Method for producing lipid particles conjugated with ligand specific to target cell

Abstract

The present invention provides a method for efficiently producing lipid particles that contain an active component and in which the surfaces of the lipid particles are conjugated with a ligand specific to a target cell. The method for producing lipid particles with a ligand conjugated to the surface thereof includes: (1) a step for mixing an organic solvent phase (a) containing a lipid component, an aqueous phase (b) containing an active component, and an organic solvent phase or an aqueous phase (c) containing a molecule (targeting lipid molecule) in which a ligand specific to a target cell and a lipid are covalently bonded; and (2) a step for purifying the mixture obtained in step (1).

Description

Method for producing lipid particles conjugated with ligands specific to target cells.

[0001] The present invention relates to lipid particles that enable the introduction of active ingredients (e.g., nucleic acids) into various types of cells, tissues, or organs, and to compositions containing the lipid particles and nucleic acids.

[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] 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.

[0008] Patent Document 3 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, the LNP further comprising a nucleic acid placed therein (Claim 1, etc.). Patent Document 3 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 a range of 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 3, 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).

[0009] Special Publication No. 2024-529343 (corresponding to WO2023 / 287861 pamphlet), WO2023 / 196445 pamphlet, WO2022 / 120388 pamphlet

[0010] Kheirolomoom et al., In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift, Biomaterials, 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.

[0011] A method for producing lipid particles containing an active ingredient, conjugated with a ligand specific to target cells (e.g., an antibody or its antigen-binding fragment), is described as follows: (1) a production method (hereinafter referred to as the "Post method"), in which an organic solvent phase containing a total lipid component, which includes a lipid (reactive lipid) having a site (functional group, etc.) capable of forming a covalent bond by reacting with an antibody or its antigen-binding fragment, is mixed with an aqueous phase containing an active ingredient to obtain a lipid particle preparation (dispersion), and then the reactive lipid in the lipid particle preparation is reacted with the ligand to form a covalent bond; and (2) a production method described as follows, in Non-Patent Document As described in Patent Documents 2 and 3, the manufacturing method can be broadly classified into two types (referred to as the "Pre method" herein): one in which a reactive lipid is reacted with a ligand to obtain a molecule in which the reactive lipid and ligand are covalently bonded (targeted lipid molecule); the other in which an organic solvent phase (solvent is, for example, ethanol) containing lipid components that constitute lipid particles other than the targeted lipid molecule is mixed with an aqueous phase containing an active ingredient to obtain a lipid particle preparation (dispersion) that does not contain the targeted lipid molecule; the organic solvent is removed by dialysis or the like; and then the organic solvent phase or aqueous phase (solvent is, for example, a mixture of alcohol and a buffer) containing the previously prepared targeted lipid molecule is mixed to incorporate it into the lipid components that constitute the lipid particle.

[0012] However, in the Post method, for example, the reaction efficiency between reactive lipids and ligands is a problem, and reactive lipids that do not react with the ligand tend to remain in the lipid particles and be wasted. On the other hand, in the Pre method, in order to prevent the added targeted lipid molecules from being denatured, it is common to remove the organic solvent by dialysis or the like after obtaining a lipid particle composition (dispersion) that does not contain the targeted lipid molecules, which makes the process complicated.

[0013] The present invention aims to provide an efficient method for producing lipid particles containing an active ingredient, on which a ligand specific to target cells is conjugated to the surface.

[0014] Firstly, the inventors have found that the above problems can also be solved by a manufacturing method (sometimes referred to herein as the "One Step method") in which, unlike the conventional Pre method, the lipid particle preparation (dispersion) is mixed with the organic solvent phase or aqueous phase containing the targeted lipid molecule before the step of removing the organic solvent by dialysis or the like. In other words, the function of ligands specific to target cells (e.g., antibodies or their antigen-binding fragments) is not impaired, and the active ingredient can be delivered to various cells with good efficiency. Furthermore, the inventors have found that even when targeted lipid molecules are incorporated into an organic solvent phase containing lipid components or an aqueous phase containing active ingredients, it may be possible to obtain a product that does not impair the function of the ligand specific to target cells and can deliver the active ingredient to various cells with good efficiency.

[0015] Secondly, the present inventors have found that the above problems can also be solved by a manufacturing method (sometimes referred to herein as the "Quick Prep method") for obtaining targeted, non-encapsulating lipid particles (targeted cavity lipid particles) by introducing a ligand specific to target cells, and by a manufacturing method for obtaining lipid particles (targeted loaded lipid particles) having a ligand specific to target cells on its surface and encapsulating the active ingredient by mixing a mixture containing the cavity lipid particles obtained thereby with an aqueous phase containing an active ingredient. In addition, the present inventors have also found several preferred embodiments as steps for obtaining targeted cavity lipid particles.

[0016] In other words, the present invention encompasses at least the following: [Item 1] A method for producing lipid particles on which the ligand is conjugated on the surface, comprising the steps of: (1) mixing an organic solvent phase (a) containing a lipid component, an aqueous phase (b) containing an active ingredient, and an organic solvent phase or aqueous phase (c) containing a molecule in which a ligand specific to target cells and a lipid are covalently bonded (hereinafter referred to as "targeted lipid molecule"); and (2) purifying the mixture obtained in step (1). [Item 2] The method according to item 1, wherein step (1) is performed using a microfluidic mixing system. [Item 3] The method according to item 1, wherein step (1) comprises: (1-1) a process of mixing the organic solvent phase (a) and the aqueous phase (b); and (1-2) a process of mixing the mixture prepared by the process with the organic solvent phase or aqueous phase (c). [Item 4] The method for producing a lipid according to any one of items 1 to 3, wherein the targeted lipid molecule is formed by a covalent bond between a saltase recognition sequence on one of the ligand or the lipid molecule and a glycine or amino group on the other. [Item 5] The method for producing a lipid according to any one of items 1 to 4, wherein the targeted lipid molecule is a molecule in which the ligand and a lipid having a polyethylene glycol (PEG) chain (hereinafter referred to as "PEG lipid") are covalently bonded. [Item 6] The method for producing a lipid according to item 5, wherein the PEG lipid is a PEG lipid having 30 or more carbon atoms in its hydrophobic carbon chain. [Item 7] The method for producing a lipid according to item 6, wherein the PEG lipid is PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE). [Item 8] The method for producing a lipid according to any one of items 1 to 7, 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 9] The method for producing an antibody according to any one of Clauses 1 to 8, wherein the cell surface antigen is a cell surface antigen of a T cell or an NK cell. [Clause 10] The method for producing an antibody according to any one of Clauses 1 to 9, 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 11] The method for producing an antibody according to any one of Clauses 1 to 10, wherein the active ingredient contains nucleic acid.[Clause 12] The method for producing a lipid particle according to Claim 11, wherein the nucleic acid is DNA or RNA. [Clause 13] The method for producing a lipid particle according to Claim 11 or 12, wherein at least a portion of the nucleic acid is encapsulated in the lipid particle. [Clause 14] Lipid particles obtained by the method for producing a lipid particle according to any one of Claims 1 to 13. [Clause 15] A method for producing targeted, non-active lipid particles (hereinafter referred to as "cavity lipid particles"), comprising the step of introducing a ligand specific to target cells. [Clause 16] The method for producing a lipid particle according to Claim 15, comprising the steps of: (A1) mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing cavity lipid particles; and (A2) mixing the mixture obtained in step (A1) with an organic solvent phase or aqueous phase (c) containing a targeted lipid molecule to obtain targeted cavity lipid particles. [Clause 17] The manufacturing method according to Clause 15, comprising: (B1) mixing an organic solvent phase (a) containing a lipid component having a reactive polyethylene glycol (PEG) chain (hereinafter referred to as "reactive PEG lipid") with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing cavity lipid particles; (B2) reacting a ligand specific to target cells with the reactive PEG lipid contained in the lipid component constituting the lipid particles to form a covalent bond, thereby obtaining targeted cavity lipid particles. [Clause 18] The manufacturing method according to Clause 15, comprising: (C1) mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent containing a reactive PEG lipid to prepare a mixture containing cavity lipid particles; (C2) reacting a ligand specific to target cells with the reactive PEG lipid contained in the lipid component constituting the lipid particles to form a covalent bond, thereby obtaining targeted cavity lipid particles. [Clause 19] The method for producing a product according to Claim 15, comprising the step of (D1) mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent containing targeted lipid molecules to prepare a mixture containing targeted cavity lipid particles.[Clause 20] The method for producing a target cell, comprising the steps of: (E1) mixing an organic solvent phase (a) containing a lipid component including a targeted lipid molecule with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing targeted cavity lipid particles. [Clause 21] The method for producing a target cell, comprising the steps of: (F1) mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing cavity lipid particles; (F2) mixing the mixture obtained in step (F1) with an organic solvent phase or aqueous phase (c) containing a reactive PEG lipid; and (F3) reacting a ligand specific to target cells with the reactive PEG lipid contained in the lipid component constituting the lipid particle to form a covalent bond, thereby obtaining targeted cavity lipid particles. [Clause 22] The method for producing an aqueous solution according to any one of claims 16 to 21, further comprising the step of (G) replacing the solvent of the mixture containing the cavity lipid particles or targeted cavity lipid particles obtained in any of the steps above with a second aqueous solvent (b2). [Clause 23] The method for producing an aqueous solution according to claim 22, wherein the first aqueous solvent and the second aqueous solvent are aqueous solvents having different components. [Clause 24] The method for producing an aqueous solution according to claim 22 or 23, wherein the second aqueous solvent is an aqueous solvent containing 2-morpholinoethanesulfonic acid (MES) buffer or water. [Clause 25] The method for producing an aqueous solution according to any one of claims 16 to 21, wherein the mixing is carried out using a microfluidic mixing system. [Clause 26] A method for producing targeted-loaded lipid particles, comprising the step of (H) mixing a mixture containing targeted-loaded cavity lipid particles with an aqueous phase (b3) containing an active ingredient and a third aqueous solvent to prepare a mixture containing lipid particles having ligands specific to target cells on their surface and encapsulating the active ingredient (hereinafter referred to as "targeted-loaded lipid particles"). [Clause 27] The method for producing targeted-loaded lipid particles according to Claim 26, wherein the step of replacing the solvent in the mixture obtained in step (H) with an aqueous solvent after step (H). [Clause 28] The method for producing targeted-loaded lipid particles according to Claim 26 or 27, wherein the active ingredient contains nucleic acid. [Clause 29] The method for producing targeted-loaded lipid particles according to Claim 28, wherein the nucleic acid is DNA or RNA. [Clause 30] Lipid particles having ligands specific to target cells on their surface and not encapsulating the active ingredient (targeted-loaded cavity lipid particles).[Item 31] A kit comprising lipid particles (targeted cavity lipid particles) that have a ligand specific to target cells on their surface and do not contain an active ingredient.

