which may be substituted with a hydroxyl group. In a case where R5is an alkyl group, R5may be linked to R4, R6, R10, and R12to form a ring which may contain an O atom. Particularly, R5is preferably an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43, an alkyl group having 1 to 12 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group, and more preferably an alkyl group having 1 to 18 carbon atoms or an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43.

[0044] R7and R8preferably each independently represent a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43, an alkyl group having 1 to 8 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group. Alternatively, it is preferable that R7and R8be linked to each other to form a 4- to 7-membered ring which may contain an O atom.

[0045] R5is not linked to R7or R8and does not form a ring with R7or R8.

[0046] a + b is preferably 1 or 2, and more preferably 1. c + d is preferably 1 or 2, and more preferably 1.

[0047] In preferable embodiment, the lipid resented by Formula (2) is preferably a compound represented by Formula (21). In the formula, R2and R3each independently represent a hydrocarbon group containing one or more unsaturated bond and having 3 to 24 carbon atoms, or R2and R3each independently represent a group represented by R31-L2-R32-, or one of R2and R3represents a group represented by R31-L2-R32- and the other represents a hydrocarbon group having 3 to 24 carbon atoms, R31represents a hydrocarbon group having 1 to 24 carbon atoms, L2represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula, , and R32represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms, R5represents an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R42or -(CO)O-R43where R42and R43each independently represent a hydrocarbon group having 1 to 18 carbon atoms, R7and R8each independently represent an alkyl group having 1 to 4 carbon atoms e represents 2 or 3.

[0048] In formula (21), preferably one of R2and R3is a group represented by R31-L2-R32-, and the other is a hydrocarbon group having 3 to 24 carbon atoms. In formula (21), L2 preferably represents -O (CO)- or - (CO) O-.

[0049] The compound represented by Formula (2) may form a salt. Examples of the salt in 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. Examples of the salt in an acidic group include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; 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, and the like. Among the above salts, for example, pharmacologically acceptable salts are preferable.

[0050] The lipid represented by the formula (2) and a method for producing the same are described in WO2019 / 235635A and WO2021 / 095876A.

[0051] As another example of ionizable lipids, a lipid represented by Formula (3) or a salt thereof may be used as the ionizable lipid. In the formula, R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with one or more substituents selected from -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, and -O-R156, R104represents a hydrocarbon group having 1 to 8 carbon atoms, R105and R106each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R108-L101-R109, excluding a case that both R105and R106are hydrocarbon groups having 1 to 8 carbon atoms, R107represents -R110-L102-R111-L103-R112, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R153, R154, R155, and R156may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, R158represents a hydrocarbon group having 1 to 12 carbon atoms, and R157represents -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, or -O-R166. R161and R162each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R163, R164, R165, and R166each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R163, R164, R165, and R166may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R168, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, -O-R166, or -(hydrocarbon group having 1 to 12 carbon atoms), R168represents a hydrocarbon group having 1 to 12 carbon atoms, and L101, L102, and L103each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-. R108represents a hydrocarbon group having 1 to 12 carbon atoms, R109represents a hydrocarbon group having 1 to 24 carbon atoms, R110represents a hydrocarbon group having 1 to 8 carbon atoms, R111represents a hydrocarbon group having 1 to 24 carbon atoms, R112represents a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R109and R112may be substituted with an aryl group, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, where definitions of R153, R154, R155, and R158are as described above, and the hydrocarbon group represented by R111may be substituted with -OC(O)O-R153, -C(O)O-R154, or -OC(O)-R155, where the definitions of R153, R154, and R155are as described above.

[0052] A hydrocarbon group having 1 to 24 carbon atoms, a hydrocarbon group having 1 to 18 carbon atoms, a hydrocarbon group having 1 to 12 carbon atoms, a hydrocarbon group having 2 to 8 carbon atoms, and a hydrocarbon group having 1 to 8 carbon atoms are each preferably an alkyl group, an alkenyl group, or an alkynyl group.

[0053] The alkyl group may be linear or branched, or may be chainlike or cyclic. Specifically, 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, a 2-butyloctyl group, a 1-pentylhexyl group, a 2-pentylheptyl group, a 3-pentyloctyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 2-hexyloctyl group, a 2-hexyldecyl group, a 3-hexylnonyl group, a 1-heptyloctyl group, a 2-heptylnonyl group, a 2-heptylundecyl group, a 3-heptyldecyl group, a 1-octylnonyl group, a 2-octyldecyl group, a 2-octyldodecyl group, a 3-octylundecyl group, a 2-nonylundecyl group, a 3-nonyldodecyl group, a 2-decyldodecyl group, a 2-decyltetradecyl group, a 3-decyltridecyl group, a 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like.

[0054] The alkenyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of the alkenyl group include an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably a tetradec-9-enyl group), a pentadecenyl group (preferably a (Z)-pentadec-8-enyl group), a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-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.

[0055] The alkynyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of alkynyl group 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, an octadecynyl group, and the like.

[0056] All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0057] The hydrocarbon group having 1 to 12 carbon atoms in -(hydrocarbon group having 1 to 12 carbon atoms)-R67is preferably an alkylene group having 1 to 12 carbon atoms or an alkenylene group having 2 to 12 carbon atoms. The alkylene group having 1 to 12 carbon atoms and the alkenylene group having 2 to 12 carbon atoms may be linear or branched, or may be chainlike or cyclic. Specifically, examples thereof include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a decamethylene group, an undecamethylene group, and the like.

[0058] The aryl group preferably has 6 to 20 carbon atoms, more preferably has 6 to 18 carbon atoms, and even more preferably 6 to 10 carbon atoms. Specifically, examples of the aryl group include a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, and the like.

[0059] R101and R102each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms. R103preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms. The hydrocarbon groups represented by R101, R102, and R103may be preferably substituted with -OH.

[0060] L101and L103each independently preferably represent -C(O)O- or -OC(O)-. L102preferably represents -OC(O)O-, -C(O)O-, or -OC(O)-.

[0061] R108preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms. R109preferably represents a hydrocarbon group having 1 to 20 carbon atoms and more preferably represents a hydrocarbon group having 1 to 16 carbon atoms. R111preferably represents a hydrocarbon group having 1 to 16 carbon atoms and more preferably represents a hydrocarbon group having 1 to 9 carbon atoms. R112preferably represents a hydrocarbon group having 1 to 20 carbon atoms and more preferably represents a hydrocarbon group having 1 to 16 carbon atoms. The hydrocarbon groups represented by R109and R112may be preferably substituted with an aryl group or -S-R158. Here, R158preferably represents a hydrocarbon group having 1 to 8 carbon atoms. The hydrocarbon group represented by R111may be preferably substituted with -C(O)O-R155or -OC(O)-R156, where R155and R156each independently represent a hydrocarbon group having 1 to 16 carbon atoms. The hydrocarbon groups represented by R155and R156may be preferably substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, and the definition of R158is as described above.

[0062] The compound represented by Formula (3) is preferably a compound represented by Formula (3-1) as a first example. In the formula, R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156, R104represents a hydrocarbon group having 1 to 8 carbon atoms, R105and R106each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R108-L101-R109, excluding a case that both R105and R106are hydrocarbon groups having 1 to 8 carbon atoms, L101represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, R108represents a hydrocarbon group having 1 to 12 carbon atoms, R109represents a hydrocarbon group having 1 to 24 carbon atoms, where the hydrocarbon group represented by R109may be substituted with an aryl group, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon groups represented by R153, R154, R155, and R156may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, R158represents a hydrocarbon group having 1 to 12 carbon atoms, and R157represents -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156. R113represents a hydrocarbon group having 1 to 8 carbon atoms, R114represents -R115-L105-R116, where R115represents a hydrocarbon group having 1 to 24 carbon atoms, L105represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, and R116represents a hydrocarbon group having 1 to 24 carbon atoms, the hydrocarbon group having 1 to 24 carbon atoms represented by R115may be substituted with -OC(O)O-R153, -C(O)O-R154, or -OC(O)-R155, where definitions of R153, R154, and R155are as described above, and the hydrocarbon group having 1 to 24 carbon atoms represented by R116may be substituted with an aryl group having 6 to 20 carbon atoms, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155or -S-R158, where the definitions of R153, R154, R155, and R158are as described above. 　　　L104represents -OC(O)O-, -C(O)O-, -OC(O)-, or -O-.

[0063] In Formula (3-1), R101and R102each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms. R103preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms. The hydrocarbon groups represented by R101, R102, and R103may be preferably substituted with -OH.

[0064] L101preferably represents -C(O)O- or -OC(O)-. R108preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms. R109preferably represents a hydrocarbon group having 1 to 18 carbon atoms, and the hydrocarbon group represented by R109may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R58. R114preferably represents -R115-L105-R116, where R115represents a hydrocarbon group having 1 to 18 carbon atoms, L115represents -OC(O)O-, and R116represents a hydrocarbon group having 1 to 18 carbon atoms. The hydrocarbon group having 1 to 18 carbon atoms represented by R115may be preferably substituted with -C(O)O-R154or -OC(O)-R155. R154and R155each independently represent a hydrocarbon group having 1 to 16 carbon atoms, and the hydrocarbon groups represented by R154and R155may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158, where the definition of R158is as described above. The hydrocarbon group having 1 to 18 carbon atoms represented by R116may be preferably substituted with an aryl group or -S-R158, where the definition of R158is as described above.

[0065] The compound represented by Formula (3) is preferably a compound represented by Formula (3-2) as a second example. In the formula, R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156, R104and R108each independently represent a hydrocarbon having 1 to 8 carbon atoms, R121and R122each independently represent a hydrocarbon group having 1 to 18 carbon atoms, R123and R124each independently represent a hydrocarbon group having 1 to 12 carbon atoms, R125and R126each independently represent a hydrocarbon group having 1 to 24 carbon atoms, L121and L122each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, the hydrocarbon groups represented by R125and R126may be substituted with an aryl group having 6 to 20 carbon atoms, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 18 carbon atoms, the above-described aryl group having 6 to 20 carbon atoms may be substituted with OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, and R157represents -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156. R158represents a hydrocarbon group having 1 to 12 carbon atoms.

[0066] In Formula (3-2), R101and R102each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms. The hydrocarbon groups represented by R101and R102may be preferably substituted with -OH, but has more preferably a hydrocarbon having no substituent.

[0067] R103preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0068] R121and R122each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 6 carbon atoms. R123and R124each independently preferably represent a hydrocarbon group having 1 to 10 carbon atoms and more preferably represent a hydrocarbon group having 1 to 8 carbon atoms. R125and R126each independently preferably represent a hydrocarbon group having 1 to 20 carbon atoms, more preferably represent a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 12 carbon atoms. L121and L122each independently preferably represent -C(O)O- or -OC(O)-.

[0069] The compound represented by Formula (3) is preferably a compound represented by Formula (3-3) as a third example. In the formula, R101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156, R104and R108each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R131, R132, R133, and R134each independently represent a hydrocarbon group having 1 to 12 carbon atoms, R135, R136, R137, and R138each independently represent a hydrocarbon group having 1 to 24 carbon atoms, L131, L132, L133, and L134each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-, the hydrocarbon groups represented by R135, R136, R137, and R138may be substituted with an aryl group having 6 to 20 carbon atoms, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or S-R158, R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms, R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 18 carbon atoms, the above-described aryl group having 6 to 20 carbon atoms may be substituted with OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157, and R157represents -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -O-R156. R158represents a hydrocarbon group having 1 to 12 carbon atoms.

[0070] In Formula (3-3), R101and R102each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms, more preferably represent a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms. The hydrocarbon groups represented by R101and R102may be preferably substituted with -OH, but has more preferably a hydrocarbon having no substituent.

[0071] R103preferably represents a hydrocarbon group having 2 to 6 carbon atoms and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.

[0072] R131, R132, R133, and R134each independently preferably represent a hydrocarbon group having 1 to 10 carbon atoms, more preferably represent a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 3 carbon atoms.

[0073] R135, R136, R137, and R138each independently preferably represent a hydrocarbon group having 1 to 20 carbon atoms, more preferably represent a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably represent a hydrocarbon group having 1 to 12 carbon atoms. The hydrocarbon groups represented by R135, R136, R137, and R138may be preferably substituted with an aryl group having 6 to 20 carbon atoms or S-R158. More preferably, these may be substituted with -S-R158. R135, R136, R137, and R138each independently particularly preferably represent a hydrocarbon group having 1 to 12 carbon atoms substituted with -S-R158, or a hydrocarbon group having 1 to 12 carbon atoms.

[0074] L131, L132, L133, and L134each independently preferably represent -C(O)O-, or -OC(O)-.

[0075] R158preferably represents a hydrocarbon group having 1 to 10 carbon atoms and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.

[0076] The compound according to the embodiment of the present invention may form a salt. Examples of the salt in 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. Examples of the salt in an acidic group include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; 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; and the like. Among the above-described salts, for example, pharmacologically acceptable salts are preferable.

[0077] The compound represented by the formula (3) is described in WO2022 / 230964A, and can be made by the production method shown in WO2022 / 230964A .

[0078] As another example of ionizable lipids, a lipid represented by Formula (4) or a salt thereof may be used. In the formula, R201, R202, R203, and R204each independently represent a hydrogen, a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, the substituted or unsubstituted hydrocarbon groups represented by R201, R202, R203, and R204each independently represent -C(O)O-R2011, -OC(O)-R2012, -O-R2013, -CO-R2014-, -OC(O)O-R2015, or -S-S-R2016, R2011, R2012, R2013, R2014-, R2015, and R2016each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R2017, and R2017represents a hydrocarbon group having 1 to 12 carbon atoms, R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 18 carbon atoms, the substituted groups on the substituted hydrocarbon groups having 1 to 18 carbon atoms represented by R205and R206each represent -OH, -COOH, -NR2021R2022, -OC(O)O-R2023, -C(O)O-R2024, -OC(O)-R2025, -O-R2026, -C(O)NR2027R2028, -NR2029C(O)R2030, -N(R2031)S(O)2R2032, -N(R2033)C(O)N(R2034)R2035, -N(R2036)C(S)N(R2037)R2038, -OC(O)N(R2039)R2040, or -N(R2041)C(O)OR2042, R2021and R2022each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030, R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042each independently represent a hyrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, where the substituted groups on the substituted hydrocarbon group having 1 to 24 carbon atoms represented by R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030,R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042represent an aryl group having 6 to 20 carbon atoms, a heterocyclic group, -OH, -COOH, or NR2051R2052, and R2051and R2052each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms, R207, R208, and R209each independently represent a hydrocarbon group having 2 to 8 carbon atoms, R205and R206, or R205and R207may form a 4 to 7-membered ring together.

[0079] 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 each preferably an alkyl group, an alkenyl group, or an alkynyl group.

[0080] The alkyl group may be linear or branched, or may be chainlike or cyclic. Specifically, 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, a 2-butyloctyl group, a 1-pentylhexyl group, a 2-pentylheptyl group, a 3-pentyloctyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 2-hexyloctyl group, a 2-hexyldecyl group, a 3-hexylnonyl group, a 1-heptyloctyl group, a 2-heptylnonyl group, a 2-heptylundecyl group, a 3-heptyldecyl group, a 1-octylnonyl group, a 2-octyldecyl group, a 2-octyldodecyl group, a 3-octylundecyl group, a 2-nonylundecyl group, a 3-nonyldodecyl group, a 2-decyldodecyl group, a 2-decyltetradecyl group, a 3-decyltridecyl group, a 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like.

[0081] The alkenyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of the alkenyl group include an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably a tetradec-9-enyl group), a pentadecenyl group (preferably a (Z)-pentadec-8-enyl group), a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadec-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.

[0082] The alkynyl group may be linear or branched, or may be chainlike or cyclic. Specifically, examples of alkynyl group 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, an octadecynyl group, and the like.

[0083] All of the above alkenyl groups preferably have one double bond or two double bonds. All of the above alkynyl groups preferably have one triple bond or two triple bonds.

[0084] The hydrocarbon group having 2 to 8 carbon atoms represented by R207, R208, and R209is preferably an alkylene group, an alkenylene group, or an alkynylene group. The alkylene group having 2 to 8 carbon atoms, the alkenylene group having 2 to 8 carbon atoms or the alkynylene group having 2 to 8 carbon atoms may be linear or branched, or may be chainlike or cyclic. Specifically, examples thereof include an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, and the like.

[0085] The aryl group having 6 to 20 carbon atoms preferably has 6 to 18 carbon atoms, and more preferably 6 to 10 carbon atoms. Specifically, examples of the aryl group include a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, and the like.

[0086] A heterocyclic group means a heteroaryl group or a heteroaliphatic ring group.

[0087] A heteroaryl group means an aromatic heterocyclic group, may be an aromatic heterocyclic group fused with an aromatic hetero ring, an aromatic hydrocarbon ring, a heteroaliphatic 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 and oxygen-containing heteroaryl group, a monocyclic nitrogen and sulfur-containing heteroaryl group, a bicyclic nitrogen-containing heteroaryl group, a bicyclic oxygen heteroaryl group, a bicyclic sulfur heteroaryl group, a bicyclic nitrogen and oxygen-containing heteroaryl group or a bicyclic nitrogen and sulfur-containing heteroaryl group. A 5 membered ring heteroaryl group is a monocyclic heteroaryl group with five atoms constituting ring thereof.

