HLA gene modified cell

Abstract

The present invention provides a cell having improved tissue compatibility with a transplantation recipient. Specifically, a polynucleotide encoding a single-stranded fusion peptide that mimics HLA-G and / or HLA-E which functions in a suppressing manner against the activity (cytotoxicity) of NK cells or macrophages is knocked into a region encoding the α-chain of the HLA class I molecule, thereby suppressing the expression of the α-chain of the HLA class I molecule while expressing the single-stranded fusion peptide.

Description

HLA gene modified cells

[0001] The present invention relates to HLA gene modified cells that have improved histocompatibility with recipients.

[0002] In cell-based regenerative medicine, cells are transplanted into a patient's body to repair and regenerate the function of organs and tissues damaged by disease or injury. Immune rejection, which occurs when the patient's HLA (Human Leukocyte Antigen) does not match the transplanted cells, significantly affects the success of treatment. Autologous transplants, which use cells harvested from the patient's own body, are advantageous in terms of suppressing immune rejection, but the cost of producing the transplanted cells is high. Allogeneic transplants, which use cells from another person's body, are advantageous in terms of low production costs, but require treatment to suppress immune rejection (e.g., administration of immunosuppressants). For allogeneic transplants using iPS cells, one possible strategy is to prepare multiple types of transplanted cells in advance from multiple iPS cell lines obtained from an iPS cell bank and select those with high HLA compatibility with the patient. However, this approach also faces challenges: production costs increase depending on the number of iPS cell lines used, and it is difficult to match pre-prepared transplanted cells to all patient HLA types.

[0003] Immune rejection occurs when the patient's immune cells (e.g., T cells) recognize transplanted cells with a different HLA type from the patient's as non-self and attack them. Therefore, attention has been focused on a technology called hypoimmunogenic technology, which improves tissue compatibility with the recipient by genetically modifying transplanted cells to suppress HLA expression. By applying hypoimmunogenic technology to a single type of iPS cell, transplant cells suitable for a variety of patients can be produced at low cost.

[0004] Patent Literature 1 discloses stem cells in which the expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) and HLA class II molecules has been reduced by genetic modification. Patent Literature 2 discloses primate cells in which β2 microglobulin (B2M) has been deleted by genetic modification. Cells lacking B2M, a component (β chain) of HLA class I molecules, do not form HLA class I molecules capable of antigen presentation. Patent Literature 2 further discloses that, in creating B2M-deficient cells, a polynucleotide encoding either (1) a single-chain fusion protein (heterodimeric protein) comprising B2M and the α chain of an HLA class I molecule, or (2) a single-chain fusion protein (heterotrimeric protein) comprising a peptide presented on the cell surface by HLA class I molecules (e.g., the signal peptide of HLA-G), B2M, and the α chain of an HLA class I molecule, is knocked into the B2M locus. Non-Patent Document 1 discloses that when a polynucleotide encoding (1) a single-chain fusion protein comprising B2M and the α chain of HLA-E or (2) a single-chain fusion protein comprising a peptide antigen, B2M, and the α chain of HLA-E was knocked into the B2M locus of human pluripotent stem cells to forcibly express HLA-E, the cells exhibited resistance to natural killer (NK) cell-mediated lysis. However, because B2M is involved in the expression and function of various cell surface molecules in addition to the expression of HLA class I molecules, knocking out B2M posed a problem, potentially affecting cellular function. When HLA-G binds to KIR2DL4 and LILRB1, which are expressed as inhibitory receptors on NK cells and macrophages, their cytotoxicity is suppressed. When HLA-E binds to CD94 / NKG2A or CD94 / NKG2B, which are expressed as inhibitory receptors on NK cells or macrophages, their cytotoxicity is suppressed (Non-Patent Documents 2 and 3).

[0005] Special table 2022-526218 publication Special table 2014-513948 publication

[0006] Nature Biotechnology volume 35, pages765-772 (2017)Arch Immunol Ther Exp (Warsz). 2016; 64(6): 505-514.Nature. 1998; 391: 795-799

[0007] The goal was to provide cells with improved tissue compatibility with the recipient.

[0008] A polynucleotide encoding an effector cell inhibitory factor (particularly a single-chain fusion peptide mimicking HLA-G or HLA-E) that functions to suppress the activity (cytotoxicity) of effector cells (particularly NK cells and macrophages) is knocked into a region encoding the α chain of an HLA class I molecule, thereby suppressing the expression of the α chain of an HLA class I molecule while expressing the effector cell inhibitory factor (particularly the single-chain fusion peptide). That is, the present invention relates to the following [1] to

[47] . [1] An HLA gene-modified cell, in which the following polynucleotides (1) and (2) have been knocked into a region containing the α-chain genes of HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G: (1) a polynucleotide encoding a single-chain dimeric fusion peptide (Single Chain Dimer: SCD) comprising β2 microglobulin and the α-chain of HLA-G; (2) a polynucleotide encoding a single-chain trimeric fusion peptide (Single Chain Trimer: SCT) comprising the signal peptide of HLA-A, HLA-B, HLA-C, or HLA-G, β2 microglobulin, and the α-chain of HLA-E. [2] The cell according to [1] above, in which expression of the α-chain genes of HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G is reduced compared to a wild-type cell. [3] The cell according to [1] above, in which the α-chain genes of HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G have been deleted from the cell before genetic modification. [4] The cell according to [1] above, in which (1) a polynucleotide encoding SCD has been knocked into a region containing the α-chain genes of HLA-F, HLA-G, and HLA-A, and (2) a polynucleotide encoding SCT has been knocked into a region containing the α-chain genes of HLA-C and HLA-B. [5] The cell according to [1] above, in which (2) a polynucleotide encoding SCT has been knocked into a region containing the α-chain genes of HLA-F, HLA-G, and HLA-A, and (1) a polynucleotide encoding SCD has been knocked into a region containing the α-chain genes of HLA-C and HLA-B.[6] An HLA gene-modified cell, wherein the polynucleotide (1) below has been knocked into a region containing the α-chain genes of HLA-F, HLA-G, and HLA-A: (1) a polynucleotide encoding a single-chain dimer fusion peptide (Single Chain Dimer: SCD) containing the α-chain of β2-microglobulin and HLA-G. [7] The cell according to [6] above, in which expression of the α-chain genes of HLA-F, HLA-G, and HLA-A is reduced compared to that of a wild-type cell. [8] The cell according to [6] above, in which the α-chain genes of HLA-F, HLA-G, and HLA-A have been deleted in the cell before gene modification. [9] An HLA gene-modified cell, wherein the polynucleotide of (2) below has been knocked into a region containing the α-chain genes of HLA-C and HLA-B: (2) A polynucleotide encoding a single chain trimer (SCT) comprising a signal peptide of HLA-A, HLA-B, HLA-C, or HLA-G, β2-microglobulin, and the α-chain of HLA-E.

[10] The cell according to [9] above, in which expression of the α-chain genes of HLA-C and HLA-B is reduced compared to that of a wild-type cell.

[11] The cell according to [9] above, in which the α-chain genes of HLA-C and HLA-B have been deleted from the cell before gene modification.

[12] The cell according to [1] or [6] above, wherein the region containing the α-chain genes of HLA-F, HLA-G, and HLA-A is a chromosomal region corresponding to chr6: 29,704,906-29,958,642 of the hg38 genome sequence.

[13] The cell according to [1] or [9] above, wherein the region containing the α-chain genes of HLA-C and HLA-B is a chromosomal region corresponding to chr6: 31,224,837-31,378,196 of the hg38 genome sequence.

[14] The cell according to [1] or [9] above, wherein the region containing the α-chain genes of HLA-C and HLA-B is a chromosomal region corresponding to chr6: 31,227,286-31,378,780 of the hg38 genome sequence.

[15] A cell described in [1], [6] or [9], in which (1) a polynucleotide encoding SCD and / or (2) a polynucleotide encoding SCT is knocked in only one allele of the target cell.

[16] The cell according to [1], [6] or [9] above, further comprising a modification that reduces expression of an HLA class II molecule gene compared to a wild-type cell.

[17] The cell according to [1], [6] or [9] above, further comprising a deletion of the HLA class II molecule gene by modification.

[18] The cell according to

[16] above, wherein the HLA class II molecule gene is the CIITA gene.