[0017] The present invention makes it possible to efficiently produce lipid particles containing an active ingredient, on which a ligand specific to target cells is conjugated to the surface.

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

[0019] In this specification, PEG lipids are sometimes 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 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, "lipids having a reactive PEG chain" are referred to as "reactive PEG lipids," and "lipids having a non-reactive PEG chain" are referred to as "non-reactive PEG lipids." A "reactive PEG chain" is a PEG chain that has a site (functional group, etc.) capable of forming a covalent bond in reaction with a ligand (e.g., an antibody or its antigen-binding fragment), while a "lipid having a non-reactive PEG chain" is a PEG chain that does not have such a site (functional group, etc.). Reactive PEG lipids can also be described as compounds obtained by chemical synthesis or other means from PEG lipids so that the PEG chain of the PEG lipid becomes reactive, that is, has a site (functional group, etc.) capable of forming a covalent bond in reaction with a ligand; therefore, they are also called "reactive derivatives" of PEG lipids. Reactive PEG lipids are sometimes expressed by combining the notation of the original PEG lipid with the notation (abbreviation) of the introduced site. For example, "DSPE-PEG(5000)Azide" represents a compound in which an azide group for a click reaction has been introduced into DSPE-PEG(5000).

[0021] In this specification, a "molecule in which a PEG lipid and a ligand are covalently bonded" is referred to as a "targeted PEG lipid."

[0022] In this specification, lipid particles that do not contain an active ingredient are referred to as "cavity lipid particles," and lipid particles that contain an active ingredient are referred to as "carrying lipid particles." Furthermore, cavity lipid particles and carrying lipid particles that have a ligand specific to target cells conjugated on their surface are referred to as "targeted cavity lipid particles" and "targeted carrying lipid particles," respectively.

[0023] 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.

[0024] —Lipid Particles— The targeted lipid particles in this invention are lipid particles conjugated with a ligand specific to target cells. Regardless of the form, embodiment, or manufacturing method, conjugation refers to the fact that the lipid particles possess a ligand specific to target cells so that the lipid particles can be introduced into the target cells.

[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 lipid particles of the present invention are used to introduce and express genes encoding CAR or exogenous TCR in immune cells, more specifically T cells responsible for cellular immunity among acquired immunity, NK cells responsible for innate immunity, monocytes, macrophages, dendritic cells, etc., and NK T cells which are T cells having the properties of NK cells, particularly in vivo. In such embodiments, the lipid particles of the present invention can be conjugated with antibodies or antigen-binding fragments that specifically bind to the cell surface antigens of immune cells, preferably T cells or NK cells. 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 targeted, anti-CD3 antibodies, anti-CD7 antibodies, or anti-CD8 antibodies or their antigen-binding fragments are preferred, and when NK cells are targeted, anti-CD7 antibodies, anti-CD56 antibodies, or anti-CD16 antibodies or their antigen-binding fragments are preferred.

[0029] Antibodies or antigen-binding fragments that specifically bind to a desired target cell surface antigen can be prepared by known methods and used in the present invention. When introducing a functional group capable of forming a covalent bond with a functional group of a reactive lipid, such as DBCO, into an antibody or antigen-binding fragment using a transpeptidase for conjugation to lipid particles, the antibody or antigen-binding fragment must first possess an oligopeptide corresponding to a saltase recognition motif. Such antibodies or antigen-binding fragments possessing a saltase recognition motif can also be prepared by known methods, such as genetic engineering.

[0030] "Lipid particles" can encapsulate (enclose) various substances according to their uses, especially active ingredients, or form complexes through interactions. For example, when the lipid component constituting the lipid particles contains ionized lipids (e.g., cationized lipids), it can form complexes with various substances (e.g., negatively charged nucleic acids) that exhibit electrostatic interactions with it. The shape of the lipid particles is not particularly limited. For example, it includes complexes in which lipid components aggregate to form a substantially spherical shape, complexes that aggregate without forming a specific shape, complexes dissolved in a solvent, and complexes uniformly or non-uniformly dispersed in a dispersion medium. Conventionally, various embodiments of "lipid particles" are known or common in this technical field. In the present invention as well, embodiments of lipid particles similar to the conventional ones can be used, except for introducing the configurations necessary to achieve the effects of the present invention.

[0031] - Lipid component Lipid particles are usually composed of a lipid component containing two or more types of lipids. The composition of the lipid component constituting the lipid particles (the types of lipids and their blending amounts) is not particularly limited, and various lipid components known or common in this technical field can be adopted, except for introducing the configurations necessary to achieve the effects of the present invention (satisfying the specific conditions described in this specification).

[0032] As the lipid for constituting the lipid particles, for example, at least one selected from the group consisting of sterols, phospholipids, lipids having a polyethylene glycol chain (PEG lipids), and ionized lipids can be used, and it is preferable to use all four of these types.

[0033] Examples of sterols include cholesterol, cholesterol ester, cholesterol hemisuccinate, etc. As one embodiment of the present invention, it is preferable that the lipid component contains cholesterol as a sterol.

[0034] As phospholipids, for example, phosphatidylcholine (e.g., dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), lysophosphatidylcholine, dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), dilinolenoylphosphatidylcholine (DLPC), dieleoylphosphatidylcholine (DEPC), MC-1010 (NOF CORPORATION), MC-2020 (NOF CORPORATION), MC-4040 (NOF CORPORATION), MC-6060 (NOF CORPORATION), MC-8080 (NOF CORPORATION), etc.), 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. are exemplified. As one embodiment of the present invention, the lipid component preferably contains DSPC, DPPC or other phosphatidylcholine as the phospholipid.

[0035] Examples of lipids having polyethylene glycol chains (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-dimyristoylglycerol (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-phosphoethanolamineExamples include PEG-1,2-distearyl-sn-glycero-3-phosphoethanolamine [C18 x 2], PEG-ceramide, PEG-cholesterol, PEG-C-DOMG, 2KPEG-CMG, etc.

[0036] In one embodiment of the present invention, the lipid component preferably includes a reactive PEG lipid, such as a reactive derivative of PEG-DSPE or another reactive derivative of PEG-phospholipid.

[0037] In one embodiment of the present invention, the lipid component preferably includes PEG-DMG as a PEG lipid.

[0038] <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.