[0088] An aromatic heterocyclic ring means an aromatic ring having heteroatoms as ring members thereof, may be fused with an aromatic heterocyclic ring, an aromatic hydrocarbon ring, a heteroaliphatic ring, or an aliphatic hydrocarbon ring, and is preferably a monocyclic nitrogen-containing aromatic heterocyclic ring, a monocyclic oxygen-containing aromatic heterocyclic ring, a monocyclic sulfur-containing aromatic heterocyclic ring, a monocyclic nitrogen and oxygen-containing aromatic heterocyclic ring, a monocyclic nitrogen and sulfur-containing aromatic heterocyclic ring, a bicyclic nitrogen-containing aromatic heterocyclic ring, a bicyclic oxygen-containing aromatic heterocyclic ring, a bicyclic sulfur-containing aromatic heterocyclic ring, a bicyclic nitrogen-containing sulfur-containing aromatic heterocyclic ring, a bicyclic nitrogen and oxygen-containing aromatic heterocyclic ring or a bicyclic nitrogen and sulfur-containing aromatic heterocyclic ring.

[0089] A monocyclic nitrogen-containing heteroaryl group means a heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one nitrogen atom, such as a pyrrolinyl, pyrrolyl, tetrahydropyridyl, pyridyl, imidazolinyl, imidazolyl, pyrazolinyl, pyrazolyl, pyrazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazolyl and tetrazolyl groups, is aromatic. The heteroaryl group may be further fused with other aromatic ring or aliphatic ring. A monocyclic oxygen-containing heteroaryl group means a heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one oxygen atom, such as a furanyl or pyranyl group, is aromatic. The heteroaryl group may be further fused with other aromatic ring or aliphatic ring. A monocyclic nitrogen and oxygen-containing heteroaryl group means an oxazolyl, isoxazolyl or oxadiazolyl group, and the like. The heteroaryl group may be further fused with other aromatic ring or aliphatic ring. A monocyclic nitrogen and sulfur-containing heteroaryl group means a thiazolyl, isothiazolyl or thiadiazolyl group, and the like. The heteroaryl group may be further fused with other aromatic ring or aliphatic ring.

[0090] A bicyclic nitrogen-containing heteroaryl group means a bicyclic heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one nitrogen atom, such as an indolyl, isoindolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pyrrolopyridyl, imidazopyridyl, pyrazolopyridyl, pyridopyrazyl, purinyl, pteridinyl, 5,6,7,8-tetrahydrophthalazinyl, 5,6,7,8-tetrahydrocinnolinyl, 1,2,3,4-tetrahydropyrido[2,3-d]pyridazinyl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, 5,6,7,8-tetrahydropyrido[3,4-d]pyridazinyl, 5,6,7,8-tetrahydropyrido[3,2-c]pyridazinyl, 5,6,7,8-tetrahydropyrido[4,3-c]pyridazinyl, 6,7-dihydro-5H-cyclopenta[d]pyridazinyl, 6,7-dihydro-5H-cyclopenta[c]pyridazinyl, 2,3-dihydro-1H-pyrrolo[2,3-d]pyridazinyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyridazinyl, 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, is aromatic.

[0091] A bicyclic oxygen-containing heteroaryl group means a bicyclic heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one oxygen atom, such as a benzofuranyl, isobenzofuranyl and chromenyl groups, is aromatic.

[0092] A bicyclic nitrogen and oxygen-containing heteroaryl group means a bicyclic heteroaryl group (This heteroaryl group may be partially saturated.) in which the ring containing at least one nitorogen atom and at least one oxygen atom, such as a benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, dihydropyranopyridyl, dihydrodioxinopyridyl, dihydropyrido oxazienyl, 3,4-dihydro-2H-pyrano[2,3-d]pyridazinyl, 7,8-dihydro-5H-pyrano[3,4-d]pyridazinyl, 7,8-dihydro-6H-pyrano[3,2-c]pyridazinyl, 7,8-dihydro-5H-pyrano[4,3-c]pyridazinyl, 2,3-dihydrofuro[2,3-d]pyridazinyl, 5,7-dihydrofuro[3,4-d]pyridazinyl, 6,7-dihydrofuro[3,2-c]pyridazinyl, 5,7-dihydrofuro[3,4-c]pyridazinyl and 5,6-dihydrofluoro[2,3-c]pyridazinyl groups, is aromatic.

[0093] A heteroaliphatic ring group means a nitrogen-containing heteroaliphatic ring group, an oxygen-containing heteroaliphatic ring group, a sulfur-containing heteroaliphatic ring group, a nitrogen and oxygen-containing heteroaliphatic ring group, a nitrogen and sulfur-containing heteroaliphatic ring group, a hetero cross-linked ring group or a heterospiro ring group. A heteroaliphatic ring also means an aliphatic ring having heteroatoms as ring members thereof, and includes a nitrogen-containing heteroaliphatic ring, an oxygen-containing heteroaliphatic ring, a sulfur-containing heteroaliphatic ring, a nitrogen and oxygen-containing heteroaliphatic ring, a nitrogen and sulfur-containing heteroaliphatic ring, a hetero cross-linked ring, and heterospiro ring as a preferred example.

[0094] A nitrogen-containing heteroaliphatic ring group means a heteroaliphatic ring group in which the ring containing at least one nitrogen atom, such as an azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, octahydroazocinyl, imidazolidinyl, pyrazolidinyl, piperazinyl and homopiperazinyl groups, is not aromatic. This nitrogen-containing heteroaliphatic ring group may be further fused with other aromatic ring or aliphatic ring. An oxygen-containing heteroaliphatic ring group means a tetrahydrofuranyl, tetrahydropyranyl, oxetanyl or 1,3-dioxanyl group, and the like. The oxygen-containing heteroaliphatic ring group may be further fused with other aromatic ring or aliphatic ring. A nitrogen and oxygen-containing heteroaliphatic ring group means a morpholinyl or 1,4-oxazepanyl group, and the like. The nitrogen and oxygen-containing heteroaliphatic ring group may be further fused with other aromatic ring or aliphatic ring.

[0095] A heteroaliphatic ring C1-8alkyl group means a linear, branched-chain or cyclic C1-8alkyl group to which a heteroaliphatic ring group such as a pyrrolidinyl methyl group, pyrrolidinyl ethyl group, pyrrolidinyl propyl group, pyrrolidinyl octyl group, piperidinyl methyl group and tetrahydrofuranyl methyl group is attached.

[0096] In Formula (4), preferably, R201represents -R201a-L201-R201b, R201arepresents a hydrocarbon group having 1 to 18 carbon atoms, L201represents -C(O)O-, -OC(O)-, -OC(O)O-, or -S-S-, R201brepresents a hydrocarbon group having 1 to 18 carbon atoms, R203represents -R203a-L203-R203b, R203arepresents a hydrocarbon group having 1 to 18 carbon atoms, L203represents -C(O)O-, -OC(O)-, -OC(O)O-, or -S-S-, R203brepresents a hydrocarbon group having 1 to 18 carbon atoms, R202and R204each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 18 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 18 carbon atoms represented by R202and R204each independently represent -C(O)O-R2011, -OC(O)-R2012, -O-R2013, -CO-R2014, -OC(O)O-R2015, or -S-S-R2016, R2011, R2012, R2013, R2014, R2015, and R2016each independently represent a hydrocarbon group having 1 to 18 carbon atoms, R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 12 carbon atoms represented by R205and R206each independently represent -OH, -O-R2026, -C(O)NR2027R2028, or -NR2029C(O)R2030, R2026, R2027, R2028, R2029, and R2030each independently represent a hydrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 12 carbon atoms represented by R2026, R2027, R2028, R2029, and R2030each independently represent an aryl group having 6 to 10 carbon atoms, or a heterocyclic group, and R207, R208, and R209each independently represent -(CH2)n-, where n represents an integer of 2 to 8.

[0097] In Formula (4), more preferably, R201represents -R201a-L201-R201b, R201arepresents a hydrocarbon group having 1 to 18 carbon atoms, L201represents -C(O)O-, or -OC(O)-, R201brepresents a hydrocarbon group having 1 to 18 carbon atoms, R203represents -R203a-L203-R203b, R203arepresents a hydrocarbon group having 1 to 18 carbon atoms, L203represents -C(O)O-, or -OC(O)-, R203brepresents a hydrocarbon group having 1 to 18 carbon atoms, R202and R204each independently represent a hydrocarbon group having 1 to 10 carbon atoms, R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 6 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 6 carbon atoms represented by R205and R206each independently represent -OH, -O-R2026, -C(O)NR2027R2028, or -NR2029C(O)R2030, R2026, R2027, R2028, R2029, and R2030each independently represent a hydrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms, the substituted groups on the substituted hydrocarbon group having 1 to 12 carbon atoms represented by R2026, R2027, R2028, R2029, and R2030each independently represent an aryl group having 6 to 10 carbon atoms, and R207, R208, and R209each independently represent -(CH2)n-, where n represents an integer of 2 to 8.

[0098] In Formula (4), most preferably, R201represents -R201a-L201-R201b, R201arepresents a hydrocarbon group having 1 to 5 carbon atoms, L201represents -C(O)O-, R201brepresents a hydrocarbon group having 7 to 14 carbon atoms, R203represents -R203a-L203-R203b, R203arepresents a hydrocarbon group having 1 to 5 carbon atoms, L203represents -C(O)O-, R203brepresents a hydrocarbon group having 7 to 14 carbon atoms, R202and R204each independently represent a hydrocarbon group having 3 to 8 carbon atoms, R205and R206each independently represent a hydrocarbon group having 2 carbon atoms, R207, R208, and R209each independently represent -(CH2)n-, where n represents an integer of 2 to 4.

[0099] The compound of the present invention may form a salt. Examples of the salt in a basic group include, for example, 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 methane sulfonic acid, benzenesulfonic acid, p-toluene sulfonic acid, mesitylene sulfonic acid and naphthalene sulfonic acid. Examples of the salt in an acidic group 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. Among the above salts, preferred salts include pharmacologically acceptable salts.

[0100] Further, for example, a lipid represented by Formula (1) or a salt thereof may be used as the ionizable lipid.

[0101] wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, the substituent A represents a hydroxyl group, or a group represneted by -G20-CH(R55)(R56), -N(R58)(R59) or -G20-R60, G20represents -O(CO)-, or-(CO)O-, R55and R56each independently represent a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R58and R59each independently represent a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B, the substituent B is-N(R61)(R62), R61and R62each independently represent a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R60represents a hydrocarbon group having 1 to 18 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G30indicates-S-(CO)-NR64, R64represents a group represented by-L30-G20-CH(R55)(R56), a represents 0 or 1, L30represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G10represents -O(CO)-, -(CO)O-, -O(CO)O-, or -N(C(O)R63)-, R63represents a hydrocarbon group having 1 to 18 carbon atoms, L20represents a hydrocarbon group having 1 to 6 carbon atoms, b represents 0 or 1, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, the substituent C represents a group represented by-(CO)O R65or-O(CO)-R65, R65represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by-L40-CH(R66)(R67), L40represents a hydrocarbon group having 1 to 6 carbon atoms, R66and R67represent a hydrocarbon group having 1 to 10 carbon atoms or an alkoxy group.

[0102] The compound represneted by Formula (1) may be a compound represneted by Formula (1A): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A, the substituent A represents a hydroxyl group, or a group represneted by -G20-CH(R55)(R56), G20represents -O(CO)-, or-(CO)O-, R55and R56each independently represent a hydrocarbon group having 1 to 18 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G10represents -O(CO)-, or -(CO)O-, R63represents a hydrocarbon group having 1 to 18 carbon atoms, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0103] The compound represneted by Formula (1) may preferably be a compound represneted by Formula (1B): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G10represents -O(CO)O-, L20represents a hydrocarbon group having 1 to 6 carbon atoms, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, the substituent C represents a group represented by -O(CO)-R65, R65represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by-L40-CH(R66)(R67), L40represents a hydrocarbon group having 1 to 6 carbon atoms, R66and R67represent an alkoxy group having 1 to 10 carbon atoms.

[0104] The compound represneted by Formula (1) may preferably be a compound represneted by Formula (1C): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms L10represents a hydrocarbon group having 1 to 18 carbon atoms, G10represents -N(C(O)R63)-, R63represents a hydrocarbon group having 1 to 18 carbon atoms, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C, the substituent C represents a group represented by-(CO)O R65, R65represents a group represented by-L40-CH(R66)(R67), L40represents a hydrocarbon group having 1 to 6 carbon atoms, R66and R67represent a hydrocarbon group having 1 to 10 carbon atoms.

[0105] The compound represneted by Formula (1) may preferably be a compound represneted by Formula (1D): wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms, L10represents a hydrocarbon group having 1 to 18 carbon atoms, G30indicates-S-(CO)-NR64, R64represents a group represented by-L30-G20-CH(R55)(R56), L30represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms, G20represents -(CO)O-, R55and R56each independently represent a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, G10represents -(CO)O-, R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms.

[0106] The hydrocarbon group having 1 to 21 carbon atoms is preferably an alkyl group having 1 to 21 carbon atoms, an alkenyl group having 2 to 21 carbon atoms, or an alkynyl group having 2 to 21 carbon atoms, more preferably an alkyl group having 1 to 21 carbon atoms, or an alkenyl group having 2 to 21 carbon atoms. The alkyl group having 1 to 21 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21 carbon atoms. Examples include propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, tert-butyl group, cyclobutyl group, pentyl group, cyclopentyl group, hexyl group, cyclohexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group and octadecyl group. The alkenyl group having 2 to 18 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Examples include allyl group, prenyl group, pentanyl group, hexenyl group, heptenyl group, octenyl group, nonenyl group (preferably (Z) -2-nonenyl group or (E) -2-nonenyl group), decenyl group, undecenyl group, dodecenyl group, dodecadienyl group, tridecenyl group (preferably (Z) -trideca-8-enyl group), tetradecenyl group (preferably tetradeca-9-enyl group), pentadecenyl group (preferably (Z)-pentadeca-8-enyl group), hexadecenyl group (preferably (Z)-hexadeca-9-enyl group), hexadecadienyl group, heptadecenyl group (preferably (Z)-heptadeca-8-enyl group), heptadecadienyl group (preferably (8Z, 11Z)-heptadeca-8,11-dienyl group), octadecenyl group (preferably (Z)-octadeca-9-enyl group), octadecadienyl Groups (preferably (9Z, 12Z)-octadeca-9,12-dienyl group). The alkynyl group having 2 to 21 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 21, and more preferably 5 to 21 carbon atoms. Examples include propargyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group, nonynyl group, decynyl group, undecynyl group, dodecynyl group, tetradecynyl group, pentadecynyl group, hexadecynyl group, heptadecynyl group, octadecynyl group and the like. Examples of the hydrocarbon group having 1 to 18 carbon atoms include those having 1 to 18 carbon atoms among the hydrocarbon groups having 1 to 21 carbon atoms.

[0107] As the cyclic hydrocarbon group, a cycloalkyl group having 3 to 10 carbon atoms, a cycloalkenyl group having 3 to 10 carbon atoms, a cycloalkynyl group having 3 to 10 carbon atoms, and an aryl group having 6 to 10 carbon atoms are preferable.

[0108] The hydrocarbon group having 1 to 6 carbon atoms is preferably an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms or an alkynyl group having 2 to 6 carbon atoms, and more preferably an alkyl group having 1 to 6 carbon atoms or an alkenyl group having 2 to 6 carbon atoms. The alkyl group having 1 to 6 carbon atoms may be linear or branched, and may be chain or cyclic. Specific examples thereof include propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, tert-butyl group, cyclobutyl group, pentyl group, cyclopentyl group and hexyl group. The alkenyl group having 2 to 6 carbon atoms may be linear or branched, and may be chain or cyclic. Specific examples thereof include allyl group, prenyl group, pentenyl group, and hexenyl group. The alkynyl group having 2 to 6 carbon atoms may be linear or branched, and may be chain or cyclic. Specific examples thereof include propargyl group, butynyl group, pentynyl group, and hexynyl group.

[0109] The hydrocarbon group having 1 to 10 carbon atoms is preferably an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, and preferably an alkyl group having 1 to 10 carbon atoms or an alkenyl group having 2 to 10 carbon atoms. The alkyl group having 1 to 10 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, and more preferably 5 to 10 carbon atoms. Specific example include propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, tert-butyl group, cyclobutyl group, pentyl group, cyclopentyl group, hexyl group,cyclohexyl group, heptyl group, octyl group, nonyl group, and decyl group. The alkenyl group having 2 to 10 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, more preferably 5 to 10. Specific examples include allyl group, prenyl group, pentenyl group, hexenyl group, heptenyl group, octenyl group, a nonenyl group (preferably (Z)-2-nonenyl group or (E)-2-nonenyl group), and decenyl group. The alkynyl group having 2 to 10 carbon atoms may be linear or branched, and may be chain or cyclic. The number of carbon atoms is preferably 3 to 10, and more preferably 5 to 10 carbon atoms. Specific examples thereof include propargyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group, noninyl group and a decynyl group.

[0110] The compound represented by Formula (1) may form a salt. Examples of the salt in 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. Among the above salts, for example, pharmacologically acceptable salts are preferable.