[19] The cell according to

[17] above, wherein the HLA class II molecule gene is the CIITA gene.

[20] The cell according to [1], [6] or [9] above, which is a human pluripotent stem cell.

[21] The cell according to [1], [6] or [9] above, which is a human iPS cell or an ES cell.

[22] The cell according to [1], [6] or [9] above, which is a human iPS cell line 253G4 or a human iPS cell line 201B7.

[23] The cell according to [1], [6] or [9] above, which is a cell differentiated from a human pluripotent stem cell.

[24] The cell according to

[23] above, wherein the human pluripotent stem cell is a human iPS cell or an ES cell.

[25] The cell according to

[23] above, wherein the human pluripotent stem cell is a human iPS cell 253G4 strain, a human iPS cell or an ES cell.

[26] The cell according to

[23] above, wherein the cell differentiated from a human pluripotent stem cell is an immune cell, a tissue stem cell or a somatic cell.

[27] The cell according to

[26] above, wherein the immune cell is a T cell or an NK cell.

[28] The cell according to [1] or [6] above, wherein the SCD comprises the amino acid sequence shown in SEQ ID NO: 1 or 4, or an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 4.

[29] The cell according to [1] or [9], wherein the SCT comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 6.

[30] The cell according to [1] or [6], wherein the polynucleotide of (1) comprises the nucleotide sequence shown in SEQ ID NO: 2 or 5 or a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2 or 5.

[31] The cell according to [1] or [9], wherein the polynucleotide of (2) comprises the nucleotide sequence shown in SEQ ID NO: 7 or a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 7.

[32] The cell according to [1] or [6], wherein the SCD further comprises a linker sequence between the β2 microglobulin and the α chain of HLA-G.

[33] The cell according to [1] or [9], wherein the SCT further comprises a linker sequence between the signal peptide and β2 microglobulin.

[34] The cell according to [1] or [9], wherein the SCT further comprises a linker sequence between the β2 microglobulin and the α chain of HLA-E.

[35] The cell according to

[32] , wherein the linker sequence comprises a peptide consisting of glycine and serine.

[36] The cell according to

[33] , wherein the linker sequence comprises a peptide consisting of glycine and serine.

[37] The cell according to

[34] , wherein the linker sequence comprises a peptide consisting of glycine and serine.

[38] The cell according to

[32] , wherein the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 3.

[39] The cell according to

[33] above, wherein the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 8.

[40] The cell according to

[34] above, wherein the linker sequence comprises the amino acid sequence shown in SEQ ID NO: 9.

[41] The cell according to [1] or [6] above, wherein a promoter sequence of the HLA-A α chain gene has been knocked in together with the polynucleotide of (1), and the promoter sequence is operably linked to a sequence encoding SCD.

[42] The cell according to

[41] above, wherein the promoter sequence of the HLA-A α chain gene comprises the nucleotide sequence shown in SEQ ID NO: 10 or a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 10.

[43] The cell according to [1] or [9] above, wherein a promoter sequence of the HLA-B α chain gene has been knocked in together with the polynucleotide of (2), and the promoter sequence is operably linked to a sequence encoding SCT.

[44] The cell according to

[43] above, wherein the promoter sequence of the HLA-B α-chain gene comprises the nucleotide sequence shown in SEQ ID NO: 11 or a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 11.

[45] The cell according to [1], [6] or [9] above, wherein the rejection reaction by T cells and / or NK cells is reduced compared to wild-type cells.

[46] The T cells are CD8. + T cells and / or CD4+

[47] The cell according to

[45] above, which is a T cell.

[47] A cell derived from the HLA gene modified cell according to [1], [6] or [9] above.

[0009] In the cells of the present invention, expression of the α chain of a specific HLA class I molecule (and thus expression of functional HLA class I molecules with antigen-presenting ability) is suppressed, thereby avoiding attack by recipient T cells and the like. Furthermore, the cells of the present invention avoid attack by NK cells and macrophages by expressing effector cell inhibitors (particularly single-chain fusion peptides). These mechanisms improve histocompatibility with the recipient, making the cells of the present invention useful as transplant cells for regenerative medicine (particularly allogeneic transplantation). In HLA gene modification using the Cre / loxP system (Patent Document 2), foreign genes used as tools (e.g., loxP sequences and marker genes) may remain in the genome after modification, which is undesirable for transplantation purposes. In the present invention, the cells of the present invention can be produced efficiently without using loxP sequences and without leaving traces of marker genes by using a homologous recombination method using a site-specific nuclease (described below).

[0010] Figure 1 shows the results of expression analysis of the SCD coding sequence in the genetically modified cells of Example 1. Figure 2 shows the results of expression analysis of HLA-A, HLA-B, HLA-C, SCT, and SCD in the genetically modified cells of Example 2. Figure 3 shows T cell responsiveness to the genetically modified cells of Example 2. Figure 4 shows the cytotoxicity of T cells against the genetically modified cells of Example 2. Figure 5 shows the cytotoxicity of NK cells against the genetically modified cells of Example 2.

[0011] [Cells Subject to Genetic Modification] Genetic modification according to the present invention can be applied to cells having HLA genes without any particular limitations. The cells may have a genome of haploid or more, with diploid cells (cells with two alleles) being preferred. The cells may be mammalian (e.g., human) cells, non-human mammalian (e.g., monkey, dog, cat, cow, horse, sheep, goat, llama, rodent) cells, or other vertebrate cells, and can be selected appropriately depending on the transplant recipient. The cells may be either proliferative or non-proliferative cells, with proliferative cells being preferred. Non-proliferative cells include neurons and cardiomyocytes. Proliferative cells include pluripotent stem cells (iPS cells and embryonic stem cells (ES cells)), hematopoietic stem cells, T cells, NK cells, and B cells, with iPS cells being preferred. In a preferred embodiment, the cells subject to genetic modification (target cells) are selected from the public interest incorporated foundation. Human iPS cell lines available from the iPS cell stock derived from HLA-homologous donors at the Kyoto University iPS Cell Research Foundation (e.g., human iPS cell line 253G1, human iPS cell line 253G4, human iPS cell line 201B7, human iPS cell line 409B2, human iPS cell line 454E2, human iPS cell line 585A1, human iPS cell line 585B1, human iPS cell line 606A1) strain, human iPS cell strain 610B1, human iPS cell strain 648A1, human iPS cell strain 1201C1, human iPS cell strain 1210B2, human iPS cell strain 1231A3, human iPS cell strain 1383D2, human iPS cell strain 1383D6, HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 strains. In a more preferred embodiment, the cells subjected to genetic modification are human iPS cell strain 253G4 or human iPS cell strain 201B7. The cells may be differentiated cells that express more HLA than undifferentiated cells (e.g., iPS cells). The cells may also be cells differentiated from pluripotent stem cells (e.g., human T cells or NK cells differentiated from human pluripotent stem cells).The differentiated cells and cells differentiated from pluripotent stem cells may be, for example, somatic cells such as immune cells such as T cells, NK cells, macrophages, B cells, dendritic cells, monocytes, eosinophils, neutrophils, basophils, innate lymphocytes, and mast cells, hematopoietic stem cells, mesenchymal stem cells (including bone marrow-derived and adipose-derived mesenchymal stem cells), and tissue stem cells such as neural stem cells, adipocytes, epidermal cells, endothelial cells, chondrocytes, skeletal myoblasts, retinal cells, retinal pigment epithelial cells, corneal epithelial cells, fibroblasts, muscle cells, cardiac muscle cells, nerve cells, astrocytes, hepatocytes, pancreatic β cells, kidney cells, and adrenal cells, or precursor cells thereof.

