Method for producing cell in which exogenous nucleic acid is incorporated

Abstract

Disclosed is a method for producing a cell in which an exogenous nucleic acid has been incorporated into the genome of the cell, the method comprising: (1) a step for introducing a nuclease and a donor nucleic acid into a cell; and (2) a step for placing the cell obtained in step (1) at a temperature of at least 18°C and less than 28°C. Also disclosed is a method for improving the recombination efficiency of exogenous nucleic acid with the genome of a cell, the method comprising: (1) a step for introducing a nuclease and a donor nucleic acid into a cell; and (2) a step for placing the cell obtained in step (1) at a temperature of at least 18°C and less than 28°C. Also disclosed is a method for producing a cell in which exogenous nucleic acid has been incorporated into the genome of the cell, the method comprising: (A) a step for introducing a nuclease and a donor nucleic acid into a cell; and (B) a step for placing the cell obtained in step (A) at a temperature of not more than 37°C in the presence of a ROCK inhibitor and at least one selection from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamines, and trans-ISRIB.

Description

Method for producing cells incorporating exogenous nucleic acid

[0001] The present invention relates to a method for producing a cell in which an exogenous nucleic acid has been integrated into the genome of the cell, and a method for improving the efficiency of recombination of an exogenous nucleic acid into the genome of a cell.

[0002] In recent years, research and development has been progressing on genome editing methods, such as the CRISPR (clustered, regularly interspaced, short palindromic repeats) system, to perform genetic modification in various cells. Class 1 and class 2 CRISPR systems are known, with type I, type III, and type IV being known in class 1, and type II, type V, and type VI being known in class 2. In performing genetic modification, class 2 type II Cas9, which binds to and cleaves DNA, is widely used, but class 2 type V Cpf1 (Cas12a) and C2c1 (Cas12b), which also bind and cleave DNA, are also used. In addition, class 2 type VI Cas13a (C2c2) and Cas13b, which bind to and cleave RNA, have also been reported.

[0003] Genome editing involves introducing a DNA double-strand break (DSB) at a specific site in the genome using a site-specific nuclease, and then inserting (knock-in) the target gene at the site of the nuclease break via the DSB repair mechanism of homologous recombination (HR). HR-mediated knock-in involves using a targeting vector (also known as a "donor vector") carrying the gene to be inserted, double-stranded DNA, single-stranded oligo DNA, etc. The targeting vector has a structure in which a targeting cassette containing the gene to be inserted is sandwiched between two homology arms for initiating HR. Knock-in via DSB and HR can be achieved by transfecting cells with the targeting vector together with an expression vector incorporating an RNA-guided nuclease and guide RNA (gRNA). When such a DSB induced by genome editing technology occurs, repair in a manner that recognizes the homologous sequence of the donor vector and incorporates the vector sequence is called homology-directed repair (HDR).

[0004] Therefore, there is still a need for a method for increasing knock-in efficiency in a cellular genome. As a method for improving such knock-in efficiency (HDR frequency), cold shock treatment has been reported in Patent Literature 1 and Non-Patent Literature 1.

[0005] Non-Patent Document 1 discloses that the efficiency of HDR in iPS cells increases by 1.4 times by performing a cold shock at 32°C for 48 hours after electroporation. Patent Document 1 also discloses a method for increasing the efficiency of HDR in the genome of a cell by introducing a nuclease and a donor nucleic acid into the cell and then shifting the temperature of the cell from 37°C to a lower temperature (e.g., 28°C to 35°C).

[0006] Furthermore, Patent Document 2 discloses that CEPT, which is a combination of chroman I, emricasan, trans-ISRIB and polyamines, significantly improves the survival of pluripotent stem cells in culture.

[0007] International Publication No. WO 2018 / 119060 International Publication No. WO 2020 / 077266

[0008] Nat Commun 11, 2876 (2020)

[0009] An object of the present invention is to provide a method for producing a cell in which an exogenous nucleic acid has been incorporated into the genome of the cell, a method for improving the efficiency of recombination of an exogenous nucleic acid into the genome of a cell, and the like.

[0010] As a result of extensive research to achieve the above object, the present inventors have discovered that the recombination efficiency of exogenous nucleic acids can be improved by introducing a nuclease and a donor nucleic acid into cultured cells by electroporation and then performing cold shock at a temperature below 28° C., and that the recombination efficiency of exogenous nucleic acids is particularly increased by performing cold shock at a temperature of 18° C. or higher but less than 28° C. Furthermore, they have found that performing such cold shock in the presence of a CEPT cocktail allows cells to grow stably while maintaining recombination efficiency.

[0011] The present invention was completed based on these findings and through further investigation, and provides a method for producing cells in which exogenous nucleic acid has been incorporated into the genome of the following cells, a method for improving the efficiency of recombination of exogenous nucleic acid into the genome of a cell, etc.

[0012] [1] A method for producing a cell having an exogenous nucleic acid integrated into its genome, comprising: (1) introducing a nuclease and a donor nucleic acid into the cell; and (2) placing the cell obtained in step (1) at a temperature of 18°C ​​or higher and lower than 28°C. [2] The method according to [1], wherein the temperature in step (2) is 18 to 27°C. [3] The method according to [1], wherein the temperature in step (2) is 21 to 26°C. [4] The method according to any one of [1] to [3], wherein the cell is placed at the temperature for 20 to 80 hours in step (2). [4a] In step (2), the cell is placed under the temperature for 20 to 80 hours in an atmosphere of 0.04 to 5% by volume of CO ２The method according to any one of [1] to [4], wherein the cells are placed at a concentration of 800 μg / mL or less in step (1). [5] The method according to any one of [1] to [4a], wherein the concentration of the donor nucleic acid in step (1) is 800 μg / mL or less. [6] The method according to any one of [1] to [5], wherein the cells are mammalian cells. [7] The method according to [6], wherein the cells are pluripotent stem cells or immune cells. [8] The method according to [7], wherein the cells are induced pluripotent stem cells or embryonic stem cells. [9] The method according to any one of [1] to [8], wherein the nuclease is Cas9 nuclease or Cpf1 nuclease.

[10] The method according to any one of [1] to [9], wherein in step (1), a guide RNA is further introduced into the cells.

[11] The method according to [1] to

[10] , wherein in step (1), the nuclease and the donor nucleic acid are introduced by electroporation.

[12] The method according to any one of [1] to

[11] , wherein step (2) is carried out in the presence of a ROCK inhibitor.

[13] The method according to

[12] , wherein step (2) is further carried out in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamine, and trans-ISRIB.

[14] A method for improving the efficiency of recombination of an exogenous nucleic acid into a cellular genome, comprising: (1) introducing a nuclease and a donor nucleic acid into the cell; and (2) placing the cell obtained in step (1) at a temperature of 18°C ​​or higher and lower than 28°C. [14a] The method according to

[14] , wherein the temperature in step (2) is 18 to 27°C. [14b] The method according to

[14] , wherein the temperature in step (2) is 21 to 26°C. [14c] The method according to any one of

[14] to [14b], wherein the cell is placed at the temperature for 20 to 80 hours in step (2). [14d] The method according to any one of

[14] to [14b], wherein the cell is placed at the temperature for 20 to 80 hours in step (2). ２The method according to any one of

[14] to [14c], wherein the cells are placed at a concentration of 800 μg / mL or less in step (1). [14e] The method according to any one of

[14] to [14d], wherein the concentration of the donor nucleic acid in step (1) is 800 μg / mL or less. [14f] The method according to any one of

[14] to [14e], wherein the cells are mammalian cells. [14g] The method according to [14f], wherein the cells are pluripotent stem cells or immune cells. [14h] The method according to [14g], wherein the cells are induced pluripotent stem cells or embryonic stem cells. [14i] The method according to any one of

[14] to [14h], wherein the nuclease is Cas9 nuclease or Cpf1 nuclease. [14j] The method according to any one of

[14] to [14i], wherein a guide RNA is further introduced into the cells in step (1). [14k] The method according to

[14] to [14j], wherein the nuclease and donor nucleic acid are introduced by electroporation in step (1). [14l] The method of any one of

[14] to [14k], wherein step (2) is carried out in the presence of a ROCK inhibitor. [14m] The method of [14l], wherein step (2) is further carried out in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamines, and trans-ISRIB.