[0111] <<Examples of ionizable lipids>> Examples of ionizable lipids include the following lipids. FL-A FL-B MC3 ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate) L-319 (bis[(Z)-non-2-enyl] 9-[4-(dimethylamino)butanoyloxy]heptadecanedioate) ALC-0315 (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate) SM-102 (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) Lipid 5 (nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate) Lipid 29 (undecan-3-yl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-[3-[[2-(methylamino)-3,4-dioxocyclobuten-1-yl]amino]propyl]amino]octanoate) ATX-100 (pentadecan-8-yl 4-[3-(dimethylamino)propylsulfanylcarbonyl-(4-oxo-4-pentadecan-8-yloxybutyl)amino]butanoate) Lipid A9 (bis(2-butyloctyl) 10-[3-(dimethylamino)propyl-nonanoylamino]nonadecanedioate) Lp01 ([2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate) GCL1 ([(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate)

[0112] In the lipid composition of the present invention, the content of the ionizable lipid or a salt thereof with respect to the total lipids is preferably 10 mol% to 80 mol%, more preferably 20 mol% to 80 mol%, still more preferably 30 mol% to 70 mol%, further more preferably 40 mol% to 60 mol%.

[0113] <Phospholipid> The lipid composition of the present invention may contain a phospholipid. The phospholipid is preferably Zwitterionic phospholipid. Examples of the zwitterionic phospholipid include phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and the like. As the phospholipid, a phospholipid having a choline group such as phosphatidylcholine is preferable. The zwitterionic lipid may be used alone or in combination of a plurality of different phospholipids.

[0114] The phosphatidylcholine is not particularly limited, and examples thereof include soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated egg yolk lecithin (HEPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylcholine (DLPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and the like. Among these, dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC) and dilauroylphosphatidylcholine (DLPC) are preferable. Particularly, distearoylphosphatidylcholine (DSPC) is preferable.

[0115] DSPC: 1,2-Distearoyl-sn-glycero-3-phosphocholine

[0116] 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, diphytanylphosphatidylethanolamine, and the like. The phospholipid is preferably phosphatidylcholine having a fatty acid tail of 12 to 20 carbon atoms, and is more preferably phosphatidylcholine having a saturated fatty acid tail of 14 to 18 carbon atoms, and is further preferably phosphatidylcholine having a saturated fatty acid tail of 18 carbon atoms.

[0117] The sphingomyelin (SM) is not particularly limited, and examples thereof include egg yolk-derived sphingomyelin, milk-derived sphingomyelin, and the like.

[0118] In the lipid composition of the present invention, the amount of the phospholipid is preferably 1 to 30 mol%, more preferably 5 to 25 mol%, still more preferably 7 to 23 mol% with respect to the total amount of the constituent lipid components.

[0119] <Polymer-conjugated lipid> The lipid composition of the present invention comprises a polymer-conjugated lipid which is selected from; (a) aceramide-polyethylene glycol, a phospholipid-polyethylene glycol, or a glyceride- polyethylene glycol with average polyethylene glycol molecular weight of 550-1100; (b) acholesterol-polyethylene glycol with average polyethylene glycol molecular weight of 350-5000 or 550-2000; (c) a polyethylene glycol fatty acid ester with average polyethylene glycol molecular weight of 350-2000, or 550-2000, or 1000-2000; (d) a polyoxyethylene alkyl ether with the number of ethylene oxide units of 10-25; (e) a polyethylene glycol hydrogenated castor oil or a polyethylene glycol castor oil with an average ethoxylation degree of 30-50; or (f) a polyethylene glycol(20) sorbitan fatty acid monoester.

[0120] The polymer-conjugated lipid is preferably selected from; (b) acholesterol-polyethylene glycol with average polyethylene glycol molecular weight of 350-5000 or 550-2000 or 1000-2000; or (d) a polyoxyethylene alkyl ether with the number of ethylene oxide units of 10-25;

[0121] The polymer-conjugated lipid is more preferably selected from; (d) polyoxyethylene (21) lauryl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (25) lauryl ether, polyoxyethylene (10) cethyl ether, polyoxyethylene (15) cethyl ether, polyoxyethylene (20) cethyl ether, polyoxyethylene (23) cethyl ether, polyoxyethylene (25) cethyl ether, polyoxyethylene (12) cetostearyyl ether, polyoxyethylene (20) cetostearyyl (25) ether, polyoxyethylene (10) stearyl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (10) oleyl ether, or polyoxyethylene (20) oleyl ether.

[0122] The polymer-conjugated lipid is present in the lipid composition in a range of preferably 0.25-10 mol %, or more preferably 0.5-10 mol %, or further preferably 1-10 mol %, or particularly preferably 1-5 mol %, or most preferably 2-4 mol % at a mol ratio to total lipids.

[0123] In a preferable embodiment, the lipid composition of the present invention comprises 10-55 mol % ionizable lipid, 5-25 mol % phospholipid, 30 to 70 mol % sterol, and 0.25-10 mol % polymer-conjugated lipid at a mol ratio to total lipids. In a preferable embodiment, the lipid composition of the present invention comprises 15-50 mol % ionizable lipid, 10-20 mol % phospholipid, 40 to 65 mol % sterol, and 0.5-10 mol % polymer-conjugated lipid at a mol ratio to total lipids. In a further preferable embodiment, the lipid composition of the present invention comprises 20-40 mol % ionizable lipid, 10-20 mol % phospholipid, 40 to 60 mol % sterol, and 1-5 mol % polymer-conjugated lipid at a mol ratio to total lipids. In a particularly preferable embodiment, the lipid composition of the present invention comprises 20-40 mol % ionizable lipid, 15-20 mol % phospholipid, 40 to 55 mol % sterol, and 2-4 mol % polymer-conjugated lipid at a mol ratio to total lipids.

[0124] <Payload> The lipid composition contains a payload. The payload may be a therapeutic agent or other drug, preferably a nucleic acid molecule such as a polynucleotide. The nucleic acid molecule such as a polynucleotide may be either DNA or RNA, and may be plasmid (including nanoplasmid), single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (micro RNA), mRNA, antisense oligonucleotide (also known as ASO), ribozyme, aptamer, decoy nucleic acid, gRNA used in genome editing and the like. The mRNA may encode DNA binding proteins. Examples of the DNA binding protein includes TALEN, ZFN, CRISPR-Cas system, transposase or their derivatives. RNA is the preferred nucleic acid molecule. It may also contain modified nucleic acids. Also, the nucleic acid molecule may be a DNA. The DNA may be a single strand DNA. The single strand DNA may be a linear or circular single strand DNA. The linear single strand DNA may be hybridized with a short single strand DNA to form guide RNA-Cas enzyme complex binding domain. Also, the DNA may be a double strand DNA. The double strand DNA may be a circular or linear double strand DNA.

[0125] Examples of modified nucleic acids include 5-methoxyuridine (5moU), N1-methylpseudouridine (N1mΨ), pseudouridine (Ψ), 2-thiouridine (s2U), and 5-methylcytidine (5mC). Such modifications can be incorporated into RNA to achieve improved therapeutic properties and reduce innate immune activation. Cas9-based gene editing is a versatile and widely used method for precise genome manipulation. This system, derived from the bacterial adaptive immune CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, utilizes the Cas9 protein as a DNA endonuclease to introduce site-specific double-strand breaks (DSBs) guided by a customizable single guide RNA (sgRNA). The DSB can then be repaired by cellular DNA repair pathways, including non-homologous end joining (NHEJ) or homology-directed repair (HDR). Through this mechanism, CRISPR-Cas9 enables a variety of genetic modifications, including targeted gene knockouts, insertions, deletions, and precise sequence corrections. The payload may include Cas9 as mRNA, DNA, ribonucleoprotein complexes (RNPs), or derivatives such as base editors (e.g., cytosine or adenine base editors) or prime editors, which allow for site-specific nucleotide changes or sequence insertions without introducing DSBs.

[0126] Additionally, Cas9 can be engineered to enhance HDR efficiency by incorporating additional DNA-binding motifs. These motifs enable the Cas9 protein to bind and stabilize the HDR donor template in proximity to the site of repair, increasing the likelihood of template-directed repair. For instance, Cas9 can be fused with DNA-binding domains or protein domains that interact with single-stranded or double-stranded HDR donor DNA, thereby forming a complex with both the Cas9 protein and the repair template. The system may also include one or more guide RNAs (gRNAs), which serve to direct Cas9 to its target genomic loci. By delivering these components using lipid-based compositions, therapeutic gene-codingtic modifications can be achieved with high specificity and efficiency. The gRNA may be provided in the format of a single guide RNA (sgRNA) or as separate components comprising a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). When provided as an sgRNA, it is a single RNA molecule that combines the functions of crRNA and tracrRNA into one structure. The gRNA includes a target-specific sequence complementary to the target DNA, which enables recognition of the target site, and a scaffold region that associates with the Cas9 protein to form a functional ribonucleoprotein complex. This ribonucleoprotein complex facilitates Cas9-mediated DNA cleavage at the specified genomic location, thereby enabling precise genetic modifications. The target-specific sequence of the gRNA, whether in sgRNA form or as separate crRNA and tracrRNA components, may be engineered to correspond to the desired genomic locus for targeted applications.

[0127] In the lipid composition of the present invention, the weight ratio of the lipid to the payload is preferably 5 to 100, more preferably 5 to 70, still more preferably 5 to 40, and particularly preferably 5 to 35.

[0128] As DNA insertion, a site-specific insertion and a semi-random insertion are known (Patent Literatures 7 and 8). In the site-specific insertion, for example, a first LNP containing Cas9 mRNA and sgRNA and a second LNP containing a linear single strand DNA and short double strand DNA (Cas9 binding site) are used. NHEJ inhibitors are also used as DNA repair inhibitor. In the semi-random insertion, for example, a first LNP containing transposase mRNA and a second LNP containing a circular double strand DNA or nanoplasmid DNA (transposase binding site) are used. Kinase inhibitors (Optional) may also be used as an immune modulating agent.

[0129] Circular double-stranded DNA (circular dsDNA) refers to a DNA molecule in which the two complementary strands form a continuous double helix structure without free ends, resulting in a closed, circular topology. Circular dsDNA can exist in naturally occurring forms, such as plasmids and bacterial genomes, or can be synthetically engineered.

[0130] Circular dsDNA constructs include specialized vectors like nanoplasmid DNA and minicircle DNA, which are engineered to eliminate unnecessary bacterial sequences, minimize the size of the DNA backbone, and optimize gene expression and stability. These features are particularly beneficial for applications such as gene therapy, vaccine development, and recombinant protein production.

[0131] Nanoplasmid DNA is a type of plasmid vector engineered to have several advantageous features compared to conventional plasmid DNA. Firstly, nanoplasmid vectors have a minimized bacterial backbone (<500 bp), which reduces the presence of unnecessary sequences and potential immunogenic elements. This can be particularly beneficial for therapeutic applications. To minimize the backbone sequence, nanoplasmid DNAs use non-antibiotic selection markers encoding a 150 bp antisense RNA instead of traditional antibiotic resistance genes (800-1000 bp). This is known as “RNA-OUT platform” which works by repressing the expression of a counter-selectable marker (SacB) from the host chromosome. SacB encodes for the levansucrase enzyme, which creates a toxic environment in the presence of sucrose, leading to cell death. Transforming the antibiotic-free Nanoplasmid in the host expresses a 150bp RNA-OUT antisense RNA (ROUT) that binds the SacB mRNA, represses levansucrase expression, prevents toxicity, and maintains cell survival.

[0132] Minicircle DNA is another specialized form of plasmid DNA removing unnecessary bacterial sequences by using recombination. In the host bacteria, site-specific recombination is induced, often by using inducible recombinase enzymes. This recombination event excises the bacterial backbone sequence such as origins of replication (ori) and selection marker gene, leaving only promoter sequence, the gene of interest, and essential regulatory elements.

[0133] A nuclear localization signal (NLS) is a short peptide sequence within a protein that facilitates its transport into the nucleus. This signal serves as a "nuclear address tag," enabling the protein to interact with nuclear transport receptors, such as importins, which mediate translocation through the nuclear pore complex (NPC). The NLS is a critical regulatory element for ensuring proper subcellular localization of proteins, particularly those involved in essential nuclear functions such as transcription, replication, DNA repair, and chromatin remodeling.

[0134] Linear double-stranded DNA (linear dsDNA) refers to a molecule of genomic or synthetic DNA that comprises two complementary strands forming a double helix, where the DNA is in a linear, non-circular state. Linear dsDNA can encompass various forms of DNA, including constructs generated through distinct biological or synthetic processes. Examples include doggyboneTMDNA, which is a linear closed-end DNA construct designed to lack free ends, DNA linearized by enzymatic digestion of circular double-stranded DNA, and DNA produced through polymerase chain reaction (PCR), which generates defined linear DNA fragments with precise sequences. In certain contexts, linear dsDNA refers to constructs designed to be shorter than specific length thresholds, such as less than 4500 base pairs, 4000 base pairs, or 3500 base pairs, to suit particular applications. Such linear dsDNA constructs are widely suited for genetic engineering, therapeutic delivery, and molecular biology applications that require high precision in length and sequence. The term encompasses molecules prepared by various methodologies, ensuring flexibility for different experimental or therapeutic needs.

[0135] Transposase refers to an enzyme responsible for mediating the mobilization of genetic elements, known as transposons, within a genome. These enzymes recognize specific DNA sequences, such as terminal inverted repeats (TIRs) or related transposable motifs, flanking the transposable element. By catalyzing the excision and reintegration of these elements at new genomic locations, transposases facilitate genetic rearrangements and have become critical tools in molecular biology and therapeutic applications. The natural ability of transposases to insert transposons into the genome has been harnessed for genetic engineering, non-viral gene delivery, and genome modification technologies.

[0136] In an example of a clinically relevant application, synthetic mRNA encoding the transposase enzyme is co-transfected with a DNA encoding the transposon, which includes the gene of interest flanked by TIRs. This method is advantageous because the transposase mRNA is transiently expressed, reducing the risk of long-term off-target integrations and increasing biosafety. Several transposase systems have been developed and optimized for diverse applications in biotechnology and therapeutic contexts. These systems originate from naturally occurring transposons observed in different species, but they have been engineered to enhance their activity, specificity, and utility in laboratory and clinical settings.

[0137] Examples include the followings: The PiggyBac transposase originates from a transposable element initially identified in the cabbage looper moth (Trichoplusia ni). Engineered versions of PiggyBac, including Super PiggyBac and Hyper PiggyBac, were developed to further enhance transposition efficiency and reduce off-target effects. Super PiggyBac features optimized activity for large DNA cargos and long-term genomic expression. Meanwhile, Hyper PiggyBac incorporates mutations that significantly increase its transposition rate while maintaining high genomic stability. The Sleeping Beauty transposase was reconstructed from an ancient, inactive Tc1-like transposon isolated from a salmonid fish species. . To improve its functionality, an engineered version known as SB100X was developed, representing a hyperactive variant of the Sleeping Beauty transposase. SB100X exhibits significantly enhanced transposition activity and integration efficiency, making it particularly well-suited for applications requiring high levels of stable gene transfer. The TcBuster transposase originates from the Tc1 / mariner family of transposons, which are widely distributed across many species. An engineered version, known as TcBusterM, was introduced to enhance the system's integration efficiency and reduce any potential biases in insertion site selection. TcBusterM represents a hyperactive and highly optimized variant of the original TcBuster transposase, designed for improved performance in mammalian cells and increased specificity in targeted gene delivery.

[0138] Retrotransposase is a specialized enzyme associated with retrotransposons, which move via an RNA intermediate. These enzymes mediate the reverse transcription of the transposon's RNA into DNA, followed by its integration into a target sequence within the genome. Advances in molecular engineering have enabled the effective use of retrotransposase mRNA and retrotransposon RNA for gene integration.

[0139] Examples of retrotransposon sysmet are LINE-1 (Long Interspersed Nuclear Element-1), Ty1, Ty3 Copia, Gypsy, HERVs (Human Endogenous Retroviruses), R2, BEL / Pao, CR1 (Chicken Repeat 1).

[0140] Examples of the immune modulating agent are mentioned below. 1. cGAS-STING-TBK1 signaling pathway inhibitor (a)cyclic GMP-AMP (cGAMP) synthase (cGAS) inhibitor G140 (CAS 2369751-07-3) G150 (CAS No. 2369751-30-2) CU-32(CAS No. 2400954-16-5) CU-76 (CAS No. 2400954-58-5) RU-521 (CAS No. 2262452-06-0) PF-06928215 (CAS No. 2378173-15-8), Preferrbly (CU32)

[0141] (b)Stimulator of Interferon Genes (STING) inhibitor C-176(CAS No. 314054-00-7), H-151 (CAS No. 941987-60-6) C-178 (CAS No. 329198-87-0) C-170 (CAS No. 346691-38-1) C-171 (CAS No. 2244881-69-2) SN-001 (CAS No. 727699-84-5) Astin C Preferably (C-176)

[0142] (c) TRAF-associated NF-κB activator (TANK)-binding kinase (TBK) inhibitor BX-795, MRT67307, GSK8612, BAY-985, Amlexanox, AZD6738, SBI-0206965, CYT387 (Momelotinib) Preferably (BX-795, MRT67307, GSK8612,)

[0143] (d) IκB kinase (IKK) inhibitor BI605906, etc.