[0012] [Single-chain fusion peptide mimicking HLA-G or HLA-E] The single-chain fusion peptide is configured to exhibit the function of HLA-G or HLA-E (the function of binding to inhibitory receptors expressed on the surface of NK cells or macrophages). HLA-G is a ligand for the inhibitory receptors KIR2DL4 and LILRB1. HLA-E is a ligand for the inhibitory receptors CD94 / NKG2A and CD94 / NKG2B. In the present invention, the single-chain fusion peptide is introduced into cells and expressed to achieve an inhibitory receptor-mediated inhibitory receptor-mediated cytotoxicity of NK cells or macrophages. The HLA-G-mimicking single-chain fusion peptide binds to the inhibitory receptor KIR2DL4 or LILRB1, thereby inhibiting inhibitory receptor-mediated cytotoxicity of NK cells or macrophages. Single-chain fusion peptides mimicking HLA-E inhibit the inhibitory receptor-mediated cytotoxicity of NK cells or macrophages by binding to the inhibitory receptor CD94 / NKG2A or CD94 / NKG2B. Because both HLA-G and HLA-E are HLA class I molecules, three components (α chain, β2 microglobulin, and an endogenous peptide presented on each HLA molecule) are required for their function to be fulfilled. The single-chain fusion peptide is preferably one or more selected from a single-chain fusion peptide comprising β2 microglobulin (hereinafter also referred to as B2M) and the α chain of HLA-G, and a single-chain fusion peptide comprising β2 microglobulin and the α chain of HLA-E. The single-chain fusion peptide may also be a single-chain fusion peptide further comprising an endogenous peptide presented on an HLA molecule.

[0013] [HLA-G Mimetic Single-Chain Fusion Peptide (SCD)] The HLA-G mimetic single-chain fusion peptide is a single-chain dimer (SCD) comprising "β2 microglobulin (hereinafter also referred to as B2M)" and "HLA-G α chain." Unlike the HLA-G mimetic single-chain fusion peptide (SCT) described below, the endogenous peptide presented on SCD is supplied from within the cell. In SCD, "B2M" and "HLA-G α chain" are preferably present in this order from the N-terminus to the C-terminus. In the present invention, a publicly known amino acid sequence of B2M can be used. The amino acid sequence of B2M is preferably a wild-type sequence, but one or more amino acids may be deleted, substituted, or added as long as the function of SCD is not impaired. In the present invention, a publicly known amino acid sequence of HLA-G α chain can be used. The amino acid sequence of the HLA-G α chain is preferably a wild-type sequence, but one or more amino acids may be deleted, substituted, or added as long as the function of SCD is not impaired. In a preferred embodiment, the amino acid sequence of SCD may be a sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with SEQ ID NO: 1 below. In a particularly preferred embodiment, the amino acid sequence of SCD is the sequence shown in SEQ ID NO: 1 below. In SEQ ID NO: 1, the amino acid sequence of positions 1 to 119 corresponds to B2M, the amino acid sequence of positions 120 to 134 corresponds to the linker, and the amino acid sequence of positions 135 to 448 corresponds to the HLA-G α chain. The amino acid sequences of B2M, the linker, and the α chain of HLA-G contained in the SCD may be sequences that have 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with the amino acid sequence of amino acids 1 to 119, the amino acid sequence of amino acids 120 to 134, and the amino acid sequence of amino acids 135 to 448 of SEQ ID NO: 1 below, respectively.

[0014] [SEQ ID NO: 1] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEA SSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLR CWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD

[0015] The polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2. In a preferred embodiment, the polynucleotide sequence encoding SCD may be a sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with SEQ ID NO: 2 below. In a particularly preferred embodiment, the polynucleotide sequence encoding SCD is the sequence shown in SEQ ID NO: 2 below.

[0016]

[0017] In a preferred embodiment, the linker sequence present between B2M and the α chain of HLA-G in SCD is a peptide (GS linker) composed of glycine and serine. The GS linker preferably has a chain length of 15 amino acids or more, and more preferably contains three or more GGGGS. Use of a GS linker with a long chain length improves the expression level of SCD and improves the inhibitory effect of SCD on NK cells and macrophages. In a particularly preferred embodiment, the amino acid sequence of the linker sequence present between B2M and the α chain of HLA-G in SCD is the sequence shown in SEQ ID NO: 3 below. [SEQ ID NO: 3] GGGGSGGGGSGGGGS

[0018] In another preferred embodiment, the amino acid sequence of SCD may comprise a sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with SEQ ID NO: 4 below. In a particularly preferred embodiment, the amino acid sequence of SCD is the sequence shown in SEQ ID NO: 4 below. In SEQ ID NO: 4, the amino acid sequence of amino acids 1 to 119 corresponds to B2M, the amino acid sequence of amino acids 120 to 134 corresponds to the linker, and the amino acid sequence of amino acids 135 to 477 corresponds to the α chain of HLA-G. The amino acid sequences of B2M, the linker, and the α chain of HLA-G contained in SCD may comprise sequences having 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with the amino acid sequence of amino acids 1 to 119, the amino acid sequence of amino acids 120 to 134, and the amino acid sequence of amino acids 135 to 477 of SEQ ID NO: 4 below, respectively.

[0019] [SEQ ID NO: 4] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSMKTPRMVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPQRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDR MNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWIHRYLENGKEMLQRADPPKTHVTHHPV FDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVIWRKKSSD

[0020] The polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 5. In a preferred embodiment, the polynucleotide sequence encoding SCD may be a sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with SEQ ID NO: 5 below. In a particularly preferred embodiment, the polynucleotide sequence encoding SCD is the sequence shown in SEQ ID NO: 5 below.

[0021]

[0022] [HLA-E Mimetic Single-Chain Fusion Peptide (SCT)] The HLA-E mimetic single-chain fusion peptide is a single-chain trimer (SCT) comprising an "HLA-A, HLA-B, HLA-C, or HLA-G signal peptide," "B2M," and the "HLA-E α chain." The "HLA-A, HLA-B, HLA-C, or HLA-G signal peptide" (hereinafter also referred to as "signal peptide") is an endogenous peptide presented on HLA-E. In the SCT, the "signal peptide," "B2M," and the "HLA-E α chain" are preferably present in this order from the N-terminus to the C-terminus. In the present invention, the amino acid sequence of a known signal peptide can be used. The HLA-G signal peptide is preferred as the signal peptide. The amino acid sequence of the signal peptide is preferably a wild-type sequence, but may have one or more amino acids deleted, substituted, or added, as long as the function of SCT is not impaired. In the present invention, a known amino acid sequence of B2M can be used. The amino acid sequence of B2M is preferably a wild-type sequence, but may have one or more amino acids deleted, substituted, or added, as long as the function of SCT is not impaired. In the present invention, a known amino acid sequence of the HLA-E α chain can be used. The amino acid sequence of the HLA-E α chain is preferably a wild-type sequence, but may have one or more amino acids deleted, substituted, or added, as long as the function of SCT is not impaired. In a preferred embodiment, the amino acid sequence of SCT may be a sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with SEQ ID NO: 6 below. In a particularly preferred embodiment, the amino acid sequence of SCT is the sequence shown in SEQ ID NO: 6 below.In SEQ ID NO: 6, the amino acid sequence at positions 1 to 20 corresponds to the 20 amino acid sequence at the N-terminus of B2M, the amino acid sequence at positions 21 to 29 corresponds to the signal peptide of HLA-G, the amino acid sequence at positions 30 to 44 corresponds to the linker (first linker), the amino acid sequence at positions 45 to 143 corresponds to the 99 amino acid sequence at the C-terminus of B2M, the amino acid sequence at positions 144 to 163 corresponds to the linker (second linker), and the amino acid sequence at positions 164 to 500 corresponds to the α chain of HLA-E. The amino acid sequences of the HLA-G signal peptide, first linker, B2M, second linker, and HLA-E α chain contained in the SCT may be sequences that have 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with the amino acid sequence of positions 21 to 29, the amino acid sequence of positions 30 to 44, a sequence combining the amino acid sequence of positions 1 to 20 and the amino acid sequence of positions 45 to 143, the amino acid sequence of positions 144 to 163, and the amino acid sequence of positions 164 to 500 of SEQ ID NO: 6 below, respectively.

[0023] [SEQ ID NO: 6] MSRSVALAVLALLSLSGLEAVMAPRTLFLGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQ SEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAE ITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL

[0024] A polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 7. In a preferred embodiment, the polynucleotide sequence encoding SCT may be a sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with SEQ ID NO: 7 below. In a particularly preferred embodiment, the polynucleotide sequence encoding SCT is the sequence shown in SEQ ID NO: 7 below.