[15] A method for producing a cell having an exogenous nucleic acid integrated into its genome, comprising: (A) introducing a nuclease and a donor nucleic acid into the cell; and (B) placing the cell obtained in step (A) at a temperature of 37°C or less in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamines, and trans-ISRIB, and a ROCK inhibitor. [15a] The method of

[15] , wherein the temperature in step (B) is 18 to 27°C. [15b] The method of

[15] , wherein the temperature in step (B) is 21 to 26°C. [15c] The method according to any one of

[15] to [15b], wherein in step (B), the cells are kept at the temperature for 20 to 80 hours. [15d] In step (B), 0.04 to 5% by volume of CO ２The method according to any one of

[15] to [15c], wherein the cells are placed at a concentration of 800 μg / mL or less in step (A). [15e] The method according to any one of

[15] to [15d], wherein the concentration of the donor nucleic acid in step (A) is 800 μg / mL or less. [15f] The method according to any one of

[15] to [15e], wherein the cells are mammalian cells. [15g] The method according to [15e], wherein the cells are pluripotent stem cells or immune cells. [15h] The method according to [15g], wherein the cells are induced pluripotent stem cells or embryonic stem cells. [15i] The method according to any one of

[15] to [15h], wherein the nuclease is Cas9 nuclease or Cpf1 nuclease. [15j] The method according to any one of

[15] to [15i], wherein a guide RNA is further introduced into the cells in step (A). [15k] The method according to

[15] to [15j], wherein the nuclease and donor nucleic acid are introduced by electroporation in step (A). [15l] The method according to any one of

[15] to [15k], wherein step (B) is carried out in the presence of at least two or three selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, a polyamine, and trans-ISRIB. [15m] The method according to [15l], wherein step (B) is carried out in the presence of chroman 1 or a derivative thereof, emricasan or a derivative thereof, a polyamine, trans-ISRIB, and a ROCK inhibitor.

[0013] According to the present invention, it is possible to improve the efficiency of recombination of exogenous nucleic acids into the genome of cells.

[0014] 1 is a graph showing the effects of cold shock at 32°C and 27°C on the knock-in efficiency of exogenous nucleic acid. The vertical axis shows the HLA-E expression rate (%) of iPS cells recovered after cold shock. EP (electroporation) only shows the results when electroporation was performed without adding nuclease, gRNA, or plasmid, and QHJI only shows the results when electroporation was not performed. This graph shows the effect of cold shock at 25°C on the knock-in efficiency of exogenous nucleic acid. The vertical axis shows the HLA-E expression rate (%) of iPS cells recovered after cold shock. This graph shows the effects of Y27632 and CEPT cocktail as medium additives on the knock-in efficiency of exogenous nucleic acid and cell proliferation. The vertical axis shows the HLA-E expression rate (%) of iPS cells recovered after cold shock. This graph shows the effect of cold shock at 22°C on the knock-in efficiency of exogenous nucleic acid. The bar graph shows the tEGFR expression rate (%) of iPS cells recovered after cold shock, the solid line shows the viability (%) of cells recovered at passage 1 after electroporation, and the dotted line shows the viability (%) of cells recovered at passage 2 after electroporation. QHJI only, 37°C, QHJI only, 25°C cold shock, and QHJI only, 22°C cold shock represent the results without electroporation or cold shock, with 25°C and 48 hours of cold shock, and with 22°C and 48 hours of cold shock, respectively.

[0015] Hereinafter, embodiments of the present invention will be described in detail.

[0016] "Comprise(s)" or "comprising" means the inclusion of, but is not limited to, the elements that follow the phrase. Thus, it implies the inclusion of the elements that follow the phrase, but not the exclusion of any other elements. "Consist(s) of" or "consisting of" means inclusive of and limited to any elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, with other elements being substantially absent. "Consist(s) essentially of" or "consisting essentially of" means inclusive of any elements that follow the phrase, and is limited to other elements that do not affect the activity or function of the element identified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0017] As used herein, "culturing" refers to maintaining and / or growing cells in an in vitro environment. "Culturing" refers to maintaining and / or growing cells outside a tissue or body, for example, in a cell culture dish or flask.

[0018] As used herein, "placing in the presence of a substance" refers to, for example, placing cells in a medium containing the substance. Examples of such a medium include a medium containing only the substance, or a medium containing the substance together with other medium components. When the substance is added to the medium, it can be added directly to the medium, or the substance can be dissolved in an appropriate solvent just before use and then added to the medium. The substance can also be used by immobilizing it on the surface of a substrate or carrier.

[0019] As used herein, "placing cells" means causing cells to exist (be placed) in a medium under specific culture conditions (e.g., temperature conditions), in the presence of a specific substance, etc., particularly culturing cells in a medium under specific culture conditions (e.g., temperature conditions), in the presence of a specific substance, etc. Furthermore, in the case of a step of placing cells at a specific temperature, it means setting the environment in which the cells are placed to a specific temperature and placing the cells in that environment for a certain period of time or more (e.g., 10 hours or more, 20 hours or more, or 24 hours or more).

[0020] As used herein, "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter within a cell.

[0021] As used herein, "pluripotent stem cells" refers to embryonic stem cells (ES cells) and cells that have the same pluripotency, i.e., the potential to differentiate into various tissues in the body (all of the endoderm, mesoderm, and ectoderm). Cells that have the same pluripotency as ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in this specification). When pluripotent stem cells are ES cells or any cells derived from a human embryo, the cells may be cells produced by destroying an embryo or cells produced without destroying an embryo, and are preferably cells produced without destroying an embryo.

[0022] As for "ES cells," in the case of mouse ES cells, various mouse ES cell lines established by inGenious targeting laboratory, RIKEN (Institute of Physical and Chemical Research), etc. can be used, and in the case of human ES cells, various human ES cell lines established by University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, etc. can be used. For example, human ES cell lines that can be used include CHB-1 to CHB-12 strains, RUES1 strain, RUES2 strain, HUES1 to HUES28 strains, etc. distributed by ESI Bio, H1 strain, H9 strain, etc. distributed by WiCell Research, and KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. distributed by RIKEN.

[0023] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells," including iPS cells established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al., Cell, (2007) 131: 861-872), and Nanog-iPS cells established by selecting cells using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells produced by a method that does not contain c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), and iPS cells established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May; 8(5): 409-12, Okita K et al. Stem Cells. 31(3): 458-66.) can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (Japanese Patent Laid-Open Publication No. 2008-307007), and the like can also be used.

[0024] In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholeer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7,795-797), or patents (e.g., JP 2008-307007 A, JP 2008-283972 A, U.S. Patent Application Publication No. 2008 / 2336610, U.S. Patent Application Publication No. 2009 / 047263, WO 2007 / 069666, WO 2008 / 118220, WO 2008 / 124133, WO 2008 / 151058, WO 2009 / 006930, WO 2009 / 006997, WO 2009 / 007852) Any of the induced pluripotent stem cells known in the art can be used.