[0144] (e)Nuclear factor kappa B (NF-κB) inhibitor Sulfasalazine, Withaferin A, BAY 11-7082, Caffeic Acid Phenethyl Ester, QNZ (Dehydroxymethylepoxyquinomicin, CAS No. 545380-34-5), PPM-18 (CAS No. 65240-86-0), JSH-23

[0145] 2. IFN-JAK-STAT pathway inhibitor (f)JAK inhibitor JAK1 inhibitor, JAK2 inhibitor, JAK3 inhibitor, pan-JAK inhibitor JAK Inhibitor I, Peficitinib, Baricitinib, Oclacitinib, Gusacitinib, SAR20347, Delgocitinib, Ruxolitinib, Tofacitinib Preferrably (JAK inhibitor I, peficitinib)

[0146] References: WO2010133369 Transfection method for nonviral gene delivery systems with improved activity by blocking the innate immune system WO2018019341 Transfection method comprising nonviral gene delivery systems WO2021141020A1: NUCLEIC ACID INTRODUCTION USING TWO TYPES OF TBK1 / IKKe INHIBITORS iScience 23, 101026, April 24, 2020 (https: / / doi.org / 10.1016 / j.isci. 2020.101026)

[0147] DNA repair inhibitors are explained below. The primary mechanism of action for CRISPR-Cas9 involves generating a double-strand break (DSB) at a specific DNA target site. Depending on the cellular repair pathways engaged following the DSB, this can result in error-prone repair via non-homologous end joining (NHEJ) or precise genome editing via homology-directed repair (HDR). While NHEJ remains the dominant repair pathway in most mammalian cells due to its rapid and template-independent nature, HDR is often desired for precise genome edits, such as gene replacements, insertions, or corrections. However, achieving efficient HDR poses a challenge due to the inherently lower activity of this repair pathway compared to NHEJ.

[0148] To enhance HDR, various DNA repair inhibitors can be used. DNA-dependent protein kinase (DNA-PK) inhibitor: NU7441, M3814 (Peposertib),CC-115, KU-0060648, NU7026, VX-984, AZD7648, IC87361, SCR7, LY294002, and Nedisertib, DNA polymerase theta (Polθ) inhibitor Novobiocin) ART558, RP-6685 poly(ADP-ribose) polymerase (PARP) Inhibitors: Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib

[0149] Reference: Simultaneous inhibition of DNA-PK and Polθimproves integration efficiency and precision of genome editing | Nature Communications

[0150] A Nuclear Localization Signal (NLS) is a short peptide sequence within a protein that facilitates its transport into the cell nucleus. The NLS serves as a critical "nuclear address tag," allowing the protein to interact with nuclear transport receptors, such as importins, which guide the protein through the nuclear pore complex (NPC). This translocation process is essential for proteins involved in vital nuclear functions such as transcription, replication, DNA repair, and chromatin remodeling, for which correct subcellular localization is of paramount importance.

[0151] NLSs are commonly characterized by sequences that are rich in positively charged amino acids, particularly lysine (K) and arginine (R), and may vary in structure depending on their functional mechanism. Classical Nuclear Localization Signals are divided into two major types: monopartite and bipartite. A monopartite NLS consists of a single continuous stretch of basic amino acids, as exemplified by the well-characterized sequence PKKKRKV found in the SV40 large T-antigen. In contrast, bipartite NLSs contain two clusters of basic amino acids separated by a spacer region of approximately 10 to 12 amino acids, an example of which is found in the Xenopus nucleoplasmin NLS with the sequence KRPAATKKAGQAKKKK.

[0152] In addition to these classical forms, there are non-classical NLSs, which do not conform to the monopartite or bipartite structure but still mediate nuclear import through alternative mechanisms. One example of a non-classical NLS is the M9 NLS found in hnRNP A1, which displays a unique sequence that facilitates both nuclear import and export.

[0153] Protein / Origin Type NLS Sequence SV40 Large T-Antigen Monopartite PKKKRKV c-Myc Monopartite PAAKRVKLD Xenopus Nucleoplasmin Bipartite KRPAATKKAGQAKKKK hnRNP A1 (M9 NLS) Non-Classical RGFGGRGGFGGSGGNRGSGGRR p53 Minimal Motif PPKKKPLD Retinoblastoma (Rb) Monopartite KRRLFD NF-κB Monopartite KRKR HIV-1 Rev Non-Classical RQARRNRRRRWR

[0154] Cas9 mRNA encodes the Cas9 protein with an N and C terminal nuclear localization signal (NLS). The incorporation of two NLS signals within the mRNA increases the frequency of delivery to the nucleus, thus increasing the rate of DNA cleavage.

[0155] SpCas9 mRNA AUGGCCCCCAAGAAGAAGCGGAAGGUGGGCAUCCACGGCGUGCCCGCCGCCGACAAGAAGUACAGCAUCGGCCUGGACAUCGGCACCAACAGCGUGGGCUGGGCCGUGAUCACCGACGAGUACAAGGUGCCCAGCAAGAAGUUCAAGGUGCUGGGCAACACCGACCGGCACAGCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUUCGACAGCGGCGAGACCGCCGAGGCCACCCGGCUGAAGCGGACCGCCCGGCGGCGGUACACCCGGCGGAAGAACCGGAUCUGCUACCUGCAGGAGAUCUUCAGCAACGAGAUGGCCAAGGUGGACGACAGCUUCUUCCACCGGCUGGAGGAGAGCUUCCUGGUGGAGGAGGACAAGAAGCACGAGCGGCACCCCAUCUUCGGCAACAUCGUGGACGAGGUGGCCUACCACGAGAAGUACCCCACCAUCUACCACCUGCGGAAGAAGCUGGUGGACAGCACCGACAAGGCCGACCUGCGGCUGAUCUACCUGGCCCUGGCCCACAUGAUCAAGUUCCGGGGCCACUUCCUGAUCGAGGGCGACCUGAACCCCGACAACAGCGACGUGGACAAGCUGUUCAUCCAGCUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCAGCGGCGUGGACGCCAAGGCCAUCCUGAGCGCCCGGCUGAGCAAGAGCCGGCGGCUGGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGUUCGGCAACCUGAUCGCCCUGAGCCUGGGCCUGACCCCCAACUUCAAGAGCAACUUCGACCUGGCCGAGGACGCCAAGCUGCAGCUGAGCAAGGACACCUACGACGACGACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCUGUUCCUGGCCGCCAAGAACCUGAGCGACGCCAUCCUGCUGAGCGACAUCCUGCGGGUGAACACCGAGAUCACCAAGGCCCCCCUGAGCGCCAGCAUGAUCAAGCGGUACGACGAGCACCACCAGGACCUGACCCUGCUGAAGGCCCUGGUGCGGCAGCAGCUGCCCGAGAAGUACAAGGAGAUCUUCUUCGACCAGAGCAAGAACGGCUACGCCGGCUACAUCGACGGCGGCGCCAGCCAGGAGGAGUUCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGGCACCGAGGAGCUGCUGGUGAAGCUGAACCGGGAGGACCUGCUGCGGAAGCAGCGGACCUUCGACAACGGCAGCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUCCUGCGGCGGCAGGAGGACUUCUACCCCUUCCUGAAGGACAACCGGGAGAAGAUCGAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUGGCCCGGGGCAACAGCCGGUUCGCCUGGAUGACCCGGAAGAGCGAGGAGACCAUCACCCCCUGGAACUUCGAGGAGGUGGUGGACAAGGGCGCCAGCGCCCAGAGCUUCAUCGAGCGGAUGACCAACUUCGACAAGAACCUGCCCAACGAGAAGGUGCUGCCCAAGCACAGCCUGCUGUACGAGUACUUCACCGUGUACAACGAGCUGACCAAGGUGAAGUACGUGACCGAGGGCAUGCGGAAGCCCGCCUUCCUGAGCGGCGAGCAGAAGAAGGCCAUCGUGGACCUGCUGUUCAAGACCAACCGGAAGGUGACCGUGAAGCAGCUGAAGGAGGACUACUUCAAGAAGAUCGAGUGCUUCGACAGCGUGGAGAUCAGCGGCGUGGAGGACCGGUUCAACGCCAGCCUGGGCACCUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGACCCUGACCCUGUUCGAGGACCGGGAGAUGAUCGAGGAGCGGCUGAAGACCUACGCCCACCUGUUCGACGACAAGGUGAUGAAGCAGCUGAAGCGGCGGCGGUACACCGGCUGGGGCCGGCUGAGCCGGAAGCUGAUCAACGGCAUCCGGGACAAGCAGAGCGGCAAGACCAUCCUGGACUUCCUGAAGAGCGACGGCUUCGCCAACCGGAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACCUUCAAGGAGGACAUCCAGAAGGCCCAGGUGAGCGGCCAGGGCGACAGCCUGCACGAGCACAUCGCCAACCUGGCCGGCAGCCCCGCCAUCAAGAAGGGCAUCCUGCAGACCGUGAAGGUGGUGGACGAGCUGGUGAAGGUGAUGGGCCGGCACAAGCCCGAGAACAUCGUGAUCGAGAUGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGAUGAAGCGGAUCGAGGAGGGCAUCAAGGAGCUGGGCAGCCAGAUCCUGAAGGAGCACCCCGUGGAGAACACCCAGCUGCAGAACGAGAAGCUGUACCUGUACUACCUGCAGAACGGCCGGGACAUGUACGUGGACCAGGAGCUGGACAUCAACCGGCUGAGCGACUACGACGUGGACCACAUCGUGCCCCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUGCUGACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGUGCCCAGCGAGGAGGUGGUGAAGAAGAUGAAGAACUACUGGCGGCAGCUGCUGAACGCCAAGCUGAUCACCCAGCGGAAGUUCGACAACCUGACCAAGGCCGAGCGGGGCGGCCUGAGCGAGCUGGACAAGGCCGGCUUCAUCAAGCGGCAGCUGGUGGAGACCCGGCAGAUCACCAAGCACGUGGCCCAGAUCCUGGACAGCCGGAUGAACACCAAGUACGACGAGAACGACAAGCUGAUCCGGGAGGUGAAGGUGAUCACCCUGAAGAGCAAGCUGGUGAGCGACUUCCGGAAGGACUUCCAGUUCUACAAGGUGCGGGAGAUCAACAACUACCACCACGCCCACGACGCCUACCUGAACGCCGUGGUGGGCACCGCCCUGAUCAAGAAGUACCCCAAGCUGGAGAGCGAGUUCGUGUACGGCGACUACAAGGUGUACGACGUGCGGAAGAUGAUCGCCAAGAGCGAGCAGGAGAUCGGCAAGGCCACCGCCAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACCGAGAUCACCCUGGCCAACGGCGAGAUCCGGAAGCGGCCCCUGAUCGAGACCAACGGCGAGACCGGCGAGAUCGUGUGGGACAAGGGCCGGGACUUCGCCACCGUGCGGAAGGUGCUGAGCAUGCCCCAGGUGAACAUCGUGAAGAAGACCGAGGUGCAGACCGGCGGCUUCAGCAAGGAGAGCAUCCUGCCCAAGCGGAACAGCGACAAGCUGAUCGCCCGGAAGAAGGACUGGGACCCCAAGAAGUACGGCGGCUUCGACAGCCCCACCGUGGCCUACAGCGUGCUGGUGGUGGCCAAGGUGGAGAAGGGCAAGAGCAAGAAGCUGAAGAGCGUGAAGGAGCUGCUGGGCAUCACCAUCAUGGAGCGGAGCAGCUUCGAGAAGAACCCCAUCGACUUCCUGGAGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCUGAUCAUCAAGCUGCCCAAGUACAGCCUGUUCGAGCUGGAGAACGGCCGGAAGCGGAUGCUGGCCAGCGCCGGCGAGCUGCAGAAGGGCAACGAGCUGGCCCUGCCCAGCAAGUACGUGAACUUCCUGUACCUGGCCAGCCACUACGAGAAGCUGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCUGUUCGUGGAGCAGCACAAGCACUACCUGGACGAGAUCAUCGAGCAGAUCAGCGAGUUCAGCAAGCGGGUGAUCCUGGCCGACGCCAACCUGGACAAGGUGCUGAGCGCCUACAACAAGCACCGGGACAAGCCCAUCCGGGAGCAGGCCGAGAACAUCAUCCACCUGUUCACCCUGACCAACCUGGGCGCCCCCGCCGCCUUCAAGUACUUCGACACCACCAUCGACCGGAAGCGGUACACCAGCACCAAGGAGGUGCUGGACGCCACCCUGAUCCACCAGAGCAUCACCGGCCUGUACGAGACCCGGAUCGACCUGAGCCAGCUGGGCGGCGACAGCGGCGGCAAGCGGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCAGCUACCCCUACGACGUGCCCGACUACGCCUGA

[0156] SpCas9 amino acid sequence Bold: NLS(PKKKRKV and KRPAATKKAGQAKKKK) Italic: SpCas9 MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGKRPAATKKAGQAKKKKGSYPYDVPDYA-

[0157] SB100X mRNA AUGGGAAAAUCAAAAGAAAUCAGCCAAGACCUCAGAAAAAGAAUUGUAGACCUCCACAAGUCUGGUUCAUCCUUGGGAGCAAUUUCCAAACGCCUGGCGGUACCACGUUCAUCUGUACAAACAAUAGUACGCAAGUAUAAACACCAUGGGACCACGCAGCCGUCAUACCGCUCAGGAAGGAGACGCGUUCUGUCUCCUAGAGAUGAACGUACUUUGGUGCGAAAAGUGCAAAUCAAUCCCAGAACAACAGCAAAGGACCUUGUGAAGAUGCUGGAGGAAACAGGUACAAAAGUAUCUAUAUCCACAGUAAAACGAGUCCUAUAUCGACAUAACCUGAAAGGCCACUCAGCAAGGAAGAAGCCACUGCUCCAAAACCGACAUAAGAAAGCCAGACUACGGUUUGCAACUGCACAUGGGGACAAAGAUCGUACUUUUUGGAGAAAUGUCCUCUGGUCUGAUGAAACAAAAAUAGAACUGUUUGGCCAUAAUGACCAUCGUUAUGUUUGGAGGAAGAAGGGGGAGGCUUGCAAGCCGAAGAACACCAUCCCAACCGUGAAGCACGGGGGUGGCAGCAUCAUGUUGUGGGGGUGCUUUGCUGCAGGAGGGACUGGUGCACUUCACAAAAUAGAUGGCAUCAUGGACGCGGUGCAGUAUGUGGAUAUAUUGAAGCAACAUCUCAAGACAUCAGUCAGGAAGUUAAAGCUUGGUCGCAAAUGGGUCUUCCAACACGACAAUGACCCCAAGCAUACUUCCAAAGUUGUGGCAAAAUGGCUUAAGGACAACAAAGUCAAGGUAUUGGAGUGGCCAUCACAAAGCCCUGACCUCAAUCCUAUAGAAAAUUUGUGGGCAGAACUGAAAAAGCGUGUGCGAGCAAGGAGGCCUACAAACCUGACUCAGUUACACCAGCUCUGUCAGGAGGAAUGGGCCAAAAUUCACCCAAAUUAUUGUGGGAAGCUUGUGGAAGGCUACCCGAAACGUUUGACCCAAGUUAAACAAUUUAAAGGCAAUGCUACCAAAUACUA

[0158] SB100X amino acid sequence Bold: NLS(KRVLYRHNLKGHSARKK) MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY

[0159] <Method for manufacturing lipid composition> The method for manufacturing the lipid composition of the present invention will be described. The method for manufacturing the lipid composition is not limited. For example, the lipid composition can be manufactured by a method in which all of the constituent components of the lipid particles or some of oil-soluble components of the lipid particles are dissolved in an organic solvent or the like such that an oil phase is formed, water-soluble components of the lipid particles are dissolved in water such that a water phase is formed, and the oil phase and the water phase are mixed together. A micromixer may be used for mixing, or an emulsifying machine such as a homogenizer, an ultrasonic emulsifying machine, or a high-pressure injection emulsifying machine may be used for emulsification.

[0160] Alternatively, the lipid composition can also be manufactured by a method in which a lipid-containing solution is subjected to evaporation to dryness using an evaporator under reduced pressure or subjected to spray drying using a spray drier such that a dried mixture containing a lipid is prepared, and the mixture is added to an aqueous solvent and further emulsified using the aforementioned emulsifying machine or the like.

[0161] One of the examples of the method for manufacturing the lipid composition containing is a method including a step (a) of dissolving the lipid components in an organic solvent so as to obtain an oil phase; a step (b) of mixing the oil phase obtained in the step (a) with a water phase containing a payload; a step (c) of diluting the mixed solution containing the oil phase and the water phase obtained in step (b) so as to obtain a dispersion liquid of payload-containing lipid composition; and a step (d) of removing the organic solvent from the dispersion liquid of the lipid composition obtained in the step (c).

[0162] In the step (a), the lipid components are dissolved in an organic solvent (an alcohol such as ethanol, an ester, or the like). The total lipid concentration is not particularly limited, but is generally 1 mmol / L to 100 mmol / L, preferably 3 mmol / L to 50 mmol / L, and more preferably 5 mmol / L to 30 mmol / L.

[0163] In the step (b), the water phase can be obtained by dissolving a payload (for example, nucleic acid molecule or the like) in water or a buffer. If necessary, a component such as an antioxidant can be added. The mixing ratio (volume ratio) of water phase:oil phase is preferably 5:1 to 1:1 and more preferably 4:1 to 2:1.

