Targeted lipid molecules and production method therefor

Abstract

The present invention provides an efficient method for producing lipid molecules to which a ligand is bound. One aspect of the present invention provides molecules in which a ligand specific to a target cell and a lipid molecule are covalently bonded (hereinafter referred to as "targeted lipid molecules"), wherein a covalent bond (amide bond) is formed between a carboxy group of a threonine residue contained in a sortase recognition motif possessed by one of the ligand and the lipid molecule and a glycine residue or an amino group possessed by the other, and the glycine residue or the amino group is directly covalently bonded to the ligand or the lipid molecule. Another aspect of the present invention provides a production method for said targeted lipid molecules, the method comprising a step for reacting the ligand and the lipid in the presence of a sortase to covalently bond (amide bond) a carboxy group of a threonine residue contained in a sortase recognition sequence with the glycine residue or the amino group, wherein one of the ligand and the lipid molecule has the sortase recognition sequence and the other has the glycine residue or the amino group.

Description

Targeted lipid molecules and methods for producing the same

[0001] The present invention relates to targeted lipid molecules that can be used to produce lipid particles that enable the introduction of active ingredients (e.g., nucleic acids) into various types of cells, tissues, or organs, and to a method for producing the same.

[0002] [Background of the Invention] In recent years, research and development of nucleic acid drugs containing nucleic acids as active ingredients has been actively pursued. For example, numerous studies have been conducted on nucleic acid drugs containing nucleic acids such as siRNA, miRNA, miRNA mimic, or antisense nucleic acids that have the effect of degrading or inhibiting the function of target mRNA. Research is also being conducted on nucleic acid drugs containing mRNA encoding the target protein, etc., for expressing the target protein in cells. In connection with this research and development, technologies for efficiently introducing nucleic acids into cells, tissues, or organs are being developed as drug delivery system (DDS) technologies.

[0003] The above-mentioned DDS technology is conventionally known to involve mixing nucleic acids and lipids to form a complex, and then allowing the nucleic acids to be taken up by cells via this complex. Conventionally known lipids used for the formation of the above complex include cationic lipids, hydrophilic polymer lipids, and helper lipids. As for the cationic lipids, for example, compounds described in the following prior art documents are known.

[0004] Patent Document 1 describes a pharmaceutical composition comprising lipid nanoparticles (LNPs), therapeutic nucleic acids (TNAs), and at least one pharmaceutically acceptable excipient, wherein the LNPs include single-stranded variable region fragments (scFvs) linked to the LNPs, and the scFvs target antigens present on the surface of cells. Patent Document 1 also describes that scFv may be chemically conjugated to LNP via an incleavable linker, such as a maleimide-containing linker (Claim 5, etc.), that scFv may be chemically conjugated to or covalently linked to PEGylated lipids of LNP, such as DSPE [*DSPE = distearoylphosphoethanolamine]-PEG2000, DSPE-PEG5000, to form a PEGylated lipid conjugate (Claim 42, 44, 45, etc.), and that the PEGylated lipids may constitute 0-20% (mol) of the total lipids present in the LNP (Paragraph

[0384] , etc.). In Example 2 of Patent Document 1, α-HER2 scFv (SEQ ID NO: 2) derived from trastuzumab was prepared, with its C-terminus modified by a Myc tag, a His tag, and a cysteine ​​residue; the cysteine ​​residue of the modified scFv was reduced, and it was incubated with LNPs prepared separately using DSPE-PEG-maleimide in different molar percentages (0.1%, 0.5%, 0.75%, 1%, 1.25%) and PEG lengths (2k, 5k) from lipid A (scFv / maleimide ratio was 0.05). Modified scFv that did not react with the LNPs were removed by dialysis to obtain α-HER2. The document describes the production of LNPs (maleimide conjugate LNPs) in which scFv is conjugated by an inescapable covalent bond; and the evaluation of the inclusion efficiency (Figures 4A and 4B) and uptake into cells (Figures 8A and 8B) of the maleimide conjugate LNPs. Example 2 of Patent Document 1 also describes the production of scFv (SEQ ID NO: 3) for HER2 targeting, in which the N-terminus is modified with a His tag and the C-terminus is modified with an LLQGA polypeptide.

[0005] Non-patent document 1 describes anti-CD3F(ab') for introduction into T cells. ２By reducing the fragment to generate an SH group and incubating it with a mixed lipid containing 0.5% DSPE-PEG5k-maleimide and LNPs separately prepared from nucleic acids (mRNA), (F(ab') ２ Fragment: Maleimide (molar ratio = 1:1), F(ab') ２ The report describes the creation of conjugated LNPs (aCD3-LNPs) from the fragments, and the accumulation of aCD3-LNPs in the spleen after systemic administration.

[0006] Patent Document 2 describes PEG lipids (claims 1, 3, 6, 9, etc.) having a PEG chain having a predetermined functional group (maleimide, azide, dibenzocyclooctin (DBCO), etc.) and a hydrophobic carbon chain having a predetermined structure, and LNPs (claim 16, etc.) containing said PEG lipids. Example 44 of Patent Document 2 describes the preparation of LNPs containing a PEG lipid having a predetermined functional group such as maleimide (0.1% mol / mol) and several other types of lipids in a predetermined ratio; and the incubation of an anti-CD5 antibody modified with N-succinimidyl-S-acetylthioacetate (SATA) with the LNPs, and the conjugation of the antibody to the LNPs by a reaction between SATA and maleimide.

[0007] Patent Document 3 describes lipid nanoparticles having proteins in which multiple (e.g., 20 or more) DBCOs are covalently bonded per molecule, and a method for producing the same. DBCOs can be covalently bonded by a reaction that does not use azide groups and copper catalysts (click chemistry method), and the target lipid nanoparticles can be obtained by reacting DBCO-modified IgG with lipid nanoparticles containing PEG lipids having azide groups at their ends.

[0008] Non-patent document 2 describes how LNPs were prepared by mixing a lipid membrane containing predetermined lipids in predetermined ratios with an aqueous phase containing plasmids (CAR19, shIL6), and then mixing it with PSPE-PEG2000-anti-CD3 antibody (in a 1:10 ratio to the plasmid) to produce LNPs conjugated with the antibody.

[0009] Patent Document 4 describes a lipid nanoparticle (LNP) comprising a lipid blend for targeted delivery of nucleic acids to immune cells, wherein the LNP further comprises a lipid-immune cell targeting group conjugate containing a compound represented by a predetermined formula: [lipid]-[optional linker]-[immune cell targeting group], and an ionizable cationic lipid represented by a predetermined formula, wherein the nucleic acid is placed therein (Claim 1, etc.). Patent Document 4 also describes that the immune cell targeting group (e.g., an antibody) may be covalently bonded to the lipid in the lipid blend via a PEG-containing linker (Claim 6, 30, etc.), the lipid-immune cell targeting group conjugate may be present in the lipid blend in an amount ranging from 0.001 to 0.5 mole percent (Claim 9, etc.), and the lipid blend may contain free PEG-lipid (e.g., PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE)) (Claim 10, 16, etc.). In the example of Patent Document 4, LNPs were prepared using an mRNA solution and an ethanolic lipid solution (cationic lipids, cholesterol, DSPC, and DMG-PEG(2000)) (Example 2). DSPE-PEG(2000)-maleimide was conjugated with a Fab as a T cell targeting group such as an anti-CD3 Fab, resulting in DSPE-PEG-Fab:DSPE-PEG-maleimide(cysteine ​​terminus):DSPE-PEG-OCH ３ The document describes the preparation of a micelle composition consisting of a mixture in a molar ratio of 1:2.45:3.45 to 10.35 (Example 4), and the preparation of LNPs containing T cell targeting groups by combining (mixing) the LNPs and the conjugate (Example 5).

[0010] On the other hand, as a DDS technology, the following techniques are known for conjugating drugs to antibodies, etc. Patent Document 5 describes a method for producing an immune ligand / payload complex in which an immune ligand (e.g., an antibody) and a payload (a substance that confers a new function to the immune ligand, e.g., a marker, a processing tag, a drug) are conjugated using a sequence-specific transpeptidase (e.g., a saltase enzyme) or its catalytic domain. The saltase enzyme has the effect of conjugating a specific amino acid sequence motif (for example, LPXTG for saltase A derived from Staphylococcus aureus) with glycine residues, etc., and can conjugate an immune ligand having the motif or glycine residue to a payload modified with the motif or glycine residue.

[0011] Pamphlet WO2023 / 287861 (Publication No. 2024-529343), Pamphlet WO2023 / 196445, Pamphlet WO2021 / 113519, Pamphlet WO2022 / 120388, Pamphlet WO2014 / 140317 (Publication No. 2016-511279)

[0012] Kheirolomoom et al., In situ T-cell transfection by anti-CD3-ated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift, Bioconjugates, Volume 281, 2022, 121339, https: / / doi.org / 10.1016 / j.biomaterials.2021.121339.Zhou et al., Lipid nanoparticles produce Chimeric antigen receptor T cells with interleukin-6 knockdown in vivo, Journal of Controlled Release, Volume 350, October 2022, Pages 298-307, https: / / doi.org / 10.1016 / j.jconrel.2022.08.033.

[0013] One method for producing lipid particles conjugated on the surface with ligands specific to target cells is described in Non-Patent Document 2 and Patent Document 4, in which lipid molecules conjugated with ligands such as antibodies are prepared in advance and then mixed with lipid particles prepared using other lipid molecules to incorporate them into the lipid particles. However, one method for preparing ligand-conjugated lipid molecules involves reacting a ligand having a thiol group with a PEG lipid having a maleimide group at its terminus. For example, when using an antigen-binding fragment of an antibody as the ligand, the antibody is degraded with pepsin and then treated with protein A to form F(ab'). ２ Furthermore, the cross-linked structure is cleaved by reduction to obtain Fab' having a thiol group, and then a multi-step reaction is required to react it with a PEG lipid having a maleimide group at its terminus, which tends to complicate the process.

[0014] The object of this invention is to provide an efficient method for producing ligand-bound lipid molecules.

[0015] The inventors have discovered that a lipid molecule with a covalently bonded ligand can be easily produced by having a saltase recognition motif (a specific amino acid sequence) on one of the ligand and lipid molecules, and a glycine residue or amino group on the other. For example, by modifying an antibody as a ligand with a saltase recognition motif, and modifying the tip of the PEG chain of a lipid, preferably a PEG lipid, with a glycine residue or amino group, and then reacting them in the presence of saltase.

[0016] In other words, the present invention encompasses at least the following matters. As can be understood from this specification, the targeted lipid molecule in the manufacturing method of [Item 12] may be the targeted lipid molecule according to [Items 1] to [Item 8], and the matters specified for the inventions of [Items 1] to [Item 8] may be applied to the inventions of [Items 12] to [Item 13]. Similarly, the matters concerning "PEG lipid molecules" in [Items 3] and [Item 4] may be applied to the invention concerning "PEG lipids" in [Item 15]. [Item 1] A molecule in which a ligand specific to target cells and a lipid molecule are covalently bonded (hereinafter referred to as "targeted lipid molecule"), wherein a covalent bond (amide bond) is formed between the carboxyl group of a threonine residue contained in a saltase recognition motif of either the ligand or the lipid molecule and a glycine residue or amino group of the other, and the glycine residue or amino group is directly covalently bonded to the ligand or the lipid molecule. [Clause 2] The targeted lipid molecule according to Claim 1, wherein the targeted lipid molecule is a molecule in which the ligand and a lipid molecule having a polyethylene glycol (PEG) chain (hereinafter referred to as "PEG lipid molecule") are covalently bonded. [Clause 3] The targeted lipid molecule according to Claim 1 or 2, wherein the PEG lipid molecule is a PEG lipid molecule having 30 or more carbon atoms in its hydrophobic carbon chain. [Clause 4] The targeted lipid molecule according to Claim 3, wherein the PEG lipid molecule is PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE). [Clause 5] The targeted lipid molecule according to any one of Claims 1 to 4, wherein the ligand is an antibody or an antigen-binding fragment thereof that specifically binds to the cell surface antigen of the target cell. [Clause 6] The targeted lipid molecule according to Claim 5, wherein the antibody or an antigen-binding fragment has the saltase recognition motif at its C-terminus. [Clause 7] The targeted lipid molecule according to any one of Claims 1 to 6, wherein the target cell is a T cell or an NK cell. [Clause 8] The targeted lipid molecule according to any one of Clauses 5 to 7, wherein the antibody or its antigen-binding fragment is an anti-CD3 antibody or an anti-CD7 antibody, or an antigen-binding fragment thereof. [Clause 9] Lipid particles containing a mixed lipid component comprising the targeted lipid molecule according to any one of Clauses 1 to 8.[Clause 10] A nucleic acid introduction composition comprising nucleic acids and lipid particles as described in Clause 9. [Clause 11] The composition according to Clause 10, wherein the nucleic acid is DNA or RNA. [Clause 12] A method for producing a molecule (hereinafter referred to as "targeted lipid molecule") in which a ligand specific to target cells and a lipid molecule are covalently bonded, wherein one of the ligand or the lipid molecule has a saltase recognition sequence and the other has a glycine residue or an amino group, and the method comprises the step of reacting the ligand and the lipid molecule in the presence of saltase to covalently bond (amide bond) the carboxyl group of the threonine residue contained in the saltase recognition motif to the glycine residue or the amino group (hereinafter referred to as "saltase reaction step"). [Clause 13] The method for producing the product according to Clause 12, further comprising the step of purifying the reaction mixture obtained by the saltase reaction step by chromatography using a mobile phase containing water or a buffer and an organic solvent. [Clause 14] The method for producing the product according to Clause 13, wherein the organic solvent is an alcohol. [Item 15] A PEG lipid having an amino acid residue directly bound to the PEG chain at the tip of the PEG chain. [Item 16] The PEG lipid according to item 15, wherein the amino acid residue includes a glycine residue.