[0025] As induced pluripotent stem cell lines, various iPS cell lines established by the NIH, RIKEN, Kyoto University, etc. can be used. Examples of human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain, and Kyoto University's Ff-I01s04 strain, QHJI strain, RWMH strain, DRXT strain, RJWI strain, YZWJ strain, ILCL strain, GLKV strain, 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, and 648A1 strain.

[0026] From the viewpoint of therapeutic application, the various cells used in the present invention are preferably cells that comply with GMP (Good Manufacturing Practice) standards.

[0027] The term "nucleic acid" refers to any molecule formed by polymerizing nucleotides and molecules having functions equivalent to those nucleotides, such as RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; a mixed polymer of ribonucleotides and deoxyribonucleotides; and a nucleotide polymer containing a nucleotide analogue. Nucleic acids may also be single-stranded or double-stranded nucleic acids. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes to the other strand under stringent conditions.

[0028] The nucleotide analogue may be any molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve or stabilize nuclease resistance, increase affinity with a complementary nucleic acid strand, increase cell permeability, or enable visualization, compared to RNA or DNA. The nucleotide analogue may be a naturally occurring molecule or a non-natural molecule, and examples thereof include sugar-modified nucleotide analogues (e.g., nucleotide analogues substituted with 2'-O-methylribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, nucleotide analogues substituted with 2'-O-methoxyethylribose, nucleotide analogues substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogues substituted with 2'-fluororibose, bridged artificial nucleic acid (BNA), locked artificial nucleic acid (LNA), ethylene bridged artificial nucleic acid (ENA), and the like. acid), peptide nucleic acid (PNA), oxypeptide nucleic acid (OPNA), peptide ribonucleic acid (PRNA)), nucleotide analogs modified with a phosphodiester bond (e.g., nucleotide analogs substituted with a phosphorothioate bond, nucleotide analogs substituted with an N3'-P5' phosphoamidate bond), etc.

[0029] The nucleic acid derivative may be any molecule in which another chemical substance is added to the nucleic acid in order to improve nuclease resistance, stabilization, affinity with a complementary nucleic acid strand, cell permeability, or visualization, compared to nucleic acids. 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.

[0030] The method of the present invention for producing cells having exogenous nucleic acid integrated into their genome is characterized by comprising the following steps (1) and (2): (1) introducing a nuclease and a donor nucleic acid into the cells; and (2) placing the cells obtained in step (1) at a temperature of 18°C ​​or higher but lower than 28°C.

[0031] The method of the present invention for improving the efficiency of recombination of an exogenous nucleic acid into the genome of a cell is characterized by comprising the above steps (1) and (2).

[0032] The type of cell is not particularly limited, and a wide variety of animal cells can be used, including, for example, spleen cells, nerve cells, glial cells, pancreatic beta cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, myoblasts, and muscle satellite cells), adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells (NK cells), mast cells, neutrophils, basophils, eosinophils, monocytes, and megakaryocytes), synoviocytes, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes, stromal cells, egg cells, and sperm cells, as well as stem cells that can be induced to differentiate into these cells (including pluripotent stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, iPS cells, and ES cells), progenitor cells, blood cells, oocytes, and fertilized eggs. T cells include αβ T cells, γδ T cells, helper T cells, cytotoxic T cells, regulatory T cells, suppressor T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NKT cells, TCR-T cells, STAR receptor T cells, CAR-T cells, etc. Furthermore, animal cells also include primary cells and the above-mentioned cells produced by inducing differentiation of the above-mentioned stem cells (e.g., iPS cells) in vitro. Cells also include various cancer cells. The cells may be of only one type, or may contain two or more types.

[0033] The origin of the cells is not particularly limited, and they are particularly mammalian cells. Mammalian cells may be cells of human origin or mammalian cells of non-human mammals. Examples of non-human mammals include mice, rats, cows, horses, pigs, rabbits, dogs, cats, goats, monkeys, rhesus monkeys, cynomolgus monkeys, and chimpanzees. Of these, humans are preferred as mammals.

[0034] An "exogenous nucleic acid" is a nucleic acid introduced from the outside, and is a nucleic acid having a sequence that does not naturally exist in a cell, or a nucleic acid that exists at a location different from its natural location in the cell genome. An "exogenous nucleic acid" is, for example, a gene introduced from the outside to cause a cell to express a desired protein (e.g., an enzyme, transcription factor, cytokine, tissue growth factor, antibody, therapeutic protein, receptor, or antigen), and can be appropriately selected depending on the intended use of the cell. Examples of exogenous nucleic acids include genes for expressing HLA-C, HLA-E, HLA-F, and HLA-G, which are important for evading NK cell attack. Other exogenous nucleic acids include, for example, genes for expressing CARs (chimeric antigen receptors), which may further contain genes for expressing cytokines and / or chemokines. CARs are basically composed of peptides linked, optionally via spacers, at each of the following sites: (i) an antigen recognition site (e.g., a single-chain antibody) that recognizes a cell surface antigen on a cancer cell; (ii) a transmembrane domain; and (iii) a signal transduction domain that induces T cell activation. The exogenous nucleic acid may also be, for example, a gene for expressing an exogenous T cell receptor (TCR). The exogenous TCR means that the nucleic acid encoding the exogenous TCR is exogenous to the T cell into which it is introduced, and the amino acid sequence of the exogenous TCR may be the same as or different from the endogenous TCR of the T cell.

[0035] Other exogenous nucleic acids include, for example, reporter genes (e.g., genes encoding fluorescent proteins of various colors), drug selection genes (e.g., kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene), suicide genes (e.g., diphtheria A toxin, herpes simplex thymidine kinase (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella-zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyltransferase (XGPRT), inducible caspase 9 (inducible caspase 9)), and the like. Examples of exogenous nucleic acids include nucleic acids (base sequences) encoding "functional genes" such as genes encoding the nucleotide sequences of ...

[0036] Step (1): In step (1), a nuclease and a donor nucleic acid are introduced into cells. The cells are preferably mammalian cells, more preferably pluripotent stem cells or immune cells, and particularly preferably induced pluripotent stem cells or embryonic stem cells. Examples of immune cells include macrophages, T cells, B cells, natural killer cells (NK cells), mast cells, neutrophils, basophils, eosinophils, monocytes, and megakaryocytes. Examples of immune cells include macrophages, T cells, and natural killer cells (NK cells).

[0037] Nucleases used in step (1) include nucleases used in genome editing systems, such as RNA-guided nucleases (particularly RNA-guided endonucleases), TAL effector nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0038] An "RNA-guided endonuclease" is a protein that contains at least one nuclease domain and at least one domain that interacts with a gRNA, and is guided to a target site by forming a complex with the gRNA.

[0039] The RNA-guided endonuclease may be derived from a CRISPR system, which may be a Type I, Type III, or Type IV system within Class 1, or a Type II, Type V, or Type VI system within Class 2. Examples of CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a (or Cpf1), Cas12b (or C2c1), Cas12c, Cas13a1 (or C2c2), Cas13a2, Cas13b, CasF, CasG, CasH, Csy1, Csy2, Csy3, and Csf. Examples include se1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966.

[0040] Examples of RNA-guided endonucleases include those derived from class 2 type II CRISPR systems, particularly those derived from Cas9 proteins. Examples of RNA-guided endonucleases include those derived from class 2 type V CRISPR-Cas12a / Cpf1 systems, particularly those derived from Cpf1 proteins. Nucleases used in the present invention are preferably Cas nucleases, more preferably Cas9 nucleases and Cpf1 nucleases (including Cas12a family nucleases, Cas12a modified nucleases, etc.), and even more preferably Cpf1 nucleases.