[0039] 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.

[0040] 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%.

[0041] In the present invention, the form of covalent bonding between a ligand, such as an antibody or its antigen-binding fragment, and a lipid is not particularly limited, and various forms of covalent bonding known or common in the art can be employed. For example, the ligand may form a covalent bond with a lipid (reactive PEG lipid) having a PEG chain comprising a maleimide group, a thiol group, an azide group, a DBCO or other alkyne group, a saltase recognition sequence, an amino group, glycine, etc.

[0042] <Reaction between maleimide group and thiol group> Reactive PEG lipids having a maleimide group can react with antibodies or antigen-binding fragments having a thiol group, such as Fab' produced by treating F(ab')2 with a reducing agent to cleave the disulfide bond, or with antigens or antigen-binding fragments treated with sulfhydrylation reagents (e.g., SATA), to form a covalent bond (thioether bond). Bromomaleimide groups or bromoacetamide groups can also be used instead of maleimide groups.

[0043] <Click Chemistry: Reaction of Azide Groups with DBCO, etc.> Reactive PEG lipids having azide groups can react with antibodies or antigen-binding fragments having DBCO (dibenzocyclooctin), for example, derivatives obtained by treating antibodies, etc., with DBCO-NHS ester to bind NHS groups to amino groups (e.g., lysine residues) of the antibodies, etc., and form covalent bonds. Such reactions between azide groups and DBCO are preferable because they do not require a copper catalyst. In click chemistry, compounds having other alkyne groups (carbon-carbon triple bonds) can be used instead of DBCO, but a copper catalyst may be required. In addition, to introduce DBCO, etc., into antibodies, etc., a method using a transpeptidase such as saltase, as described below, can also be used. For example, a method in which an antibody, etc., to which a saltase recognition motif (LPXTG, etc.) has been introduced to the C-terminus beforehand is reacted with DBCO to which a glycine residue (GG, etc.) is bound, in the presence of saltase.

[0044] <Saltase Recognition Motif: Reaction of LPXTG etc. with Glycine Residues, etc.> Saltase is a transpeptidase known for protein modification, and saltase A (SrtA) and saltase B (SrtB) derived from Staphylococcus aureus can be used. Saltase A recognizes the Leu-Pro-Xxx-Thr-Gly motif (where Xxx is any amino acid; hereafter referred to as "LPXTG") near the carboxyl terminus (C-terminus) of one molecule, such as a protein or peptide, thereby mediating a transpeptidase reaction between a glycine residue (G) in the motif and a glycine residue at the amino terminus (N-terminus) of the other molecule. Therefore, in the present invention, for example, an antibody or its antigen-binding fragment having a recognition motif of saltase A at its C-terminus can react with a reactive PEG lipid having a glycine residue at its telogen in the presence of saltase A, and form a covalent bond more directly (without the need for specific reactive groups such as the azide group and DBCO as described above). The glycine residue may be one, two, three or more consecutively, but one or two is preferred. The glycine residue may exist as a peptide having a glycine residue at its terminus (for example, in the molecular structure of Gly-Xxx-Xxx-Xxx-Xxx-Xxx-PEG lipid). The telogenous residue of the reactive PEG lipid is not limited to a glycine residue, but may be any other N-terminal amino acid residue or a functional group having an amino group. Saltase A may be a mutant (for example, one with an improved reaction rate than the wild type), and instead of saltase A, saltase B, which recognizes NPQTN, NPKTG, etc. as its recognition motif, can also be used.

[0045] Purification of targeted lipid molecules from reaction products of lipids (e.g., PEG lipids) and ligands (e.g., antibodies or their antigen-binding fragments) can be performed by extracting the targeted lipid molecules from the reaction product or by removing molecules other than the targeted lipid molecules (i.e., lipids that did not covalently bind to the ligand, ligands that did not covalently bind to the lipid, etc.) from the reaction product. For such purification of targeted lipid molecules, methods such as dialysis filtration, ultrafiltration, and chromatography can be used. Examples of ultrafiltration include normal flow filtration and tangential flow filtration. Examples of chromatography include size exclusion chromatography (gel filtration chromatography), hydrophobic interaction chromatography, ion exchange chromatography, and affinity chromatography. As the mobile phase used in chromatography, for example, water, or organic solvents described later, or buffers described later, or mixtures thereof (e.g., a mixed solvent containing 0-50% alcohols and water or buffer) can be used.

[0046] The active ingredients encapsulated within the lipid particles are not particularly limited, and any substance or molecule common in the field of lipid particle technology that exerts a predetermined function upon delivery to target cells can be used. Such active ingredients are preferably nucleic acids that exert function within target cells.

[0047] 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.

[0048] "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.

[0049] 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.

[0050] 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)].

[0051] 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)].

[0052] 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.

[0053] 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.

[0054] 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.).

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] 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.

[0062] —Composition for introducing nucleic acids— In one aspect of the present invention, a composition for introducing nucleic acids is provided, which contains the lipid particles and nucleic acids of the present invention. In the composition for introducing nucleic acids, 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.

[0063] 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).

[0064] (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.

[0065] (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.

[0066] (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.

[0067] (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.

[0068] (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.

[0069] (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.

[0070] (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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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).

[0075] • 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] ―First Method for Producing Lipid Particles (One Step Method)― The present invention provides a method for producing lipid particles on which a ligand specific to target cells is conjugated on the surface (targeted) (hereinafter also referred to as the "first method"), which includes: (1) a step of mixing an organic solvent phase (a) containing a lipid component, an aqueous phase (b) containing an active ingredient, and an organic solvent phase or aqueous phase (c) containing a molecule in which a ligand specific to target cells and a lipid are covalently bonded (targeted lipid molecule) (hereinafter referred to as the "mixing step"), and (2) a step of purifying the mixture obtained in step (1) (hereinafter referred to as the "purification step"). The first method for producing lipid particles is similar to the fourth embodiment of the second method for producing lipid particles described later in that it uses an organic solvent phase containing a lipid component and an aqueous phase containing a targeted lipid molecule during mixing, but differs from the fourth embodiment of the second method for producing lipid particles, which does not use an aqueous phase containing an active ingredient (used in step (H) of the method for producing targeted lipid particles), in that it also uses an aqueous phase containing an active ingredient.

[0093] (1) Mixing step: In the mixing step, an organic solvent phase containing lipid components constituting the lipid particles (excluding targeted lipid molecules), an aqueous phase containing the active ingredient, and an organic solvent phase or aqueous phase containing targeted lipid molecules are prepared in advance and used.

[0094] • Targeted lipid molecules (e.g., targeted PEG lipids) used in the preparation and mixing step of targeted lipid molecules can be prepared in advance by reacting a reactive lipid with a ligand (e.g., an antibody or its antigen-binding fragment) and covalently bonding them together. Embodiments for preparing such targeted lipid molecules can be adapted to the form of covalent bonding between the reactive lipid and the ligand, and for example, the following embodiments can be used.

[0095] As the reactive lipid, preferably the reactive PEG lipid, the reactive lipids described herein in relation to lipid particles can be used. The concentration of the reactive lipid in the reaction solvent is preferably 0.5 to 100 mg / mL.

[0096] When covalently bonding an antibody or its antigen-binding fragment containing a thiol group to a reactive lipid containing a maleimide group, an appropriate amount of the antibody, etc., should be added to a solution of the reactive lipid prepared using an appropriate solvent (organic solvent and / or buffer), and the reaction should be carried out under appropriate conditions (e.g., pH 6.5 to 7.5, approximately 20 to 25°C (room temperature), several minutes to several hours).

[0097] When covalently bonding an antibody containing DBCO or its antigen-binding fragment to a reactive lipid having an azide group, an appropriate amount of the antibody, etc., should be added to a solution of the reactive lipid prepared using an appropriate solvent (organic solvent and / or buffer), and the reaction should be carried out under appropriate conditions (e.g., pH 7.0 to 8.5, approximately 20 to 25°C (room temperature), several minutes to several hours).

[0098] When covalently binding an antibody or its antigen-binding fragment containing the recognition sequence (LPXTG) for saltase A to a reactive lipid having a glycine residue or an amino group, the antibody, etc., dissolved in a suitable buffer, a suitable amount of the reactive lipid solution prepared using a suitable solvent (organic solvent and / or buffer), and a suitable amount of saltase A prepared using a suitable buffer are mixed under suitable conditions (e.g., pH 7.0-8.5, calcium (e.g., CaCl)). ２ The reaction should be carried out in the presence of the following: at 4 to 40°C, preferably about 20 to 25°C (room temperature), for several minutes to 24 hours.