[0017] The present invention enables the efficient production of ligand-bound lipid molecules that can be used to produce lipid particles in which a ligand specific to a target cell is conjugated on the surface.

[0018] —Terminology— In this specification, "lipid molecules having polyethylene glycol chains" are referred to as "PEG lipid molecules." PEG lipid molecules are derivatives of general lipid molecules in which PEG chains are attached. They have hydrophobic carbon chains and hydrophilic polyethylene glycol chains, but the molecule (polymer) as a whole is hydrophilic.

[0019] In this specification, PEG lipid molecules may be referred to as the abbreviation of the original lipid molecule - PEG, or PEG - the abbreviation of the original lipid molecule, to indicate that they are molecules formed by the bonding of a PEG chain to a pre-existing lipid molecule. For example, a PEG lipid molecule formed by the bonding of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine to a PEG chain may be referred to as "DSPE-PEG" or "PEG-DSPE," using the abbreviation "DSPE" for the former and "PEG" for the latter. In such notations, the number in parentheses after PEG represents the average molecular weight (usually the number-average molecular weight) of the PEG chain. For example, "DSPE-PEG (5000)" represents a compound formed by the bonding of DSPE to a PEG chain with an average molecular weight of 5000.

[0020] In this specification, a "lipid molecule having a reactive PEG chain" is referred to as a "reactive PEG lipid molecule," and a "lipid molecule having a non-reactive PEG chain" is referred to as a "non-reactive PEG lipid molecule." A "reactive PEG chain" is a PEG chain that has a site (functional group, etc.) capable of forming a covalent bond by reacting with a ligand (e.g., an antibody or its antigen-binding fragment), and a "lipid having a non-reactive PEG chain" is a PEG chain that does not have such a site (functional group, etc.). A reactive PEG lipid molecule can also be described as a compound obtained by chemical synthesis or other means of derivatization of a PEG lipid molecule so that its PEG chain becomes reactive, that is, so that it has a site (functional group, etc.) capable of forming a covalent bond by reacting with a ligand; therefore, it is also called a "reactive derivative" of a PEG lipid molecule. Reactive PEG lipid molecules may be expressed by combining the notation of the original PEG lipid molecule with the notation (abbreviation) of the introduced site, for example, "DSPE-PEG(5000)Amine(-NH ２ ") represents a compound in which an amino group has been introduced into DSPE-PEG (5000).

[0021] In this specification, "approximately" refers to any number that falls within ±10%, ±5%, or ±1% of the number to which it is attached. Numbers described herein may be marked with "approximately" as needed.

[0022] —Targeted Lipid Molecules— The targeted lipid molecules of the present invention are molecules in which a ligand specific to target cells and a lipid molecule are covalently bonded, wherein a covalent bond (amide bond) is formed between the carboxyl group of a threonine residue contained in a saltase recognition motif of either the ligand or the lipid molecule and a glycine residue or amino group of the other, and the glycine residue or amino group is directly covalently bonded to the ligand or the lipid molecule. Such targeted lipid molecules of the present invention can be produced by reacting the carboxyl group of a threonine residue contained in a saltase recognition sequence of either the ligand, such as an antibody or its antigen-binding fragment, and the lipid molecule with a glycine residue or amino group of the other in the presence of saltase to form a covalent bond (amide bond). Details of such a production method will be described separately in this specification.

[0023] In the present invention, "directly covalently bonded" of a glycine residue or amino group to a ligand or lipid molecule means that the ligand and the glycine residue or amino group, or the lipid molecule and the glycine residue or amino group, are not bonded via specific reactive groups such as a reaction using a maleimide group (with a thiol group), a reaction using an N-hydroxysuccinimidyl group (with an amino group), or a reaction using an azide group and DBCO or other alkyne group (click chemistry). In other words, the targeted lipid molecule of the present invention does not contain a chemical structure resulting from a reaction using specific reactive groups such as a maleimide group, an N-hydroxysuccinimidyl group, or an azide group and DBCO or other alkyne group between the ligand and the glycine residue or amino group, or between the lipid molecule and the glycine residue or amino group. The "lipid molecule" to which the glycine residue or amino group is directly covalently bonded may also be a "PEG lipid molecule," in which case it means that the glycine residue or amino group is directly covalently bonded to the PEG chain of the PEG lipid molecule (not bonded via specific reactive groups). Furthermore, the amino group attached to the end of the PEG chain, which is used to introduce glycine residues or other amino acid residues in the method for producing the compound of the present invention described later, does not fall under the category of "specific reactive groups" as described above.

[0024] Sortase is a transpeptidase known in protein modification, and sortase A (SrtA) and sortase B (SrtB) derived from Staphylococcus aureus can be used. Sortase A mediates a transpeptidase reaction between the glycine residue in the motif Leu-Pro-Xxx-Thr-Gly (where Xxx is any amino acid; herein denoted as "LPXTG") and, on the other hand, a glycine residue present at the end of the reactive PEG chain of, for example, a reactive PEG lipid molecule. Therefore, a ligand (such as an antibody or an antigen-binding fragment thereof) or a lipid molecule having the recognition motif of sortase A can react with a lipid molecule (such as a reactive PEG lipid molecule) or a ligand having a glycine residue in the presence of sortase A and can form a covalent bond more directly (without passing through the azide group and DBCO used in the prior art). The glycine residue may be one, or two, three or more may be consecutive, but one or two are preferred. The glycine residue may be present as a peptide having a glycine residue at the end (for example, with a structure such as Gly-Gly-PEG lipid, Gly-Xxx-Xxx-Xxx-Xxx-Xxx-PEG lipid). Instead of the glycine residue, an amino group (for example, with a molecular structure of NH ２ -CH ２ -CH ２ - can also be used to react with the sortase recognition motif. When the ligand is an antigen or an antigen-binding fragment thereof, since an antigen recognition site (heavy chain and / or light chain variable region) is present on the N-terminal side, it preferably has a sortase recognition motif or a glycine residue or an amino group on the C-terminal side. Sortase A may be a mutant (for example, one with a higher reaction rate than the wild type), and instead of sortase A, sortase B having a recognition motif such as NPQTN, NPKTG, etc. can also be used.

[0025] The ligand specific to the target cell is not particularly limited, and any substance or molecule common in the field of lipid particle technology that specifically recognizes and binds to proteins, peptides, etc., expressed on the surface of the target cell can be used. As such ligands, for example, antibodies or antigen-binding fragments thereof that specifically bind to cell surface antigens are preferred.

[0026] • Antibodies or their antigen-binding fragments: Depending on the application, antibodies or their antigen-binding fragments that can be conjugated as ligands to the lipid particles of the present invention may be those that specifically bind to the target cell surface antigen. The target cell surface antigen is not particularly limited, but surface molecules that are specifically or highly expressed in the target cell are preferred.

[0027] Examples of antigen-binding fragments of antibodies include Fab, F(ab')2, Fab', Fv, reductive antibody (rIgG), disulfide-stabilized Fv (dsFv), single-chain Fv (single-chain antibody, scFv), dibody, tribody, HCAb, and VHH. When the lipid particles of the present invention target immune cells, as described below, Fab, Fab', and VHH are preferred as antibodies or their binding fragments.

[0028] In one embodiment of the present invention, the targeted lipid molecule of the present invention (and the lipid particle of the present invention containing the same) is used to introduce and express a gene encoding a CAR or an exogenous TCR into immune cells, more specifically, T cells responsible for cellular immunity among acquired immunity, NK cells, monocytes, macrophages, dendritic cells, etc. responsible for innate immunity, and NKT cells, which are T cells having the properties of NK cells, particularly in vivo. In such an embodiment, the targeted lipid molecule of the present invention can be one in which an antibody or an antigen-binding fragment thereof that targets immune cells, preferably T cells or NK cells, and specifically binds to their cell surface antigens is covalently bound. Examples of cell surface antigens of immune cells include CD3, CD4, CD5, CD7, CD8, CD16, CD28, CD56, etc. For example, when cytotoxic T cells are used as target cells, an anti-CD3 antibody, an anti-CD7 antibody, or an anti-CD8 antibody or their antigen-binding fragments are preferred, and when NK cells are used as target cells, an anti-CD7 antibody, an anti-CD56 antibody, or an anti-CD16 antibody or their antigen-binding fragments are preferred.

[0029] An antibody or an antigen-binding fragment thereof that specifically binds to a desired target cell surface antigen can be prepared by known methods and used in the present invention. In addition, ligands other than the antibody or its antigen-binding fragment can also be prepared by known methods and used in the present invention.

[0030] In the present invention, for covalent bonding using sortase with a lipid molecule, it is necessary to modify (generate internally or introduce externally) an antibody or an antigen-binding fragment thereof, or other ligands with a sortase recognition motif or a glycine residue or an amino group. Such modified ligands can also be prepared by known methods, for example, by using genetic recombination techniques for an antigen or its antigen-binding fragment. In the present invention, it is preferable to use an antibody or an antigen-binding fragment thereof having a sortase recognition motif on the C-terminal side as a ligand for covalent bonding with a lipid molecule.

[0031] The lipid molecule having a saltase recognition motif or a glycine residue or amino group (the target of modification thereto) for covalent bonding with the lipid molecule ligand can be selected from various lipid molecules used to constitute lipid particles, but a lipid molecule having a polyethylene glycol chain (PEG lipid molecule) is preferred. In other words, in the present invention, it is preferable to use a reactive PEG lipid molecule to which reactivity has been conferred by a saltase recognition motif or a glycine residue or amino group as the lipid molecule for covalent bonding with the ligand, and it is more preferable to use a PEG lipid molecule having a PEG chain to which a glycine residue or amino group is directly bonded.

[0032] Examples of PEG lipids include PEG-dialkyloxyalkyl (PEG-DAA) (e.g., PEG-dilauryloxypropyl [C12 x 2], PEG-dimyristyloxypropyl [C14 x 2], PEG-dipalmityloxypropyl [C16 x 2], PEG-distearyloxypropyl [C18 x 2]), PEG-diacylglycerol (PEG-DAG) (e.g., PEG-dilauroylglycerol (PEG-DLG) [C12 x 2], PEG-dimiristoylglycerol (PEG-DMG) [C14 x 2], PEG-dipalmitoylglycerol (PEG-DPG) [C16 x 2], PEG-distearoylglycerol (PEG-DSG) [C18 x 2]), and SUNBRIGHT GM-020 (NOF CORPORATION), SUNBRIGHT GS-050 (NOF CORPORATION), PEG-phospholipids (e.g., PEG-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (PEG-DMPE) [C14 x 2] such as N-(carbonyl-methoxypolyethylene glycol)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, PEG-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (PEG-DPPE) [C16 x 2] such as N-(carbonyl-methoxypolyethylene glycol)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine) PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE) [C18 x 2], PEG-1,2-dimyristyl-sn-glycero-3-phosphoethanolamine [C14 x 2], PEG-1,2-dipalmityl-sn-glycero-3-phosphoethanolamine [C16 x 2], N-(carbonyl-methoxypolyethylene glycol)-1,2-dipalmityl-sn-glycero-3-phosphoethanolaminePEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine such as 2-distearoyl-sn-glycero-3-phosphoethanolamine [C18×2]), PEG-ceramide, PEG-cholesterol, PEG-C-DOMG, 2KPEG-CMG, and the like.

[0033] The average molecular weight of the PEG chain contained in the reactive PEG lipid molecule is preferably 2000 or more (average number of repeating units of -C2H4-O- is 44 or more), more preferably 3400 or more (same 77 or more), and even more preferably 5000 or more (same 113 or more). The average molecular weight of the PEG chain contained in the non-reactive PEG lipid molecule described later is preferably 1000 or more (average number of repeating units of -C2H4-O- is 22 or more), more preferably 2000 or more (same 44 or more).