[0025]

[0026] In a preferred embodiment, the linker sequence present between the signal peptide and B2M in SCT is a peptide composed of glycine and serine (GS linker). The GS linker preferably has a chain length of 15 amino acids or more, and more preferably contains three or more GGGGS. The use of a GS linker with a long chain length improves the expression level of SCT and improves the inhibitory effect of SCT on NK cells and macrophages. In a particularly preferred embodiment, the amino acid sequence of the linker sequence present between the signal peptide and B2M in SCT is the sequence shown in SEQ ID NO: 8 below. [SEQ ID NO: 8] GGGGSGGGGSGGGGS

[0027] In a preferred embodiment, the linker sequence present between B2M and the α chain of HLA-E in SCT is a peptide (GS linker) composed of glycine and serine. The GS linker in this embodiment preferably has a chain length of 15 amino acids or more, and more preferably contains three or more GGGS. The use of a GS linker with a long chain length improves the expression level of SCT and improves the inhibitory effect of SCT on NK cells and macrophages. Furthermore, in a particularly preferred embodiment, the amino acid sequence of the linker sequence present between B2M and the α chain of HLA-E in SCT is the sequence shown in SEQ ID NO: 9 below. [SEQ ID NO: 9] GGGGSGGGGSGGGGS

[0028] In the present invention, at least one of SCD or SCT, and preferably both SCD and SCT, is knocked in.

[0029] [Knock-in of polynucleotides encoding SCD and / or SCT] In this specification, when identifying a location in a genome, the location in the hg38 genome sequence is used as a reference genome. hg38 is a reference genome released by the University of California, Santa Cruz (UCSC) in December 2013. A reference genome is a reference genome created by combining various genomes, and it does not necessarily mean that there is a human being that possesses this genome. However, by comparing fragmentary sequence information decoded from the genomic DNA of a human individual with the reference genome, the decoded fragmentary sequence information can be linked to construct a continuous sequence on a computer, thereby estimating the sequence of the genomic DNA of the human individual. In this way, the genomic DNA of an individual, such as a human individual, is usually decoded by correlating the sequence of the genomic DNA of the human individual with the reference genome. A position or region corresponding to a specific position or region in the hg38 genome sequence refers to a position or region linked to the specific position or region in the genome of another individual with a different specific sequence. Specifically, a position or region having a sequence characteristic of that position or region based on sequence identity corresponds to a specific position or region in the hg38 genome sequence. Corresponding positions can be determined by aligning the partial sequences of two genomic DNAs. Even if there are differences in specific sequences, the correspondence between the two genomic DNAs can be determined by aligning them based on an orthologous relationship or sequence identity. In regions rich in paralogs resulting from gene duplication, simply determining the correspondence between sequences based on individual sequences may not be sufficient to determine the true correspondence between two genomes. When determining corresponding sequences, high sequence identity can be required to clarify the correspondence between the two genomes. Furthermore, when a specific region is large and contains multiple genes, synteny can be considered. Synteny refers to the conservation of the physical positional relationship between orthologs on a genome. Synteny can exist between individuals and between organisms.Therefore, a specific region can be determined taking synteny into consideration.

[0030] In one embodiment of the present invention, a "region comprising the α-chain genes of HLA-F, HLA-G, and HLA-A" of a cell is replaced (knocked in) with a "polynucleotide encoding SCD (hereinafter also referred to as "SCD coding sequence")." The "region comprising the α-chain genes of HLA-F, HLA-G, and HLA-A" includes a "region corresponding to the HLA-F α-chain gene region on chr6 of the hg38 genomic sequence," a "region corresponding to the HLA-G α-chain gene region on chr6 of the hg38 genomic sequence," and a "region corresponding to the HLA-A α-chain gene region on chr6 of the hg38 genomic sequence," as shown below. The "region containing the α-chain genes of HLA-F, HLA-G, and HLA-A" may include the upstream region of the HLA-F α-chain gene (a region corresponding to a region upstream of 29,723,657 on chr6 of the hg38 genomic sequence) and / or the downstream region of the HLA-A α-chain gene (a region corresponding to a region downstream of 29,945,281 on chr6 of the hg38 genomic sequence). The length of the upstream region of the HLA-F α-chain gene is not particularly limited as long as the method for knocking in the "SCD coding sequence" described below can be carried out, but is, for example, 800 to 20,000 nucleotides, preferably 1,000 to 5,000 nucleotides. The length of the upstream region can be appropriately set taking into consideration factors such as the design position of the guide RNA and whether the homology arm has a specific sequence. The length of the downstream region of the HLA-A α-chain gene is not particularly limited as long as it allows the knock-in method of the "SCD coding sequence" described below to be carried out, but is, for example, 800 to 20,000 nucleotides, preferably 1,000 to 5,000 nucleotides. The length of the downstream region can be appropriately set taking into consideration the design position of the guide RNA, whether the homology arm has a specific sequence, and the like. In a preferred embodiment of the present invention, the "region containing the HLA-F, HLA-G, and HLA-A α-chain genes" is a chromosomal region (target region 1) corresponding to the chromosomal region chr6:29,704,906-29,958,642 of the hg38 genome sequence. In another preferred embodiment of the present invention, the "region containing the HLA-F, HLA-G, and HLA-A α-chain genes" is a chromosomal region (target region 2) corresponding to the chromosomal region chr6:29,707,019-29,956,470 of the hg38 genome sequence.

[0031] In another embodiment of the present invention, a "region comprising the HLA-C and HLA-B α-chain genes" of a cell is replaced (knocked in) with a "polynucleotide encoding SCT (hereinafter also referred to as an "SCT coding sequence")." The "region comprising the HLA-C and HLA-B α-chain genes" includes a "region corresponding to the HLA-C α-chain gene region on chr6 of the hg38 genomic sequence" and a "region corresponding to the HLA-B α-chain gene region on chr6 of the hg38 genomic sequence" shown below. The "region containing the HLA-C and HLA-B α-chain genes" may include the upstream region of the HLA-C α-chain gene (a region corresponding to a region upstream of 31,269,338 on chr6 of the hg38 genomic sequence) and / or the downstream region of the HLA-B α-chain gene (a region corresponding to a region downstream of 31,356,958 on chr6 of the hg38 genomic sequence). The length of the upstream region of the HLA-C α-chain gene is not particularly limited as long as the method for knocking in the "SCT coding sequence" described below can be carried out, but is, for example, 800 to 60,000 nucleotides, preferably 1,000 to 5,000 nucleotides. The length of the upstream region can be appropriately set taking into consideration the design position of the guide RNA, whether the homology arm has a specific sequence, and the like. The length of the downstream region of the HLA-B α-chain gene is not particularly limited as long as it allows the knock-in method of the "SCT coding sequence" described below to be carried out, but is, for example, 800 to 35,000 nucleotides, preferably 1,000 to 5,000 nucleotides. The length of the downstream region can be appropriately set taking into consideration the design position of the guide RNA, whether the homology arm has a specific sequence, and the like. In a preferred embodiment of the present invention, the "region containing the HLA-C and HLA-B α-chain genes" is a chromosomal region corresponding to the chromosomal region chr6:31,224,837-31,378,196 of the hg38 genome sequence. In another preferred embodiment of the present invention, the "region containing the HLA-C and HLA-B α-chain genes" is a chromosomal region corresponding to the chromosomal region chr6:31,227,286-31,378,780 of the hg38 genome sequence.

[0032] In a preferred embodiment of the present invention, the "region containing the α-chain genes of HLA-F, HLA-G, and HLA-A" of a cell is replaced (knocked in) with the "SCD coding sequence," and the "region containing the α-chain genes of HLA-C and HLA-B" is replaced (knocked in) with the "SCT coding sequence."

[0033] These gene modifications can be performed using gene modification methods using site-specific nucleases (e.g., the methods described in eLife, 2017, 6, e27873, FEBS Letters, 591, 2017, 903-913, and WO 2021 / 206054). Below, we will explain a method of genetically modifying diploid cells having two alleles using the CRISPR / Cas system as a site-specific nuclease. Note that the gene modification method can also be performed using site-specific nucleases other than the CRISPR / Cas system (e.g., TALEN, ZFN, etc.).