[0099] The organic solvents and buffers used for preparing the targeted lipid molecules can be the same as those described below in relation to the mixing step.

[0100] After reacting a reactive lipid with a ligand and covalently bonding them, it is desirable to further purify the mixture to remove molecules other than the reactive lipid to which the ligand has covalently bonded (i.e., reactive lipids that did not covalently bond with the ligand, and ligands that did not covalently bond with the reactive lipid, etc.). Such purification processes can utilize, for example, dialysis filtration, ultrafiltration (normal flow filtration, tangential flow filtration, etc.), and chromatography (size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, etc.), as will be described later in relation to the purification process of the present invention.

[0101] Examples of organic solvents for preparing the organic solvent phase (a) containing lipid components other than the targeted lipid molecule include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, N,N-dimethylacetamide, or mixtures thereof. The organic solvent may contain 0-20% water or buffer solution.

[0102] Examples of buffers for preparing the aqueous phase (b) containing the active ingredient 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)). Acidic buffers are preferred as the buffer for preparing the aqueous phase (b) containing the active ingredient. Additives such as glycerol or salts such as NaCl may be further added to the buffer.

[0103] For the preparation of the organic solvent phase or aqueous phase (c) containing the targeted lipid molecules, water, the above-mentioned organic solvent, the above-mentioned buffer, or a mixture thereof (for example, a mixed solvent containing alcohols and 0-20% water or buffer) can be used. 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.

[0104] The mixing process can be carried out by various emulsification methods or by using a microfluidic mixing system (e.g., Asia microfluidic system (Syrris) or Nanoassemblr (Precision Nanosystems)). The mixing process of the present invention can be in various embodiments in which lipid particles are formed by mixing predetermined liquid phases (a) to (c). For example, the mixing of predetermined liquid phases (a) to (c) can be carried out in the order of first mixing liquid phases (a) and (b), and then mixing the resulting mixture with the organic solvent layer or aqueous phase (c). Alternatively, the organic solvent layer or aqueous phase (c) can be mixed with either liquid phase (a) or (b), and then mixed with the other.

[0105] In one embodiment of the first manufacturing method of the present invention, the mixing step includes (1-1) a process of mixing the organic solvent phase (a) with the aqueous phase (b), and (1-2) a process of mixing the mixture prepared by the above process with the organic solvent phase or the aqueous phase (c). The mixing step in such an embodiment is preferably carried out using a microfluidic mixing system.

[0106] When performing the above mixing process (1-1) in a microfluidic mixing system, it is preferable to mix 1 to 5 volumes of aqueous phase (b) with 1 volume of organic solvent phase layer (a). In addition, in the mixing process (1-1), the flow rate of the mixture of organic solvent layer (a) and aqueous phase (b) (a+b) is, for example, 0.01 to 115 mL / min, preferably 0.1 to 115 mL / min, and the temperature is, for example, 5 to 60°C, preferably 15 to 45°C.

[0107] Next, when performing the above mixing process (1-2) in the same microfluidic mixing system, it is preferable to mix 1 to 5 volumes of the organic solvent phase or aqueous phase (c) with 1 volume of the mixed liquid (a + b) obtained in mixing process (1-1). Furthermore, in mixing process (1-2), the flow rate of the mixed liquid of the organic solvent phase (a), aqueous phase (b), and organic solvent phase or aqueous phase (c) is, for example, 0.01 to 345 mL / min, preferably 0.1 to 345 mL / min, and the temperature is, for example, 5 to 60°C, preferably 15 to 45°C.

[0108] The mixing process in the microfluidic mixing system of the above embodiment can be carried out using appropriately designed microchannels. For example, the microfluidic mixing system may be configured such that a supply section (α) for an organic solvent phase (a), a supply section (β) for an aqueous phase (b), and a supply section (γ) for either an organic solvent phase or an aqueous phase (c), and a channel communicating with supply section (α) and a channel communicating with supply section (β) merge at a first confluence, and a channel communicating with the first confluence and a channel communicating with supply section (γ) merge at a second confluence downstream of the first confluence (corresponding to the "in-line dilution" in the embodiments described later), with mixing (1-1) performed at the first confluence and mixing (1-2) performed at the second confluence.

[0109] (2) Purification step The first method for producing lipid particles of the present invention involves a step to purify the obtained lipid particle preparation solution (dispersion) after the mixing step, that is, a step to reduce the amount of organic solvent contained in the lipid particle preparation solution (dispersion).

[0110] 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.

[0111] 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.

[0112] 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).

[0113] (3) Other steps (purification steps, etc.) The first method for producing lipid particles of the present invention may further include steps other than the mixing step and the purification step, for example, a purification step, as needed. In the purification step, for example, chromatography (size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, etc.) can be used. Such a purification step may be an embodiment that is integrated with the purification step (2), that is, an embodiment in which solvent replacement and impurity removal are performed simultaneously.

[0114] —Method for producing a nucleic acid-introducing composition— The nucleic acid-introducing composition of the present invention can be produced by using an aqueous phase (b) containing nucleic acid as an active ingredient in the mixing step included in the first method for producing lipid particles of the present invention as described above (i.e., a nucleic acid-introducing composition is obtained as equivalent to a dispersion of lipid particles). In such a production method (step), it is preferable to add nucleic acid in an amount such that its concentration in water or buffer solution is 0.05 to 2.0 mg / mL.

[0115] 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.

[0116] 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%.

[0117] 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 ＴＭ Nucleic 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).

[0118] —Second Method for Producing Lipid Particles (Quick Prep Method)— The second method for producing lipid particles in which a ligand specific to target cells is conjugated on the surface (hereinafter referred to as the "second production method") according to the present invention includes a step of introducing a ligand specific to target cells into untargeted cavity lipid particles (hereinafter referred to as the "cavity lipid particle targeting step").

[0119] The second method for producing lipid particles, particularly the step of targeting the cavity lipid particles, can be, for example, one of the following first to sixth embodiments. Of these embodiments, the first, fourth, and fifth embodiments can be classified as methods for forming lipid particles containing a targeted lipid molecule after preparation (Pre-type), while the second, third, and sixth embodiments can be classified as methods for targeting lipid particles that do not contain a targeted lipid molecule after preparation (Post-type). Unlike the first method for producing lipid particles described above, none of the first to sixth embodiments of the second method for producing lipid particles use an aqueous phase containing an active ingredient.

[0120] The first embodiment of the second method for producing lipid particles includes: (A1) a step of mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing cavity lipid particles; and (A2) a step of mixing the mixture obtained in step (A1) with an organic solvent phase or aqueous phase (c) containing a targeted lipid molecule to obtain targeted cavity lipid particles.

[0121] A second embodiment of the second method for producing lipid particles includes: (B1) a step of mixing an organic solvent phase (a) containing a lipid component having a reactive polyethylene glycol (PEG) chain (hereinafter referred to as "reactive PEG lipid") with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing cavity lipid particles; and (B2) a step of reacting a ligand specific to target cells with the reactive PEG lipid contained in the lipid component constituting the lipid particles to form a covalent bond, thereby obtaining targeted cavity lipid particles.

[0122] A third embodiment of the second method for producing lipid particles includes: (C1) a step of mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent containing a reactive PEG lipid to prepare a mixture containing cavity lipid particles; and (C2) a step of reacting a ligand specific to target cells with the reactive PEG lipid contained in the lipid component constituting the lipid particles to form a covalent bond, thereby obtaining targeted cavity lipid particles.

[0123] A fourth embodiment of the second method for producing lipid particles includes the step of (D1) mixing an organic solvent phase (a) containing lipid components with an aqueous phase (b1) containing a first aqueous solvent containing targeted lipid molecules to prepare a mixture containing targeted cavity lipid particles.

[0124] A fifth embodiment of the second method for producing lipid particles includes the step of (E1) mixing an organic solvent phase (a) containing a lipid component including a targeted lipid molecule with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing targeted cavity lipid particles.

[0125] A sixth embodiment of the second method for producing lipid particles includes: (F1) a step of mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing cavity lipid particles; (F2) a step of mixing the mixture obtained in step (F1) with an organic solvent phase or aqueous phase (c) containing a reactive PEG lipid; and (F3) a step of reacting a ligand specific to target cells with the reactive PEG lipid contained in the lipid component constituting the lipid particles to form a covalent bond, thereby obtaining targeted cavity lipid particles.