[0034] The reactive PEG lipid molecule preferably has 30 or more, 32 or more, 34 or more, or 36 or more carbon atoms in the hydrophobic carbon chain. "The number of carbon atoms in the hydrophobic carbon chain is 30 or more / 32 or more / 34 or more / 36 or more" means that the total number of carbon atoms in the carbon chains contained in the hydrophobic part of the PEG lipid, for example, in PEG-dialkyloxyalkyl (PEG-DAA), PEG-diacylglycerol (PEG-DAG), and PEG-phospholipid, is 30 or more / 32 or more / 34 or more / 36 or more. As an example, PEG-DSPE has two stearoyl groups (-O-CO-(CH2) 16 -CH3, 18 carbon atoms), that is, it is a PEG-phospholipid with 36 carbon atoms in the hydrophobic carbon chain, and it corresponds to any of the PEG lipid molecules with 30 or more / 32 or more / 34 or more / 36 or more carbon atoms in the hydrophobic carbon chain.

[0035] As one embodiment of the present invention, the reactive PEG lipid molecule preferably includes a reactive derivative of PEG-DSPE or a reactive derivative of other PEG-phospholipids.

[0036] In this invention, in order to covalently bond with a ligand using saltase, it is necessary to modify (either internally generated or externally introduced) a reactive PEG lipid molecule or other reactive lipid molecule with a saltase-recognizing motif, a glycine residue, or an amino group. Such modified lipid molecules can be prepared by known methods and are also available as general products.

[0037] In one aspect, the present invention provides a PEG lipid molecule (hereinafter referred to as "the compound of the present invention") in which an amino acid residue is directly bound to the tip of the PEG chain. The amino acid residue on the PEG chain of the compound of the present invention is typically a glycine residue suitable for producing the targeted lipid molecule of the present invention, i.e., for reacting with the saltase recognition motif of the ligand. However, by the method for producing the compound of the present invention described below, it is also possible to obtain a PEG lipid molecule in which an amino acid residue other than a glycine residue is directly bound to the tip of the PEG chain.

[0038] The method for producing the compound of the present invention will be described below.

[0039] The raw materials and reagents used in each step of the following manufacturing method, as well as the resulting compounds, may each form salts. Examples of such salts include those similar to those found in ionized lipids that can be incorporated into the lipid components constituting the lipid particles of the present invention as described above.

[0040] If the compounds obtained in each step are free compounds, they can be converted to the desired salt by known methods. Conversely, if the compounds obtained in each step are salts, they can be converted to free compounds or other types of salts of the desired type by known methods.

[0041] The compounds obtained in each step can be used in subsequent reactions as reaction solutions or as crude products. Alternatively, the compounds obtained in each step can be isolated and / or purified from the reaction mixture by conventional methods such as concentration, crystallization, recrystallization, distillation, solvent extraction, fractional distillation, chromatography, and dialysis.

[0042] If the raw materials and reagent compounds for each step are commercially available, the commercially available products can be used as is.

[0043] In each step of the reaction, the reaction time may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually 1 minute to 72 hours, preferably 10 minutes to 48 hours.

[0044] In the reaction of each step, the reaction temperature may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually -78°C to 300°C, preferably -78°C to 150°C.

[0045] In each step of the reaction, the pressure may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually between 1 atmosphere and 20 atmospheres, preferably between 1 atmosphere and 3 atmospheres.

[0046] In the reactions of each step, a Microwave synthesis apparatus such as a Biotage Initiator may be used. The reaction temperature may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually room temperature to 300°C, preferably room temperature to 250°C, and more preferably 50°C to 250°C. The reaction time may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually 1 minute to 48 hours, preferably 1 minute to 8 hours.

[0047] In each step of the reaction, unless otherwise specified, 0.5 to 20 equivalents, preferably 0.8 to 5 equivalents, of the reagent are used relative to the substrate. When the reagent is used as a catalyst, 0.001 to 1 equivalent, preferably 0.01 to 0.2 equivalents, of the reagent are used relative to the substrate. When the reagent also acts as the reaction solvent, the amount of the reagent equal to the solvent is used.

[0048] Unless otherwise specified, the reactions in each step are carried out without a solvent, or by dissolving or suspending the product in a suitable solvent. Specific examples of solvents include those described in the examples, or the following:

[0049] Alcohols: methanol, ethanol, isopropanol, isobutanol, tert-butyl alcohol, 2-methoxyethanol, etc.; Ethers: diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, 1,2-dimethoxyethane, cyclopentyl methyl ether, etc.; Aromatic hydrocarbons: chlorobenzene, toluene, xylene, etc.; Saturated hydrocarbons: cyclohexane, hexane, heptane, etc.; Amides: N,N-dimethylformamide, N-methylpyrrolidone, etc.; Halogenated hydrocarbons: dichloromethane, carbon tetrachloride, etc.; Nitriles: acetonitrile, etc.; Sulfoxides: dimethyl sulfoxide, etc.; Aromatic organic bases: pyridine, etc.; Acid anhydrides: acetic anhydride, etc.; Organic acids: formic acid, acetic acid, trifluoroacetic acid, etc.; Inorganic acids: hydrochloric acid, sulfuric acid, etc.; Esters: ethyl acetate, isopropyl acetate, etc.; Ketones: acetone, methyl ethyl ketone, etc.; Water. The above solvents may be mixed in appropriate proportions of two or more types.

[0050] When a base is used in the reaction of each step, for example, the bases shown below, or the bases described in the examples, may be used.

[0051] Inorganic bases: sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.; Basic salts: sodium carbonate, calcium carbonate, sodium bicarbonate, etc.; Organic bases: triethylamine, diethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, N,N-dimethylaniline, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene, imidazole, piperidine, etc.; Metal alkoxides: sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, etc.; Alkali metal hydrides: sodium hydride, etc.; Metal amides: sodium amide, lithium diisopropylamide, lithium hexamethyldisilazide, etc.; Organolithium compounds: n-butyllithium, sec-butyllithium, etc.

[0052] When an acid or acidic catalyst is used in the reaction of each step, for example, the acids and acidic catalysts shown below, or the acids and acidic catalysts described in the examples, may be used.

[0053] Inorganic acids: hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, etc.; Organic acids: acetic acid, trifluoroacetic acid, citric acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, etc.; Lewis acids: boron trifluoride diethyl ether complex, zinc iodide, anhydrous aluminum chloride, anhydrous zinc chloride, anhydrous iron chloride, etc.

[0054] Unless otherwise specified, the reactions in each step are based on known methods, e.g., the 5th edition of the Experimental Chemistry Course, Volumes 13-19 (edited by the Chemical Society of Japan); the New Experimental Chemistry Course, Volumes 14-15 (edited by the Chemical Society of Japan); the Revised 2nd Edition of Precision Organic Chemistry (L. F. Tietze, Th. Eicher, Nankodo); Revised Organic Named Reactions: Their Mechanisms and Key Points (by Hideo Togo, Kodansha); ORGANIC SYNTHESES Collective Volumes I-VII (John Wiley & Sons Inc.); Modern Organic Synthesis in the Laboratory: A Collection of Standard Experimental Chemistry The process is carried out according to the methods described in Procedures (by Jie Jack Li, published by Oxford University); Comprehensive Heterocyclic Chemistry III, Vol. 1-14 (Elsevier Japan Co., Ltd.); Organic Synthesis Strategies Learned from Named Reactions (supervised translation by Kiyoshi Tomioka, published by Kagaku Dojin); Comprehensive Organic Transformations (VCH Publishers Inc.), 1989, or according to the methods described in the examples.

[0055] In each step, the functional group protection or deprotection reaction is carried out according to known methods, such as those described in "Protective Groups in Organic Synthesis, 4th Edition" (Theodora W. Greene, Peter G. M. Wuts), published by Wiley-Interscience in 2007; "Protecting Groups, 3rd Edition" (P.J. Kocienski), published by Thiemé in 2004; or according to the methods described in the examples.

[0056] Examples of protecting groups for hydroxyl groups of alcohols and other substances, or phenolic hydroxyl groups, include ether-type protecting groups such as methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, and tetrahydropyranyl ether; carboxylic acid ester-type protecting groups such as acetate esters; sulfonic acid ester-type protecting groups such as methanesulfonic acid esters; and carbonate ester-type protecting groups such as t-butyl carbonate.

[0057] Examples of protecting groups for carboxyl groups include ester-type protecting groups such as methyl esters and benzyl esters; and amide-type protecting groups such as N,N-dimethylamide.

[0058] Examples of protecting groups for amino groups and aromatic heterocycles such as imidazole, pyrrole, and indole include carbamate-type protecting groups such as benzylcarbamate; amide-type protecting groups such as acetamide; alkylamine-type protecting groups such as N-triphenylmethylamine; and sulfonamide-type protecting groups such as methanesulfonamide.

[0059] Deprotection of protecting groups can be carried out using known methods, such as methods using acids, bases, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylsilyl halides (e.g., trimethylsilyl iodide, trimethylsilyl bromide), or reduction methods.

[0060] When esterification, amidation, carbamate, or urea formation reactions are carried out in each step, the reagents used include acyl halogenated compounds such as esters, carbonates, acid chlorides, and acid bromides; and activated carboxylic acids such as acid anhydrides, activated esters, and sulfated esters. Examples of carboxylic acid activators include carbodiimide-based condensing agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N,N'-dicyclohexylcarbodiimide (DCC); triazine-based condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride-n-hydrate (DMT-MM); carbonate ester-based condensing agents such as 1,1-carbonyldiimidazole (CDI); diphenyl phosphate azide (DPPA); benzotriazole-1-yloxy-trisdimethylaminophosphonium salt (BOP reagent); 2-chloro-1-methylpyridinium iodide (Mukoyama reagent); thionyl chloride; lower alkyl halomates such as ethyl chloroformate; and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium Examples include hexafluorophosphate (HATU); sulfuric acid; or combinations thereof. Additives such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), 4-nitrophenol, and dimethylaminopyridine (DMAP) may be added to the reaction. The carbonate compound can be obtained by reacting the alcohol compound with bis(4-nitrophenyl) carbonate or the like.

[0061] Scheme 1 below shows an example of a method for producing compound (I), which corresponds to the compound of the present invention. In Scheme 1, P represents a protecting group, X represents a PEG lipid, and H ２ The amino group of N-X is covalently bonded to the PEG terminus. Y is appropriately adjusted to match the structure of an N-terminally protected amino acid or N-terminally protected peptide. For example, if the compound of the present invention has a glycine residue, Y is -CH ２ - is H ２N-X can be obtained as a commercially available product, such as DSPE-PEG(5000)Amine(AVANTI), but it can also be synthesized using Scheme 2, which is described below.

[0062]

[0063] The following scheme 2 is H in scheme 1. ２ The synthesis method for N-X is shown. In Scheme 2, P represents a protecting group, L represents a releasing group, and Z represents the lipid portion of X, which are adjusted as appropriate according to the structure of X. ２ N-Z can be obtained commercially, and examples include phospholipids such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0064]

[0065] —Lipid Particles— The lipid particles in the present invention are lipid particles conjugated with a ligand specific to target cells, and contain a mixed lipid component including the targeted lipid molecule of the present invention. Conjugation refers to the fact that the lipid particles are equipped with a ligand specific to target cells, regardless of their form, embodiment, or manufacturing method, so that the lipid particles can be introduced into the target cells.

[0066] "Lipid particles" can encapsulate (contain) various substances, especially active ingredients, depending on the application, or form complexes through interactions. For example, if the lipid components constituting the lipid particles include ionized lipids (e.g., cationized lipids), they can form complexes with various substances (e.g., negatively charged nucleic acids) that have electrostatic interactions with them. The shape of the lipid particles is not particularly limited and includes, for example, complexes in which lipid components are aggregated to form a substantially spherical shape, complexes aggregated without forming a specific shape, complexes dissolved in a solvent, and complexes uniformly or heterogeneously dispersed in a dispersion medium. In the art, various embodiments of "lipid particles" have been known or common, and in the present invention, embodiments of lipid particles similar to those in the past can be used, except for introducing the configuration necessary to achieve the effects of the present invention.

[0067] Lipid components: Lipid particles are typically composed of lipid components containing two or more types of lipids. The composition of the lipid components constituting the lipid particles (types of lipid molecules and their proportions) is not particularly limited, and various lipid components known or common in the art can be used, as long as the necessary configuration for achieving the effects of the present invention is introduced (satisfying the specific conditions described herein), and in particular, the mixed lipid components containing the targeted lipid molecules of the present invention are included.