[0164] In the step (b), the mixed solution can be diluted with water or a buffer (for example, phosphate buffered saline (PBS) or the like).

[0165] In the step (c), as the method of removing the organic solvent from the dispersion liquid of the lipid composition, a general method can be used without particular limitation. For example, by dialyzing the dispersion liquid with the phosphate buffered saline, the organic solvent can be removed.

[0166] If necessary, the lipid composition can be subjected to sizing. Although the sizing method is not particularly limited, an extruder or the like can be used to reduce the particle size.

[0167] <Composition> The lipid composition of the present invention may be lipid particle. The lipid particle means a particle composed of a lipid, and includes a composition having any structure selected from a lipid aggregate in which the lipid is aggregated, a micelle, a liposome, a lipid nanoparticle (LNP), and lipoplex. However, the structure of the lipid particles is not limited to these as long as the composition contains lipids. The lipid composition of the present invention is preferably lipid nanoparticles (LNPs).

[0168] The form of the lipid particles can be checked by electron microscopy, structural analysis using X-rays, and the like. For example, by a method using Cryo transmission electron microscopy (CryoTEM method), it is possible to check, for example, whether a lipid particle such as a liposome has a structure composed of a bimolecular lipid membrane structure (lamella structure) and an inner water layer or a structure composed of an inner core with a high electron density and packed with constituent components including a lipid. The X-ray small angle scattering (SAXS) analysis also makes it possible to check whether or not a lipid particle has a bimolecular lipid membrane structure (lamella structure).

[0169] When the lipid composition of the present invention is a particle, the particle size is not particularly limited, but is preferably 10 to 1,000 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 (for example, a dynamic light scattering method, a laser diffraction method, or the like).

[0170] When the lipid composition of the present invention is a particle, the zeta potential of the particle is not particularly limited, but is preferably -20 to +20 mV, and more preferably -10 to 10 mV. The zeta potential in the present invention is a value measured by the electrophoresis method obtained by diluting the lipid composition in a phosphate buffer solution, but the method is not limited thereto.

[0171] The pKa of the lipid composition of the present invention is not particularly limited, but is preferably 9 to 4, more preferably 8 to 5, and even more preferably 7.5 to 6. The pKa of the lipid composition in the present invention adopts the value measured by the TNS assay, but is not limited to this.

[0172] <Use of lipid composition> The present invention relates to a method to deliver a payload to cells, which comprises adding at least one LDLR binding polypeptide to a culture media and contacting the cell with the lipid composition of the present invention. That is, the lipid composition of the invention can be used as compositions for delivering a payload to the cells.

[0173] Examples of the LDLR binding polypeptide include an apolipoprotein. Examples of the apolipoprotein include ApoE1, ApoE2, ApoE3 or ApoE4, or their fragment. The apolipoprotein is preferably human ApoE1, human ApoE2, human ApoE3 or human ApoE4, or fragments thereof. The amount of LDLR-binding polypeptide added in the medium is not limited, and is generally 1 ng / mL to 100 μg / mL, preferably 10 ng / mL to 10 μg / mL, and more preferably 100 ng / mL to 1 μg / mL.

[0174] The method of the present invention as mentioned above may further comprise inducing expression of low density lipoprotein receptor (LDLR). The methods to induce expression of low-density lipoprotein receptor (LDLR) include stimulation of cells with cytokine, stimulation of cells with agonist, or stimulation with cytokine and agonist. For T cells, examples of cytokines include IL-2, IL-7, IL-15, and IL-21, and examples of agonists include CD2 agonist antibody, CD3 agonist antibody, and CD28 agonist antibody. For B cells, examples of cytokines include IL-2, IL-4, IL-15, IL-10, IL-21, and B cell activation factor (BAFF), and examples of agonists include CD40 agonists, Toll-like receptors (TLRs) agonists, B cell receptor agonists. For NK cells, examples of cytokines include IL-2, IL-7, IL-15, and IL-21, and examples of agonists include 4-1BB agonist, OX40 agonist (ligand), and GITR agonist. For hematopoietic stem cells, examples of cytokines include thrombopoietin (TPO), stem cell factor (SCF), IL-3, and IL-6, and examples of agonists include Flt3 agonists (ligand). For monocytes and macrophages, examples of cytokines include GM-CSF.

[0175] In the present invention, the cell type is not limited, but is preferably an immune cell, a stem cell, or a progenitor cell. Examples of the imune cell include cells which are selected from lymphocytes (for example, T cells, B cells, natural killer cells ("NK cells"), and NKT cells or iNKT cells), monocytes, macrophages, mast cells, dendritic cells, granulocytes (for example, neutrophils, eosinophils, basophils), primary immune cells, CD3+ cells, CD4+ cells, CD8+ T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DC). Examples of the stem cell include celles which are selected from mesenchymal stem cells (MSCs); hematopoietic stem cells (HSCs); endothelial progenitor cell (EPCs); neural stem cell (NSCs); limbal stem cell (LSCs); induced pluripotent stem cell (iPSCs); ocular stem cells; pluripotent stem cell (PSCs); or embryonic stem cell (ESCs).

[0176] The cells to which a payload was delivered by the method of the present invention can be used for transplantation.

[0177] The payload may be delivered to the cells in vitro or in vivo. Preferably, the payload is delivered to the cells in vitro.

[0178] In one embodiment of the invention, there is provided a method for generating one or more genetically modified cells, comprising: (a) contacting the cells with a lipid nanoparticle (LNP) composition comprising a first nucleic acid encoding a DNA binding protein with at least one nuclear localization signal sequence, (b) contacting the cells with an LNP composition comprising a second nucleic acid encoding a sequence for insertion, and the second nucleic acid comprises a sequence which bind to the DNA binding protein, wherein the LNP composition is a lipid composition of the present invention.

[0179] Preferably, (a) the first nucleic acid is mRNA, and (b) the second nucleic acid may be DNA or RNA. Preferably, the method may further comprise (c) loading the nucleic acid into pre-formed empty LNPs.

[0180] Preferably, (a) the DNA binding protein may be a transposase. Preferably, (b) the DNA may be a circular double strand DNA, or a linear double strand DNA. Preferably, (b) the DNA may comprise: Terminal repeat, a promoter region, therapeutic gene-coding region, poly A signal region, terminal repeat, wherein the terminal repeat can bind to the transposase.

[0181] Preferably, the method may further comprise (d) treating the cells with an immune modulating agent. Preferably, (d) the immune modulating agent may be selected from cGASinhibitor, STING inhibitor, TBK1 inhibitor, IKKε inhibitor, JAK inhibitor, TYK2 inhibitor, interferon inhibitor. Preferably, (d) the immune modulating agent is selected from BX-795, MRT67307, GSK8612, Peficitinib, JAK Inhibitor I, or Baricitinib. Preferably, (a) the DNA binding protein may be an RNA-guided DNA binding protein, zinc-finger nuclease, meganuclease, or TALEN nuclease. Preferably, (a) the RNA-guided DNA binding protein may be a Cas nuclease or its derivative.

[0182] Preferably, (b) the DNA may be a single strand DNA. Preferably, (b) the single strand DNA may be a linear or circular single strand DNA. Preferably, (b) the linear single strand DNA may be hybridized with a short single strand DNA to form guide RNA-Cas enzyme complex binding domain. Preferably, (b) the DNA may comprises a 5′ homology arm, a therapeutic gene-coding region, and a 3′ homology arm to a cell, wherein the 5′ homology arm and the 3′ homology arm are complementary to the polynucleotides in a target region of a genomic DNA in the cell.

[0183] Preferably, (a) the DNA binding protein may further comprise single strand DNA binding domain, and (b) the DNA is a circular single strand DNA. Preferably, the method may further comprises (e) treating the cells with a DNA damage repair inhibitor. Preferably, the DNA damage repair inhibitor may be selected from NHEJ inhibitors. Preferably, the NHEJ inhibitor may be DNA-dependent protein kinase (DNA-PK) inhibitor or DNA polymerase theta (Polθ) inhibitor. Preferably, the NHEJ inhibitor may be selected from Nu-7441,AZD7648, LTURM 34, Ku-0060648, Compound 401, LY294002, KU55933, Wortmannin, Nu7206, MSC2490484, VX-984, CC-115, STL127705, or SCR-7.

[0184] When the payload is delivered to the cells in vivo, the lipid composition can be administered to a living body as a nucleic acid medicine. When the lipid composition of the present invention is used as a nucleic acid drug, the lipid composition of the present invention alone may be administered to a living body, or the lipid composition may be mixed with a pharmaceutically acceptable carrier (eg, an administration medium such as saline or phosphate buffer) and administered to a living body. That is, the lipid composition of the present invention may further contain a pharmaceutically acceptable carrier.

[0185] The concentration of the lipid composition in the mixture with the pharmaceutically acceptable carrier is not particularly limited and can generally be 0.05% by weight to 90% by weight. Further, other pharmaceutically acceptable additives such as a pH adjustment buffer and an osmotic pressure adjustment agent may be added to the nucleic acid drug containing the lipid composition of the present invention.

[0186] The route of administration for administering the lipid composition of the present invention is not particularly limited. The lipid composition can be administered by any method. Examples of the administration method include oral administration and parenteral administration (intra-articular administration, intravenous administration, intra-arterial administration, subcutaneous administration, intracutaneous administration, intravitreal administration, intraperitoneal administration, intramuscular administration, intravaginal administration, intravesical administration, intrathecal administration, pulmonary administration, rectal administration, colonic administration, buccal administration, nasal administration, intracisternal administration, inhalation, and the like). Among these, parenteral administration is preferable. As the method of administration, intravenous injection, subcutaneous injection, intracutaneous injection, or intramuscular injection is preferable. Intravenous injection or intramuscular injection is particularly preferable. As the administration, nucleic acid delivery can also be performed by local administration in vivo. The lipid composition of the present invention can also be administered by direct injection into the diseased site.

[0187] The dosage form of the lipid composition according to the present invention is not particularly limited. For oral administration, the lipid composition of the present invention can be used in the form of tablets, troches, capsules, pills, suspension, syrup, and the like by being combined with an appropriate excipient. In addition, additives such as an antioxidant, a buffer, a bacteriostat, an isotonic sterile injection, a suspending agent, a solubilizer, a thickener, a stabilizer, and a preservative can be appropriately incorporated into formulations suitable for parenteral administration.

[0188] <Use of lipid composition as nucleic acid delivery carrier> The lipid composition of the present invention can retain a nucleic acid at a high encapsulation rate. Therefore, the lipid composition are extremely useful as a nucleic acid delivery carrier. According to the nucleic acid delivery carrier using the present invention, for example, by mixing the obtained composition with a nucleic acid or the like and performing transfection in vitro, ex vivo or in vivo, the nucleic acid and the like can be introduced into cells. Furthermore, the nucleic acid delivery carrier using the present invention is also useful as a nucleic acid delivery carrier in nucleic acid drugs. That is, the lipid composition of the present invention are useful as a composition for in vitro, ex vivo or in vivo delivery of a nucleic acid.

[0189] Next, the present invention will be described based on examples, but the present invention is not limited thereto.

[0190] Examples <Example 1> Materials and methods <Material> mRNA

[0191] sgRNA Media A Nucleotide monomer abbreviations used in the nucleic acid sequences. These monomers, when present in oligonucleotide, are linked by 5'-3'-phosphate ester bonds. When phosphorothioate is present, they are linked by phosphorothioate bond. rA Adenosine-3'-phosphate rC Cytidine-3'-phosphate rG Guanosine-3'-phosphate rU Uridine-3'-phosphate mA 2'-O-methyladenosine-3'-phosphate mC 2'-O-methylcytidine-3'-phosphate mG 2'-O-methylguanosine-3'-phosphate mU 2'-O-methyluridine-3'-phosphate rA* Adenosine-3'-phosphorothioate rC* Cytidine-3'-phosphorothioate rG* Guanosine-3'-phosphorothioate rU* Uridine-3'-phosphorothioate mA* 2'-O-methyladenosine-3'-phosphorothioate mC* 2'-O-methylcytidine-3'-phosphorothioate mG* 2'-O-methylguanosine-3'-phosphorothioate mU* 2'-O-methyluridine-3'-phosphorothioate

[0192] Media A

[0193] <Preparation of lipid nanoparticle> LNPs were prepared either a microfluidics method or a vortex mixing method. RNA was diluted in 10 mM citrate buffer, pH 3.0, (aqueous phase) while the appropriate amounts of lipids were co-dissolved in 200 proof ethanol (ethanol phase). In the microfluidics method, the aqueous and ethanol phases were mixed together at a 3:1 volume ratio in a microfluidic chip device using syringe pumps to a final RNA concentration of 0.1 to 0.2 mg / mL.The resultant LNPs were then dialyzed against PBS in a 10 kDa MWCO cassette at 4°C overnight. In the vortex mixing method, the ethanol phase was added to the aqueous phase under vortex mixing at a 3:1 volume ratio in a microtube to a final RNA concentration of 0.1 to 0.2 mg / mL.The resultant LNPs were then dialyzed against PBS in a 10 kDa MWCO cassette at 4°C overnight or directly diluted with PBS by 5-fold to neutralize pH and reduce ethanol concentration.

[0194] <Particle size measurement> LNP particle size, PDI(polydispersity index), and ζ potential (i.e., zeta potential) were obtained using a Zetasizer (Malvern). For size measurement, LNPs were diluted in PBS at a 1 / 200 v / v ratio and z-average values were reported. For zeta potential measurement, LNPs were diluted in 0.1X.PBS at a 1 / 200 v / v.

[0195] <Quantification of RNA concentration and encapsulation> The RNA concentration of LNPs obtained by the vortex-mixing methods and subsequent direct dilution was calculated based on the initial RNA concentration. The RNA concentration in dialyzed samples was determined via a modified Quant- iT RiboGreen RNA assay (Thermo Fisher). A nanoparticle dilution of ~l ng μL-lRNA was made in TE buffer (pH 8.5) and RNA standards were made ranging from 2 ng μL-lto 0.125 ng μL-l. 50 μL of each solution was added to separate wells in a 96-well black polystyrene plate. 50 μL of TE buffer was added to each well. The plate was incubated at 37°C for 15 minutes with shaking at 350 rpm. Following the incubation, the diluted RiboGreen reagent was added (100 μL per well), and the plate was incubated as before for 3 minutes. RiboGreen fluorescence was measured according to the supplied protocol using a Tecan plate reader, and the RNA standard was used to determine nanoparticle RNA concentration. It should be noted that two separate standards were made: one with and without Triton-X. The particles in TE buffer were used to determine un-encapsulated RNA concentration and TE-TX , and encapsulation efficiency was determined via the following equation:

[0196]

[0197] <T cell preparation> Human Peripheral Blood Mononuclear Cells (PBMCs) were obtained commercially (STEMCELL Technologies), and cells were washed and re-suspended in MACS buffer (PBS, 2% BSA, 1 mM EDTA). T cells were isolated via Pan T Cell Isolation Kit, human (Miltenyi BioTec cat. 130-096-535). Isolated T cells were cultured at a density around 500,000 cells / mL in the Media A unless otherwise specified.

[0198] <T cell activation> Isolated T cells were activated with ImmunoCultTMHuman CD3 / CD28 / CD2 T Cell Activator (25 μL / mL media, STEMCELL Technologies, Cat. 10970) for 72 hours prior to editing in the Media A.

[0199] <LNP TREATMENT OF T CELLS> Seventy two hours post activation, T cells were washed and suspended in in Media A at a density of 100,000 cells per mL, and seeded to 96-well plates. 10 μL of LNP solution was added to the each well to yield a final concentration of 1 μg / million cells. T cells were cultured overnight for luciferase assay, or for 4-5 days, with media exchanges every other day, before being evaluated by flow cytometry. All groups were done with replicate wells (n=2-3).

[0200] < LUCIFERASE ASSAY > The RNA-LNPs were added to human primary T cells or other cells seeded in 96-well plates. After overnight incubation, the firefly luciferase transfection efficiency was measured using Steady-Glo Luciferase Assay System (Promega) according to manufacturer’s instructions. The luminescence was quantified using Tecan Infinite M200 Pro plate reader (Tecan).

[0201] < FLOW CYTOMETRY> The cells of interest were phenotyped by flow cytometry to determine reporter protein expression level from mRNA (e.g. mCherry), endogenous gene knockout (e.g. B2M, TCR), and gene knockin (e.g. GFP) as well as T cell memory and exhaustion status. Briefly, cells were incubated in cocktails of antibodies targeting. Cells were subsequently washed, processed on a flow cytometry instrument and analyzed using the FlowJo software package.

[0202] <Example 2>Induction of LDLR expression on T cells LDLR expression levels on human primary T cells were assessed via flow cytometry 36 hours after initiating cell culture under three distinct conditions: (a) in the absence of activation by CD3 / CD28 agonistic antibody and cytokine stimulation, (b) with stimulation solely by IL-2, IL-7, and IL-15, and (c) with CD3 / CD28 agonistic antibody stimulation combined with IL-2, IL-7, and IL-15 stimulation. The results are shown in Figure 1. Figure 1 shows that LDLR expression level was upregulated by CD3 / CD28 agonistic antibody activation or IL-2 / IL-7 / IL-15 cytokine stimulation on human primary T Cells. Data are shown as mean ± SD. n = 3 biological replicates.