[0041] The CRISPR / Cas protein can be a wild-type CRISPR / Cas protein, a modified CRISPR / Cas protein, or a fragment of a wild-type or modified CRISPR / Cas protein. The CRISPR / Cas protein may be modified to increase nucleic acid binding affinity and / or specificity, alter enzymatic activity, or alter another property of the protein.

[0042] The RNA-guided nuclease may be a Cas nuclease or a Cas nickase, and the term Cas nuclease or Cas nickase refers to an endonuclease or nickase that has activity when it forms a complex with two RNAs, CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).

[0043] Cas9 is a preferred RNA-guided nuclease. Examples of Cas9 include Cas9 with the desired nuclease activity, such as Cas9 (SpCas9) derived from Streptococcus pyogenes (S. pyogenes) and Cas9 (SaCas9) derived from Staphylococcus aureus (S. aureus). Cas9 nuclease or Cas9 nickase derived from Streptococcus pyogenes recognizes NGG or NAG trinucleotides as PAM (protospacer adjacent motif) sequences (N is A, T, G, or C).

[0044] Cpf1 nuclease is also preferred as an RNA-guided nuclease, and examples of Cpf1 nuclease include MAD7. MAD7 requires only crRNA for genome editing and recognizes YTTV nucleotides as PAM sequences (Y is C or T, V is A or C).

[0045] The TALEN system using TALEN protein uses artificial nuclease (TALEN) that comprises DNA cleavage domain (for example, FokI domain) and the DNA binding domain of transcription activator-like (TAL) effector.By introducing this system into cells, TALEN binds to target site via DNA binding domain and cuts DNA there.The DNA binding domain that binds to target site can be designed according to known scheme.

[0046] The ZFN system using ZFN protein uses an artificial nuclease (ZFN) that comprises a nucleic acid cleavage domain conjugated to a DNA binding domain that comprises a zinc finger array.By introducing this system into cells, ZFN binds to target site via DNA binding domain and cleaves DNA there.The DNA binding domain that binds to target site can be designed according to a known scheme.

[0047] In the present invention, such nucleases may be used in the form of the nuclease protein itself, or in the form of a nucleic acid containing a sequence encoding the nuclease protein.

[0048] The donor nucleic acid is used to insert (knock-in) a desired base sequence into a target site using homology-directed repair (HDR) that occurs at the site cleaved by a nuclease. The donor nucleic acid contains two base sequences (homology arms) that are highly identical to the base sequence in the target region and the base sequence to be inserted located between them. The base sequence to be knocked-in is not particularly limited. The donor nucleic acid can be an oligo-DNA or a general-purpose vector containing an exogenous nucleic acid, with a plasmid vector containing an exogenous nucleic acid being preferred. If the sequence contained in the exogenous nucleic acid encodes a stop codon, it is possible to suppress the production of a protein that would be expressed in its natural state. The donor nucleic acid can contain a protein-coding sequence integrated under the control of an exogenous promoter. Alternatively, the exogenous nucleic acid can be integrated into a chromosomal sequence so that its expression can be controlled by an endogenous promoter.

[0049] The donor nucleic acid has a structure in which the exogenous nucleic acid to be inserted is flanked by two homology arms for inducing HR. Each homology arm has a sequence substantially identical to a sequence located upstream or downstream of the target site on the chromosome. Here, "substantially identical sequence" means a sequence having at least about 75% sequence identity. The sequence identity is preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Sequence identity can be determined by any method known to those skilled in the art (e.g., using programs such as Clustal, FASTA, and BLAST). The homology arms can range in length from 20 to 5,000 nucleotides. The homology arms are preferably 50 to 1,500 nucleotides in length, more preferably 500 to 1,500 nucleotides in length, and particularly preferably 700 to 1,000 nucleotides in length.

[0050] In step (1), the concentration of the donor nucleic acid used is not particularly limited as long as knock-in is possible, and is, for example, 800 μg / mL or less, preferably 600 μg / mL or less, more preferably 400 μg / mL or less, and for example, 10 μg / mL or more, preferably 50 μg / mL or more, more preferably 100 μg / mL or more. In one aspect, the concentration of the donor nucleic acid used is 10 to 800 μg / mL, preferably 50 to 600 μg / mL, more preferably 100 to 400 μg / mL.

[0051] When an RNA-guided nuclease (particularly an RNA-guided endonuclease) is used in step (1), it is desirable to further introduce a guide RNA (gRNA) into the cell.

[0052] The guide RNA may be in the form of a single RNA in which the crRNA and tracrRNA are linked, i.e., a chimeric RNA (single guide RNA (sgRNA)), or in the form of a single unlinked RNA. In the present invention, such a guide RNA may be used in the form of RNA itself, or in the form of a nucleic acid containing a sequence encoding the guide RNA.

[0053] The crRNA contains a nucleic acid sequence of approximately 17 to 20 bases that hybridizes to a target base sequence for genetic modification (sometimes referred to herein as the "target sequence") in the genome or gene locus within a cell. The target sequence is flanked by a short sequence (PAM) recognized by the CRISPR system. The sequence and length of the PAM vary depending on the type of nuclease used, and the PAM is typically a 2 to 5 base pair sequence adjacent to the target sequence. In order to bind to the target sequence, the crRNA contains a sequence that has, for example, at least 90%, 93%, 95%, 98%, 99%, or 100% identity with the target sequence. Furthermore, when an sgRNA is used as the gRNA, the gRNA has a tracrRNA, and this tracrRNA sequence binds to the RNA-guided nuclease, thereby guiding the RNA-guided nuclease to the target site in the genomic DNA.

[0054] The target sequence is not particularly limited as long as it satisfies the above-mentioned PAM conditions, and can be appropriately selected depending on the purpose. For example, the target sequence can include at least one HLA (human leukocyte antigen) gene from the viewpoint of reducing rejection in allogeneic transplantation. Specific examples of HLA genes include class I HLA (i.e., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G) genes and class II HLA (i.e., HLA-DR, HLA-DQ, and HLA-DP) genes. Alternatively, the expression of a gene important for the expression of the HLA genes and their presentation on the cell surface can be suppressed. Examples of such genes include the B2M gene encoding B2M, a protein important for presenting HLA class I HLA on the cell surface, and the CIITA gene encoding CIITA, a protein important for the expression of class II HLA genes. Other target sequences include, for example, T-cell receptor alpha chain C region (TRAC), which contributes to the onset of graft-versus-host disease.

[0055] In the present invention, when both the guide RNA and the nuclease are in the form of a gene construct such as an expression plasmid, the sequence encoding the guide RNA and the sequence encoding the nuclease protein may both be contained in a single gene construct, or these sequences may be contained in separate gene constructs. Furthermore, the gene construct may contain sequences such as a promoter, enhancer, start codon, stop codon, polyadenylation signal, nuclear localization signal (NLS), drug selection gene, reporter gene, etc., as necessary.

[0056] In step (1), the method for introducing the nuclease and donor nucleic acid (and gRNA) into the cells can be an appropriate method depending on the embodiment. For example, viral infection, calcium phosphate method, lipofection, microinjection, electroporation (e.g., nucleofection), etc. are mentioned, preferably electroporation and lipofection, and particularly preferably electroporation. These methods can be carried out by methods known to those skilled in the art. Furthermore, the electroporation conditions can be appropriately adjusted depending on the embodiment.