[0068] As lipid molecules for constituting lipid particles, for example, at least one molecule selected from the group consisting of cholesterols, phospholipids, PEG lipids, and ionized lipids can be used, and it is preferable to use all four of these types.

[0069] Examples of sterols include cholesterol, cholesterol esters, and cholesterol hemisuccinate. In one embodiment of the present invention, the lipid component preferably contains cholesterol as a sterol.

[0070] Phospholipids include, for example, phosphatidylcholine (e.g., dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), lysophosphatidylcholine, dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), dilinolenoylphosphatidylcholine (DLPC), diecoylphosphatidylcholine (DEPC), MC-1010 (NOF CORPORATION), MC-2020 (NOF CORPORATION), MC-4040 (NOF CORPORATION), MC-6060 (NOF CORPORATION), MC-8080 (NOF CORPORATION), etc.). Examples include phosphatidylserine (e.g., dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), palmitoyloleoylphosphatidylserine (POPS)), phosphatidylethanolamine (e.g., dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine, lysophosphatidylethanolamine), phosphatidylinositol, phosphatidic acid, etc. In one embodiment of the present invention, the lipid component preferably contains DSPC, DPPC or other phosphatidylcholine as a phospholipid.

[0071] Examples of PEG lipids include those exemplified in relation to the targeted lipid molecules of the present invention.

[0072] In one embodiment of the present invention, the lipid component preferably includes PEG-DMG as a non-reactive PEG lipid molecule (PEG lipid molecule other than a reactive PEG lipid molecule).

[0073] <Ionized Lipids> Ionized lipids are lipids that have a site that ionizes in a solvent. Various structures of ionized lipids that can be incorporated into lipid components constituting lipid particles are known, but for example, ionized lipids (cationic lipids, etc.) described in WO2016 / 021683, WO2019 / 131770, WO2019 / 131839, WO2020 / 032184, WO2023 / 085299, etc. can be used in the present invention.

[0074] Furthermore, the ionized lipids used as raw materials for the production of lipid particles may form salts with inorganic bases, organic bases, inorganic acids, organic acids, basic or acidic amino acids, and preferably pharmacologically acceptable salts.

[0075] The proportions of each lipid included in the lipid component can be appropriately adjusted depending on the type of lipid used, its chemical structure, and the intended use of the resulting lipid particles. For example, the ratio (mol%) of sterols:phospholipids:PEG lipids:ionized lipids in the total lipids present in the lipid particles is typically 10-60%:0-50%:0.1-10%:10-80%, preferably 15-55%:5-40%:0.2-5%:20-70%, and more preferably 20-50%:5-30%:0.5-2%:30-60%.

[0076] When reactive PEG lipid molecules are used to produce targeted lipid molecules, the proportion (mol%) of unreacted (not covalently bonded to the ligand) relative to the total lipid components constituting the lipid particle should be as low as possible, for example, preferably 0.1 mol% or less, more preferably 0.01 mol% or less, and even more preferably substantially zero. Such lipid particles can be produced by using a purified targeted PEG lipid molecule obtained from the reaction product of a reactive PEG lipid molecule and a ligand.

[0077] —Composition for introducing nucleic acids— The active ingredient encapsulated (encapsulated) in the lipid particles of the present invention is not particularly limited, and any substance or molecule common in the art of lipid particles that exerts a predetermined function upon delivery to target cells can be used. As such an active ingredient, nucleic acids that exert function within target cells are preferred, for example.

[0078] In other words, in one aspect of the present invention, a nucleic acid delivery composition is provided, which contains the lipid particles and nucleic acids of the present invention. In the nucleic acid delivery composition, the nucleic acids are preferably encapsulated within substantially spherical lipid particles, but a portion of the nucleic acids may be complexed in other ways or present in the composition.

[0079] In one aspect of the present invention, the lipid particles of the present invention can be used to prepare the nucleic acid introduction composition of the present invention, that is, to encapsulate (encapsulate) nucleic acids as active ingredients or to form complexes through interactions.

[0080] "Nucleic acid" refers to any molecule formed by the polymerization of nucleotides and molecules having equivalent functions to nucleotides. Examples include RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; polymers of a mixture of ribonucleotides and deoxyribonucleotides; and nucleotide polymers containing nucleotide analogs. Furthermore, nucleotide polymers containing nucleic acid derivatives may also be included. Nucleic acids may be single-stranded or double-stranded. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes with the other under stringent conditions.

[0081] Nucleotide analogs can be any molecule that has been modified from ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve nuclease resistance or stability compared to RNA or DNA, to increase affinity with complementary nucleic acids, to increase cell permeability, or to make them visible. Nucleotide analogs can be naturally occurring or unnatural molecules, such as sugar-modified nucleotide analogs or phosphate diester-modified nucleotide analogs.

[0082] As sugar-modified nucleotide analogs, any chemical structural substance can be added to or substituted for part or all of the chemical structure of the sugar of a nucleotide. Specific examples include nucleotide analogs substituted with 2'-O-methylribose, nucleotide analogs substituted with 2'-O-propylribose, nucleotide analogs substituted with 2'-methoxyethoxyribose, nucleotide analogs substituted with 2'-O-methoxyethylribose, nucleotide analogs substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogs substituted with 2'-fluororibose, nucleic acid analogs in which the sugar portion is replaced with a morpholino ring (morpholino nucleic acids), bridged nucleotides (BNA) having two cyclic structures by introducing a cross-linking structure to the sugar portion, more specifically, locked nucleotides (LNA) in which the oxygen atom at the 2' position and the carbon atom at the 4' position are cross-linked via methylene, and ethylene cross-linked nucleotides (Ethylene Examples include bridged nucleic acids (ENA) [Nucleic Acid Research, 32, e175 (2004)], amide-bridged nucleic acids (AmNA) in which the 2' and 4' carbon atoms are bridged via an amide bond, as well as peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].

[0083] Phosphate diester bond-modified nucleotide analogs can be any nucleotide in which any chemical substance is added to or substituted for part or all of the phosphate diester bond in the chemical structure of the nucleotide. Specific examples include nucleotide analogs substituted with phosphorothioate bonds and nucleotide analogs substituted with N3'-P5' phosphoamide bonds [Cell Engineering, 16, 1463-1473 (1997)] [RNAi and Antisense Methods, Kodansha (2005)].

[0084] Nucleic acid derivatives can be any molecule to which another chemical substance has been added to the nucleic acid in order to improve nuclease resistance, stabilize it, increase affinity with complementary nucleic acid chains, increase cell permeability, or make it visible. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0085] The nucleic acids in the present invention are not particularly limited and may include, for example, nucleic acids intended for the improvement of diseases, symptoms, disorders, or pathological conditions, and for the alleviation or prevention of the onset of diseases, symptoms, disorders, or pathological conditions (which may be referred to as "treatment of diseases, etc." in this specification), or nucleic acids for regulating the expression of desired proteins that are useful for research purposes but do not contribute to the treatment of diseases, etc.

[0086] Information on disease-related genes or polynucleotides (hereinafter sometimes referred to as "disease-related genes") is available, for example, from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.).

[0087] Specific examples of nucleic acids in the present invention include, for example, single-stranded DNA, double-stranded DNA, siRNA, miRNA, miRNA mimic, antisense nucleic acids, ribozymes, mRNA, circRNA, self-replicating RNA, gRNA, decoy nucleic acids, aptamers, etc., and may also be analogs or derivatives that have been artificially modified. Furthermore, nucleic acids may be linear or covalently closed circular. Preferred nucleic acids are DNA, RNA, such as single-stranded DNA, double-stranded DNA, siRNA, mRNA, circRNA, self-replicating RNA, and gRNA, or analogs or derivatives of these that have been artificially modified.

[0088] In the present invention, "siRNA" means a double-stranded RNA or its analogues having 10 to 30 bases, preferably 15 to 25 bases, and containing a complementary sequence. siRNA preferably has 1 to 3 overhanging bases, more preferably 2 bases, at its 3' end. The complementary sequence portion may be perfectly complementary or may contain non-complementary bases, but is preferably perfectly complementary.

[0089] The siRNA used in this invention is not particularly limited, and for example, siRNA for knocking down the expression of disease-related genes can be used. Disease-related genes refer to any genes or polynucleotides that produce transcription or translation products at abnormal levels or in abnormal forms in cells derived from affected tissue compared to non-disease control tissue or cells. In addition, the siRNA used in this invention can also be siRNA for regulating the expression of a desired protein useful for research.

[0090] In the present invention, "mRNA" means RNA containing a base sequence that can be translated into a protein. The mRNA in the present invention is not particularly limited as long as it is mRNA that can express a desired protein in a cell. Preferably, the mRNA is mRNA that is useful for pharmaceutical uses (e.g., disease treatment) and / or research purposes, and such mRNA is, for example, mRNA for expressing a marker protein such as luciferase in a cell.

[0091] In the present invention, "gRNA" means a guide RNA corresponding to the CRISPR system. The gRNA in the present invention may be in the form of a single RNA formed by the ligation of crRNA and tracrRNA, i.e., a chimeric RNA (sometimes called a single guide RNA, sgRNA, etc.), or it may be in the form of two unligated RNAs (a combination of two RNAs, or a combination of more than two RNAs).

[0092] In the present invention, "DNA" means DNA containing a base sequence that can be transcribed into mRNA. The DNA in the present invention is not particularly limited as long as it is DNA that can be transcribed into a desired mRNA within a cell. Preferably, the DNA is useful for pharmaceutical applications (e.g., gene therapy applications) and / or research purposes. Examples of such DNA include plasmid DNA (pDNA), single-stranded DNA (ssDNA), nanoplasmids, minicircle DNA (Minicircle), closed-end DNA (ceDNA), doggybone DNA (dbDNA), ministring DNA (msDNA), and linear DNA (linDNA). Examples of such DNA include DNA used to express marker proteins such as luciferase within a cell.

[0093] The DNA in this invention may include an enhancer or a promoter. The enhancer or promoter in this invention is not particularly limited as long as it can control the transcription to a desired mRNA within the cell. Examples of the above promoters or enhancers include the ApoE / hAAT enhancer or promoter, CAG promoter, CMV (Cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter or enhancer, SV40 promoter, DHFR (Dihydrofolate reducase) promoter, EF1α promoter, EF and CBA (Chicken β-Actin) promoter, PGK (Phosphoricerate Kinase) promoter, hSYN (human synapsin) promoter, MND promoter, RSV (Rous Sarcoma Virus LTR) promoter, and Chicken beta actin + intron promoter, TRE (tetracycline-responsive element) promoter, UBC (Ubiquitin C) promoter, MSCV U3 (Murine stem cell virus LTR) promoter, GALV U3 (Gibbon ape leukemia virus LTR) promoter, GUSB (Beta gluturonidase) promoter, MeCP2 promoter, GFAP (glial fibrillary acid protein) promoter, Human beta actin promoter, EBV (Epstein-Barr virus) promoter, SFFV (Spleen Focus) Examples include the Forming Virus LTR promoter. The CMV promoter or CAG promoter is preferred as the enhancer or promoter.

[0094] Nucleic acid introduction compositions may take the form of pharmaceutical compositions that have applications depending on the nucleic acid used, for example, for treating specific diseases. Diseases targeted for treatment or other procedures are not particularly limited, and examples include the diseases listed below (1) to (7). Unless otherwise specified, the information in parentheses indicates examples of disease-related genes. The nucleic acids used in this invention also include nucleic acids that regulate the expression levels of these disease-related genes (or the proteins they encode).

[0095] (1) Hematological disorders: Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT), lymphocyte insufficiency syndromes (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5), hemorrhagic disorders (TBXA2R, P2RX1, P2X1), H factor and Factor H-like factor 1 deficiency (HF1, CFH, HUS), factor V and factor VIII deficiency (MCFD2), factor VII deficiency (F7), factor X deficiency (F10), factor XI deficiency (F11), factor XII deficiency (F12, HAF), factor XIIIA deficiency (F13A1, F13A), factor XIIIB deficiency (F13B), Fanconi anemia (FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064, FANCB, FAN CC, FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FACE, FACE, FANCF, XRCC9, FANCG, BRIP1, BACH1, FANCJ, PHF9, FANCL, FAN CM, KIAA1596), hemophagocytic lymphohistiocytosis (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3), hemophilia A (F8, F8C, HEMA), hemophilia B (F9, HEMB), Blood disorders (PI, ATT, F5), leukocyte deficiencies (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4), sickle cell anemia (HBB), thalassemia (HBA2, HBB, HBD, LCRB, HBA1), von Willebrand disease (VWF), hypoalbuminemia, hypovolemia, severe congenital protein C deficiency, prothrombin deficiency, etc.