[0034] 1. Method for knocking in SCD [Step 1] Substitution of a region containing the α-chain genes of HLA-F, HLA-G, and HLA-A with a selection marker gene The "region containing the α-chain genes of HLA-F, HLA-G, and HLA-A" (hereinafter also referred to as "target region") to be substituted in this step is, for example, a chromosomal region corresponding to chr6:29,704,906-29,958,642 (target region 1) of the hg38 genome sequence. [1-1] Site-specific cleavage of DNA using the CRISPR / Cas system When the target region is target region 1, a guide RNA (gRNA) targeting the upstream region of the HLA-F α-chain gene (chr6 of the hg38 genome sequence: 29,707,002-29,707,024) and a gRNA targeting the downstream region of the HLA-A α-chain gene (chr6 of the hg38 genome sequence: 29,956,465-29,956,487) are used in conjunction with the CRISPR / Cas9 system to create two double-stranded breaks per allele. The sequence of the gRNA does not have to be completely identical to the target sequence, as long as it can bind to the target sequence. A polynucleotide encoding the gRNA (e.g., an expression vector) may be used to introduce the gRNA. The method for introducing gRNA and Cas9 into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. Note that step [1-1] may be performed using other site-specific nucleases, such as TALENs or ZFNs, instead of the CRISPR / Cas system.

[0035] [1-2] Introduction of a Different Selectable Marker Gene into the Target Region of Each Allele To introduce a selectable marker gene, a selectable marker donor DNA is used. The "selectable marker donor DNA" contains a "selectable marker gene" between an "upstream homology arm sequence" and a "downstream homology arm sequence." The "upstream homology arm sequence" has a sequence capable of homologous recombination with an upstream sequence within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination) (e.g., chr6:29,704,906-29,707,018 of the hg38 genome sequence when the target region is target region 1). The upstream homology arm sequence does not need to be completely identical to the upstream sequence within the target region as long as homologous recombination is possible, but preferably has a sequence identity of 90% or more, and more preferably 95% or more. The "downstream homology arm sequence" has a sequence capable of homologous recombination with a downstream sequence within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination) (e.g., chr6:29,956,471-29,958,642 of the hg38 genome sequence when the target region is target region 1). The downstream homology arm sequence does not need to be completely identical to the downstream sequence within the target region as long as homologous recombination is possible, but preferably has a sequence identity of 90% or more, and more preferably 95% or more. The lengths of the upstream homology arm and downstream homology arm can be, for example, approximately 500 bp to 3 kbp, but can be appropriately designed by those skilled in the art, similar to the design of conventional donor DNA. The "donor DNA for selection marker" contains a positive selection marker (e.g., a drug resistance gene) used in this step and a negative selection marker (e.g., a fluorescent protein) used in steps 2 and 3 described below. The positive selection marker allows selection of target cells based on its expression. The negative selection marker allows selection of target cells based on its non-expression. The selection marker can be appropriately selected depending on the type of cell to be used, etc. The selection marker may be a marker that serves as both a positive selection marker and a negative selection marker (dual-purpose marker). An example of a dual-purpose marker is a fluorescent protein.

[0036] In this step, two types of selectable marker donor DNAs are used. The two types of selectable marker donor DNAs each contain a mutually distinguishably different positive selectable marker gene. "Mutually distinguishably different" means that they can be distinguished from each other based on the physiological properties (e.g., drug resistance) conferred to cells by the positive selectable marker genes. For example, if two types of drug resistance genes that exhibit resistance to different types of drugs are used as positive selectable marker genes, only cells in which the target regions of the two alleles have been replaced with different drug resistance genes will exhibit resistance to the two types of drugs. Furthermore, the two types of selectable marker donor DNAs each contain a mutually distinguishably different negative selectable marker gene. Below are examples of combinations of selectable marker genes contained in two types of selectable marker donor DNAs.

[0037] The donor DNA for a selection marker may be either linear or circular, but is preferably circular. Preferably, the donor DNA for a selection marker is a plasmid. The donor DNA for a selection marker may contain a spacer sequence between the above sequences.

[0038] The method for introducing the selectable marker donor DNA into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. It is preferable to simultaneously introduce gRNA, Cas9, and the selectable marker donor DNA into cells, and then simultaneously perform steps [1-1] and [1-2]. Homologous recombination (HDR) occurs between the selectable marker donor DNA having the above-mentioned "upstream homology arm sequence" and "downstream homology arm sequence" and the cell chromosome. Sequence replacement by homologous recombination can be performed using well-known conventional techniques. After homologous recombination, cells expressing two types of positive selectable markers are selected and used in step 2. In the selected cells, the target regions of the two alleles are replaced with different types of selectable marker genes. In other words, in the selected cells, the α-chain genes of the two alleles of HLA-F, HLA-G, and HLA-A are knocked out, and instead, different types of selectable marker genes are knocked into each allele.

[0039] [Step 2] Replacement of a selectable marker gene in one allele with a polynucleotide encoding SCD [2-1] Site-specific cleavage of DNA in one allele using the CRISPR / Cas system. A gRNA targeting one of the two positive selectable marker genes used in step [1-2] and a Cas9 protein are used to create a double-stranded break at one site per allele using the CRISPR / Cas9 system. The gRNA sequence does not need to be completely identical to the target sequence as long as it can bind to the target sequence. A polynucleotide (e.g., an expression vector) encoding the gRNA may be used to introduce the gRNA. The method for introducing the gRNA and Cas9 into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. Note that step [2-1] may also be performed using other site-specific nucleases, such as TALENs or ZFNs, instead of the CRISPR / Cas system.

[0040] [2-2] Introduction of a Polynucleotide Encoding an SCD into Only One Allele To introduce a polynucleotide encoding an SCD (hereinafter also referred to as an "SCD coding sequence"), a donor DNA for SCD is used. The "donor DNA for SCD" contains an "SCD coding sequence" (e.g., SEQ ID NO: 2) between an "upstream homology arm sequence" and a "downstream homology arm sequence." The "upstream homology arm sequence" and "downstream homology arm sequence" of the donor DNA for SCD are the same as the "upstream homology arm sequence" and "downstream homology arm sequence," respectively, of the donor DNA for the selectable marker used in step [1-2]. Therefore, homologous recombination occurs between the "donor DNA for SCD" and the "allele containing the selectable marker gene," and the selectable marker gene is replaced with the SCD coding sequence. Sequence replacement by homologous recombination can be carried out using well-known, conventional techniques. As long as homologous recombination occurs, the "upstream homology arm sequence" and "downstream homology arm sequence" of the donor DNA for SCD may be different from the "upstream homology arm sequence" and "downstream homology arm sequence" of the donor DNA for the selection marker used in step [1-2]. For the purpose of increasing the expression level of SCD, the donor DNA for SCD preferably contains a promoter sequence of the HLA-A α-chain gene (preferably a sequence corresponding to chr6:29,938,509-29,942,553 of the hg38 genome sequence) upstream of the SCD coding sequence. Since SCD mimics HLA-G, it is conceivable to use a promoter of the HLA-G α-chain gene. However, HLA-G is known to be expressed in limited cells (e.g., placental cells). On the other hand, HLA-A is known to be stably expressed in many types of cells. When applying the present invention to human pluripotent stem cells, it is expected that the resulting genetically modified cells will be differentiated into various types of cells before transplantation, and therefore it is preferable to use a promoter sequence for the HLA-A α-chain gene. Furthermore, by using a promoter sequence for the HLA-A α-chain gene that is inducible by interferon-γ stimulation, the timing of SCD expression can also be controlled.In the present invention, a known promoter sequence of the HLA-A α chain gene can be used. In a preferred embodiment, the promoter sequence of the HLA-A α chain gene may be a sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with SEQ ID NO: 10 below. In a particularly preferred embodiment, the promoter sequence of the HLA-A α chain gene is the sequence shown in SEQ ID NO: 10 below.

[0041]

[0042] The promoter sequence of the HLA-A α chain gene is knocked in operably linked to the SCD coding sequence. "Operably linked" means that the promoter sequence of the HLA-A α chain gene is positioned relative to the SCD coding sequence in a manner that controls the expression of the SCD coding sequence.

[0043] In another aspect of the present invention, the promoter sequence of a gene encoding the α chain of an HLA class I molecule, excluding HLA-G, may be knocked in operably linked to the SCD coding sequence.

[0044] The donor DNA for SCD may be either linear or circular, but is preferably circular. Preferably, the donor DNA for SCD is a plasmid. The donor DNA for SCD may contain a spacer sequence between the above sequences.