[0057] Step (2) In step (2), the cells obtained in step (1) are placed at a temperature of 18°C ​​or higher and lower than 28°C.

[0058] The medium used in step (2) can be a medium used for culturing animal cells as the basal medium. The basal medium is not particularly limited as long as it can be used for culturing animal cells, and examples thereof include AIMV, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, improved MEM with zinc option, IMDM, 199 medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, Fischer's medium, a medium for primate ES cells (a culture medium for primate ES / iPS cells, Reprocell Inc.), a medium for mouse ES cells (TX-WES culture medium, ThromboX), and a serum-free medium (mTeSR, Stemcell). Technology), ReproFF, StemSpan (trade name) SFEM, StemSpan (trade name) H3000, Stemline II, ESF-B medium, ESF-C medium, CSTI-7 medium, Neurobasal medium (Thermo Fisher Scientific), StemPro-34 medium, StemFit (trade name) (e.g., StemFit AK03N, StemFit AK02N), various commercially available products for T cell culture (e.g., CTS ＴＭ OpTmizer ＴＭT-Cell Expansion Basal Medium (Thermo Fisher Scientific), CTS OpTmizer Pro Serum Free Medium (Thermo Fisher Scientific) Scientific), CTS OpTMizer Pro ＴＭ (Thermo Fisher Scientific), CTS ＴＭ OpTmizer ＴＭ T-Cell Expansion Supplement (Thermo Fisher Scientific), 4Cell Nutri-T GMP Lymphocyte Medium (Sartorius), PRIME-XV T Cell Series (Fujifilm Wako Pure Chemical Corporation), ImmunoCult-XF T cell expansion medium (Veritas Corporation), RPMI 1640 Medium Series (Thermo Fisher Scientific), iMedium for T Medium (GC Lymphotec), TexMACS ＴＭ Medium (Miltenyi Biotec), Excelrate ＴＭ T Cell Expansion Media, Xeno-Free (R&D Systems), etc.), commercially available NK cell culture media (e.g., LGM-3 (Lonza), X-VIVO ＴＭ 15 Serum-free lymphocyte medium (Lonza), Advanced RPMI 1640 Medium (Thermo Fisher Scientific), EALI ＴＭ 515-NK Series (Bold Biotechnology Co., Ltd.), Excellerate ＴＭExamples of such a medium include Human NK Cell Expansion Media, Xeno-Free (R&D Systems), NK Cell Initial Medium and NK Cell Subculture Medium in the BINKIT, an NK cell expansion culture kit (Japan BioTherapy Research Institute, Inc.), and KBM NKCC-1, -2 in the KBN NK Kit (Kohjin Bio Co., Ltd.). The medium may contain serum or may be serum-free. The medium may also contain a serum substitute (e.g., albumin, transferrin, Knockout Serum Replacement (KSR), CTS Immune Cell SR (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, ITS supplement, B27™ supplement, etc.). Furthermore, the medium may also contain one or more substances such as lipids, amino acids (e.g., non-essential amino acids), L-glutamine, vitamins, growth factors, cytokines, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. The pH of the medium at the start of culture is usually 7.0 to 7.8, preferably 7.2 to 7.6. After the start of culture, the pH varies depending on the type of cells being cultured and the culture period, but is, for example, 6.8 to 7.8. Before use, the medium is preferably sterilized by filtration, ultraviolet irradiation, heat sterilization, radiation exposure, or the like to prevent contamination.

[0059] Step (2) is preferably carried out in the presence of a ROCK inhibitor (Rho kinase inhibitor). Examples of ROCK inhibitors include Y-27632 ((R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate), fasudil (HA1077) (5-(1,4-diazepan-1-ylsulfonyl)isoquinoline), and H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride). The ROCK inhibitor is preferably added within the first 1 to 3 days, preferably 1 day, after seeding the cells obtained in step (1).

[0060] The concentration of the ROCK inhibitor in the medium is not particularly limited and is adjusted appropriately depending on the type of ROCK inhibitor used, etc. The concentration of the ROCK inhibitor is, for example, 0.1 to 100 μM, preferably 1 to 10 μM.

[0061] Step (2) is preferably carried out in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, a polyamine, and trans-ISRIB, in addition to a ROCK inhibitor. This makes it possible to achieve both high knock-in efficiency and stable cell proliferation (improved survival rate). Preferably, at least two, more preferably at least three, and particularly preferably all four of chroman 1 or a derivative thereof, emricasan or a derivative thereof, a polyamine, and trans-ISRIB are used. The combination of chroman 1 or a derivative thereof, emricasan or a derivative thereof, a polyamine, and trans-ISRIB is sometimes referred to as "CEPT" or a "CEPT cocktail."

[0062] Chroman 1 is (3S)-N-{2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl}-6-methoxy-3,4-dihydro-2H-1-benzopyran-3-carboxamide. Derivatives of chroman 1 are structurally related compounds, and such derivatives are described in Chen et al., "Chroman-3-amides as potent Rho kinase inhibitors," Bioorganic and Medicinal Chemistry Letters 18:6406-6409 (2008) and LoGrasso et al., "Rho Kinase (ROCK) Inhibitors and Their Application to Inflammatory Disorders," Current Topics in Medicinal Chemistry 9:704-723 (2009).

[0063] Emricasan is 3-(2-(2-tert-butylphenylaminooxalyl)aminopropionylamino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid. Derivatives of emricasan are structurally related compounds (such as Q-VD-OPh), and such derivatives are described in Linton et al., "First-in-Class Pan Caspase Inhibitor Developed for the Treatment of Liver Disease," J. Med. Chem. 48:6779-6782, (2005).

[0064] Trans-ISRIB is N,N'-((1r,4r)-cyclohexane-1,4-diyl)bis(2-(4-chlorophenoxy)acetamide).

[0065] Examples of polyamines include polycations such as putrescine, spermidine, and spermine, which are known to interact with negatively charged macromolecules such as DNA, RNA, and proteins.

[0066] The concentration of chroman 1 or a derivative thereof in the medium is not particularly limited, and examples thereof include about 4 nM to about 80 μM, about 10 nM to about 20 μM, about 20 nM to about 10 μM, and about 30 nM to about 500 nM.

[0067] The concentration of emricasan or a derivative thereof in the medium is not particularly limited, and examples include about 5 nM to about 100 μM, about 5 nM to about 80 μM, about 200 nM to about 30 μM, and about 300 nM to about 20 μM.

[0068] The concentration of trans-ISRIB in the medium may be about 5 nM to about 80 μM, about 5 nM to about 50 μM, about 100 nM to about 6.25 μM, or about 200 nM to about 6.25 μM.

[0069] The concentration of polyamine in the medium is, for example, about 0.5 nM to 1 mM.

[0070] Furthermore, ROCK inhibitors, chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamines, trans-ISRIB, and the like can be used in the free state or in the form of a salt. Examples of salts include salts with inorganic bases such as sodium salt, magnesium salt, potassium salt, calcium salt, and aluminum salt; salts with organic bases such as methylamine salt, ethylamine salt, and ethanolamine salt; salts with basic amino acids such as lysine, ornithine, and arginine; and ammonium salts. The salts may be acid addition salts, and specific examples of such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. The ROCK inhibitors, Chroman 1 or a derivative thereof, Emricasan or a derivative thereof, polyamines, trans-ISRIB, etc. also include hydrates, solvates, crystalline polymorphs, etc.