[0096] (2) Inflammatory and immune diseases: AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1), autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95, ALPS1A), combined immunodeficiency (IL2RG, SCIDX1, SCIDX, IMD4), HIV infection (CCL5, SCYA5, D17S135E, TCP228, IL10, CSIF, CMKBR2, CCR2, DMKBR5, CCCKR5, CCR5), immunodeficiency (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPE Inflammation (IL10, IL-1, IL-13, IL-17, IL-23, CTLA4), severe combined immunodeficiency (JAK3, JAKL, DCLRE1C, ATREMI) S, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4), primary immunodeficiency, secondary immunodeficiency, multifocal motor neuropathy, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, rheumatoid arthritis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, etc.), Sjögren's syndrome, Behçet's disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis pus nephritis, discoid lupus erythematosus, Castleman's disease, ankylosing spondylitis, polymyositis, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, scleroderma, deep lupus erythematosus, chronic thyroiditis, Graves' disease, autoimmune gastritis, type I and type II sugars. Urinary disease, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, atopic dermatitis, chronic active hepatitis, myasthenia gravis, graft-versus-host disease, Addison's disease, abnormal immune response, arthritis, dermatitis, radiation dermatitis, primary biliary cirrhosis, etc.

[0097] (3) Metabolic, hepatic, and renal diseases: Amyloid neuropathy (TTR, PALB), amyloidosis (APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB), non-alcoholic steatohepatitis and hepatic fibrosis (COL1A1), cirrhosis (KRT18, KRT8, CIRH1A, NAIC, TEX292, KI AA1988), cystic fibrosis (CFTR, ABCC7, CF, MRP7), glycogen storage disorders (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM), hepatocellular adenoma (TCF1, HFN1A, MODY3), liver failure (SCOD1, S CO1), hepatic lipase deficiency (LIPC), hepatoblastoma (CTNNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5), medullary polycystic kidney disease (UMOD, HNFJ, FJHN, MCKD2, ADMKD2), phenylketonuria (PAH) PKU1, QDPR, DHPR, PTS), polycystic kidney and liver diseases (FCYT, PKHD1, APKD, PDK1, PDK2, PDK4, PDKTS, PRKCSH, G19P1, PCLD, SEC63), Hunter syndrome, lysosomal storage diseases, Fabry disease, Pompe disease, Gaucher disease, mucopolysaccharidosis, hypoparathyroidism, Wilson's disease, etc.

[0098] (4) Neurological disorders: ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF), Alzheimer's disease (APP, AAA, CVAP, AD1, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3), Autism (BZRAP1, MDGA2, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2), Fragile X syndrome ( FMR2, FXR1, FXR2, mGLUR5), Huntington's disease (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17), Parkinson's disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, DBH, NDUFV2), Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9), schizophrenia (GSK3, 5-HTT, COMT, DRD, SLC6A3, DAOA, DTNBP1), secretase-related disorders (APH-1), etc.

[0099] (5) Eye diseases: macular degeneration (Abcr, Ccl2, cp, Timp3, カテプシンD, Vld lr, Ccr2), cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, B FSP2, CP49, CP47, PAX6, AN2, MGDA, CRYBA1, CRYB1, CR YGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BSFP2, CP4 9. CP47, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1), corneal turbidity (APOA1, TGFFB1, CSD2, CDGG1, CSD, BIGH3, CDG2, TASTD2, TROP2, M1S) 1. VSX1, RIX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD), congenital hereditary flat cornea (KERA, CNA2), green cataract (MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A), leaver Congenital agrarian syndrome (CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, C ORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6、RDH12、LCA3)、Macula ジストロフCー(ELOVL4、ADMD、STGD2 , STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2)なI.

[0100] (6) Neoplastic diseases: malignant tumors, neovascular glaucoma, infantile hemangioma, hereditary angioedema, multiple myeloma, chronic sarcoma, metastatic melanoma, Kaposi's sarcoma, vascular proliferation, cachexia, metastasis of breast cancer, etc., cancer (e.g., colorectal cancer (e.g., familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumors, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, etc.), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous cell carcinoma, etc.), breast Cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer, etc.), ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, low-grade ovarian tumor, etc.), prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), liver cancer (e.g., primary liver cancer, extrahepatic cholangiocarcinoma, etc.), thyroid cancer (e.g., medullary thyroid carcinoma, etc.), kidney cancer (e.g., renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, etc.), uterine cancer, brain tumor (e.g., pineal astrocytic tumor) (Piliocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, etc.), melanoma, sarcoma, bladder cancer, hematological cancers including multiple myeloma, pituitary adenoma, glioma, acoustic neuroma, retinal sarcoma, pharyngeal cancer, laryngeal cancer, tongue cancer, thymoma, esophageal cancer, duodenal cancer, colon cancer, rectal cancer, hepatocellular carcinoma, pancreatic endocrine tumor, bile duct cancer, gallbladder cancer, penile cancer, ureteral cancer, testicular tumor, vulvar cancer, cervical cancer, uterine body cancer, uterine sarcoma, gestational trophoblastic disease, vaginal cancer, skin cancer, mycosis fungoides, basal cell tumor, soft tissue sarcoma, malignant lymphoma Hodgkin's disease, myelodysplastic syndrome, adult T-cell leukemia, chronic myeloproliferative disorders, pancreatic endocrine tumors, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, cancer of unknown primary origin, etc.), leukemia (e.g., acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, etc.), chronic leukemia (e.g., chronic lymphoblastic leukemia, chronic myeloid leukemia, etc.), myeloplastic syndromes, etc.), uterine sarcoma (e.g., mixed mesodermal tumor, uterine leiomyosarcoma, endometrial stromal tumor, etc.), myelofibrosis, etc.

[0101] (7) Other diseases: IgA nephropathy, aplastic anemia, sarcoidosis, Williams syndrome, Marfan syndrome, muscular dystrophy, spinocerebellar degeneration, hypoparathyroidism, pemphigus, bullous pemphigoid, amyotrophic lateral sclerosis, spina bifida, hypertrophic cardiomyopathy, idiopathic thrombocytopenic purpura, ankylosing spondylitis, osteomalacia, dermatomyositis, IgG4-related disease, Usher syndrome, Apert syndrome, Alport syndrome, Angelman syndrome, West syndrome, spinal muscular atrophy, Werner syndrome, Osler disease, Crouzon syndrome, Creutzfeldt-Jakob disease, POEMS syndrome Group, prion disease, Shy-Drager syndrome, Charcot-Marie-Tooth disease, Sturge-Weber syndrome, Stevens-Johnson syndrome, SMON, Sotos syndrome, Dravet syndrome, Noonan syndrome, Buerger disease, Hirschsprung's disease, Pfeiffer syndrome, Tetralogy of Fallot, phenylketonuria, Prader-Willi syndrome, porphyria, mitochondrial disease, maple syrup urine disease, familial hypercholesterolemia, familial Mediterranean fever, Kabuki syndrome, fulminant hepatitis, tuberous sclerosis, polyarteritis nodosa, thrombotic thrombocytopenic purpura, microscopic Polyangiitis, primary sclerosing cholangitis, primary biliary cholangitis, eosinophilic sinusitis, Takayasu's arteritis, osteogenesis imperfecta, mixed connective tissue disease, neuromyelitis optica, autoimmune hepatitis, autoimmune hemolytic anemia, xeroderma pigmentosum, progressive supranuclear palsy, adult Still's disease, syringomyelia, congenital myopathy, systemic sclerosis, multiple system atrophy, aortitis syndrome, corticobasal degeneration, biliary atresia, fatal familial insomnia, toxic epidermal necrolysis, idiopathic interstitial pneumonia, achondroplasia, pustular psoriasis, pulmonary arterial hypertension, inclusion body myositis, chronic inflammatory demyelinating polyneuropathy, chronic active EB virus infection, Retinitis pigmentosa, Cushing's disease, familial chronic pyoderma, autosomal dominant polycystic kidney disease, 1p36 deletion syndrome, 22q11.2 deletion syndrome, HTLV-1 associated myelopathy, Aicardi syndrome, Weaver syndrome, granulomatosis with polyangiitis, Ehlers-Danlos syndrome, Emanuel syndrome, Klippel-Trenaunay-Weber syndrome, Cockayne syndrome, Costello syndrome, Coffin-Siris syndrome, Coffin-Lowry syndrome, Smith-Maginis syndrome, thanatophoric dysplasia, Tangier disease, CHARGE syndrome, Budd-Chiari syndrome, peroxisomal disease,Myoclonic absence epilepsy, Moebius syndrome, Menkes disease, lymphangioleiomyomatosis, Rubinstein-Taybe syndrome, Leber's hereditary optic neuropathy, subacute sclerosing panencephalitis, ossification of the ligamentum flavum, familial benign chronic pemphigus, oculocutaneous albinism, giant cell arteritis, ossification of the posterior longitudinal ligament, extensive spinal stenosis, hypertrichosis IgD syndrome, relapsing polychondritis, tricuspid atresia, congenital ichthyosis, polysplenia syndrome, pseudoxanthoma elastica, delayed endolymphatic hydrops, Nakajo-Nishimura syndrome, hypophosphatasia, idiopathic portal hypertension, Nasu-Hakola disease, refractory frequent partial seizure status epilepticus, urea cycle disorders, pulmonary alveolar proteinosis, paroxysmal nocturnal hemoglobinuria, hypertrophic dermatoperiosteopathy, bronchiolitis obliterans Arima syndrome, status epilepticus (biphasic) acute encephalopathy, Epstein syndrome, Fanconi anemia, 4p deletion syndrome, 5p deletion syndrome, Ulrich disease, Occipital-Horn syndrome, Carney complex, galactose-1-phosphate uridyltransferase deficiency, Galloway-Mowat syndrome, Mowat-Wilson syndrome, Young-Simpson syndrome, Landau-Kleffner syndrome, Rossmund-Thomson syndrome, suppurative aseptic arthritis, pyoderma gangrenosum, acne syndrome, interstitial cystitis, megalymphatic malformation, eosinophilic granulomatosis with polyangiitis, autoimmune hemorrhagic disease XIII, congenital erythrodysplasia anemia, septal optic nerve malformation, branchio-otorenephrosis, etc.

[0102] The nucleic acid delivery composition of the present invention, as a pharmaceutical composition, can be manufactured by methods known in the pharmaceutical technology field using a pharmaceutically acceptable carrier. Examples of dosage forms of the pharmaceutical composition include parenteral administration formulations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.) and topical formulations such as ointments, creams, solutions, and plasters. Parenteral administration formulations such as injections may contain conventional adjuvants such as buffers and / or stabilizers, and topical formulations may contain conventional pharmaceutical carriers.

[0103] The nucleic acid delivery composition of the present invention can be used to introduce active ingredients into a wide variety of cells, tissues, or organs. Examples of cells to which the composition of the present invention can be applied include spleen cells, nerve cells, glial cells, pancreatic B cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrous cells, muscle cells (e.g., skeletal muscle cells, cardiomyocytes, myoblasts, muscle satellite cells), adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes, megakaryocytes), synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, egg cells, spermatocytes, or progenitor cells that can be differentiated into these cells, stem cells (e.g., induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells)), hematopoietic cells, oocytes, and fertilized eggs. Furthermore, tissues or organs to which the composition of the present invention can be applied include any tissue or organ in which the above-mentioned cells exist, such as the brain, various parts of the brain (e.g., olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, corpus callosum, substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonads, thyroid gland, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (e.g., large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testes, ovaries, placenta, uterus, bone, joints, and skeletal muscle. These cells, tissues, or organs may also be cancerous cancer cells or cancerous tissue.

[0104] When the nucleic acid delivery composition of the present invention is used in vivo, typically as a pharmaceutical composition administered into the body, the target and dosage are not particularly limited and can be adjusted according to the application. The target may be a human or a non-human mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey). The dosage can be adjusted so that an effective amount of nucleic acid is delivered to the target cells and the desired effect is achieved.

[0105] In one embodiment of the present invention, the nucleic acid introduction composition of the present invention may be a composition comprising the lipid particles of the present invention and a nucleic acid encoding a chimeric antigen receptor (CAR) or a nucleic acid encoding an exogenous T cell receptor (TCR).

[0106] • Nucleic acid encoding CAR CAR is an artificially constructed hybrid protein containing an antigen-binding domain of an antibody (e.g., scFv) linked to a T cell signaling domain. Generally, CAR includes an antigen-binding domain of an antibody, an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain that can specifically recognize a surface antigen (e.g., cancer antigen peptide, surface receptors that are upregulated in cancer cells, etc.) that the target immune cells (e.g., T cells, NK cells) should recognize. The amino acid sequence of CAR and the base sequence of the nucleic acid encoding it are not particularly limited and can be adapted to the application of the nucleic acid delivery composition of the present invention.