[0045] The method for introducing the SCD donor DNA into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. It is preferable to simultaneously introduce gRNA, Cas9, and the SCD donor DNA into cells, and then simultaneously perform steps [2-1] and [2-2]. Sequence replacement by homologous recombination can be performed using well-known conventional techniques. After homologous recombination, cells that meet the following two conditions are selected and used in step 3. Condition 1: The negative selection marker present in the allele targeted for cleavage in step [2-1] (the allele containing the positive selection marker gene targeted by gRNA) is not expressed. Condition 2: The negative selection marker present in an allele other than the allele targeted for cleavage in step [2-1] is expressed. In the selected cells, the target region of one allele is replaced with the SCD coding sequence, and the target region of the other allele remains replaced with the selection marker gene. In other words, in the selected cells, the α chain genes of one allele of HLA-F, HLA-G and HLA-A are knocked out, and instead, the SCD coding sequence is knocked in.

[0046] [Step 3] Deletion of the selection marker gene from the other allele. The other allele refers to the allele into which the SCD coding sequence was not introduced in step 2. [3-1] Site-specific cleavage of DNA in the other allele using the CRISPR / Cas system. A double-stranded break is created at one site per allele using the CRISPR / Cas9 system, which uses a gRNA targeting a positive selection marker gene not targeted in step [2-1] and the Cas9 protein. The sequence of the gRNA does not need to be completely identical to the target sequence, as long as it can bind to the target sequence. A polynucleotide (e.g., an expression vector) encoding the gRNA may be used to introduce the gRNA. The method for introducing the gRNA and Cas9 into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. In addition, step [3-1] may be performed using other site-specific nucleases, such as TALENs and ZFNs, instead of the CRISPR / Cas system.

[0047] [3-2] Deletion of the selectable marker gene from the other allele To delete the selectable marker, a selectable marker deleting DNA is used. In the selectable marker deleting DNA, an "upstream homology arm sequence" and a "downstream homology arm sequence" are linked, with no other sequences present between the two homology arm sequences. The "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker deleting DNA are the same as the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker donor DNA used in step [1-2], respectively. Therefore, homologous recombination occurs between the "selectable marker deleting DNA" and the "allele containing the selectable marker gene," resulting in the removal of the selectable marker gene from the allele. Homologous recombination can be carried out using well-known conventional techniques. Note that, as long as homologous recombination occurs, the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker deleting DNA may be different from the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker donor DNA used in step [1-2], respectively.

[0048] The DNA for deleting the selection marker may be either linear or circular, but is preferably circular. Preferably, the DNA for deleting the selection marker is a plasmid.

[0049] The method for introducing the selection marker deletion DNA into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. It is preferable to simultaneously introduce gRNA, Cas9, and SCD donor DNA into cells, and then simultaneously perform steps [3-1] and [3-2]. After homologous recombination, cells that do not express either of the two types of negative selection markers introduced in step [1-2] are selected. In the selected cells, the selection marker gene is deleted from the other allele. In other words, in the selected cells, the "HLA-F, HLA-G, and HLA-A α-chain genes" are deleted in one allele, and instead, an "SCD coding sequence" is knocked in, and further, the "HLA-F, HLA-G, and HLA-A α-chain genes" are deleted in the other allele. In other words, for example, if the target region is target region 2, the selected cells have target region 2 (a chromosomal region corresponding to chr6:29,707,019-29,956,470 of the hg38 genome sequence) deleted in both alleles. Cells in which the "HLA-F, HLA-G, and HLA-A α-chain genes" have been deleted from both alleles have reduced expression of the genes compared to wild-type cells. "Wild-type cells" refer to cells that have not been subjected to steps 1 to 3 above. Comparison of gene expression can be performed using known gene expression analysis techniques (e.g., reverse transcription quantitative PCR). Cells in which the "HLA-F, HLA-G, and HLA-A α-chain genes" have been deleted from both alleles may lack expression of the genes. Cells in which the "HLA-F, HLA-G, and HLA-A α-chain genes" have been deleted from both alleles may lack expression of proteins encoded by the genes, or may lack expression of proteins encoded by the genes on the cell membrane.

[0050] The reason why SCD is introduced into only one allele in steps 1 to 3 is that it is easy to confirm whether the intended gene introduction has been achieved. Generally, genotype confirmation is performed using PCR, but when the aim is to introduce SCD into both alleles, it is difficult to distinguish by PCR between "cells in which SCD has been introduced into both alleles (target cells)" and "cells in which SCD has been introduced into only one allele (non-target cells)." In contrast, when the aim is to introduce SCD into only one allele, it is easy to distinguish by PCR between "cells in which SCD has been introduced into only one allele (target cells)" and "cells in which SCD has been introduced into both alleles (non-target cells)." However, the present invention also encompasses genetically modified cells in which SCD has been introduced into both alleles.

[0051] 2. Method for knocking in SCT [Step 1] Substitution of a region containing the HLA-C and HLA-B α-chain genes with a selection marker gene The "region containing the HLA-C and HLA-B α-chain genes" (hereinafter also referred to as "target region") to be substituted in this step is, for example, a chromosomal region corresponding to chr6:31,224,837-31,378,196 of the hg38 genome sequence (target region 1), or a chromosomal region corresponding to chr6:31,227,286-31,378,780 of the hg38 genome sequence (target region 2). [1-1] Site-specific cleavage of DNA using the CRISPR / Cas system When the target region is target region 1, a guide RNA (gRNA) targeting the upstream region of the HLA-C α-chain gene (chr6:31,225,752-31,225,771 of the hg38 genome sequence) and a gRNA targeting the downstream region of the HLA-B α-chain gene (chr6:31,377,258-31,377,277 of the hg38 genome sequence) are used in combination with the Cas9 protein to create two double-stranded breaks per allele using the CRISPR / Cas9 system. When the target region is target region 2, a guide RNA (gRNA) targeting the upstream region of the HLA-C α-chain gene (chr6:31,228,800-31,228,822 of the hg38 genome sequence) and a gRNA targeting the downstream region of the HLA-B α-chain gene (chr6:31,377,258-31,377,277 of the hg38 genome sequence) are used in combination with the CRISPR / Cas9 system to create two double-stranded breaks per allele. The sequence of the gRNA does not have to be completely identical to the target sequence, as long as it can bind to the target sequence. A polynucleotide (e.g., an expression vector) encoding the gRNA may be used to introduce the gRNA. The method for introducing the gRNA and Cas9 into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. In addition, step [1-1] may be performed using other site-specific nucleases, such as TALENs and ZFNs, instead of the CRISPR / Cas system.

[0052] [1-2] Introduction of Different Selectable Marker Genes into the Target Region of Each Allele To introduce a selectable marker gene, a selectable marker donor DNA is used. The "selectable marker donor DNA" contains a "selectable marker gene" between an "upstream homology arm sequence" and a "downstream homology arm sequence." The "upstream homology arm sequence" has a sequence capable of homologous recombination with an upstream sequence within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination) (e.g., if the target region is target region 1, chr6:31,224,837-31,225,754 of the hg38 genomic sequence. For example, if the target region is target region 2, chr6:31,227,286-31,228,805 of the hg38 genomic sequence). The upstream homology arm sequence does not need to be completely identical to the sequence adjacent to the upstream side of the target region, as long as homologous recombination is possible, but preferably has a sequence identity of 90% or more, and more preferably 95% or more. The "downstream homology arm sequence" has a sequence capable of homologous recombination with a downstream sequence within a target region present in one of two chromosomes (the chromosome to be subjected to homologous recombination) (for example, when the target region is target region 1, chr6:31,377,261-31,378,196 of the hg38 genome sequence; for example, when the target region is target region 2, chr6:31,377,261-31,378,780 of the hg38 genome sequence). The downstream homology arm sequence does not have to be completely identical to the sequence adjacent to the downstream side of the target region, as long as homologous recombination is possible, but preferably has a sequence identity of 90% or more, and more preferably 95% or more. The lengths of the upstream homology arm and downstream homology arm can be, for example, about 500 bp to 3 kbp, but can be appropriately designed by those skilled in the art, similar to the design of ordinary donor DNA. The "donor DNA for selection marker" contains a positive selection marker (e.g., a drug resistance gene) used in this step and a negative selection marker (e.g., a fluorescent protein) used in steps 2 and 3 described below. The selection marker can be selected appropriately depending on the type of cells used, etc. The selection marker may be one that serves as both a positive selection marker and a negative selection marker (a dual-purpose marker).An example of a dual-purpose marker is a fluorescent protein.