[0126] Technical matters concerning the "lipid components," "targeted lipid molecules," and "reactive PEG lipids" used in the first to sixth embodiments of the second method for producing lipid particles can be adapted by referring to those described herein in relation to the first method for producing lipid particles. For example, the targeted lipid molecules used in the first, fourth, and fifth embodiments (Pre type) of the second method for producing lipid particles can be prepared in the same way as the targeted lipid molecules described in relation to the first method for producing lipid particles.

[0127] Furthermore, in the second, third, and sixth embodiments (Post type) of the second method for producing lipid particles, the technical matters (reaction mode, conditions, etc.) related to targeting untargeted cavity lipid particles can be adapted by referring to the technical matters described as necessary for producing targeted lipid molecules in relation to the first method for producing lipid particles.

[0128] Technical matters concerning the "organic solvent phase" and "aqueous phase" used in the first to sixth embodiments of the second method for producing lipid particles can also be adapted by referring to those described herein in relation to the first method for producing lipid particles. Furthermore, the "aqueous solvent" used in the first to sixth embodiments of the second method for producing lipid particles may be, for example, water or a buffer that can be used to prepare the aqueous phase (such as an acidic buffer), or a solvent obtained by mixing water or a buffer with an organic solvent that has appropriate compatibility with water (such as alcohols) that can be used to prepare the organic solvent phase in an appropriate ratio (for example, a mixed solvent with a volume ratio of alcohols:water or buffer = 100 to 80:0 to 20).

[0129] In the first to sixth embodiments of the second method for producing lipid particles, "mixing" can be performed using a microfluidic mixing system. Technical matters concerning the microfluidic mixing system in the second method for producing lipid particles should be referred to in relation to the first method for producing lipid particles, and for example, the mixing ratio of the organic solvent phase and the aqueous phase, the flow rate, the temperature, etc., in each embodiment can be adjusted as appropriate.

[0130] The second method for producing lipid particles may further include, if necessary, the step of (G) replacing the solvent in the mixture containing the cavity lipid particles or targeted cavity lipid particles obtained in any of the above steps with a second aqueous solvent (b2).

[0131] In the first to sixth embodiments of the second method for producing lipid particles, the "first aqueous solvent mixture" and the "second aqueous solvent" can be aqueous solvents with different components. For example, the second aqueous solvent can be an aqueous solvent containing 2-morpholinoethanesulfonic acid (MES) buffer or water, and the first aqueous solvent can be a different aqueous solvent.

[0132] Technical matters relating to solvent replacement in step (G) above can be adapted by referring to and adapting the matters described in this specification in relation to the replacement of the dispersion medium in the "purification step" in relation to the first method for producing lipid particles, and in relation to the pH adjustment and osmotic pressure adjustment that may be performed as needed.

[0133] —Method for producing targeted lipid particles— The method for producing targeted lipid particles of the present invention includes the steps of: (H) mixing a mixed solution containing targeted cavity lipid particles with an aqueous phase (b3) containing an active ingredient and a third aqueous solvent to prepare a mixed solution containing lipid particles (i.e., targeted lipid particles) having a ligand specific to target cells on its surface and encapsulating the active ingredient.

[0134] The "targeted cavity lipid particles (including the mixed solution)" used in the method for producing targeted mounted lipid particles is typically obtained by the second method for producing lipid particles described herein, and the technical matters concerning the targeted cavity lipid particles (including the mixed solution) can be adapted by referring to those described in relation to the second method for producing lipid particles.

[0135] Technical matters concerning the "active ingredient" used in the method for producing targeted lipid particles can be adapted by referring to those described herein in relation to lipid particles, the first method for producing lipid particles, or the nucleic acid introduction composition. For example, the active ingredient used in the method for producing targeted lipid particles preferably contains nucleic acid (DNA or RNA).

[0136] Technical matters concerning the "aqueous phase" and "aqueous solvent" used in the method for producing targeted lipid particles can be adapted by referring to those described herein in relation to the second method for producing lipid particles (first to sixth embodiments) or those described in relation to the first method for producing lipid particles.

[0137] The present invention's method for producing targeted-loaded lipid particles can be an embodiment that does not include a step of replacing the solvent in the mixture obtained in step (H) with an aqueous solvent after step (H). If the targeted-cavity lipid particles (including the mixture) used in step (H) are obtained by the second method for producing lipid particles described herein, and step (G) is performed in that second method, then it is not necessary to perform the step of replacing the solvent with an aqueous solvent again after step (H).

[0138] ―Kit― The kit of the present invention includes lipid particles (targeted cavity lipid particles) that have a ligand specific to target cells on their surface and do not contain an active ingredient. The use of the kit of the present invention is not particularly limited, but for example, embodiments for use in the method of producing targeted loaded lipid particles of the present invention may include components for preparing a “mixture containing targeted cavity lipid particles” in step (H) (targeted cavity lipid particles, solvent, equipment for preparing the mixture, etc.), preferably components for preparing an “aqueous phase (b3) containing an active ingredient and a third aqueous solvent” (active ingredient, aqueous solvent, equipment for mixing these, etc.), and equipment for mixing the “mixture containing targeted cavity lipid particles” and the “aqueous phase (b3) containing an active ingredient and a third aqueous solvent,” and may further include instructions for carrying out the method of producing targeted loaded lipid particles of the present invention as needed.

[0139] In this specification, matters described in relation to a certain category of the present invention (e.g., lipid particles) may be referred to with appropriate modifications as to matters relating to other categories (e.g., methods for producing lipid particles, compositions for introducing nucleic acids).

[0140] The present invention will be further described in detail by the following examples, manufacturing 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.

[0141] In the following examples (production examples), "DSPE-PEG(5000)Azide" (AVANTI) is a compound having 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 azide group as a reactive group for covalent bonding with antibodies, etc. "SUNBRIGHT GM-020" (NOF CORPORATION) is a non-reactive PEG lipid having a total of 28 carbon atoms in its hydrophobic carbon chain and an average molecular weight of 2000 in the PEG chain. Furthermore, the "lipid solution," "nucleic acid solution," and "antibody solution" in each example correspond to the "organic solvent phase containing lipid components (a)," the "aqueous phase containing active ingredients (b)," and the "organic solvent phase or aqueous phase containing targeted lipid molecules (c)" in the present invention, respectively.

[0142] [Preparation Example 1-1] Preparation of mCD3Fab-DBCO, hCD3Fab-DBCO, and hCD7Fab-DBCO Fab-DBCO was prepared by performing the following two steps. DBCO / Antibody ratio (DAR) and protein concentration were quantified by LC-MS and BCA methods, respectively. The results of the physical property evaluation are shown in Table 1.

[0143] Preparation process for Fabs having LPETGG-His6 at the C-terminus: Plasmid pMG2.2 vector encoding mCD3Fab (clone: ​​145-2C11), hCD7Fab (Fab fragment of grisnilimap), or hCD3 (clone: ​​OKT3) 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 samples were purified using a compacte Ni column and a Superdex 200 size exclusion column to prepare LPETGG-His-tagged Fab.

[0144] Preparation process for Fab-DBCO: LPETGG-His-tagged Fab is mixed with CaCl in a pH 7.4 HEPES buffer. ２ The mixture was then miscible with 5-(glycylglycyl-beta-alanyl)-11,12-didehydro-5,6-dihydrodibenzo[b,f]azosin and treated with Sortase A (P94S / D160N / D165A / K196T, Processings of the National Academy of Sciences of the United States of America. 2011;108:11399-11404) (Current Protocols in Protein Science, 89, 15.3.1-15.3.19). The treated product was purified by Ni column and dialysis to obtain Fab-DBCO.

[0145]

[0146] [Production Example 1-2] Preparation of mCD3Fab-PEG(5000)-DSPE, hCD3Fab-PEG(5000)-DSPE, and hCD7Fab-PEG(5000)-DSPE A Fab-DBCO solution, an aqueous solution of DSPE-PEG(5000) Azide, and SUNBRIGHT GM-020 were mixed in amounts such that the molar ratio of each compound was 1:1:10, and reacted at 25 °C for 24 hours to obtain each Fab-PEG(5000)-DSPE.

[0147] [Example 1-1] Preparation of LNP1 A lipid mixture (ionizable lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60:10.6:27.99:1.3, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.3 mg / ml. As the ionizable lipid, 3-(((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl 3-pentyloctanoate described in WO2016 / 021683 was used.

[0148] mRNA (TriLink) encoding a CD19-targeted CAR having CD28 and CD3ζ as intracellular signaling domains was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0149] Also, mCD3Fab-PEG(5000)-DSPE (Production Example 1-2) at 0.01 mol% with respect to the total lipid was diluted with water to obtain an antibody solution of 0.01 mg / ml (concentration as Fab).