[0107] Surface antigens specifically recognized by the antigen-binding domain of CAR include, for example, acute lymphoblastic carcinoma, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, vulvar cancer, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, and leukemia (e.g., acute lymphoblastic leukemia, acute lymphoblastic leukemia). Examples of surface antigens that are upexpressed in various cancer cells include lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), humoral neoplasms, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, cancers of the peritoneum, retinoplasm, and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer. Specific examples of such surface antigens include CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, B-cell mutation antigen (BCMA), CD123, CD3, prostate specific embrasure antigen (PSMA), CD33, MUC-1, CD138, CD22, GD2, PD- Examples include surface receptors such as L1, CEA, chondroitin sulfate protein glycycan-4, IL-13 receptor α chain, and IgG κ light chain; cancer antigen peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4, etc.; and extracellular domains of transmembrane proteins such as Claudin (CLDN) 3, CLDN4, CLDN6, and CLDN18.2.

[0108] The antigen-binding domain of a CAR can be any antibody fragment capable of specifically recognizing a target antigen, and is not particularly limited. However, considering the ease of CAR production, it is preferable to use a single-chain antibody (scFv) in which the light chain variable region and the heavy chain variable region are linked via a linker peptide. The arrangement of the light chain variable region and the heavy chain variable region in a single-chain antibody is not particularly limited as long as both can reconstruct a functional antigen-binding domain, but it can usually be designed in the order of light chain variable region - linker peptide - heavy chain variable region from the N-terminus. It is preferable that a leader sequence is further added to the N-terminus of the antigen-binding domain to present the CAR on the surface of immune cells.

[0109] The base sequences of nucleic acids encoding the light chain variable region and heavy chain variable region can be obtained based on the amino acid sequence information of the light chain variable region and heavy chain variable region of an antibody or its antigen-binding fragment that specifically binds to a target cell surface antigen, or they can be obtained by cloning the light chain gene and heavy chain gene from the antibody-producing cell.

[0110] As the linker peptide, known linker peptides commonly used in the production of single-chain antibodies can be used. Based on the amino acid sequence of the linker peptide, the base sequence of the nucleic acid encoding it can also be designed.

[0111] For the extracellular hinge domain and transmembrane domain of CAR, domains derived from T cell surface molecules, which are common in CAR construction, can be used. Examples of such extracellular hinge domains and transmembrane domains include domains derived from CD8α or CD28.

[0112] As the intracellular signaling domain of a CAR, various domains commonly used in CAR construction can be appropriately combined and used. Examples of such intracellular signaling domains include those having a CD3ζ chain, those further having co-stimulus transmission motifs such as CD28, CD134, CD137, Lck, DAP10, ICOS, and 4-1BB between the transmembrane domain and the CD3ζ chain, and those having two or more co-stimulus transmission motifs.

[0113] The base sequences of nucleic acids encoding extracellular hinge domains, transmembrane domains, and intracellular signaling domains can be designed to correspond to the amino acid sequences of each domain, and the commonly known amino acid sequences of each domain and the nucleic acid sequences encoding them are publicly available.

[0114] The nucleic acid sequence encoding the entire CAR can be designed by concatenating the nucleic acid sequences encoding the antigen-binding domain (heavy chain variable region, light chain variable region, linker peptide, etc.), the extracellular hinge domain, the transmembrane domain, and the intracellular signal transduction domain.

[0115] Nucleic acids encoding exogenous TCRs T cell receptors (TCRs) are composed of dimers of TCR chains (α-chain and β-chain) that can specifically recognize surface antigens (e.g., cancer antigen peptides, etc.) that target T cells should recognize. They are receptors that recognize antigens or antigen-HLA (human leukocyte antigen) (MHC; major histocompatibility complex) complexes and transmit stimulating signals to T cells. Each TCR chain consists of a variable region and a constant region, and the variable region contains three complementarity-determining regions (CDR1, CDR2, CDR3). In this invention, TCRs include not only those in which the α-chain and β-chain constitute a heterodimer, but also those in which they constitute a homodimer. Furthermore, TCRs also include those with a partial or complete deletion of the constant region, those with rearranged amino acid sequences, and soluble TCRs.

[0116] Furthermore, "exogenous TCR" means that it is exogenous to T cells, which are the target cells of the lipid particles of the present invention. The amino acid sequence of the exogenous TCR may be the same as or different from the endogenous TCR expressed by T cells, which are the target cells of the lipid particles of the present invention.

[0117] The amino acid sequence of the exogenous TCR and the base sequence of the nucleic acid encoding it are not particularly limited and can be adapted to the application of the nucleic acid introduction composition of the present invention.

[0118] The base sequence of the nucleic acid encoding the exogenous TCR can be obtained based on the amino acid sequence information of the TCR chain (α-chain and β-chain), or it can be obtained by cloning the gene of the T cell expressing the target TCR.

[0119] Preparation of nucleic acids encoding CAR or exogenous TCR Nucleic acids encoding CAR or exogenous TCR can be prepared by general methods based on their base sequence, for example, by chemical synthesis as DNA or RNA strands, or by joining partially overlapping oligoDNA short chains using PCR or Gibson Assembly.

[0120] The nucleic acid encoding the CAR or exogenous TCR obtained in this manner may be used directly for the preparation of the nucleic acid delivery composition, or it may be converted into an expression vector (preferably a plasmid vector) before being used for the preparation of the nucleic acid delivery composition. When preparing an expression vector, the nucleic acid encoding the CAR or exogenous TCR is preferably DNA. When preparing the nucleic acid encoding the CAR or exogenous TCR as RNA, for example mRNA, it can also be obtained by first creating an expression vector using DNA as described above, and then using that as a template in an in vitro transcription system.

[0121] Functional expression vectors for T cells, NK cells, and other immune cells can be produced using common methods. For example, functional promoters in T cells can include the SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Morony's mouse leukemia virus) LTR, and HSV-TK (herpes simplex virus thymidine kinase) promoter, which are constitutive in mammalian cells, or gene promoters such as CD3, CD4, and CD8 that are specifically expressed in T cells.

[0122] In the case of nucleic acids encoding exogenous TCRs, the DNA encoding the α-chain and the DNA encoding the β-chain may be inserted into the same expression vector or into separate expression vectors. When inserted into the same expression vector, the expression vector may express both chains polycistronically (in this case, it is appropriate to insert an intervening sequence that allows polycistronic expression, such as IRES or FMV2A, between the DNAs encoding the two chains), or it may express them monocistronically.

[0123] —Method for producing targeted lipid molecules— The present invention provides a method for producing targeted lipid molecules, comprising the steps of: reacting a ligand or lipid molecule specific to target cells, in which one of the molecules has a saltase recognition motif and the other has a glycine residue or an amino group, in the presence of saltase, to form a covalent bond (amide bond) between the carboxyl group of the threonine residue contained in the saltase recognition motif and the glycine residue or amino group (hereinafter referred to as the "saltase reaction step").

[0124] Ligands having a saltase recognition motif or a glycine residue or amino group, and lipid molecules having a glycine residue, an amino group, or a saltase recognition motif can be prepared by known methods. Lipid molecules having (directly bound) a glycine residue or amino group can be prepared by the production method described herein (method for producing the compound of the present invention).

[0125] Ligands having a saltase recognition motif, such as antigen-binding fragments of antibodies, contain a peptide moiety (e.g., LPETGGH) that includes the saltase recognition motif and a histidine tag. ６This can be done by creating an expression vector containing a nucleic acid sequence encoding the amino acid sequence of the antigen-binding fragment, including the N-terminus, introducing the expression vector into a suitable host cell and culturing it, and then recovering the produced antigen-binding fragment having the saltase recognition motif using a Ni column or the like. Similarly, antigen-binding fragments of ligands having a glycine residue at the N-terminus, such as antibodies, can be produced by using an expression vector containing a nucleic acid sequence encoding the amino acid sequence of the antigen-binding fragment, including those ligands.

[0126] Lipid molecules containing glycine residues, such as PEG lipid molecules, are generally commercially available or can be prepared by well-known and conventional methods. These include reactive lipid molecules, such as reactive PEG lipid molecules having a reactive group such as an amino group at the free end of the PEG chain (e.g., DSPE-PEG(5000)-NH ２ A saltase-recognizing motif can be prepared by reacting a saltase-recognizing motif with a glycine derivative that can react with its reactive group under appropriate conditions, and deprotecting the protecting group used in the glycine derivative as needed. A saltase-recognizing motif-containing lipid molecule, such as a PEG lipid molecule, can be prepared by reacting a commercially available or commonly prepared reactive lipid molecule, such as a reactive PEG lipid molecule having a reactive group such as a carboxyl group at the free end of the PEG chain (e.g., DSPE-PEG(5000)-COOH), with a peptide containing a saltase-recognizing motif that can react with its reactive group under appropriate conditions, and deprotecting the protecting group used in the saltase-recognizing motif as needed.

[0127] As the reactive lipid molecule, those described above in this specification, preferably reactive PEG lipid molecules, can be used. The concentration of the reactive lipid molecule in the solvent is preferably 0.5 to 100 mg / mL.

[0128] The reaction to covalently bond ligands and lipid molecules is carried out according to known methods, for example, the method described in Current Protocols in Protein Science, 89, 15.3.1-15.3.19, or the method described in the examples.

[0129] For example, when covalently attaching a ligand (e.g., an antibody or its antigen-binding fragment) having the recognition motif (LPXTG) of saltase A to a reactive lipid molecule having a glycine residue or an amino group, the ligand dissolved in a suitable buffer, a solution of a suitable amount of the reactive lipid molecule prepared using a suitable solvent (organic solvent and / or buffer), and a suitable amount of saltase prepared using a suitable buffer are prepared under suitable conditions (e.g., pH 7.0-8.5, calcium (e.g., CaCl)). ２ The reaction may be carried out in the presence of ) at 4 to 40°C, preferably about 20 to 25°C (room temperature), for several minutes to 24 hours. In such a reaction, the reactive lipid molecule can be used in amounts of, for example, 1 to 50 equivalents, preferably 3 to 30 equivalents, relative to the ligand; the saltase can be used in amounts of, for example, 0.001 to 2 equivalents, preferably 0.005 to 0.2 equivalents, relative to the ligand; and the calcium can be used in amounts of, for example, 0.1 to 200 equivalents, preferably 0.5 to 20 equivalents, relative to the ligand.

[0130] Examples of organic solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or mixtures thereof. The organic solvent may contain 0-20% water or buffer solution.

[0131] Examples of buffer solutions include acidic buffers (e.g., acetate buffer, citrate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, phosphate buffer) and neutral buffers (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate buffer, phosphate-buffered saline (PBS)). Additives such as glycerol or salts such as NaCl may be added to the buffer solution.

[0132] The method for producing targeted lipid molecules of the present invention preferably further includes a purification step after the saltase reaction step to remove molecules other than the reactive lipid molecule to which the ligand is covalently bound (i.e., reactive lipid molecules that did not covalently bind to the ligand, ligands that did not covalently bind to the reactive lipid molecule, etc.). The purification step in the method for producing targeted lipid molecules can be carried out, for example, using dialysis filtration, ultrafiltration (normal flow filtration, tangential flow filtration, etc.), or chromatography (size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, etc.). As the mobile phase used in chromatography, for example, water, or the aforementioned organic solvent, or the aforementioned buffer solution, or a mixture thereof (for example, a mixed solvent containing 50 v / v% or less of alcohols and water or a buffer solution) can be used.

[0133] In one aspect of the present invention, the method for producing targeted lipid molecules of the present invention further comprises a purification step in which the reaction mixture obtained by the saltase reaction step is purified by chromatography using a mobile phase (at least one of which is a mobile phase if there are multiple mobile phases) containing water or a buffer and an organic solvent, preferably alcohols. For example, the purification step performed by chromatography (e.g., size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography) using a mixed solvent containing 10 to 50 v / v% alcohols (e.g., about 50 v / v% ethanol) and water or a buffer (e.g., PBS, Tris buffer, acetate buffer) as the mobile phase is excellent in removing reactive lipid molecules from a reaction mixture (fraction) containing reactive lipid molecules (e.g., PEG lipid molecules having glycine residues or amino groups) used for reaction with a ligand (e.g., an antibody having a saltase recognition motif or its antigen-binding fragment), and targeted lipid molecules obtained by the reaction (e.g., PEG lipid molecules to which an antibody or its antigen-binding fragment is covalently bonded), and is preferred for increasing the purity of the targeted lipid molecules.

[0134] —Method for producing lipid particles— The lipid particles of the present invention can be produced by using the targeted lipid molecule of the present invention and modifying the process, conditions, and other embodiments to suit various methods for producing lipid particles, particularly various methods that use molecules in which a specific lipid molecule and a ligand are covalently bonded beforehand.