[0053] In this step, two types of donor DNA for selection markers are used. The two types of donor DNA for selection markers each have a positive selection marker gene that is distinguishable from one another. Furthermore, the two types of donor DNA for selection markers each have a negative selection marker gene that is distinguishable from one another. Examples of combinations of selection marker genes carried by the two types of donor DNA for selection markers include those described in "1. Method for knocking in SCD."

[0054] The donor DNA for a selection marker may be either linear or circular, but is preferably circular. Preferably, the donor DNA for a selection marker is a plasmid. The donor DNA for a selection marker may contain a spacer sequence between the above sequences.

[0055] The method for introducing the selectable marker donor DNA into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. It is preferable to simultaneously introduce gRNA, Cas9, and the selectable marker donor DNA into cells, and then simultaneously perform steps [1-1] and [1-2]. Homologous recombination (HDR) occurs between the selectable marker donor DNA containing the "upstream homology arm sequence" and "downstream homology arm sequence" and the cell chromosome. Sequence replacement by homologous recombination can be performed using well-known conventional techniques. After homologous recombination, cells expressing two types of positive selectable markers are selected and used in step 2. In the selected cells, the target regions of the two alleles are replaced with different types of selectable marker genes. In other words, in the selected cells, the α-chain genes of the two alleles of HLA-C and HLA-B are knocked out, and instead, different types of selectable marker genes are knocked into each allele.

[0056] [Step 2] Replacement of a selection marker gene in one allele with a polynucleotide encoding SCT [2-1] Site-specific cleavage of DNA in one allele using the CRISPR / Cas system. A gRNA targeting one of the two positive selection marker genes used in step [1-2] and a Cas9 protein are used to create a double-stranded break at one site per allele using the CRISPR / Cas9 system. The gRNA sequence does not need to be completely identical to the target sequence as long as it can bind to the target sequence. A polynucleotide (e.g., an expression vector) encoding the gRNA may be used to introduce the gRNA. The method for introducing the gRNA and Cas9 into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. Note that step [2-1] may also be performed using other site-specific nucleases, such as TALENs or ZFNs, instead of the CRISPR / Cas system.

[0057] [2-2] Introduction of an SCT-encoding polynucleotide into only one allele To introduce an SCT-encoding polynucleotide (hereinafter also referred to as "SCT coding sequence"), SCT donor DNA is used. The "SCT donor DNA" contains an "SCT coding sequence" (e.g., SEQ ID NO: 7) between an "upstream homology arm sequence" and a "downstream homology arm sequence." The "upstream homology arm sequence" and "downstream homology arm sequence" of the SCT donor DNA are the same as the "upstream homology arm sequence" and "downstream homology arm sequence," respectively, of the selectable marker donor DNA used in step [1-2]. Therefore, homologous recombination occurs between the "SCT donor DNA" and the "allele containing the selectable marker gene," and the selectable marker gene is replaced with the SCT coding sequence. Sequence replacement by homologous recombination can be carried out using well-known, conventional techniques. As long as homologous recombination occurs, the "upstream homology arm sequence" and "downstream homology arm sequence" of the SCT donor DNA may be different from the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker donor DNA used in step [1-2]. For the purpose of increasing the expression level of SCT, the SCT donor DNA preferably contains a promoter sequence of the HLA-B α-chain gene (preferably a sequence corresponding to chr6:31,357,159-31,359,283 of the hg38 genome sequence) upstream of the SCT coding sequence. Here, since SCT mimics HLA-E, it is conceivable to use a promoter of the HLA-E α-chain gene. However, while knockout of the HLA-E α-chain gene is not essential in the present invention, the presence of multiple identical promoters in the genome is thought to result in competition for transcription factor binding, resulting in a decrease in the expression level of SCT. Therefore, it is preferable to use the promoter of the HLA-B α-chain gene, which is known to be stably expressed in many types of cells. Furthermore, in an embodiment in which SCD is introduced in addition to SCT, when the promoter sequence of the HLA-A α-chain gene is used to increase the expression level of SCD, promoter competition can be avoided by using the promoter sequence of the HLA-B α-chain gene to increase the expression level of SCT.In the present invention, a known promoter sequence of the HLA-B α chain gene can be used. In a preferred embodiment, the promoter sequence of the HLA-B α chain gene may be a sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with the following SEQ ID NO: 11. In a particularly preferred embodiment, the promoter sequence of the HLA-B α chain gene is the sequence shown in the following SEQ ID NO: 11.

[0058]

[0059] The promoter sequence of the HLA-B α-chain gene is knocked in operably linked to the SCT coding sequence. "Operably linked" means that the promoter sequence of the HLA-B α-chain gene is positioned relative to the SCT coding sequence in a manner that controls the expression of the SCT coding sequence. In another aspect of the present invention, the promoter sequence of a gene encoding the α-chain of an HLA class I molecule excluding HLA-E may be knocked in operably linked to the SCT coding sequence.

[0060] The donor DNA for SCT may be either linear or circular, but is preferably circular. Preferably, the donor DNA for SCT is a plasmid. The donor DNA for SCT may contain a spacer sequence between the above sequences.

[0061] The method for introducing SCT donor DNA into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. It is preferable to simultaneously introduce gRNA, Cas9, and SCT donor DNA into cells, and then simultaneously perform steps [2-1] and [2-2]. Sequence replacement by homologous recombination can be performed using well-known conventional techniques. After homologous recombination, cells that meet the following two conditions are selected and used in step 3. Condition 1: The negative selection marker present in the allele targeted for cleavage in step [2-1] (the allele containing the positive selection marker gene targeted by gRNA) is not expressed. Condition 2: The negative selection marker present in an allele other than the allele targeted for cleavage in step [2-1] is expressed. In the selected cells, the target region of one allele is replaced with the SCT coding sequence, and the target region of the other allele remains replaced with the selection marker gene. In other words, in the selected cells, the α-chain genes of one allele of HLA-C and HLA-B are knocked out, and instead, the SCT coding sequence is knocked in.

[0062] [Step 3] Deletion of the selection marker gene from the other allele. The other allele refers to the allele into which the SCT coding sequence was not introduced in step 2. [3-1] Site-specific cleavage of DNA in the other allele using the CRISPR / Cas system. A double-stranded break is created at one site per allele using the CRISPR / Cas9 system, which uses a gRNA targeting a positive selection marker gene not targeted in step [2-1] and the Cas9 protein. The sequence of the gRNA does not need to be completely identical to the target sequence, as long as it can bind to the target sequence. A polynucleotide (e.g., an expression vector) encoding the gRNA may be used to introduce the gRNA. The method for introducing the gRNA and Cas9 into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. In addition, step [3-1] may be performed using other site-specific nucleases, such as TALENs and ZFNs, instead of the CRISPR / Cas system.

[0063] [3-2] Deletion of the selectable marker gene from the other allele To delete the selectable marker, a selectable marker deleting DNA is used. In the selectable marker deleting DNA, an "upstream homology arm sequence" and a "downstream homology arm sequence" are linked, with no other sequences present between the two homology arm sequences. The "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker deleting DNA are the same as the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker donor DNA used in step [1-2], respectively. Therefore, homologous recombination occurs between the "selectable marker deleting DNA" and the "allele containing the selectable marker gene," resulting in the removal of the selectable marker gene from the allele. Homologous recombination can be carried out using well-known conventional techniques. Note that, as long as homologous recombination occurs, the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker deleting DNA may be different from the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker donor DNA used in step [1-2], respectively.

[0064] The DNA for deleting the selection marker may be either linear or circular, but is preferably circular. Preferably, the DNA for deleting the selection marker is a plasmid.