[0203] Notably, IL-2 / IL-7 / IL-15 stimulation significantly upregulated LDLR expression, and the additional inclusion of CD3 / CD28 agonistic antibody further enhanced LDLR expression levels.

[0204] <Example 3> in vitro RNA delivery to human primary T cells Next, we tested whether preincubation with ApoE could enhance LNP uptake and transfection in human primary T cells ex vivo. 5 methoxyuridine (5moU)-modified mCherry mRNA was formulated into LNPs using the original formulation parameters previously published and used in mRNA vaccines (Table 1). The resulting LNPs was preincubated with recombinant human ApoE4 (rhApoE4), and then added to human primary T cells cultured in serum-free media. 24 hours post treatment, mCherry expression level was detected in approximately 60% human primary T cells activated by CD3 / CD28 antibodies and stimulated by IL-2 / IL-7 / IL-15 when LNPs were preincubated with rhApoE4 (Figure 2).

[0205] Table 1: 5moU mCherry mRNA-LNP formulation

[0206] In Figure 2, mCherry expression in human primary T cells was detected after preincubating LNP with rhApoE4 . Human primary T cells were activated by CD3 / CD28 antibodies and stimulated by IL-2 / IL-7 / IL-15 for 3 days and then treated with LNPs for 24 h at a dose of 1 μg / 1,000,000 cells. Data are shown as mean ± SD. n = 3 biological replicates.

[0207] <Example 4> mRNA delivery to human primary T cells with various ionizable lipid To identify the most effective ionizable lipids for optimal ex vivo delivery, we employed a luciferase-based reporter assay as our screening method. Following the addition of luciferin, luminescence is exclusively generated by the translated luciferase protein from the mRNA. This luminescence is easily detectable and serves as a reliable indicator of functional mRNA delivery. For our study, we curated a library of lipid nanoparticles (LNPs) utilizing 12 distinct ionizable lipids previously reported for effective intravenous liver delivery or intramuscular mRNA vaccines. These LNPs were formulated with 5moU-modified firefly luciferase mRNA using a hand mixing method. In our experimental setup, these LNPs were introduced to human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. Luminescence, indicative of successful mRNA delivery, was quantified 24 hours post-LNP treatment. The results are shown in Table 2, Table 3,, Figure 3 and Figure 4. All LNP formulations showed luminescence comparable to or higher than MC3, which is commercially used in FDA- approved siRNA therapeutic for liver diseases.

[0208] FL-A FL-B MC3 ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate) L-319 (bis[(Z)-non-2-enyl] 9-[4-(dimethylamino)butanoyloxy]heptadecanedioate) ALC-0315 (6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate) SM-102 (heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate) Lipid 5 (nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate) Lipid 29 (undecan-3-yl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-[3-[[2-(methylamino)-3,4-dioxocyclobuten-1-yl]amino]propyl]amino]octanoate) ATX-100 (pentadecan-8-yl 4-[3-(dimethylamino)propylsulfanylcarbonyl-(4-oxo-4-pentadecan-8-yloxybutyl)amino]butanoate) Lipid A9 (bis(2-butyloctyl) 10-[3-(dimethylamino)propyl-nonanoylamino]nonadecanedioate) Lp01 ([2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate) GCL1 ([(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate)

[0209] Table 2: LNPs with various ionizable lipids encapsulating 5moU-modified firefly luciferase mRNA

[0210] In Figure 3, luciferase expression after treating with the LNP library and commercial transfection reagents (TransITRand LipofectamineTMMessengerMaxTM) identified top-performing LNPs. Human primary T cells were activated by CD3 / CD28 antibodies and stimulated by IL-2 / IL-7 / IL-15 for 3 days and then treated with LNPs for 24 h at a dose of 5 μg / 1,000,000 cells. Results were normalized to MC3 LNP. Data are shown as mean ± SD. n = 2-3 biological replicates.

[0211] Table 3: LNPs with various ionizable lipids encapsulating 5moU-modified firefly luciferase mRNA

[0212] In Figure 4, luciferase expression after treating with the LNP library identified top-performing LNPs. Human primary T cells from different donor were activated by CD3 / CD28 antibodies and stimulated by IL-2 / IL-7 / IL-15 for 3 days and then treated with LNPs for 24 h at a dose of 5 μg / 1,000,000 cells. Results were normalized to MC3 LNP. Data are shown as mean ± SD. n = 2-3 biological replicates.

[0213] <Example 5> mRNA delivery to human primary T cells with various polymer-conjugated lipids Polymer-conjugated-lipids play a critical role in the design and functionality of LNPs, contributing to their colloidal stability, circulation time, and serum protein adsorption, and these features are crucial for successful delivery of payload. Among these polymer-conjugated lipids, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], also known as DMG-mPEG2000, is the most commonly used polymer-conjugated lipid in LNPs due to its well-balanced in vivodesorption rate from the LNP surface for hepatocyte and intramuscular vaccine delivery, However, ex vivo cell culture condition, especially in serum-free medium, it is unclear whether DMG-mPEG2000 performs best.

[0214] To optimize the chemical structure of polymer-conjugated lipids for ex vivouse, we designed a library of distinct polymer-conjugated lipids and formulated with the top-performing ionizable lipids identified in the previous experiment. These LNPs were formulated with 5moU-modified firefly luciferase mRNA using the vortex mixing method, and were introduced to human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. Luminescence, indicative of successful mRNA delivery, was quantified 24 hours post-LNP treatment.

[0215] The results are shown in Table 4, Table 5, Table 6, Table 7 and Figure 5.

[0216] Polymer-conjugated lipids with two acyl groups, such as glyceride-PEG, phospholipid-PEG, and ceramide-PEG, demonstrated superior performance when their average molecular weight fell within the range of 550-1100, surpassing the commonly used DMG-mPEG2000. Cholesterol-PEG consistently exhibited successful delivery and outperformed DMG-mPEG2000, with the optimal performance observed for cholesterol-PEG possessing an average molecular weight ranging from 550 to 2000. PEG fatty acid esters showcased successful delivery, consistently surpassing DMG-mPEG2000 in performance. Polyoxyethylene alkyl ethers exhibited successful delivery and outperformed DMG-mPEG2000, with the most effective range observed for Polyoxyethylene alkyl ethers featuring a PEG chain length between 9 and 40. PEG hydrogenated castor oil or PEG castor oil, with an average ethoxylation degree of 30-50, generally demonstrated successful delivery, consistently surpassing DMG-mPEG2000. PEG(20) sorbitan fatty acid monoesters demonstrated successful delivery and consistently outperformed DMG-mPEG2000. Notably, among them, PEG(20) sorbitan palmitate and oleate esters proved to be the most effective. Crucially, these polymer-conjugated lipids exhibited superior performance compared to DMG-mPEG2000 across the board, irrespective of the ionizable lipid used in the formulations.

[0217] Glyceride PEG DMG-mPEG DMG-mPEG2000 DMG-mPEG1100 DMG-mPEG550

[0218] Pospholipid-PEG DMPE-PEG DMPE-mPEG2000 DMPE-mPEG1000 DMPE-mPEG750 DMPE-mPEG550 DMPE-mPEG350

[0219] C8 Ceramide-PEG C8 Ceramide-mPEG750 C16 Ceramide-PEG C16 Ceramide-mPEG750

[0220] Cholesterol-PEG Cholesterol-mPEG550 Cholesterol-mPEG1000 Cholesterol-mPEG2000 Cholesterol-mPEG3400 Cholesterol-mPEG5000

[0221] Fatty acid methoxy PEG ester mPEG2000 monolaurate mPEG2000 monomyristate mPEG2000 monopalmitate mPEG2000 monostearate mPEG1000 monostearate mPEG5000 monostearate

[0222] Polyoxyethylene alkyl ether (also known as BrijR.e.g. PEG-Lauryl ether) PEG(9)-lauryl ether PEG(23)-lauryl ether PEG(10)-cetyl ether PEG(20)- cetyl ether PEG(40)- cetyl ether PEG(10)-stearyl ether PEG(20)-stearyl ether PEG(10)-Oleyl ether PEG(20)-Oleyl ether

[0223] Polyoxyethelene sorbitan ester (also known as polysorbate or TweenR) Polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate) x+y+z=20 Polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate) x+y+z=20 Polysorbate 60 (polyoxyethylene (20) sorbitan monostearate) x+y+z=20 Polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) x+y+z=20

[0224] Polyoxyethylene hydrogenated castor oil x+y+z=10~60 Polyoxyethylene castor oil x+y+z=29-40 Polysarcosine lipid R=H, or C14-C18 alkyl N-octadecyl-pSar25

[0225] Table 4: FL-B LNPs with various polymer-conjugated lipids encapsulating 5moU-modified firefly luciferase mRNA

[0226] Table 5: ATX-100 LNPs with various polymer-conjugated lipids encapsulating 5moU-modified firefly luciferase mRNA

[0227] Table 6: A9 LNPs with various polymer-conjugated lipids encapsulating 5moU-modified firefly luciferase mRNA

[0228] Table 7: GCL1 LNPs with various polymer-conjugated lipids encapsulating 5moU-modified firefly luciferase mRNA

[0229] Figure 5 shows the luciferase expression in human primary T cells after treatment with LNPs for 24 h at a dose of 5 μg / million cells. Results were normalized to cells treated with the original formulation using DMG-mPEG2000.

[0230] <Example 6> mRNA delivery to human primary T cells with various phospholipids To assess the effect of phospholipid amount, we first compared three LNPs with different DSPC ratios. These LNPs were formulated with 5moU-modified firefly luciferase mRNA using the vortex mixing method, and were introduced to human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. Luminescence, indicative of successful mRNA delivery, was quantified 24 hours post-LNP treatment.

[0231] The results are shown in Table 8 and Figure 6. As DSPC ratio decreased from 10%, LNPs showed lower transfection efficiency in human primary T cells, and showed lower luminescence values.

[0232] Table 8: Phospholipid ratio screening in FL-A LNPs encapsulating 5moU-firefly luciferase mRNA

[0233] Figure 6 shows the luciferase expression in human primary T cells after treatment with LNPs for 24 h at a dose of 5 μg / million cells. Results were normalized to cells treated with the Reference Example6-1.

[0234] Next, to assess the effect of chemical structure of phospholipid, we created a library of phospholipids with different tail structure (Table 9). These LNPs were formulated with 5moU-modified firefly luciferase mRNA using the vortex mixing method, and were introduced to human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. Luminescence, indicative of successful mRNA delivery, was quantified 24 hours post-LNP treatment.

[0235] The results are shown in Table 10, Table 11, Table 12, Table 13, Table 14 and Figure 7. At a fixed ratio of 10% phospholipid, all the phospholipids tested showed comparable levels of luciferase expression.

[0236] Table 9: Phospholipids tested in this screening.

[0237] Table 10: Phospholipid screening in FL-B LNPs

[0238] Table 11: Phospholipid screening in A9 LNPs

[0239] Table 12: Phospholipid screening in ATX-100 LNPs

[0240] Table 13: Phospholipid screening in FL-A LNPs

[0241] Table 14: Phospholipid screening in GCL1 LNPs

[0242] Figure 7 shows the luciferase expression in human primary T cells after treatment with LNPs for 24 h at a dose of 5 μg / million cells. Results were normalized to the cells treated with the DSPC formulation.

[0243] <Example 7> In vitro gene editing in human primary T cells using an optimized formulation We next tested whether our LNP formulation optimized with firefly luciferase mRNA could also improve the transfection efficiency of Cas9 mRNA and sgRNA. We encapsulated 5moU-modified Cas9 mRNA and sgRNA against human B2M gene together into LNPs using the vortex mixing methodand evaluated B2M knockout efficiency as well as cell viability in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The B2Mknockout efficiency and the cell viability were measured at day 4 post-LNP treatment by FACS.

[0244] The results are shown in Table 15 and Figure 8. At a dose of 6.7 μg / million cells, while FL-A / DMG-mPEG2000 LNP showed approximately 4 % B2Mknockout, FL-B / DMG-mPEG2000 LNP showed 20 % B2M knockout, which was consistent with the luciferase mRNA-based screening results described in the Example 4. Also, by switching polymer-conjugated lipid from the conventional DMG-mPEG2000 to Chol-mPEG2000, B2M knockout efficiency was improved by approximately 2-fold, indicating that polymer-conjugated lipids have a large effect on transfection efficiency. It is noteworthy that all the groups showed comparable viability.

[0245] Table 15: LNPs tested with Cas9 mRNA and sgRNA against B2M

[0246] Figure 8 shows the quantification of gene editing efficiency and cell viability after transfecting with different LNP formulations in human primary Tcells. Human rpmary T cells were treated with LNPs encapsulating 5moU-modified Cas9 mRNA and sgRNA against B2M (1:1 mass ratio) at a dose of 6.7 μg total RNA / million cells. Gene editing efficiency (a) and the live cell population (b) was measured at day 4 post-treatment. Data were presented as mean ± SD.

[0247] <Gene editing efficiency in different T cell subsets> T cells, which are defined by CD3-positive cells, can be further classified into different subsets based on the presence of specific cell surface markers and their functions. Two prominent subsets are CD4-positive T cells (also known as helper T cells) and CD8-positive T cells (also known as cytotoxic T cells or killer T cells). CD4 T cells and CD8 T cells work together to mount an effective immune response against various pathogens and maintain immune homeostasis. To confirm whether both T cell subsets can be transfected by our LNPs, T cells were further gated on CD4 / CD8 status prior to examining gene editing. We encapsulated 5moU-modified Cas9 mRNA and sgRNA against human B2M gene together into LNPs using the vortex mixing method and evaluated B2M knockout efficiency as well as cell viability in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The B2M knockout efficiency and the cell viability were measured at day 4 post-LNP treatment by FACS.

[0248] The results are shown in Table 16 and Figure 9. High levels of B2M knockout were observed in both CD4+ and CD8+ T cells, yielding >60% of B2M knockout CD8+ and CD4+ T cells.

[0249] Table 16: LNPs tested with Cas9 mRNA and sgRNA against B2M

[0250] Figure 9 shows the gene editing efficiency in different T cell subsets. Human rpmary T cells were treated with LNPs encapsulating 5moU modified Cas9 mRNA and sgRNA against B2M (1:1 mass ratio) at a dose of 0.02-5 μg total RNA / 1,000,000 cells. Gene editing efficiency was measured at day 5 post-treatment by FACS. Data were presented as mean ± SD.

[0251] <Example 8> Effect of mRNA uridine modification Uridine modifications are known to reduce immunogenicity of exogeneous mRNA, and hence contribute to the overall efficacy and safety of mRNA-based approaches for protein expression within cells. To test whether modified uridine increases Cas9-based gene knockout efficiency, we compared 5 methoxy uridine (5moU)-modified and unmodified Cas9 mRNA in human primary T cells. We encapsulated 5moU-modified and unmodified Cas9 mRNA and sgRNA against human B2M gene together into LNPs using the vortex mixing method and evaluated B2M knockout efficiency as well as cell viability in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The B2M knockout efficiency and the cell viability were measured at day 4 post-LNP treatment.

[0252] The results are shown in Table 17 and Figure 10. Unmodified uridine showed higher gene editing efficiency without reducing cellular viability.

[0253] Table 17: LNP composition tested with 5moU-modified and unmodified Cas9 mRNA

[0254] Figure 10 shows the comparison of gene editing efficiency and cell viability after transfecting with LNPs encapsulating 5moU and unmodified Cas9 mRNA in human primary Tcells. Human rpmary T cells were treated with LNPs encapsulating 5moU-modified or unmodified Cas9 mRNA and sgRNA against B2M (1:1 mass ratio) at a dose of 6.7 μg total RNA / million cells. Gene editing efficiency (a) and the live cell population (b) was measured at day 4 post-treatment by FACS. Data were presented as mean ± SD.

[0255] <Example 9> Lipid mixture composition optimization The molar ratio of LNP components was optimized for in vitro T cell transfection. In this study, LNPs were prepared using varied lipid molar ratios to investigate the impact of LNP formulation on nucleic acid delivery to T cells. We applied Design of Experiment (DOE) to reduce the number of formulations required to establish statistically significant trends in a large multidimensional formulation design space.

[0256] For the library preparation (Library A and Library B), FL-B, DSPC, cholesterol and cholesterol-mPEG2000 were used as ionizable lipid (IL), phospholipid (PL), sterol and polymer-conjugated lipid, respectively, and mixed to contain the desired lipid ratio in ethanol phase using the vortex mixing method. For aqueous phase, unmodified Cas9 mRNA and sgRNA against B2M gene were used at 1:1 mass ratio, and diluted in 10 mM citrate buffer pH3.0. To formulate LNPs, these ethanol phases and aqueous phases were mixed by the vortex mixing method.

[0257] The results are shown in Table 18, Figure 11(Library A), and Table 19, Figure 12 (Library B).

[0258] We designed the first library, Library A, to be centered around the original LNP formulation parameters (IL : PL :Sterol : polymer conjugated lipid = 50:10:38.5:1.5, IL / total lipid mass ratio = 10) with one three-level quantitative factors (IL : total RNA mass ratio of 5, 10 and 15), and 4-component mixture design DOE within the range of IL:30-60%, PL: 10-30%, Sterol:20-60%, and polymer-conjugated lipid 1-3%.