[0135] The present invention's method for producing lipid particles preferably involves a procedure (referred to as the "Pre method" in this specification) in which targeted lipid molecules are prepared in advance, as described in, for example, Patent Document 4 and Non-Patent Document 2 cited as prior art, and lipid particles are prepared using the targeted lipid molecules and other lipid molecules. The Pre method only requires that at least a portion of the targeted lipid molecules be prepared before the formation of the lipid particles containing the targeted lipid molecules. If necessary, a further process of conjugating ligands to the lipid particles after their formation may also be used.

[0136] The targeted lipid molecules may be those that have been prepared in advance according to the method for producing targeted lipid molecules described herein, preferably purified products of targeted lipid molecules obtained by a method for producing targeted lipid molecules that includes a purification step.

[0137] In the lipid particle preparation process, a process is carried out to form ligand-conjugated lipid particles using the targeted lipid molecule and other lipid molecules contained in the lipid components that make up the lipid particle. For example, such a process involves first preparing lipid particles containing the lipid components other than the targeted lipid molecule by mixing an organic solvent solution (organic solvent phase) with water or a buffer (aqueous phase) using various emulsification methods, microfluidic mixing systems, etc., and then adding a solution of the organic solvent, water, or buffer containing the targeted lipid molecule to the dispersion of these lipid particles and mixing.

[0138] One embodiment of the Pre method is, for example, a process of mixing (a) an organic solvent solution containing lipid molecules other than reactive lipid molecules (organic solvent phase), (b) an aqueous solution or buffer solution containing an active ingredient such as nucleic acid as needed (aqueous phase), and (c) an organic solvent solution (organic solvent phase) or aqueous solution or buffer solution (aqueous phase) containing targeted lipid molecules, preferably in a microfluidic mixing system, more preferably through a flow path designed to mix the organic solvent phase (a) and aqueous phase (b) in the system before mixing the organic solvent phase or aqueous phase (c), and after the mixing process, purifying the resulting mixture by removing the organic solvent by dialysis filtration or the like. Alternatively, ligand-conjugated lipid particles can also be formed by preparing first lipid particles with the targeted lipid molecules and some of the other lipid molecules contained in the lipid component, preparing second lipid particles with the remaining lipid molecules contained in the lipid component, and mixing them. Water or an acidic buffer is preferred as the buffer for preparing the organic solvent phase or aqueous phase (c) (including when it is a mixed solvent) containing the targeted lipid molecules.

[0139] In the Pre method, the organic solvent for dissolving lipid molecules other than reactive lipid molecules, the buffer for dissolving active ingredients such as nucleic acids as needed, and the organic solvent, water, or buffer for dissolving targeted lipid molecules can be the same organic solvents and buffers as those listed in the method for producing targeted lipid molecules. For example, to prepare the organic solvent phase or aqueous phase (c) containing targeted lipid molecules, water, the aforementioned organic solvent, the aforementioned buffer, or a mixture thereof (for example, a mixed solvent containing alcohols and 0-20% water or buffer) can be used.

[0140] The present invention preferably includes a step to purify the obtained lipid particle preparation solution (dispersion) after the preparation step, that is, a step to reduce the organic solvent contained in the lipid particle preparation solution (dispersion) (hereinafter referred to as the "purification step").

[0141] Such purification processes include, for example, desalting, dialysis, and sterile filtration, preferably by dialysis, to replace the dispersion medium of lipid particles with water or a buffer solution. Dialysis can be performed, for example, using an ultrafiltration membrane with a molecular weight cutoff of 10 to 20 K at 4°C to room temperature. Dialysis may be repeated. Tangential flow filtration (TFF) may be used to replace the dispersion medium.

[0142] After replacing the dispersion medium, pH and osmotic pressure adjustments may be performed as needed. Examples of pH adjusting agents include sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate. Examples of osmotic pressure adjusting agents include inorganic salts such as sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate; polyols such as glycerol, mannitol, and sorbitol; and sugars such as glucose, fructose, lactose, and sucrose. The pH is usually adjusted to 6.5 to 8.0, preferably to 7.0 to 7.8. The osmotic pressure is preferably adjusted to 250 to 350 Osm / kg.

[0143] The organic solvent and buffer used in the targeted PEG lipid preparation step of the Pre method can be the same as those described in relation to the method for producing the targeted lipid molecule.

[0144] The average particle size of the lipid particles of the present invention is preferably 10 to 200 nm. The average particle size of the lipid particles can be calculated by performing cumulant analysis of the autocorrelation function using a particle size measuring device based on dynamic light scattering measurement technology, such as the Zetasiner Nano ZS (Malvern Instruments). Since the lipid particles of the present invention are usually on the order of nanometers, less than 1 μm, they can also be called lipid nanoparticles (LNPs).

[0145] The method for producing lipid particles of the present invention may optionally further include steps other than the preparation and purification steps, such as a purification step. In the purification step of the method for producing lipid particles, chromatography (size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, etc.) can be used. Such a purification step can be an embodiment that is integrated with the purification step, that is, an embodiment in which solvent replacement and impurity removal are performed simultaneously.

[0146] ―Method for Producing a Nucleic Acid-Introducing Composition― The nucleic acid-introducing composition of the present invention can be produced by adding nucleic acids to an aqueous phase (water or buffer solution) for mixing with an organic solvent phase containing lipid components in the step of preparing a dispersion of lipid particles, which is included in the method for producing lipid particles of the present invention as described above (i.e., obtaining a nucleic acid-introducing composition equivalent to a dispersion of lipid particles). In such a production method (step), it is preferable to add nucleic acids in an amount such that the concentration in water or buffer solution is 0.05 to 2.0 mg / mL. An acidic buffer solution is preferred as the buffer solution for preparing the aqueous phase containing nucleic acids.

[0147] Furthermore, the nucleic acid introduction composition of the present invention can also be produced by mixing a dispersion of lipid particles of the present invention obtained by the production method described above with nucleic acids using a known method.

[0148] In the nucleic acid introduction composition of the present invention, the ratio (mass ratio) of nucleic acids (whether or not they are encapsulated in the lipid particles) to the lipid particles of the present invention is preferably 1 to 20%.

[0149] The nucleic acid encapsulation rate in the nucleic acid delivery composition of the present invention, that is, the ratio (by weight) of nucleic acids encapsulated in lipid particles rather than dissolved in the solvent to the total amount of nucleic acids in the nucleic acid delivery composition, is preferably 90% or more. The nucleic acid encapsulation rate is, for example, Quant-iT ＴＭNucleic acids can be fluorescently labeled using RiboGreen® (Invitrogen), and the difference in fluorescence intensity can be calculated based on the presence or absence of the addition of a surfactant that disintegrates lipid particles (e.g., Triton-X100).

[0150] In this specification, matters described in relation to a certain category of the present invention (e.g., targeted lipid molecules) may be referred to with appropriate modifications as matters relating to other categories (e.g., methods for producing targeted lipid molecules, lipid particles and methods for producing the same, nucleic acid introduction compositions and methods for producing the same, etc.).

[0151] The present invention will be further described in detail by the following examples, reference examples, and test examples, which are not intended to limit the present invention and may be modified without departing from the scope of the present invention.

[0152] The "DSPE-PEG(5000)Amine" (AVANTI) used in Reference Examples 1-1 and 1-2 is a compound with a total of 36 carbon atoms in its hydrophobic carbon chain (2 stearoyl groups), an average molecular weight of 5000 in the PEG chain, and an amino group at the end of the PEG chain. The "DPPE-PEG(5000)Amine" used in Reference Example 1-3 is a compound with a total of 32 carbon atoms in its hydrophobic carbon chain (2 palmoyl groups), an average molecular weight of 5000 in the PEG chain, and an amino group at the end of the PEG chain.

[0153] １ 1H NMR was measured using Fourier transform NMR. １ For 1H NMR analysis, software such as ACD / SpecManager (product name) was used. Proton peaks that are very gradual, such as those of hydroxyl groups and amino groups, may not be included in the description.

[0154] MS measurements were performed using LC / MS, HRMS, or MALDI / TOFMS. ESI, APCI, or MALDI ionization methods were used. The data recorded are the measured values ​​(found). For molecules containing PEG, representative values ​​are listed as they exhibit molecular weight distribution. While molecular ion peaks are usually observed, they may also be observed as polyvalent ions or fragment ions. In the case of salts, molecular ion peaks of the free form, cation species, anionic species, or fragment ion peaks are usually observed.

[0155] Reference Example 1: Preparation of PEG Lipid Molecules Containing Glycine Residues PEG lipid molecules containing glycine residues (Reference Examples 1-1 to 1-3) shown in Table 1 below were prepared according to the manufacturing methods described herein. More specific manufacturing methods for Reference Examples 1-1 and 1-3 are shown below.

[0156]

[0157] [Reference Example 1-1] Gly-PEG(5000)-DSPE

[0158] A) A mixture of 4-nitrophenyl N-(tert-butoxycarbonyl)glycine, N-(tert-butoxycarbonyl)glycine (9 g), 4-nitrophenol (7.15 g), EDCI (12.8 g), DMAP (3.14 g), and DCM (45 mL) was stirred at room temperature for 12 hours, then poured into water and extracted with DCM. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether) and preparative HPLC (Welch Ultimate XB-CN, hexane / ethanol) to obtain the title compound (1 g). 1 H NMR (400 MHz, CDCl3) δ 8.25 (d, J=9.0 Hz, 2H), 7.27 (d, J=9.0 Hz, 2H), 5.18-5.17 (m, 1H), 4.49 (d, J=5.4 Hz, 2H), 1.43 (s, 9H).

[0159] B) Boc-Gly-PEG(5000)-DSPE A mixture of DSPE-PEG(5000)Amine (600 mg), 4-nitrophenyl N-(tert-butoxycarbonyl)glycinate (93 mg), N,N-diisopropylethylamine (54 mg), and DCM (8 mL) was stirred at 30°C for 12 hours. The mixture was concentrated under reduced pressure, MeOH was added to the residue, and then dialyzed for 24 hours using an RC dialysis membrane (1000D) with MeOH / ion-exchanged water to obtain the title compound (540 mg). 1 H NMR (400 MHz, CDCl3) δ 6.85 - 6.66 (m, 1H), 5.53 - 5.04 (m, 2H), 4.43 - 4.10 (m, 4H), 4.07 - 3.93 (m, 2H), 3.66-3.26 (m, 422H), 2.37 - 2.23 (m, 4H), 1.68 - 1.52 (m, 4H), 1.45 (s, 9H), 1.28-1.24 (m, 56H), 0.87 (t, J=6.7 Hz, 6H).

[0160] C) Gly-PEG(5000)-DSPE A mixture of Boc-Gly-PEG(5000)-DSPE (300 mg) and DCM (3 mL) was mixed with trifluoroacetic acid (3 mL) and stirred at 35°C for 2 hours. The mixture was concentrated under reduced pressure, the residue was neutralized with saturated sodium bicarbonate aqueous solution, and then acetonitrile was added. The resulting solution was dialyzed for 12 hours using an RC dialysis membrane (1000D) (with deionized water), and then purified by preparative HPLC (Welch Xtimate C4, water (HCl) / acetonitrile) to obtain the title compound (115 mg). 1 H NMR (400 MHz, CDCl3) δ 7.98 - 7.73 (m, 2H), 5.24-5.20 (m, 1H), 4.44 - 4.26 (m, 2H), 4.24 - 4.11 (m, 2H), 4.09 - 3.26 (m, 406H), 2.31 - 2.26 (m, 4H), 1.65 - 1.51 (m, 4H), 1.25-1.22 (m, 56H), 0.88 - 0.85 (m, 6H).

[0161] [Reference Example 1-3] Gly-PEG(5000)-DPPE

[0162] A) Boc-Gly-PEG(5000)-DPPE A mixture of DPPE-PEG(5000)Amine (2.34 g) and DCM (24 mL) was mixed with N,N-diisopropylethylamine (212 mg) and 4-nitrophenyl N-(tert-butoxycarbonyl)glycinate (243 mg) and stirred at 40°C for 12 hours. The mixture was concentrated under reduced pressure to obtain the title compound (2.4 g).

[0163] B) Gly-PEG(5000)-DPPE A mixture of Boc-Gly-PEG(5000)-DPPE (2.4 g) and DCM (10 mL) was mixed with trifluoroacetic acid (10 mL) and stirred at 25°C for 2 hours. The mixture was concentrated under reduced pressure, the residue was neutralized with saturated sodium bicarbonate aqueous solution, and then acetonitrile was added. The resulting solution was dialyzed for 12 hours using an RC dialysis membrane (2000D) (acetonitrile / ion-exchanged water), and then purified by preparative HPLC (CD45-Phenomenex Luna C8, water (HCl) / acetonitrile) to obtain the title compound (1.4 g). 1 H NMR (400 MHz, CDCl3) 9.15 - 7.39 (m, 4H), 5.26 - 5.14 (m, 1H), 3.63 (m, 450H), 2.38 - 2.22 (m, 4H), 1.63 - 1.53 (m, 4H), 1.24 (m, 48H), 0.86 (t, J=6.7 Hz, 6H).