[0150] The obtained lipid solution, nucleic acid solution, and antibody solution were mixed at room temperature using Nanoassembler ＴＭ Ignite ＴＭThe composition was mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion. At this time, the antibody solution was mixed through a channel for in-line dilution. 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, it was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and it was stored at -80°C.

[0151] [Examples 1-2] A lipid mixture of LNP2 (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 28.04: 1.3, 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.

[0152] An mRNA encoding a CD19-targeting CAR having CD28 and CD3ζ as intracellular signaling domains (TriLink) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0153] Furthermore, 0.005 mol% of mCD3Fab-PEG(5000)-DSPE (Preparation Example 1-2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.01 mg / ml (Fab concentration).

[0154] The resulting lipid solution, nucleic acid solution, and antibody solution were heated at room temperature using Nanoassemblr. ＴＭ Ignite ＴＭThe composition was mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion. At this time, the antibody solution was mixed through a channel for in-line dilution. 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, it was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and it was stored at -80°C.

[0155] [Examples 1-3] A lipid mixture of LNP3 (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 27.99: 1.3, 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.

[0156] The mRNA encoding GFP (TriLink) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0157] Furthermore, 0.01 mol% of mCD3Fab-PEG(5000)-DSPE (Preparation Example 1-2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0158] The resulting lipid solution, nucleic acid solution, and antibody solution were heated at room temperature using Nanoassemblr. ＴＭ Ignite ＴＭThe composition was mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion. At this time, the antibody solution was mixed through a channel for in-line dilution. 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, it was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and it was stored at -80°C.

[0159] [Examples 1-4] A lipid mixture of LNP4 (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 27.99: 1.3, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 10.3 mg / ml. As the ionized lipid, 1,1'-bis(2-butylhexyl)6,6'-[2-({[4-(dimethylamino)butanoyl]oxy}methyl)-2-{[(3-pentyloctanoyl)oxy]methyl}propane-1,3-diyl]dihexane diate described in WO2023 / 085299 was used.

[0160] The mRNA encoding GFP (TriLink) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 5.5 to obtain a nucleic acid solution of 0.2 mg / ml.

[0161] Furthermore, 0.01 mol% of mCD3Fab-PEG(5000)-DSPE (Preparation Example 1-2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0162] The resulting lipid solution, nucleic acid solution, and antibody solution were heated at room temperature using Nanoassemblr. ＴＭ Ignite ＴＭThe composition was mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion. At this time, the antibody solution was mixed through a channel for in-line dilution. 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, it was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and it was stored at -80°C.

[0163] [Examples 1-5] A lipid mixture of LNP5 (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 27.99: 1.3, 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.

[0164] An mRNA (Elixirgen) encoding a CD19-targeting CAR, which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0165] Furthermore, 0.01 mol% of hCD3Fab-PEG(5000)-DSPE (Preparation Example 1-2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0166] The resulting lipid solution, nucleic acid solution, and antibody solution were stored at room temperature in a NanoAssemblr ＴＭ Ignite ＴＭThe composition was mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion. At this time, the antibody solution was mixed through a channel for in-line dilution. 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, it was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and it was stored at -80°C.

[0167] [Examples 1-6] A lipid mixture of LNP6 (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 27.99: 1.3, 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.

[0168] An mRNA (Elixirgen) encoding a CD19-targeting CAR, which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0169] Furthermore, 0.01 mol% of hCD7Fab-PEG(5000)-DSPE (Preparation Example 1-2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0170] The resulting lipid solution, nucleic acid solution, and antibody solution were stored at room temperature in a NanoAssemblr ＴＭ Ignite ＴＭThe composition was mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion. At this time, the antibody solution was mixed through a channel for in-line dilution. 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, it was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and it was stored at -80°C.

[0171] [Examples 1-7] A lipid mixture of LNP7 (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60:10.6:27.99:1.3, 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.

[0172] An mRNA (Elixirgen) encoding a CD19-targeting CAR, which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0173] Furthermore, 0.01 mol% of mCD3Fab-PEG(5000)-DSPE (Preparation Example 1-2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0174] The resulting lipid solution, nucleic acid solution, and antibody solution were stored at room temperature in a NanoAssemblr ＴＭ Ignite ＴＭThe composition was mixed using a Precision Nanosystem at flow rates of 3 ml / min:6 ml / min:9 ml / min to obtain a dispersion. At this time, the antibody solution was mixed through a channel for in-line dilution. 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, it was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and then filtered through a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and it was stored at -80°C.

[0175] [Comparative Example 1-1] A prepared lipid mixture of control LNP1 (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 27.99: 1.3, 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.

[0176] An mRNA (Elixirgen) encoding a CD19-targeting CAR, which has CD28 and CD3ζ as intracellular signaling domains, was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a nucleic acid solution of 0.2 mg / ml.

[0177] Furthermore, 0.01 mol% of mCD3Fab-PEG(5000)-DSPE (Preparation Example 1-2) relative to total lipids was diluted with water to obtain an antibody solution with a concentration of 0.02 mg / ml (Fab concentration).

[0178] The resulting lipid solution and nucleic acid solution were stored at room temperature in a nanoassemblr ＴＭ Ignite ＴＭThe mixture was prepared using a Precision Nanosystems at a flow rate ratio of 3 ml / min:6 ml to obtain a dispersion containing the composition. The obtained dispersion was dialyzed with Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific) against water at room temperature for 1 hour and against PBS at 4°C for 24 hours. To this, 0.01 mol% of mCD3Fab-PEG(5000)-DSPE (Preparation Example 1-2) relative to the total lipids was mixed at 25°C, and after 30 minutes, it was concentrated by ultrafiltration using Amicon Ultra (30K molecular weight cutoff, Merck) and filtered using a 0.2 μm syringe filter. The final mRNA concentration was adjusted to 200 μg / mL and the sucrose concentration to 20%, and the solution was stored at -80°C.

[0179] [Characterization of LNPs] The particle size, PDI, and mRNA encapsulation rate of LNPs 1-7 and control LNP1 were measured using the following method. The results of these characterizations are shown in Table 2.

[0180] Particle size and PDI: The particle size and Polydispersity index (PDI) of LNPs were measured using a Zetasizer Nano ZS (Maivern Panalogical).

[0181] mRNA encapsulation rate: The mRNA encapsulation rate in LNPs is 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.

[0182] As shown in Table 2, LNP1 to 7 obtained by the first manufacturing method of the present invention (One Step method), which requires only one mixing step, showed good particle size, PDI, and high encapsulation rate, similar to control LNP1 obtained by the conventional Pre method which requires two mixing steps.

[0183]

[0184] [Test Example 1-1] In vivo CAR expression test in mouse T cells C57BL / 6NJcl mice were administered 0.1 mg / kg of LNP1 and LNP2 RNA via tail vein at a rate of 10 mL / kg. Four hours after administration, the spleen and blood were collected. After isolating immune cells from the spleen, these cells were stained (immunostaining for CD45, CD90.2, CD4, CD8, and CD19 CAR), and the percentage of CD19 CAR-positive cells among CD90.2-positive T cells (Pan-T cells), CD4-positive T cells, and CD8-positive T cells was evaluated using an LSR Fortessa flow cytometer (BD Biosciences). As shown in Table 3, high CAR expression levels were observed in vivo.

[0185]

[0186] [Test Example 1-2] In vivo CAR expression test in hCD3KI mouse T cells. LNP5 at a dose of 0.1 mg / kg (RNA) was administered via tail vein at a rate of 10 mL / kg to hCD3KI mice expressing the human CD3 gene. Spleen and blood samples were collected 4 hours after administration. Immune cells were isolated from the spleen and stained (immunostaining for CD45, CD90.2, CD4, CD8, and CD19 CAR). The percentage of CD19 CAR-positive cells among CD90.2-positive T cells (Pan-T cells), CD4-positive T cells, and CD8-positive T cells was evaluated using an LSR Fortessa flow cytometer (BD Biosciences). As shown in Table 4, high CAR expression levels were observed in vivo.

[0187]

[0188] [Test Example 1-3] In vivo CAR expression test in mouse T cells and NK cells. Human CD7 gene-expressing hCD7KI mice were administered 0.3 mg / kg of LNP6 RNA via tail vein at a rate of 10 mL / kg. The spleen was collected 4 hours after the final administration. Immune cells were isolated from the spleen, stained (CD45, CD3, NKp46, G4S Linker), and the percentage of CD19 CAR-positive cells among NKp46-positive NK cells was evaluated using an LSR Fortessa flow cytometer (BD Biosciences). As shown in Table 5, high CAR expression levels were observed in vivo.