[0259] The most apparent trend from Library A was IL, PL and sterol ratio-dependence of B2M knockout efficiency. LNPs with less IL within 30-60% IL ratio showed higher transfection efficiency in human primary T cells in vitro. Also, In LNPs with more than 25% PL or less than 30% sterol, their transfection efficiency was reduded.

[0260] Based on the results of Library A, we designed the second library, Library B, to be centered around the top-performing LNP formulation in the Library A (IL : PL :Sterol : polymer conjugated lipid = 30:18:49:3, IL / total lipid mass ratio = 10) to identify the best range of each component. In the Library B, the top-performing LNP formulation in Library A showed the highest B2M knockout again. We didn’t observe the strong sterol ratio-dependence between 40-70%. On the other hand, we observed best IL, PL and polymer-conjugated lipid ratio around 20-40$, 10-20%, and 1-4%, respectively.

[0261] Table 18: DOE-based LNP formulation optimization (Library A, 5.5 μg / million cells)

[0262] Figure 11 shows B2M knockout efficiency in DOE-based lipid ratio optimization (Library A)

[0263] Table 19: DOE-based LNP formulation optimization (Library B, 5.5 μg / million cells)

[0264] Figure 12 shows B2M knockout efficiency in DOE-based lipid ratio optimization (Library B)

[0265] <Example 10> mRNA delivery to human primary T cells with various polymer-conjugated lipids To test whether the polymer-conjugated lipids identified in Example 5 outperform DMG-mPEG2000 in the optimized lipid ratio, we made a library of LNPs encapsulating firefly luciferase mRNA. These LNPs were formulated with 5moU-modified firefly luciferase mRNA using the vortex mixing method, and were introduced to human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The RNA dose was set as 4.6 μg / million cells. Luminescence, indicative of successful mRNA delivery, was quantified 24 hours post-LNP treatment.

[0266] The results are shown in Table 20, Table 21, Table 22, Table 23, Table 24 and Figure 13.

[0267] Table 20: Firefly luciferase mRNA LNPs with different polymer-conjugated lipid

[0268] Figure 13 shows polymer-conjugated lipid screening in FL-B LNPs using firefly luciferase mRNA

[0269] Table 21: Firefly luciferase mRNA LNPs with different polymer-conjugated lipid

[0270] Table 22: Firefly luciferase mRNA LNPs with different polymer-conjugated lipid

[0271] Table 23: Firefly luciferase mRNA LNPs with different polymer-conjugated lipid

[0272] Table 24: Firefly luciferase mRNA LNPs with different polymer-conjugated lipid

[0273] <Example 11> Gene editing in human primary T cells with the original and sub-optimized LNPs To measure the contribution from optimization of polymer-conjugated lipid structure and lipid ratio separately, we made a library of LNPs encapsulating Cas9 mRNA and sgRNA and measured B2M knockout efficiency in human primary T cells. We encapsulated unmodified Cas9 mRNA and sgRNA against human B2M gene together into LNPs using the vortex mixing method and evaluated B2M knockout efficiency as well as cell viability in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The B2Mknockout efficiency and the cell viability were measured at day 4 post-LNP treatment.

[0274] The results are shown in Table 25 and Figure 14. These data demonstrated that combination of the optimized polymer-conjugated lipid structure and lipid ratio can maximize the transfection efficiency in human primary T cells in vitro.

[0275] Table 25: LNPs tested with Cas9 mRNA and sgRNA against B2M

[0276] Figure 14 shows dose dependent B2M knockout using optimized and unoptimized LNP formulations

[0277] <Example 12> Gene editing in human primary T cells with various polymer-conjugated lipids To test whether the polymer-conjugated lipid selected through firefly luciferase mRNA can also perform with Cas9 mRNA and sgRNA, we made a library of LNPs encapsulating Cas9 mRNA and sgRNA and measured B2M knockout efficiency in human primary T cells. We encapsulated unmodified Cas9 mRNA and sgRNA against human B2M gene together into LNPs using the vortex mixing method and evaluated B2M knockout efficiency as well as cell viability in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The total RNA dose was set as 1.4 μg / million cells. The B2M knockout efficiency and the cell viability were measured at day 4 post-LNP treatment.

[0278] The results are shown in Table 26 and Figure 34.

[0279] Table 26: LNPs tested with Cas9 mRNA and sgRNA against B2M

[0280] <Example 13> Gene editing in human primary T cells with various ionizable lipids and polymer-conjugated lipids To test whether the optimized polymer-conjugated lipid structure and lipid ratio can be generalizable to LNPs with other chemically distinct ionizable lipids, we made a library of LNPs encapsulating Cas9 mRNA and sgRNA and measured B2Mknockout efficiency in human primary T cells. We encapsulated unmodified Cas9 mRNA and sgRNA against human B2M gene together into LNPs using the vortex mixing method and evaluated B2M knockout efficiency as well as cell viability in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The total RNA dose was set as 12 μg / million cells The B2M knockout efficiency and the cell viability were measured at day 4 post-LNP treatment.

[0281] The results are shown in Table 27 and Figure 15. These data demonstrated that combination of the optimized polymer-conjugated lipid structure and lipid ratio can maximize the transfection efficiency in human primary T cells in vitro.

[0282] Table 27: LNPs tested with Cas9 mRNA and sgRNA against B2M

[0283] Figure 15 shows B2M knockout efficiency using different LNP formulations (12 μg / million cells).

[0284] <Example 14> mRNA delivery to human primary T cells with various LDLR binding polypeptide Human primary T cells were engineered with LNPs with various LDLR binding proteins. We encapsulated unmodified Cas9 mRNA and sgRNA against human B2M gene together into LNPs using the vortex mixing method and evaluated B2M knockout efficiency as well as cell viability in human primary T cells that had been activated and stimulated for 3 days in serum-free media. Before adding LNPs, various LDLR binding proteins were added to the media. This study was performed as a 4-point dose response assay using recombinant human ApoA-I, ApoB and ApoE isoforms ApoE2, ApoE3 and ApoE4. The total RNA dose was set as 2.2 μg / million cells. The B2M knockout efficiency and the cell viability were measured at day 4 post-LNP treatment.

[0285] The results are shown in Table 28, Table 29 and Figure 16.

[0286] Table 28: LNPs tested with Cas9 mRNA and sgRNA against B2M

[0287] Table 29: LNPs tested with Cas9 mRNA and sgRNA against B2M

[0288] Figure 16 shows B2M knockout efficiency using different LDLR-binding proteins (2.2 μg / million cells).

[0289] <Example 15> Gene editing in human primary T cells with various mRNA: sgRNA ratio To optimize the Cas9 mRNA and sgRNA mass ratio, we made a library of LNPs encapsulating different mass ratio of Cas9 mRNA and sgRNA and measured B2M knockout efficiency in human primary T cells. We encapsulated unmodified Cas9 mRNA and sgRNA against human B2M gene together into LNPs using the vortex mixing method and evaluated B2M knockout efficiency as well as cell viability in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The B2M knockout efficiency and the cell viability were measured at day 4 post-LNP treatment.

[0290] The results are shown in Table 30, Table 31, and Figure 17. These data demonstrated that efficient gene editing can be achieved in a wide range of Cas9 mRNA and sgRNA mass ratio.

[0291] Table 30: LNPs tested with Cas9 mRNA and sgRNA against B2M (4.8 ug / mil. Cells)

[0292] Table 31: LNPs tested with Cas9 mRNA and sgRNA against B2M (1.2 ug / mil. Cells)

[0293] Figure 17 shows effect of Cas9 mRNA: sgRNA mass ratio (Left) 4.8 μg total RNA / million cells (Right) 1.2 μg total RNA / million cells

[0294] <Example 16> In vitro gene editing in human primary hematopoietic stem cells 　We tested whether LNP formulations optimized with T cells can also improve the transfection efficiency of primary human hematopoietic stem cells. Primary human HSCs were cultured for 2 days in serum-free medium containing the following cytokine agonists and 1 μg / mL rApoE4 to induce LDLR expression.

[0295] Table 32: LDLR induction media for hematopoietic stem cells

[0296] LNPs containing unmodified Cas9 mRNA and sgRNA against human B2M gene were prepared by vortex mixing method. 10 μL of the LNP solution containing a total of 10 μg / mL Cas9 mRNA and sgRNA was added to primary human hematopoietic stem cells (approximately 50,000 cells) cultured in the above medium in 96-well plates (2 μg / 106cells). After LNP treatment, the medium was changed every 2 days and B2M knockout efficiency was measured by FACS on day 5 after LNP treatment.

[0297] The results are shown in Table 33 and Figure 18. Changing the polymer- conjugated lipid from DMG-mPEG2000 to Chol-mPEG2000 increased the B2M knockout efficiency from about 62% to 84%, indicating that the increase in transfection efficiency by optimizing the polymer-conjugated lipid shown for T cells is also applicable to hematopoietic stem cells.

[0298] Table 33：LNPs tested with Cas9 mRNA and sgRNA against B2M

[0299] Figure 18 shows gene editing efficiency after transfection with different LNP formulations in primary human HSCs. Primary human HSCs were treated with LNPs containing unmodified Cas9 mRNA and sgRNA against B2M (mass ratio 1:1) at a dose of 2 μg total RNA / 106cells. Gene editing efficiency was measured on day 5 after treatment. Data are presented as mean ± SD.

[0300] <Information for the materials used in Examples 17 to 26> Table 34: mRNA

[0301] Table 35:sgRNA

[0302] Table 36: plasmid DNA

[0303] Table 37 Sleeping Beauty transposon / transposase DNA sequence

[0304] Table 38: Promoter sequences

[0305] Table 39: Poly A signal sequence

[0306] Table 40:plasmid backbone sequences

[0307] Table 41:ssDNA sequences

[0308] <Example 17> Comparison of T cell activation method LDLR expression levels on human primary T cells were assessed via flow cytometry 36 hours after initiating cell culture under three distinct conditions: (a) in the absence of activation by CD3 / CD28 agonistic antibody and cytokine stimulation, (b) with stimulation solely by IL-2, IL-7, and IL-15, and (c) with CD3 / CD28 agonistic antibody stimulation combined with IL-2, IL-7, and IL-15 stimulation. The results are shown in Figure 19. Figure 19 shows that LDLR expression level was upregulated by CD3 / CD28 agonistic antibody activation or IL-2 / IL-7 / IL-15 cytokine stimulation on human primary T Cells. Data are shown as mean ± SD. n = 3 biological replicates.

[0309] Notably, IL-2 / IL-7 / IL-15 stimulation significantly upregulated LDLR expression, and the additional inclusion of CD3 / CD28 agonistic antibody further enhanced LDLR expression levels.

[0310] Table 42:

[0311] Table 43:

[0312] <Example 18> mRNA modification Uridine modifications are known to reduce immunogenicity of exogeneous mRNA, and hence contribute to the overall efficacy and safety of mRNA-based approaches for protein expression within cells. To test whether modified uridine increases Cas9-based gene knockout efficiency, we compared 5 methoxy uridine (5moU)-modified and unmodified Cas9 mRNA in human primary T cells. We encapsulated 5moU-modified and unmodified Cas9 mRNA and sgRNA against human B2M gene together into LNPs using the vortex mixing method and evaluated B2M knockout efficiency as well as cell viability in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The B2M knockout efficiency and the cell viability were measured at day 4 post-LNP treatment. The results are shown in Table 44 and Figure 20. Unmodified uridine showed higher gene editing efficiency without reducing cellular viability.

[0313] Table 40: LNP composition tested with 5moU-modified and unmodified Cas9 mRNA

[0314] Figure 20 shows the comparison of gene editing efficiency and cell viability after transfecting with LNPs encapsulating 5moU and unmodified Cas9 mRNA in human primary Tcells. Human rpmary T cells were treated with LNPs encapsulating 5moU-modified or unmodified Cas9 mRNA and sgRNA against B2M (1:1 mass ratio) at a dose of 6.7 μg total RNA / million cells. Gene editing efficiency (a) and the live cell population (b) was measured at day 4 post-treatment by FACS. Data were presented as mean ± SD.

[0315] <Example 19> Ionizable lipid generalization To test whether the optimized polymer-conjugated lipid structure and lipid ratio can be generalizable to LNPs with other chemically distinct ionizable lipids, we made a library of LNPs encapsulating firefly luciferase mRNA and measured luciferase expression levels in human primary T cells. We encapsulated 5moU-modified firefly luciferase mRNA into LNPs using the vortex mixing method and measured bioluminescence in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The total RNA dose was set as 5 μg / million cells The bioluminescence values were measured 24 hours post-LNP treatment The results are shown in Table 43 and Figure 21. All LNP formulations showed luminescence comparable to or higher than MC3, which is commercially used in FDA- approved siRNA therapeutic for liver diseases. In Figure 21, luciferase expression after treating with the LNP library. Human primary T cells were activated by CD3 / CD28 antibodies and stimulated by IL-2 / IL-7 / IL-15 for 3 days and then treated with LNPs for 24 h at a dose of 5 μg / 1,000,000 cells. Data are shown as mean ± SD. n = 2-3 biological replicates.

[0316] Table 41. Ionizable lipid chemical structures

[0317] Table 42: LNPs with various ionizable lipids encapsulating 5moU-modified firefly luciferase mRNA

[0318] Table 43: Bioluminescence values in human primary T cells

[0319] <Example 20> Transgene expression using nanoplasmid DNA To evaluate the effect of DNA size on transgene expression, we first compared the protein expression levels of conventional plasmid DNA (pDNA) and nanoplasmid DNA (npDNA). We transfected human primary T cells with LNPs encapsulating either pDNA or npDNA encoding the firefly luciferase gene and measured luciferase activity 24 hours post- transfection. The results are shown in Figure 22. npDNA-transfected cells exhibited approximately 3-fold higher level of protein expression compared to their pDNA counterparts. To further optimize our LNP-mediated gene delivery system, we tested various ionizable lipids using npDNA encoding the firefly luciferase gene. Human primary T cells were transfected with LNPs formulated with different ionizable lipids, and luciferase activity was measured to assess protein expression levels. The results are shown in Figure 22. The results demonstrated significant variability in protein expression among the different ionizable lipids. Notably, the ionizable lipid FL-B showed one of the highest protein expression levels, with markedly higher luciferase activity compared to a benchmark formulations using MC3 or LP01. This superior performance of FL-B highlighted its potential as an optimal component for enhancing LNP-mediated gene delivery in CAR-T cell therapy.

[0320] Table 44:plasmid DNA

[0321] Table 45:

[0322] <Example 21>In vitro gene insertion using Sleeping Beauty transposase To assess whether small-sized nanoplasmid DNA (npDNA) facilitates gene insertion into the genome, we combined npDNA encoding eGFP with mRNA encoding Sleeping Beauty transposase (SB100X) to integrate the eGFP gene into the genome of human primary T cells. npDNA and mRNA were encapsulated in LNPs (Table **), and these LNPs were added to activated T cells. We tested four conditions: (a) SB100X mRNA only, (b) npDNA encoding SB insertion template eGFP with EFS promoter only, (c) combination of SB100X mRNA and npDNA encoding SB insertion template eGFP with EFS promoter (2295 bp), and (d) combination of SB100X mRNA and pDNA encoding SB insertion template eGFP with EFS promoter (4776 bp). GFP expression levels were evaluated by flow cytometry. Figure 23 shows schematic of gene insertion using transposase mRNA and transposon DNA. The results are shown in Figure 24. The group (b) showed low level of GFP expression due to the npDNA entering the nucleus and expressing GFP without transposase. However, during the 7-day culture period, T cell proliferation diluted the npDNA, resulting in reduced expression. Therefore, we considered only high levels of GFP expression as a successful genomic integration in combination with SB100X mRNA. The group (c) exhibited high levels of GFP expression in approximately 30% of T cells while the group (d) displayed negligible GFP expression, suggesting that the larger pDNA was less effective for gene insertion.

[0323] Table 46: LNPs tested with SB100X transposase mRNA and transposon DNA

[0324] <Example 22>LNP formulation optimization Uridine modifications are known to reduce immunogenicity of exogeneous mRNA, and hence contribute to the overall efficacy and safety of mRNA-based approaches for protein expression within cells. To test whether modified uridine affects transposase-based gene insertion efficiency, we compared N1-methyl pseudouridine (N1mU)-modified and unmodified Sleeping Beauty transposase (SB100X) mRNA in human primary T cells. We encapsulated N1mU-modified and unmodified SB100X mRNA and npDNA encoding EGFP trransposon into LNPs using the vortex mixing method, respectively, and evaluated GFP expression levels in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rApoE4. The GFP expression levels were measured at day 7 post-LNP treatment. The results are shown in Figure 25. N1mU showed gene insertion efficiency.

[0325] Table 47: LNPs tested with SB100X transposase mRNA and transposon DNA

[0326] <Example 23> Polymer-conjugated-lipids play a critical role in the design and functionality of LNPs, contributing to their colloidal stability, circulation time, and serum protein adsorption, and these features are crucial for successful delivery of payload. Among these polymer-conjugated lipids, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], also known as DMG-mPEG2000, is the most commonly used polymer-conjugated lipid in LNPs due to its well-balanced in vivo desorption rate from the LNP surface for hepatocyte and intramuscular vaccine delivery, Also, most of the LNP formulations had been optimized for in vivo vaccine or liver delivery, resulting in the molar ratio of ionizable lipid : phospholipid : cholesterol : polymer-conjugated lipid = 50 : 10 : 38.5: 1.5. However, ex vivo cell culture condition, especially in serum-free medium, it is unclear whether DMG-mPEG2000 and this molar ratio performs best. We designed a library of LNPs with distinct polymer-conjugated lipids, ionizable lipids and lipid molar ratio, and tested these LNPs with npDNA encoding EGFP transposon in human primary T cells that had been activated and stimulated for 3 days in serum-free media containing 1 μg / mL rhApoE4.