[0071] Step (2) may be either adherent or suspension culture. Step (2) may be performed in the presence or absence of feeder cells. In the present invention, it is preferable to perform the process in the absence of feeder cells to prevent contamination with unknown components. As used herein, "in the absence of feeder cells" or "feeder-free" refers to the absence of feeder cells, or the absence of a medium preconditioned by culturing feeder cells. Therefore, the medium does not contain growth factors, cytokines, or other substances secreted by feeder cells. "Feeder cells" or "feeders" refer to cells that are co-cultured with other types of cells and provide an environment in which the cells can grow and support them. Feeder cells may be derived from the same or a different species as the cells they support. For example, human dermal fibroblasts or human embryonic stem cells may be used as feeders for human cells, or primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts may be used. Feeder cells can be inactivated by irradiation, mitomycin C treatment, or the like.

[0072] Examples of the culture vessel used in step (2) include a petri dish, flask, plastic bag, dish, Petri dish, tissue culture dish, multi-dish, microplate, microwell plate, multi-plate, multi-well plate, chamber slide, cell culture flask, spinner flask, tube, tray, culture bag, roller bottle, and the like, which are commonly used for cell culture.

[0073] In step (2), CO ２ The concentration is, for example, 0.01% by volume to 10% by volume, preferably 0.04% by volume to 5% by volume, and the oxygen concentration is, for example, 1% by volume to 20% by volume, preferably 5% by volume to 20% by volume. ２ This can be done using an incubator, etc. In step (2), the cells may be passaged as many times as necessary, and the medium may be added and replaced.

[0074] In step (2), the temperature at which the cells are maintained is 18°C ​​or higher and lower than 28°C. For the purpose of improving the efficiency of recombination of exogenous nucleic acids, the temperature is preferably 18 to 27°C, more preferably 18 to 26°C, even more preferably 18 to 24°C, and particularly preferably 18 to 22°C. However, since low temperatures may damage cells, a preferred temperature in another embodiment is 21 to 26°C. The period for which the cells are maintained at such low temperatures is not particularly limited as long as it improves the efficiency of recombination of exogenous nucleic acids, and examples include 10 to 80 hours, 20 to 80 hours, 20 to 60 hours, 20 to 50 hours, and 24 to 48 hours. After the cells have been maintained at such low temperatures for a certain period of time, they may be cultured at a temperature of 30°C or higher (particularly 37°C).

[0075] By placing cells at such low temperatures (cold shock), the efficiency of recombination of exogenous nucleic acids can be improved. By performing cold shock, the efficiency of recombination of exogenous nucleic acids can be improved, for example, by 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more. The improvement in the efficiency of recombination of exogenous nucleic acids can be confirmed by measuring the presence of exogenous nucleic acids integrated into the genome of cells, or by measuring the proportion of cells expressing mRNA transcribed from the exogenous nucleic acids and proteins synthesized through translation, for example.

[0076] The method of the present invention for producing a cell having an exogenous nucleic acid integrated into its genome is characterized by comprising the following steps (A) and (B): (A) introducing a nuclease and a donor nucleic acid into the cell, and (B) placing the cell obtained in step (A) at a temperature of 37°C or lower in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamine, and trans-ISRIB, and a ROCK inhibitor.

[0077] Step (A) In step (A), a nuclease and a donor nucleic acid are introduced into a cell.

[0078] The step (A) can be carried out in the same manner as in the above step (1).

[0079] Step (B) In step (B), the cells obtained in step (A) are placed at a temperature of 37°C or lower in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamines, and trans-ISRIB, and a ROCK inhibitor.

[0080] Step (B) can be carried out in the same manner as in step (2) above, except that step (B) is carried out in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamines, and trans-ISRIB, and a ROCK inhibitor, and at a temperature of 37°C or lower.

[0081] In step (B), the temperature at which the cells are maintained is 37°C or lower. For the purpose of improving the efficiency of recombination of exogenous nucleic acids, the temperature is preferably 18°C ​​to 32°C, more preferably 18°C ​​to 28°C, even more preferably 18°C ​​to 27°C, even more preferably 18°C ​​to 25°C, particularly preferably 18°C ​​to 24°C, and most preferably 18°C ​​to 22°C. However, since low temperatures may damage cells, a preferred temperature in another embodiment is 21°C to 26°C. The period for which the cells are maintained at such low temperatures is not particularly limited as long as it improves the efficiency of recombination of exogenous nucleic acids, and examples include 10 to 80 hours, 20 to 80 hours, 20 to 60 hours, 20 to 50 hours, and 24 to 48 hours. After the cells have been maintained at such low temperatures for a certain period of time, they may be cultured at a temperature of 30°C or higher (particularly 37°C).

[0082] Step (B) is carried out in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamines, and trans-ISRIB, and a ROCK inhibitor, thereby enabling stable cell growth. Preferably, at least two, more preferably at least three, and particularly preferably all four of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamines, and trans-ISRIB are used.

[0083] The cells produced by the method of the present invention can be safely administered to patients in need thereof, either directly or by mixing with a pharmacologically acceptable carrier or the like to form a medicine. The cells produced by the method of the present invention can be used to treat or prevent cancer, genetic diseases, autoimmune diseases, graft-versus-host disease, etc., as long as they are administered in a therapeutically effective amount. The dosage may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the recipient, and is not particularly limited. The patient is a mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, or human), preferably a human.

[0084] As used in this specification and claims, singular terms include plurals and plural terms include the singular, unless the context otherwise requires. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept, unless otherwise specified.

[0085] The present invention will be described in detail below using examples, but the present invention is not limited in any way. Unless otherwise specified, the reagents and materials used are commercially available or can be prepared according to known literature, etc. Furthermore, any material having the same effect or action can be substituted.

[0086] [Test Example 1] Test to verify the effect of cold shock at 32°C and 27°C on the knock-in efficiency of exogenous nucleic acid

[0087] <Method> 1. Recovery and Culture of iPS Cells Frozen iPS cells QHJI01S04 strain provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, were thawed in a water bath set at 37°C and washed with iPS maintenance culture medium (AK03N, Ajinomoto Co., Inc.). After washing, the iPS cells were seeded onto iMatrix-coated (laminin-iMatrix-511, Nippi Co., Ltd.) 6-well plates in iPS maintenance culture medium supplemented with rock inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.), and incubated at 5% CO ２The cells were cultured at 37°C. The next day, the iPS maintenance culture medium containing the Rock inhibitor was removed, and fresh iPS maintenance culture medium was added. The medium was changed on days 1 and 2 after cell seeding, and daily from day 5 onwards.

[0088] 2. After gene editing and recovery culture of iPS cells, the cells were detached with a cell scraper using a mixture of TrypLE (TrypLE select, Gibco) and EDTA solution (0.5 M EDTA, Nacalai Tesque, Inc.). 4.5 x 10 cells were detached using the 4D-Nucleofector system (Lonza) and 4D NucleofectorX Cuvette (Lonza). ６ Gene editing was performed by electroporation of the cells with Cpf1 nuclease, a guide RNA targeting B2M, and a plasmid containing HLA-E (43 μg / mL). After electroporation, the cells were cultured in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.).

[0089] 3. Cold shock of gene-edited iPS cells: For cold shock, cells were incubated at 32°C or 27°C in 5% CO for 48 hours after electroporation. ２ The culture was incubated under 5% CO ２ The 37°C control was cultured under 5% CO after electroporation. ２ The cells were cultured at 37°C for 10 min. The next day, the iPS maintenance culture medium containing the ROCK inhibitor was removed and fresh iPS maintenance culture medium was added for all conditions. The medium was changed on days 1 and 2 after cell seeding, and daily from day 5 onwards. For each condition, the cells were passaged after reaching semi-confluence, and then harvested after reaching semi-confluence again.