[0189]

[0190] [Test Example 1-4] In vivo CAR expression test in mouse T cells C57BL / 6NJcl mice were administered 0.1 mg / kg of RNA of LNP7, LNP8, and control LNP1 via tail vein at a rate of 10 mL / kg. Four hours after administration, the spleen and blood were collected. After isolating immune cells from the spleen, these cells were stained (immunostaining for CD45, CD90.2, CD4, CD8, and CD19 CAR), and the proportion of CD19 CAR-positive cells among CD90.2-positive T cells (Pan-T cells), CD4-positive T cells, and CD8-positive T cells was evaluated using an LSR Fortessa flow cytometer (BD Biosciences). Plasma was fractionated from the blood, and the concentrations of various cytokines were measured using a CUSTOMIZE U-PLEX ASSAY KIT.

[0191] As shown in Table 6, LNP7 obtained by the One Step method showed high in vivo CAR expression levels comparable to control LNP1 obtained by the conventional Pre method. On the other hand, as shown in Table 7, LNP7 showed lower cytokine production capacity compared to control LNP1, indicating that LNP obtained by the One Step method is less toxic than LNP obtained by the conventional Pre method.

[0192]

[0193]

[0194] [Preparation Example 2-1] Preparation of Cavity Lipid Particles 1 For the preparation of cavity lipid particles 1, a lipid mixture (ionized lipid: DPPC: cholesterol: SUNBRIGHT GM-020: DSPE-PEG (2000) Azide = 60: 10.6: 27: 1.4: 1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 9.4 mg / ml. As the ionized lipid (cationic lipid), 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyldidecanoate, as described in WO2020 / 032184, was used.

[0195] A lipid solution for preparing cavity lipid particles 1 and a 10 mM 2-morpholinoethanesulfonic acid solution (pH 5.5) were mixed at room temperature using a Nanoassembl™ Ignite™ instrument (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed in water at room temperature for 1 hour using a Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific) and then in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5) containing 0.9% (w / v) NaCl at 4°C for 24 hours. Subsequently, the mixture was filtered using a 0.2 μm syringe filter to obtain a dispersion of cavity lipid particles 1.

[0196] The concentration of ionized lipids in the lipid particle dispersion was determined by HPLC analysis. The analytical conditions are as follows: Mobile phase A: 0.1% trifluoroacetic acid (TFA) aqueous solution Mobile phase B: 0.1% TFA acetonitrile solution Detection method: Corona charged particle detector (CAD) Column: CERI L-column2 C8

[0197] [Comparative Example 2-1] Preparation of Targeted Loading Lipid Particle 1 (Method of Loading and Targeting Cavity Lipid Particles) A ​​dispersion of cavity lipid particle 1 (Preparation Example 2-1) (5.6 mg / ml as ionized lipid, 90 μL) was mixed with a citrate buffer (pH 6.0-6.5, 1 mg / ml as mRNA, 32 μL) of mRNA encoding the CD19 target CAR. Then, 2.09 μL of hCD3Fab-DBCO solution (Preparation Example 1) was mixed in, and the mixture was reacted at 25°C for 24 hours. After that, the sucrose concentration was adjusted to 20% using a sucrose-PBS solution to obtain a dispersion of targeted loading lipid particle 1.

[0198] [Example 2-1] Preparation of Targeted Cavity Lipid Particles 1 (Second Embodiment of the Second Manufacturing Method: Post Type) A dispersion of cavity lipid particles 1 (Preparation Example 2-1) (5.6 mg / ml as ionized lipid, 90 μL) was mixed with 2.09 μL of hCD3Fab-DBCO solution (Preparation Example 1), and the mixture was reacted at 25°C for 24 hours to obtain a dispersion of targeted cavity lipid particles 1.

[0199] [Example 2-2] Preparation of Targeted Cavity Lipid Particles 2 (Second Embodiment of the Second Manufacturing Method: Post Type, with Solvent Replacement) A dispersion of cavity lipid particles 1 (Preparation Example 2-1) (5.2 mg / ml as ionized lipid, 150 μL) was mixed with 0.62 μL of hCD7Fab-DBCO solution (Preparation Example 1-1) and reacted at 25°C for 24 hours. A 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5) containing sucrose and 0.9% (w / v) NaCl was added to obtain a dispersion of targeted cavity lipid particles 2, resulting in an ionized lipid concentration of 3.0 mg / ml and a sucrose concentration of 20%.

[0200] [Examples 2-3 and 2-4] Preparation of Targeted Cavity Lipid Particles 3 and 4 (First Embodiment of the Second Manufacturing Method: Pre-type) For the preparation of Targeted Cavity Lipid Particles 3, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 27.97: 1.1, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 13.5 mg / ml. For the preparation of Cavity Lipid Particles 4, a lipid mixture (ionized lipid: DSPC: cholesterol: SUNBRIGHT GM-020 = 60: 10.6: 27.99: 1.3, mol%) was dissolved in 90% EtOH to obtain a lipid solution of 13.6 mg / ml. As the ionized lipid (cationic lipid), we used 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyldidecanoate as described in WO2020 / 032184.

[0201] The lipid solutions for preparing targeted cavity lipid particles 3 and 4, respectively, and a 10 mM 2-morpholinoethanesulfonic acid solution (pH 5.5) were mixed at room temperature using NanoAssemblr. ＴＭ Ignite ＴＭThe mixture was prepared using a Precision Nanosystems at a flow rate ratio of 3 ml / min to 6 ml / min to obtain a dispersion containing the composition. The resulting dispersion was dialyzed in water at room temperature for 1 hour using Slide-A-Lyzer Dialesis (20K molecular weight cutoff, Thermo Fisher Scientific), and then in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5) containing 0.9% (w / v) NaCl at 4°C for 24 hours. Subsequently, hCD3Fab-PEG5000-DSPE (Preparation Example 1-2) was mixed at 25°C to a concentration of 0.03 mol% (targeted cavity lipid particle 4) or 0.01 mol% (targeted cavity lipid particle 5) relative to the total lipids. After 30 minutes, the mixture was filtered using a 0.2 μm syringe filter. Then, 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5) containing sucrose and 0.9% (w / v) NaCl was added to achieve an ionized lipid concentration of 3.8 mg / ml and a sucrose concentration of 20%, thereby obtaining dispersions of targeted cavity lipid particles 3 and 4.

[0202] [Particle Size and PDI] The particle size and Polydispersity Index (PDI) of LNPs were measured using a Zetasizer Nano ZS (Maivern Panalogical). The results are shown in Table 8.

[0203] [Test Example 2-1] In vitro CAR expression test in human T cells Preparation of cavity lipid particle 1 + mRNA A dispersion of cavity lipid particle 1 (5.6 mg / ml as ionized lipid, 90 μL) and a citrate buffer (pH 6.0-6.5, 1 mg / ml as mRNA, 32 μL) of mRNA encoding a CD19 target CAR were mixed, and then the sucrose concentration was adjusted to 20% using a sucrose-PBS solution.

[0204] Preparation of Targeted Cavity Lipid Particle 1 + mRNA: A dispersion of Targeted Cavity Lipid Particle 1 (5.5 mg / ml as ionized lipid, 92 μL) and a citrate buffer (pH 6.0-6.5, 1 mg / ml as mRNA, 32 μL) containing mRNA encoding the CD19 target CAR were mixed. The mixture was then adjusted to a sucrose concentration of 20% using a sucrose-PBS solution.

[0205] Human T cells were isolated from frozen PBMCs (Charles River Laboratories Cell Solutions) 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 of IL-2 (Milteny Biotech), mixed with 3 μg / ml of cavity lipid particles + mRNA, targeted loaded lipid particles, or targeted cavity lipid particles + mRNA, and cultured for 24 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 9.

[0206] As shown in Table 9, targeted cavity lipid particle 1 + mRNA showed higher CAR expression levels compared to untargeted cavity lipid particle 1 + mRNA. Furthermore, targeted cavity lipid particle 1 + mRNA showed higher CAR expression levels compared to targeted loaded lipid particle 1, which was targeted after mRNA was loaded onto cavity lipid particle 1. These comparative results demonstrate the effectiveness of the targeted cavity lipid particle of the present invention.

[0207]

[0208] [Test Example 2-2] In vitro CAR expression test in human T cells Preparation of targeted cavity lipid particle 2 + mRNA A citrate buffer (pH 6.0-6.5, 1 mg / ml as mRNA) of mRNA encoding a CD19 target CAR was diluted with physiological saline to a nucleic acid concentration of 0.2 mg / ml. The resulting nucleic acid solution (12 μL) was mixed with a dispersion of targeted cavity lipid particle 2 (3.0 mg / ml as ionized lipid, 12.6 μL), and then physiological saline (15.4 μL) was added.