[0164] Reference Example 2: Example of preparation of ligands (Fabs) having a saltase recognition motif (LPETGG) and a histidine tag (His6) Plasmid pMG2.2 vector encoding the genes of hCD3Fab (clone: ​​OKT3), mCD3Fab (clone: ​​145-2C11), or hCD7Fab (Fab fragment of grisnilimab) having LPETGG-His6 at the C-terminus was introduced into CHOZN cells using an electroporation device (Maxcyte), and the cells were cultured for 6-8 days using EX-CELL Advanced CHO Feed 1 (with glucose). Subsequently, the ligands (LPETGG-His-tagged Fab) for Reference Examples 2-1 to 2-3 were prepared by purifying them using a compacte Ni column and a Superdex 200 size exclusion column.

[0165] Example 1: Production of Targeted Lipid Molecules Targeted lipid molecules of Examples 1-1 to 1-5 were produced using the ligands and reactive lipid molecules shown in Table 2 below, according to the production methods described herein. Mutant saltase A (P94S / D160N / D165A / K196T, Proceedings of the National Academy of Sciences of the United States of America. 2011;108:11399-11404) was used as the saltase. More specific production methods for Examples 1-1, 1-3, and 1-5 are shown below.

[0166]

[0167] [Example 1-1] 2.66 mL of PBS solution of LPETGG-His-tagged hCD3 Fab (15 mg) was mixed with 5X conjugation buffer (250 mM HEPES, 750 mM NaCl, 50% glycerol (v / v), pH 7.4, 811 μL), Gly-PEG (5000)-DSPE N,N-dimethylacetamide solution (10 mM, 469 μL), and CaCl ２A 20 mM Tris buffer (containing 150 mM NaCl and 10% glycerol (v / v), pH 8.0, 6.7 μL) with an aqueous solution (1 equivalent relative to Fab, 15 μL), saltase (0.01 equivalent relative to Fab), and water (89 μL) were mixed at 22°C. After 18 hours, EDTA solution (0.2 M, 40.5 μL) was added, and the mixture was purified by preparative HPLC (HiTrap SP HP, mobile phase A: 20 mM acetate buffer at pH 5.0, mobile phase B: 20 ​​mM acetate buffer at pH 5.0 containing 50% ethanol (v / v) and 180 mM NaCl, gradient: 30-100% B). The obtained solution was concentrated with Amicon (10K), dialyzed against PBS, and then filtered using a 0.22 μm filter to obtain the targeted lipid molecule of Example 1-1 (5.83 mg).

[0168] [Examples 1-3] 1.78 mL of PBS solution of LPETGG-His-tagged hCD3 Fab (10 mg) was mixed with 5X conjugation buffer (250 mM HEPES, 750 mM NaCl, 50% glycerol (v / v), pH 8.5, 526 μL), N,N-dimethylacetamide solution of DSPE-PEG (5000) Amine (10 mM, 313 μL), and CaCl ２ A 20 mM Tris buffer (containing 150 mM NaCl and 10% glycerol (v / v), pH 8.0, 4.5 μL) with an aqueous solution (1 equivalent relative to Fab, 0.01 equivalent relative to Fab) was mixed at 22°C. After 18 hours, EDTA solution (0.2 M, 26.3 μL) was added, and the mixture was purified by preparative HPLC (HiTrap SP HP, mobile phase A: 20 mM acetate buffer at pH 5.0, mobile phase B: 20 ​​mM acetate buffer at pH 5.0 containing 50% ethanol (v / v) and 180 mM NaCl, gradient: 30-100% B). The obtained solution was concentrated with Amicon (10K), dialyzed against PBS, and then filtered using a 0.22 μm filter to obtain the targeted lipid molecules (1.01 mg) of Examples 1-3.

[0169] [Examples 1-5] 2.47 mL of PBS solution of LPETGG-His-tagged hCD7 Fab (15 mg) was mixed with 5X conjugation buffer (250 mM HEPES, 750 mM NaCl, 50% glycerol (v / v), pH 7.4, 750 μL), Gly-PEG(5000)-DSPE N,N-dimethylacetamide solution (10 mM, 492 μL), and CaCl ２ A 20 mM Tris buffer (containing 150 mM NaCl and 10% glycerol (v / v), pH 8.0, 7.0 μL) with an aqueous solution (1 equivalent relative to Fab, 15 μL), saltase (0.01 equivalent relative to Fab), and water (19 μL) were mixed at 22°C. After 18 hours, EDTA solution (0.2 M, 37.5 μL) was added, and the mixture was purified by preparative HPLC (HiTrap SP HP, mobile phase A: 20 mM acetate buffer at pH 5.0, mobile phase B: 20 ​​mM acetate buffer at pH 5.0 containing 50% ethanol (v / v) and 180 mM NaCl, gradient: 20-100% B). The obtained solution was dialyzed against PBS, concentrated with Amicon (10K), and then filtered through a 0.22 μm filter to obtain the targeted lipid molecules (1.35 mg) of Examples 1-5.

[0170] Table 3 shows the MS measurement results before and after the reaction. After the reaction, the introduction of PEG resulted in the display of a molecular weight distribution, so representative values ​​are shown. The fact that the MS after the reaction is larger than that of the ligand before the reaction indicates that the ligand and the reactive lipid molecule were covalently bonded.

[0171]

[0172] Example 2: Removal of unreacted reactive lipid molecules by chromatography The reaction mixture obtained in the same manner as in Example 1-1 was purified using various preparative columns. The columns and mobile phases used are shown in Tables 4-6. The content of targeted lipid molecules and reactive lipid molecules in each fraction was measured by analytical HPLC, and the peak area percentages are shown in Tables 4-6. Before column purification, the peak area percentages were 20.5% for targeted lipid molecules and 75.7% for reactive lipid molecules.

[0173] The analytical conditions for the analytical HPLC are as follows: Mobile phase A: 0.05% trifluoroacetic acid (TFA) aqueous solution Mobile phase B: 0.05% TFA acetonitrile solution Detection method: Evaporative light scattering detector (ELSD) Column: Agilent PLRP-S

[0174] As shown in Table 4, under condition 2, which included ethanol in the mobile phase, the peak area percentage of reactive lipid molecules in the fraction decreased compared to condition 1, which did not include ethanol in the mobile phase. This comparative result indicates that including ethanol in the mobile phase improves the removal efficiency of reactive lipid molecules.

[0175]

[0176] As shown in Table 5, under condition 4, which included ethanol in the mobile phase, the peak area percentage of reactive lipid molecules in the fraction decreased compared to condition 3, which did not include ethanol in the mobile phase. This comparative result indicates that including ethanol in the mobile phase improves the removal efficiency of reactive lipid molecules.

[0177]

[0178] As shown in Table 6, under condition 6, which included ethanol in the mobile phase, the peak area percentage of reactive lipid molecules in the fraction decreased compared to condition 5, which did not include ethanol in the mobile phase. This comparison indicates that including ethanol in the mobile phase improves the removal efficiency of reactive lipid molecules.

[0179]

[0180] Example 3: Preparation of a nucleic acid introduction composition containing lipid nanoparticles (LNPs) containing targeted lipid molecules and nucleic acids. For the preparation of LNPs, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60:10.6:27.99:1.4, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionized lipid, 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3-pentyloctanoate, as described in WO2016 / 021683, was used.

[0181] mRNA encoding a CD19 target CAR was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0182] The lipid solution for LNP preparation and the nucleic acid solution are mixed at room temperature using NanoAssemblr. ＴＭ Ignite ＴＭ The mixture was obtained by mixing the components at a flow rate ratio of 3 ml / min to 6 ml / min using a Precision Nanosystems. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific). Subsequently, the targeted lipid molecule (Example 1-1 or Example 1-2) was mixed at 25°C to a concentration of 0.01 mol% of the total lipids and allowed to stand for 30 minutes. Then, the mixture was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and filtered using a 0.2 μm syringe filter. The obtained nucleic acid delivery composition (Example 3-1 or 3-2) was adjusted to a final mRNA concentration of 200 μg / mL and a sucrose concentration of 20%, and stored at -80°C.

[0183] LNP particle size and Polydispersity index (PDI) were measured using a Zetasizer Nano ZS (Maivern Panalogical). mRNA encapsulation rate in LNPs was measured using Quant-it. ＴＭ Measurements were performed using the RiboGreen RNA Assay Kit (Thermo Fisher Scientific). The mRNA concentration measured after lysis of LNPs with 0.5% Triton X-100 was defined as the total mRNA concentration, and the mRNA concentration measured without the addition of Triton X-100 was defined as the mRNA concentration not encapsulated in the LNPs. The mRNA encapsulation rate in LNPs was then calculated. The results are shown in Table 7.

[0184]

[0185] Test Example 1: In vitro CAR expression test on human T cells Human T cells were isolated from frozen PBMCs (HemaCare) using a human T cell isolation kit (STEMCELL Technologies) according to the attached protocol. The isolated T cells were suspended in X-VIVOTM15 Serum-free Hematopoietic Cell Medium containing 10 ng / ml IL-2 (Milteny Biotech), mixed with a nucleic acid delivery composition of Example 3-1 or 3-2 at an RNA concentration of 3 μg / mL, and cultured for 72 hours. After culturing, cells were harvested, and CAR was stained using PE-labeled human CD19 (20-291) protein His-Tag (ACROBiossystems). Expression levels were analyzed by flow cytometry (LSRFortessa, BD Biosciences). The measurement results are shown in Table 8.

[0186] As shown in Table 8, the nucleic acid delivery compositions of Example 3-1 and Example 3-2 (containing the targeted lipid molecules of Example 1-1 and Example 1-2, respectively) showed high CAR expression levels.

[0187]

[0188] The targeted lipid molecules of the present invention can be efficiently produced with fewer reaction steps and are suitably used for producing lipid particles or nucleic acid delivery compositions that enable the efficient introduction of active ingredients such as nucleic acids into various cells, tissues, or organs.

Claims

A molecule in which a ligand specific to a target cell is covalently bonded to a lipid molecule (hereinafter referred to as "targeted lipid molecule"). A targeted lipid molecule wherein a covalent bond (amide bond) is formed between the carboxyl group of a threonine residue contained in the saltase recognition motif of one of the ligand or the lipid molecule and a glycine residue or amino group of the other, and the glycine residue or amino group is directly covalently bonded to the ligand or the lipid molecule. 　 The targeted lipid molecule according to claim 1, wherein the targeted lipid molecule is a molecule in which the ligand and a lipid molecule having a polyethylene glycol (PEG) chain (hereinafter referred to as "PEG lipid molecule") are covalently bonded. 　 The targeted lipid molecule according to claim 2, wherein the PEG lipid molecule is a PEG lipid molecule having 30 or more carbon atoms in its hydrophobic carbon chain. 　 The targeted lipid molecule according to claim 3, wherein the PEG lipid molecule is PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE). 　 The targeted lipid molecule according to claim 1, wherein the ligand is an antibody or an antigen-binding fragment thereof that specifically binds to the cell surface antigen of the target cell. 　 The targeted lipid molecule according to claim 5, wherein the antibody or its antigen-binding fragment has the saltase recognition motif at its C-terminus. 　 The targeted lipid molecule according to claim 1, wherein the target cell is a T cell or an NK cell. 　 The targeted lipid molecule according to claim 5, wherein the antibody or its antigen-binding fragment is an anti-CD3 antibody or an anti-CD7 antibody, or an antigen-binding fragment thereof. 　 Lipid particles containing a mixed lipid component including the targeted lipid molecule described in claim 1. 　 A nucleic acid introduction composition comprising nucleic acids and lipid particles according to claim 9. 　 The composition according to claim 10, wherein the nucleic acid is DNA or RNA. 　 A molecule in which a ligand specific to a target cell is covalently bonded to a lipid molecule (hereinafter referred to as "targeted lipid molecule"). A method for manufacturing ) The ligand or the lipid molecule has a saltase recognition motif, and the other has a glycine residue or an amino group. A method for producing a product, comprising the step of reacting the ligand and the lipid molecule in the presence of saltase to form a covalent bond (amide bond) between the carboxyl group of the threonine residue contained in the saltase recognition motif and the glycine residue or the amino group. 　 The production method according to claim 12, further comprising the step of purifying the reaction mixture obtained by the saltase reaction step by chromatography using a mobile phase containing water or a buffer and an organic solvent. 　 The manufacturing method according to claim 13, wherein the organic solvent is an alcohol.

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