[0090] <Results> The HLA-E expression rate of iPS cells recovered after cold shock of the gene-transfected iPS cells described in 3. above was measured using a flow cytometer, and the results are shown in Figure 1. The cells subjected to cold shock at 32°C and 27°C for 48 hours showed enhanced HLA-E transduction efficiency compared to cells cultured at 37°C (Figure 1).

[0091] [Test Example 2] Test to verify the effect of 25°C cold shock on the knock-in efficiency of exogenous nucleic acid

[0092] <Method> 1. Recovery and culture of iPS cells Frozen iPS cells QHJI01S04 strain provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, were thawed in a water bath set at 37°C and washed with iPS maintenance culture medium (AK03N, Ajinomoto Co., Inc.). After washing, the iPS cells were seeded onto iMatrix-coated (laminin-iMatrix-511, Nippi Co., Ltd.) 6-well plates in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.), and incubated at 5% CO ２ The cells were cultured at 37°C. The next day, the iPS maintenance culture medium containing the ROCK inhibitor was removed, and fresh iPS maintenance culture medium was added. The medium was changed on the first day after cell seeding, and on the second day and every day thereafter from the fifth day onwards.

[0093] 2. After gene editing and recovery culture of iPS cells, the cells were detached with a cell scraper using a mixture of TrypLE (TrypLE select, Gibco) and EDTA solution (0.5 M EDTA, Nacalai Tesque, Inc.). Using the 4D-Nucleofector system (Lonza) and 4D NucleofectorX Strip (Lonza), 9 x 10 ５ Gene editing was performed by electroporation of the cells with Cpf1 nuclease, a guide RNA targeting B2M, and a plasmid containing HLA-E (100 μg / mL). After electroporation, the cells were cultured in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.).

[0094] 3. Cold shock of gene-edited iPS cells For the 48-hour cold shock, cells were incubated at 32°C, 27°C, or 25°C in 5% CO for 48 hours after electroporation. ２ The culture was incubated under 5% CO ２For the 23-hour cold shock condition, the cells were incubated at 32°C in 5% CO for 23 hours after electroporation. ２ The culture was incubated under 5% CO ２ The cells were cultured at 37°C for 10 min. The next day, the iPS maintenance culture medium containing the ROCK inhibitor was removed and fresh iPS maintenance culture medium was added for all conditions. The medium was changed on days 1 and 2 after cell seeding, and daily from day 5 onwards. For each condition, the cells were passaged after reaching semi-confluence, and then harvested after reaching semi-confluence again.

[0095] <Results> Figure 2 shows the results of flow cytometry analysis of HLA-E expression in iPS cells recovered after cold shock of the gene-transfected iPS cells described in 3 above. Cells subjected to cold shock at 25°C for 48 hours showed enhanced HLA-E transduction efficiency compared to cells cultured at 37°C or 27°C, demonstrating that the lower the temperature, the higher the transduction efficiency of foreign genes. Furthermore, an investigation of the duration of cold shock at 32°C showed that equivalent transduction efficiencies were obtained at 48 and 23 hours (Figure 2).

[0096] [Test Example 3] Test to verify the effects of Y27632 and CEPT cocktail as medium additives on the knock-in efficiency of exogenous nucleic acids and cell proliferation

[0097] <Method> 1. Recovery and culture of iPS cells Frozen iPS cells QHJI01S04 strain provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, were thawed in a water bath set at 37°C and washed with iPS maintenance culture medium (AK03N, Ajinomoto Co., Inc.). After washing, the iPS cells were seeded onto iMatrix-coated (laminin-iMatrix-511, Nippi Co., Ltd.) 6-well plates in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.), and incubated at 5% CO ２ The cells were cultured at 37°C. The next day, the iPS maintenance culture medium containing the ROCK inhibitor was removed, and fresh iPS maintenance culture medium was added. The medium was changed on the first day after cell seeding, and on the second day and every day thereafter from the fifth day onwards.

[0098] 2. After gene editing and recovery culture of iPS cells, the cells were detached with a cell scraper using a mixture of TrypLE (TrypLE select, Gibco) and EDTA solution (0.5 M EDTA, Nacalai Tesque, Inc.). Using the 4D-Nucleofector system (Lonza) and 4D NucleofectorX Strip (Lonza), 9 x 10 ５ Gene editing was performed by electroporation of the cells after adding Cpf1 nuclease, a guide RNA targeting B2M, and a plasmid containing HLA-E (100 μg / mL). After electroporation, the cells were cultured in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Corporation), a CEPT cocktail (CultureSure CEPT cocktail, Fujifilm Wako Pure Chemical Corporation), or a ROCK inhibitor and a CEPT cocktail.

[0099] 3. Cold shock of gene-edited iPS cells: For the 48-hour cold shock, cells were incubated at 25°C in 5% CO for 48 hours after electroporation. ２ The culture was incubated under 5% CO ２ The cells were cultured at 37°C for 10 min. The next day, the iPS maintenance culture medium containing the ROCK inhibitor and CEPT cocktail was removed and fresh iPS maintenance culture medium was added. Medium changes were performed on days 1 and 2 after cell seeding, and daily from day 5 onwards. For each condition, cells were passaged on day 7, when they reached semi-confluence, and then harvested on day 13, when they reached semi-confluence again.

[0100] <Results> The HLA-E expression rate of iPS cells recovered after cold shock of the gene-transferred iPS cells described in 3 above was measured using a flow cytometer. The results are shown in Figure 3, and the cell proliferation rate after electroporation is shown in Table 1.

[0101]

[0102] Under cold shock conditions at 25°C, cells treated with CEPT cocktail were more susceptible to damage during the 1-month period after electroporation than cells treated with Y27632. ｓｔ It was shown that the proliferation rate of cells during expansion was enhanced. Furthermore, cells treated with both Y27632 and the CEPT cocktail showed a 2 ｎｄ It was confirmed that, under cold shock conditions of 25°C or below, far from 37°C, the addition of both Y27632 and the CEPT cocktail enabled stable cell proliferation while maintaining gene transfer efficiency.

[0103] [Test Example 4] The effect of the combined addition of Y27632 and CEPT cocktail on cell proliferation during cold shock at 25°C was examined for each electroporation program.

[0104] <Method> 1. Recovery and culture of iPS cells Frozen iPS cells QHJI01S04 strain provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, were thawed in a water bath set at 37°C and washed with iPS maintenance culture medium (AK03N, Ajinomoto Co., Inc.). After washing, the iPS cells were seeded onto iMatrix-coated (laminin-iMatrix-511, Nippi Co., Ltd.) 6-well plates in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.), and incubated at 5% CO ２ The cells were cultured at 37°C. The next day, the iPS maintenance culture medium containing the ROCK inhibitor was removed, and fresh iPS maintenance culture medium was added. The medium was changed on the first day after cell seeding, and on the second day and every day thereafter from the fifth day onwards.

[0105] 2. After gene editing and recovery culture of iPS cells, the cells were detached with a cell scraper using a mixture of TrypLE (TrypLE select, Gibco) and EDTA solution (0.5 M EDTA, Nacalai Tesque, Inc.). Using the 4D-Nucleofector system (Lonza) and 4D NucleofectorX Strip (Lonza), 9 x 10 ５To the cells, Cpf1 nuclease, guide RNA targeting B2M, and a plasmid containing HLA-E (100 μg / mL) were added, and gene editing was performed by electroporation using three different programs (CA137, CM138, CM150) installed in the 4D-Nucleofector system. After electroporation, the cells were cultured in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Corporation), a CEPT cocktail, or a ROCK inhibitor and a CEPT cocktail.