[0209] 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 3 μg / ml targeted cavity lipid particles + mRNA, and cultured for 72 hours. After culturing, the cells were harvested, stained for CAR using PE-labeled human CD19 (20-291) protein His-Tag (ACROBiossystems), and expression levels were analyzed by flow cytometry (LSRFortessa, BD Biosciences). The measurement results are shown in Table 10.

[0210] As shown in Table 10, targeted cavity lipid particle 2 + mRNA carrying hCD7Fab showed high CAR expression levels, similar to targeted cavity lipid particle 1 + mRNA carrying hCD3Fab.

[0211]

[0212] [Test Example 2-3] In vitro CAR expression test in human T cells Preparation of targeted cavity lipid particle 3 + mRNA A dispersion of targeted cavity lipid particle 3 (3.8 mg / ml as ionized lipid, 100 μL) and a nucleic acid solution of mRNA encoding CD19 target CAR (0.2 mg / ml, 100 μL) were mixed.

[0213] Preparation step for targeted cavity lipid particle 4 + mRNA: A dispersion of targeted cavity lipid particle 4 (3.8 mg / ml as ionized lipid, 100 μL) and a nucleic acid solution of mRNA encoding CD19 target CAR (0.2 mg / ml, 100 μL) were mixed.

[0214] 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), and then conditioned with targeted cavity lipid particles + mRNA at an RNA concentration of 1 μg / ml or 3 μg / ml, or with lipofectamine at an RNA concentration of 1 μg / ml or 3 μg / ml. （Ｒ） MessengerMax reagent + mRNA was mixed. After 72 hours of incubation, 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 11.

[0215] As shown in Table 11, the targeted cavity lipid particle 1+mRNA prepared by the Post-type embodiment, the targeted cavity lipid particle 3+mRNA and the targeted cavity lipid particle 4+mRNA prepared by the Pre-type embodiment of the second manufacturing method (Quick Prep method) of the present invention are Lipofectamine. （Ｒ） Compared to MessengerMax reagent + mRNA, it showed higher CAR expression levels.

[0216]

[0217] The present invention provides a method for producing lipid particles and nucleic acid delivery compositions that enable the efficient delivery of nucleic acids to various cells, tissues, or organs. The lipid particles and nucleic acid delivery compositions thus obtained can be used as DDS (Drug Delivery System) technology in fields such as gene therapy and nucleic acid drugs, and can also be used as nucleic acid delivery reagents for research.

Claims

1. A method for producing lipid particles on which the ligand is conjugated on the surface, comprising the steps of: (1) mixing an organic solvent phase (a) containing a lipid component, an aqueous phase (b) containing an active ingredient, and an organic solvent phase or aqueous phase (c) containing a molecule in which a ligand specific to target cells and a lipid are covalently bonded (hereinafter referred to as "targeted lipid molecule"); and (2) purifying the mixture obtained in step (1).

2. The manufacturing method according to claim 1, wherein step (1) is performed using a microfluidic mixing system.

3. The manufacturing method according to claim 1, wherein step (1) includes (1-1) a process of mixing the organic solvent phase (a) and the aqueous phase (b), and (1-2) a process of mixing the mixture prepared by the above process with the organic solvent phase or the aqueous phase (c).

4. The method for producing the target lipid molecule according to claim 1, wherein a covalent bond is formed in the target lipid molecule by a reaction between a saltase recognition sequence on one of the ligand or the lipid molecule and a glycine or amino group on the other.

5. The method for producing lipids according to claim 1, wherein the targeted lipid molecule is a molecule in which the ligand and a lipid having a polyethylene glycol (PEG) chain (hereinafter referred to as "PEG lipid") are covalently bonded.

6. The manufacturing method according to claim 5, wherein the PEG lipid is a PEG lipid having 30 or more carbon atoms in its hydrophobic carbon chain.

7. The production method according to claim 6, wherein the PEG lipid is PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE).

8. The method for producing the product 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.

9. The method for producing a cell from a cell surface antigen according to claim 1, wherein the cell surface antigen is a cell surface antigen of a T cell or an NK cell.

10. The method for producing the antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment is an anti-CD3 antibody or an anti-CD7 antibody, or an antigen-binding fragment thereof.

11. The manufacturing method according to claim 1, wherein the active ingredient contains nucleic acid.

12. The manufacturing method according to claim 11, wherein the nucleic acid is DNA or RNA.

13. The manufacturing method according to claim 11, wherein at least a portion of the nucleic acid is encapsulated in the lipid particles.

14. Lipid particles obtained by the manufacturing method described in claim 1.

15. A method for producing targeted, non-active lipid particles (hereinafter referred to as "cavity lipid particles"), comprising the step of introducing a specific ligand to target cells.

16. A manufacturing method according to claim 15, comprising the steps of: (A1) mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixed solution containing cavity lipid particles; and (A2) mixing the mixed solution obtained in step (A1) with an organic solvent phase or aqueous phase (c) containing targeted lipid molecules to obtain targeted cavity lipid particles.

17. The manufacturing method according to claim 15, comprising: (B1) a step of mixing an organic solvent phase (a) containing a lipid component having a reactive polyethylene glycol (PEG) chain (hereinafter referred to as "reactive PEG lipid") with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing cavity lipid particles; and (B2) a step of reacting a ligand specific to target cells with the reactive PEG lipid contained in the lipid component constituting the lipid particles to form a covalent bond, thereby obtaining targeted cavity lipid particles.

18. The manufacturing method according to claim 15, comprising the steps of: (C1) mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent containing a reactive PEG lipid to prepare a mixture containing cavity lipid particles; and (C2) reacting a ligand specific to target cells with the reactive PEG lipid contained in the lipid component constituting the lipid particles to form a covalent bond, thereby obtaining targeted cavity lipid particles.

19. The manufacturing method according to claim 15, comprising the step of (D1) mixing an organic solvent phase (a) containing lipid components with an aqueous phase (b1) containing a first aqueous solvent containing targeted lipid molecules to prepare a mixed solution containing targeted cavity lipid particles.

20. The manufacturing method according to claim 15, comprising the step of (E1) mixing an organic solvent phase (a) containing a lipid component including a targeted lipid molecule with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixed solution containing targeted cavity lipid particles.

21. A manufacturing method according to claim 15, comprising: (F1) a step of mixing an organic solvent phase (a) containing a lipid component with an aqueous phase (b1) containing a first aqueous solvent to prepare a mixture containing cavity lipid particles; (F2) a step of mixing the mixture obtained in step (F1) with an organic solvent phase or aqueous phase (c) containing a reactive PEG lipid; and (F3) a step of reacting a ligand specific to target cells with the reactive PEG lipid contained in the lipid component constituting the lipid particles to form a covalent bond, thereby obtaining targeted cavity lipid particles.

22. The manufacturing method according to any one of claims 16 to 21, further comprising the step of (G) replacing the solvent of a mixture containing cavity lipid particles or targeted cavity lipid particles obtained in any of the above steps with a second aqueous solvent (b2).

23. The manufacturing method according to claim 22, wherein the first aqueous solvent and the second aqueous solvent are aqueous solvents having different components.

24. The production method according to claim 22, wherein the second aqueous solvent is an aqueous solvent containing 2-morpholinoethanesulfonic acid (MES) buffer or water.

25. The manufacturing method according to any one of claims 16 to 21, wherein the mixing is carried out using a microfluidic mixing system.

26. A method for producing targeted-loaded lipid particles, comprising the step of (H) mixing a mixture containing targeted-cavity lipid particles with an aqueous phase (b3) containing an active ingredient and a third aqueous solvent to prepare a mixture containing lipid particles having a ligand specific to target cells on its surface and encapsulating the active ingredient (hereinafter referred to as "targeted-loaded lipid particles").

27. The manufacturing method according to claim 26, wherein a step of replacing the solvent of the mixed solution obtained in step (H) with an aqueous solvent is not included after step (H).

28. The manufacturing method according to claim 26, wherein the active ingredient contains nucleic acid.

29. The manufacturing method according to claim 28, wherein the nucleic acid is DNA or RNA.

30. Lipid particles that have a ligand specific to target cells on their surface and do not contain an active ingredient (targeted cavity lipid particles).

31. A kit containing lipid particles (targeted cavity lipid particles) that have a ligand specific to target cells on their surface and do not contain an active ingredient.

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