[0327] The results are shown in Figures 26 and 27. The results demonstrated significant variability in GFP insertion efficiency among the different polymer-conjugated lipids, lipid molar ratio, and ionizable lipids. Notably, the ionizable lipid FL-B showed one of the highest gene insertion levels compared to a benchmark formulations using MC3, LP01 and Lipid 5. Also, Brij L23 showed with optimized lipid molar ratio showed superior gene insertion efficiency in human primary T cells.

[0328] DMG-mPEG2000 PEG(23)-lauryl ether (BrijTML23) FL-B. Lipid 5 (nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate) MC3 ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate) Lp01 ([2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate)

[0329] Table 48

[0330] Table 49

[0331] <Example 24>Optimization of Sleeping Beauty transposase mRNA and EGFP npDNA treatment Next, we optimized the doses of mRNA and npDNA for efficient gene insertion in human primary T cells. npDNA encoding eGFP and mRNA encoding SB100X were encapsulated in LNPs and administered to T cells at varying doses. Seven days post-LNP addition, GFP expression levels were quantified by flow cytometry. The results are shown in Figure 28. The highest gene integration efficiency was observed at doses of 2.5 μg / million cells of mRNA and 10 μg / million cells of npDNA. Interestingly, higher doses resulted in lower integration efficiency, indicating an optimal dosing range for maximal gene insertion.

[0332] Table 50: LNPs tested with SB100X transposase mRNA and transposon DNA

[0333] <Example 25>Enhancement of transgene integration using immune modulating agents Based on the observation that higher doses of npDNA and mRNA resulted in lower gene insertion efficiency, we speculated that exogenous nucleic acids might trigger an innate immune response. To mitigate this, we added two small molecules, BX795 and MRT67307, to the cell culture media, respectively. These molecules are potent inhibitors of the noncanonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase-epsilon (IKKε). TBK1 and IKKε play a central role in the innate immune response, serving as essential components of the interferon regulatory factor (IRF) signaling pathway. By inhibiting these kinases, we aimed to reduce the immune response and improve gene insertion efficiency. The results are shown in Table 52 and Figure 29. As we hypothesized, BX795 and MRT67307 improved gene insertion efficiency by two-fold.

[0334] Table 51: LNPs tested with SB100X transposase mRNA and transposon DNA

[0335] Table 52

[0336] We further tested whether other kinase inhibitors can be used to enhance gene integration efficiency in human primary T cells. JAK**. The results are shown in Table 53.

[0337] Table 53

[0338] <Example 26>In vitro gene insertion using CRISPR / Cas9 In most eukaryotic cells, the non-homologous end joining (NHEJ) pathway generates insertions and deletions during double-stranded break (DSB) repair. In the presence of a DNA template with homology to the sequences flanking the DSB location, homology-directed repair (HDR) can seal the DSB in an error-free manner. Therefore, the HDR pathway is commonly used to introduce targeted sequence insertions with HDR templates (HDRTs). Several methods are used to introduce HDRTs into the target cells, including viral transduction with recombinant adeno-associated virus (rAAV) or co-electroporation with naked DNA in double-strand DNA (dsDNA), single-strand DNA (ssDNA), circular, or linear formats. However, to our knowledge, there has been no successful report of LNP-mediated HDRT delivery. While rAAV vectors have facilitated rapid advances in gene therapy, their incorporation for clinical use is hindered by the cost and complexity of manufacturing these reagents. Electroporation of naked DNA presents an alternative that could accelerate innovation in gene-modified cell therapies. However, electroporation is highly damaging to cells, often impairing cell survival and proliferation. In this study, we applied our ex vivo-optimized LNP formulation to deliver Cas9 mRNA and HDRT DNA to human primary T cells. Our goal was to test whether LNPs are a viable alternative for CRISPR-based gene insertion, potentially offering a less damaging and more efficient method for precise genetic modifications. To enhance the knock-in efficiency of HDRTs, we adopted a strategy that involves incorporating Cas9 target sequences (CTSs) into HDRTs. This method allows Cas9 protein and guide RNA complex to bind the HDRTs, facilitating their delivery into nucleus using nuclear transport signal on Cas9 protein. Recognizing that single-stranded DNA (ssDNA) exhibits less toxicity compared to dsDNA, we aimed to adapt the CTS-based enhancement strategy for use with ssDNA templates. Also, HDR-mediated gene insertion is not as efficient as NHEJ-mediated gene knockout due to its slower kinetics and restriction to the S / G2 phase of the cell cycle. A critical component of the NHEJ pathway is the DNA-dependent protein-kinase catalytic subunit (DNA-PKcs) that is recruited by the Ku heterodimer to DNA DSBs to form the DNA-PK complex, which undergoes auto-phosphorylation and activates additional NHEJ factors. To address this problem, we employed NHEJ inhibitors, Nu-7441, to inhibit unwanted NHEJ-mediated DSB repair and improve HDR-mediated gene insertion. To test this approach, we tested ssDNA HDRTs containing CTSs and delivered them along with Cas9 mRNA using our ex vivo-optimized lipid nanoparticle (LNP) formulation.

[0339] The results are shown in Table 55 and Figure 33.

[0340] Table 54: LNPs tested with SB100X transposase mRNA and transposon DNA

[0341] Table 55: Summary of the effect of NHEJi and CTS on gene insertion efficiency

Claims

A method to deliver a payload to cells, which comprises adding at least one LDLR binding polypeptide to a culture media and contacting the cell with a lipid composition, wherein the lipid composition comprises a payload, an ionizable lipid, a phospholipid, a sterol and a polymer-conjugated lipid, wherein the polymer-conjugated lipid is selected from;(a) aceramide-polyethylene glycol, a phospholipid-polyethylene glycol, or a glyceride- polyethylene glycol with average polyethylene glycol molecular weight of 550-1100;(b) acholesterol-polyethylene glycol with average polyethylene glycol molecular weight of 350-5000 or 550-2000;(c) a polyethylene glycol fatty acid ester with average polyethylene glycol molecular weight of 350-2000, or 550-2000, or 1000-2000;(d) a polyoxyethylene alkyl ether with the number of ethylene oxide units of 10-25;(e) a polyethylene glycol hydrogenated castor oil or a polyethylene glycol castor oil with an average ethoxylation degree of 30-50; or(f) a polyethylene glycol(20) sorbitan fatty acid monoester.The method of claim 1, wherein the cell is an immune cell, a stem cell, or a progenitor cell.The method of claim 2, wherein the imune cell is selected from lymphocytes, monocytes, macrophages, mast cells, dendritic cells, granulocytes, primary immune cells, CD3+ cells, CD4+ cells, CD8+ T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DC).The method of claim 2, wherein the stem cell is selected from mesenchymal stem cells (MSCs); hematopoietic stem cells (HSCs); endothelial progenitor cell (EPCs); neural stem cell (NSCs); limbal stem cell (LSCs); induced pluripotent stem cell (iPSCs); ocular stem cells; pluripotent stem cell (PSCs); or embryonic stem cell (ESCs).The method of claim 1,wherein the cell is used for transplantationThe method of claim 1, which further comprises inducing expression of low density lipoprotein receptor (LDLR).The method of claim 6, wherein the LDLR binding polypeptide is an apolipoprotein.The method of claim 7, wherein the apolipoprotein is ApoE1, ApoE2, ApoE3 or ApoE4, or their fragment.The method of claim 1, wherein the payload is delivered to the cells in vitro.A lipid composition which comprises an ionizable lipid, a phospholipid, a sterol and a polymer-conjugated lipid, wherein the polymer-conjugated lipid is selected from;(a) aceramide-polyethylene glycol, a phospholipid-polyethylene glycol, or a glyceride- polyethylene glycol with average polyethylene glycol molecular weight of 550-1100;(b) acholesterol-polyethylene glycol with average polyethylene glycol molecular weight of 350-5000 or 550-2000;(c) a polyethylene glycol fatty acid ester with average polyethylene glycol molecular weight of 350-2000, or 550-2000, or 1000-2000;(d) a polyoxyethylene alkyl ether with the number of ethylene oxide units of 10-25;(e) a polyethylene glycol hydrogenated castor oil or a polyethylene glycol castor oil with an average ethoxylation degree of 30-50; or(f) a polyethylene glycol(20) sorbitan fatty acid monoester.The lipid composition of claim 10 which further comprises a payload.The lipid composition of claim 10, wherein the composition comprises 10-55 mol % ionizable lipid, 5-25 mol % phospholipid, 30 to 70 mol % sterol, and 0.25-10 mol % polymer-conjugated lipid at a mol ratio to total lipids.The lipid composition of claim 10, wherein the ionizable lipid is a compound represented by the formula (2), the formula (3) or the formula (4):In the formula, X represents -NR1- or -O-,R1represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R21-L1-R22-, where R21represents a hydrocarbon group having 1 to 24 carbon atoms, L1represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula,, and R22represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms,R2and R3each independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R31-L2-R32-, where R31represents a hydrocarbon group having 1 to 24 carbon atoms, L2represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or a group represented by the following formula,, and R32represents a divalent hydrocarbon linking group having 1 to 18 carbon atoms,R4, R5, R6, R7, R8, R9, R10, R11, and R12each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted,groups in any one or more pairs among R4and R5, R10and R5, R5and R12, R4and R6, R5and R6, R6and R7, R6and R10, R12and R7, and R7and R8may be linked to each other to form a 4- to 7-membered ring which may contain an O atom,a substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms,the substituent on the substituted or unsubstituted aryl group and on the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR45R46, or a group represented by -O(CO)O-R41, -O(CO)-R42, -(CO)O-R43, or -O-R44, where R41, R42, R43, R44, R45, and R46each independently represent a hydrocarbon group having 1 to 18 carbon atoms, anda, b, c, and d each independently represent an integer of 0 to 3, a + b is 1 or more, and c + d is 1 or more.whereinR101and R102each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and R103represents a hydrocarbon group having 2 to 8 carbon atoms, where the hydrocarbon groups represented by R101, R102, and R103may be substituted with one or more substituents selected from -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, and -O-R156,R104represents a hydrocarbon group having 1 to 8 carbon atoms,R105and R106each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R108-L101-R109, excluding a case that both R105and R106are hydrocarbon groups having 1 to 8 carbon atoms,R107represents -R110-L102-R111-L103-R112,R151and R152each independently represent a hydrocarbon group having 1 to 8 carbon atoms,R153, R154, R155, and R156each independently represent a hydrocarbon group having 1 to 24 carbon atoms,the hydrocarbon groups represented by R153, R154, R155, and R156may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R158,the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR151R152, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, -O-R156, or -(hydrocarbon group having 1 to 12 carbon atoms)-R157,R158represents a hydrocarbon group having 1 to 12 carbon atoms, andR157represents -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, or -O-R166.R161and R162each independently represent a hydrocarbon group having 1 to 8 carbon atoms,R163, R164, R165, and R166each independently represent a hydrocarbon group having 1 to 24 carbon atoms,the hydrocarbon groups represented by R163, R164, R165, and R166may be substituted with an aryl group having 6 to 20 carbon atoms or -S-R168,the above-described aryl group having 6 to 20 carbon atoms may be substituted with -OH, COOH, -NR161R162, -OC(O)O-R163, -C(O)O-R164, -OC(O)-R165, -O-R166, or -(hydrocarbon group having 1 to 12 carbon atoms),R168represents a hydrocarbon group having 1 to 12 carbon atoms, andL101, L102, and L103each independently represent -OC(O)O-, -C(O)O-, -OC(O)-, or -O-.R108represents a hydrocarbon group having 1 to 12 carbon atoms,R109represents a hydrocarbon group having 1 to 24 carbon atoms,R110represents a hydrocarbon group having 1 to 8 carbon atoms,R111represents a hydrocarbon group having 1 to 24 carbon atoms,R112represents a hydrocarbon group having 1 to 24 carbon atoms,the hydrocarbon groups represented by R109and R112may be substituted with an aryl group, -OC(O)O-R153, -C(O)O-R154, -OC(O)-R155, or -S-R158, where definitions of R153, R154, R155, and R158are as described above, andthe hydrocarbon group represented by R111may be substituted with -OC(O)O-R153, -C(O)O-R154, or -OC(O)-R155, where the definitions of R153, R154, and R155are as described above.whereinR201, R202, R203, and R204each independently represent a hydrogen, a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms,the substituted or unsubstituted hydrocarbon groups represented by R201, R202, R203, and R204each independently represent -C(O)O-R2011, -OC(O)-R2012, -O-R2013, -CO-R2014-, -OC(O)O-R2015, or -S-S-R2016,R2011, R2012, R2013, R2014-, R2015, and R2016each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with -S-R2017, and R2017represents a hydrocarbon group having 1 to 12 carbon atoms,R205and R206each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 18 carbon atoms,the substituted groups on the substituted hydrocarbon groups having 1 to 18 carbon atoms represented by R205and R206each represent -OH, -COOH, -NR2021R2022, -OC(O)O-R2023, -C(O)O-R2024, -OC(O)-R2025, -O-R2026, -C(O)NR2027R2028, -NR2029C(O)R2030, -N(R2031)S(O)2R2032, -N(R2033)C(O)N(R2034)R2035, -N(R2036)C(S)N(R2037)R2038, -OC(O)N(R2039)R2040, or -N(R2041)C(O)OR2042,R2021and R2022each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms,R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030, R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042each independently represent a hyrogen, or a substituted or unsubstituted hydrocarbon group having 1 to 24 carbon atoms, where the substituted groups on the substituted hydrocarbon group having 1 to 24 carbon atoms represented by R2023, R2024, R2025, R2026, R2027, R2028, R2029, R2030,R2031, R2032, R2033, R2034, R2035, R2036, R2037, R2038, R2039, R2040, R2041, and R2042represent an aryl group having 6 to 20 carbon atoms, a heterocyclic group, -OH, -COOH, or NR2051R2052, and R2051and R2052each independently represent a hydrogen, or a hydrocarbon group having 1 to 8 carbon atoms,R207, R208, and R209each independently represent a hydrocarbon group having 2 to 8 carbon atoms,R205and R206, or R205and R207may form a 4 to 7-membered ring together.The lipid composition of claim 10, wherein the ionizable lipid is a compound represented by the following formula (1):wherein R51and R52each independently represent a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent A,the substituent A represents a hydroxyl group, or a group represneted by -G20-CH(R55)(R56), -N(R58)(R59) or -G20-R60,G20represents -O(CO)-, or-(CO)O-,R55and R56each independently represent a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms,R58and R59each independently represent a hydrogen atom or a cyclic hydrocarbon group having 3 to 6 carbon atoms which may have a substituent B,the substituent B is-N(R61)(R62),R61and R62each independently represent a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms,R60represents a hydrocarbon group having 1 to 18 carbon atoms,L10represents a hydrocarbon group having 1 to 18 carbon atoms,G30indicates-S-(CO)-NR64,R64represents a group represented by-L30-G20-CH(R55)(R56),a represents 0 or 1,L30represents a single bond or a hydrocarbon group having 1 to 18 carbon atoms,G10represents -O(CO)-, -(CO)O-, -O(CO)O-, or -N(C(O)R63)-,R63represents a hydrocarbon group having 1 to 18 carbon atoms,L20represents a hydrocarbon group having 1 to 6 carbon atoms,b represents 0 or 1,R53, R54and R57each independently represent a hydrogen atom or a hydrocarbon group having 1 to 21 carbon atoms which may have a substituent C,the substituent C represents a group represented by-(CO)O R65or-O(CO)-R65,R65represents a hydrocarbon group having 1 to 18 carbon atoms or a group represented by-L40-CH(R66)(R67),L40represents a hydrocarbon group having 1 to 6 carbon atoms,R66and R67represent a hydrocarbon group having 1 to 10 carbon atoms or an alkoxy group.The lipid composition of claim 10, wherein the phospholipid is a phosphatidylcholine with fatty acid tails having 12 to 20 carbon atoms, a phosphatidylcholine with saturated fatty acid tails having 14 to 18 carbon atoms, or a phosphatidylcholine with saturated fatty acid tails having 18 carbon atoms.The lipid composition of claim 10, wherein the sterol is selected from cholesterol, or phytosterol.The lipid composition of claim 10, wherein the payload is a nucleic acid molecule.A method for generating one or more genetically modified cells, comprising:(a) contacting the cells with a lipid nanoparticle (LNP) composition comprising a first nucleic acid encoding a DNA binding protein with at least one nuclear localization signal sequence,(b) contacting the cells with an LNP composition comprising a second nucleic acid encoding a sequence for insertion, and the second nucleic acid comprises a sequence which bind to the DNA binding protein,wherein the LNP composition is a lipid composition of claim 10.The method of claim 18, wherein (a) the first nucleic acid is mRNA, and (b) the second nucleic acid is DNA or RNA.The method of claim 18, further comprising (c) loading the nucleic acid into pre-formed empty LNPs.

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