[0106] 3. Cold shock of gene-edited iPS cells: For the 48-hour cold shock, cells were incubated at 25°C in 5% CO for 48 hours after electroporation. ２ The culture was incubated under 5% CO ２ The 37°C control was cultured under 5% CO after electroporation. ２ The cells were cultured at 37°C. The next day, for all conditions, the iPS maintenance culture medium containing the ROCK inhibitor and CEPT cocktail was removed and fresh iPS maintenance culture medium was added. The medium was changed on days 1 and 2 after cell seeding, and daily from day 5 onwards. After the cells reached semi-confluence under each condition (day 6 for 37°C culture and day 7 for 25°C cold shock), the cells were passaged and collected.

[0107] <Results> The HLA-E expression rate of iPS cells recovered after cold shock of the gene-transferred iPS cells described in 3 above was measured using a flow cytometer, and the cell proliferation rate was also measured. Table 2 shows the results.

[0108] In the table, EP only represents the results when electroporation was performed without adding nuclease, gRNA, or plasmid, and QHJI only represents the results when electroporation was not performed.

[0109]

[0110] Cold shock at temperatures far from 37°C, such as 25°C, enhances knock-in efficiency of exogenous nucleic acids, but poses a challenge in terms of cell damage after electroporation. In this experiment, adding only Y27632 as a medium additive resulted in poor cell growth, and 7 days after electroporation, sufficient cell numbers for analysis could not be obtained. On the other hand, adding Y27632 and a CEPT cocktail as medium additives simultaneously achieved both high knock-in efficiency and stable cell growth, demonstrating that this phenomenon is independent of the electroporation program.

[0111] Test Example 5: Test to verify the effect of 22°C cold shock on the knock-in efficiency of exogenous nucleic acids <Method> 1. Recovery and culture of iPS cells Frozen iPS cells QHJI01S04 strain provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, were thawed in a water bath set to 37°C and washed with iPS maintenance culture medium (AK03N, Ajinomoto Co., Inc.). After washing, the iPS cells were seeded onto iMatrix-coated (laminin-iMatrix-511, Nippi Co., Ltd.) 6-well plates in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.), and incubated at 5% CO ２ The cells were cultured at 37°C. The next day, the iPS maintenance culture medium containing the Rock inhibitor was removed, and fresh iPS maintenance culture medium was added. The medium was changed every day from day 1, 2, and 5 after cell seeding.

[0112] 2. Gene transfer into iPS cells After recovery culture, the cells were detached with a cell scraper using a mixture of TrypLE (TrypLE select, Gibco) and EDTA solution (0.5 M EDTA, Nacalai Tesque, Inc.). 4 x 10 cells were detached using the 4D-Nucleofector system (Lonza) and 4D NucleofectorX Cuvette (Lonza). ６Gene editing was performed by electroporation of the cells after adding Cpf1 nuclease, a guide RNA targeting CIITA, and a plasmid (200 μg / ml) containing the tEGFR sequence. After electroporation, the cells were cultured in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Corporation) at 37°C, and in iPS maintenance culture medium supplemented with a ROCK inhibitor (Y-27632, Fujifilm Wako Pure Chemical Corporation) and a CEPT cocktail (CultureSure CEPT cocktail, Fujifilm Wako Pure Chemical Corporation) at 25°C and 22°C.

[0113] 3. Cold shock of gene-edited iPS cells For the 48-hour cold shock, cells were incubated at 25°C or 22°C in 5% CO for 48 hours after electroporation. ２ The culture was incubated under 5% CO ２ For the 72-hour cold shock condition, the cells were cultured at 25°C or 22°C in 5% CO for 72 hours after electroporation. ２ The culture was incubated under 5% CO for 72 hours. ２ The 37°C control was cultured under 5% CO after electroporation. ２ The cells were cultured at 37°C for 1 min. The next day, the medium was removed and fresh iPS maintenance culture medium was added for all conditions. The medium was changed on days 1 and 2 after cell seeding, and daily from day 5 onwards. For each condition, the cells were passaged after reaching semi-confluence, and then harvested after reaching semi-confluence again.

[0114] <Results> Figure 4 shows the results of flow cytometry analysis of tEGFR expression in iPS cells recovered after cold shock of the gene-transfected iPS cells described in 3 above. Cells cold-shocked at 22°C for 48 hours showed enhanced tEGFR transduction efficiency compared to cells cultured at 37°C or 25°C, demonstrating that the lower the temperature, the higher the transduction efficiency of exogenous genes. Furthermore, when cold shock durations at 25°C and 22°C were examined, the 48-hour cold shock tended to result in slightly higher transduction efficiency than the 48-hour cold shock duration at 72 hours. However, in both cases, the transduction efficiency was significantly higher than under the 37°C control condition. Furthermore, cold shock reduced cell viability at the first passage after electroporation, but fully recovered at the second passage ( Figure 4 ).

[0115] In addition, CO ２ It is necessary to adjust the concentration, but it was found that placing cells at low temperatures reduces the gas exchange rate. ２ A concentration of 1.9% by volume was preferred.

[0116] Furthermore, when the same experiment as above was carried out at a temperature of 18°C, the efficiency of tEGFR transfection was enhanced.

[0117] This application is based on Japanese Patent Application No. 2024-130212 filed on August 6, 2024, the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing cells in which exogenous nucleic acid has been integrated into the genome of the cells, comprising: (1) introducing a nuclease and a donor nucleic acid into the cells; and (2) placing the cells obtained in step (1) at a temperature of 18°C ​​or higher but lower than 28°C.

2. The method according to claim 1, wherein the temperature in step (2) is 18 to 27°C.

3. The method according to claim 1, wherein the temperature in step (2) is 21 to 26°C.

4. The method according to claim 1, wherein in step (2), the cells are kept at said temperature for 20 to 80 hours.

5. The method according to claim 1, wherein in step (1), the concentration of the donor nucleic acid is 800 μg / mL or less.

6. The method of claim 1, wherein the cell is a mammalian cell.

7. The method of claim 6, wherein the cells are pluripotent stem cells or immune cells.

8. The method of claim 7, wherein the cells are induced pluripotent stem cells or embryonic stem cells.

9. The method of claim 1, wherein the nuclease is a Cas9 nuclease or a Cpf1 nuclease.

10. The method according to claim 1, wherein in step (1), a guide RNA is further introduced into the cell.

11. The method according to claim 1, wherein in step (1), the nuclease and the donor nucleic acid are introduced by electroporation.

12. The method according to claim 1, wherein step (2) is carried out in the presence of a ROCK inhibitor.

13. The method of claim 12, wherein step (2) is further carried out in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, a polyamine, and trans-ISRIB.

14. A method for improving the efficiency of recombination of an exogenous nucleic acid into a cellular genome, comprising: (1) introducing a nuclease and a donor nucleic acid into a cell; and (2) placing the cell obtained in step (1) at a temperature of 18°C ​​or higher but lower than 28°C.

15. A method for producing a cell having an exogenous nucleic acid integrated into its genome, comprising: (A) introducing a nuclease and a donor nucleic acid into the cell; and (B) placing the cell obtained in step (A) at a temperature of 37°C or lower in the presence of at least one selected from the group consisting of chroman 1 or a derivative thereof, emricasan or a derivative thereof, polyamines, and trans-ISRIB, and a ROCK inhibitor.

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