[0065] The method for introducing the DNA for deleting the selection marker into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. It is preferable to simultaneously introduce gRNA, Cas9, and SCD donor DNA into cells, and perform steps [3-1] and [3-2] simultaneously. After homologous recombination, cells that do not express either of the two types of negative selection markers introduced in step [1-2] are selected. In the selected cells, the selection marker gene is deleted from the other allele. In other words, in the selected cells, the "HLA-C and HLA-B α chain genes" are deleted in one allele, and instead an "SCT coding sequence" is knocked in, and further, the "HLA-C and HLA-B α chain genes" are deleted in the other allele. In other words, for example, if the target region is target region 2, the chromosomal region corresponding to chr6:31,228,806-31,377,277 of the hg38 genome sequence (the region obtained by excluding the upstream and downstream homology arms from target region 2) is deleted in both alleles in the selected cells. Cells in which the "HLA-C and HLA-B α-chain genes" have been deleted from both alleles have reduced expression of the genes compared to wild-type cells. "Wild-type cells" refer to cells that have not been subjected to the above-mentioned steps 1 to 3. Comparison of gene expression can be performed using known gene expression analysis techniques (e.g., reverse transcription quantitative PCR). Cells in which the "HLA-C and HLA-B α-chain genes" have been deleted from both alleles may lack expression of the genes. Cells in which the "HLA-C and HLA-B α-chain genes" have been deleted from both alleles may lack expression of the proteins encoded by the genes, or may lack expression of the proteins encoded by the genes on the cell membrane.

[0066] The reason why SCT is introduced into only one allele in steps 1 to 3 is that it is easy to confirm whether the intended gene introduction has been achieved. Generally, genotype confirmation is performed using PCR, but when the aim is to introduce SCT into both alleles, it is difficult to distinguish by PCR between "cells in which SCT has been introduced into both alleles (target cells)" and "cells in which SCT has been introduced into only one allele (non-target cells)." In contrast, when the aim is to introduce SCT into one allele, it is easy to distinguish by PCR between "cells in which SCT has been introduced into only one allele (target cells)" and "cells in which SCT has been introduced into both alleles (non-target cells)." However, the present invention also encompasses genetically modified cells in which SCT has been introduced into both alleles.

[0067] 3. Method for knocking in SCD and SCT SCD and SCT can be knocked in by performing the above-mentioned "1. Method for knocking in SCD" followed by "2. Method for knocking in SCT", or by performing "2. Method for knocking in SCT" followed by "1. Method for knocking in SCD".

[0068] Up to this point, we have explained an embodiment in which a "polynucleotide encoding SCD" is knocked into a "region comprising the α-chain genes of HLA-F, HLA-G, and HLA-A" of a target cell, and a "polynucleotide encoding SCT" is knocked into a "region comprising the α-chain genes of HLA-C and HLA-B." However, in another embodiment of the present invention, a "polynucleotide encoding SCT" may be knocked into a "region comprising the α-chain genes of HLA-F, HLA-G, and HLA-A" of a target cell, and a "polynucleotide encoding SCD" may be knocked into a "region comprising the α-chain genes of HLA-C and HLA-B."

[0069] Therefore, the promoters used in each of the above-mentioned aspects of the present invention in a manner operably linked to SCD or SCT can also be used for knock-in in a manner operably linked to any sequence containing the SCD coding sequence or SCT coding sequence, respectively. For example, in another aspect of the present invention, the promoter sequence of the HLA-A α-chain gene can be knocked in operably linked to the SCT coding sequence. Also, in another aspect of the present invention, the promoter sequence of the HLA-B α-chain gene can be knocked in operably linked to the SCD coding sequence.

[0070] Although the above describes a procedure in which (i) deletion (deletion) of the α chain gene of an HLA class I molecule and (ii) introduction (knock-in) of an SCD and / or SCT are carried out simultaneously, the following different procedure may also be adopted: A truncation is introduced into a region (hereinafter also referred to as the "flanking region") adjacent to the α chain gene cluster region of an HLA class I molecule (such as a series of regions including the α chain genes of HLA-F, HLA-G, and HLA-A, or a series of regions including the α chain genes of HLA-C and HLA-B), and a polynucleotide encoding an SCD and / or SCT and a selection marker gene (selected from a positive selection marker gene and a negative selection marker gene, or a marker gene that can be used for both positive and negative selection) are introduced into the flanking region so that the selection marker gene is located closer to the α chain gene cluster region of the HLA class I molecule than one or both of the polynucleotides encoding the SCD and / or SCT. As described above, one of the selectable marker donor DNAs carries a polynucleotide encoding SCD and / or SCT and a selectable marker gene, and the other selectable marker donor DNA carries a marker gene distinct from the selectable marker gene. Cells expressing each selectable marker gene can be obtained by introducing a polynucleotide encoding SCD and / or SCT and a selectable marker gene into one allele of a target cell and introducing the selectable marker gene into the other allele. Subsequently, a contiguous region containing the selectable marker gene and the α-chain gene cluster region of an HLA class I molecule can be deleted from the one allele to delete the region containing the α-chain gene of an HLA class I molecule. Preferably, an additional region containing the selectable marker gene can be deleted from the other allele to prevent the region containing the α-chain gene of an HLA class I molecule from remaining in the genome of the target cell. Deletion of the contiguous region and the region containing the selectable marker gene can be achieved by sequence-specific cleavage at two sites flanking the region. This deletion does not require the use of donor DNA.Cells in which the above-mentioned series of regions have been deleted can be obtained as cells that do not express a selection marker gene (selected from a positive selection marker gene and a negative selection marker gene, or a marker gene that can be used for both positive and negative selection). Genetic analysis of the obtained cells can be performed to confirm that SCD and / or SCT have been introduced and that the series of regions have been deleted. Gene editing can also be performed in a variety of different orders.

[0071] [Modifications for Reducing CIITA Gene Expression] CIITA (MHC class II transactivator) is a transcription factor that regulates the expression of HLA class II molecules. HLA class II molecules are expressed exclusively in immune cells such as monocytes, macrophages, dendritic cells, and B cells, and activate the immune system by presenting extracellular non-self-derived peptides to helper T cells. When the presented antigen is recognized as non-self, the helper T cells initiate an immune response by releasing interferon, which activates killer T cells and damages the transplanted cells (Transplant Direct. 2017 May; 3(5): e152). Therefore, in a preferred embodiment of the present invention, the CIITA gene is deleted (knocked out) to suppress the expression of HLA class II molecules. Below, a method for knocking out the CIITA gene in diploid cells with two alleles is described.

[0072] 4. Method for knocking out the CIITA gene [Step 1] Replacement of a region containing the CIITA gene with a selection marker gene The "region containing the CIITA gene" (hereinafter also referred to as the "target region") replaced in this step contains the "region corresponding to the CIITA gene region on chr16 of the hg38 genome sequence" shown below. The "region containing the CIITA gene" may include the upstream region of the CIITA gene (a region corresponding to a region upstream of 10,877,202 on chr16 of the hg38 genomic sequence) and / or the downstream region of the CIITA gene (a region corresponding to a region downstream of 10,936,394 on chr16 of the hg38 genomic sequence). The length of the upstream region of the CIITA gene is not particularly limited as long as the method for knocking out the CIITA gene described below can be carried out, but is, for example, 800 to 20,000 nucleotides, preferably 1,000 to 5,000 nucleotides. The length of the upstream region can be appropriately set taking into consideration the design position of the guide RNA and whether the homology arm has a specific sequence. The length of the downstream region of the CIITA gene is not particularly limited as long as the method for knocking out the CIITA gene described below can be carried out, but is, for example, 800 to 20,000 nucleotides, preferably 1,000 to 5,000 nucleotides. The length of the downstream region can be appropriately set taking into consideration the designed position of the guide RNA, whether the homology arm has a specific sequence, etc. In a preferred embodiment of the present invention, the "region containing the CIITA gene" is a chromosomal region (target region 1) corresponding to chr16: 10,872,423-10,926,506 of the hg38 genome sequence, or a chromosomal region (target region 2) corresponding to chr16: 10,873,026-10,925,966 of the hg38 genome sequence.

[0073] [1-1] Site-specific cleavage of DNA using the CRISPR / Cas system A guide RNA (gRNA) targeting the upstream region of the CIITA gene (when the target region is target region 1 and 2, chr16 of the hg38 genome sequence: 10,873,824-10,873,846) and a gRNA targeting the downstream region of the CIITA gene (when the target region is target region 1 and 2, chr16 of the hg38 genome sequence: 10,925,149-10,925,171) are used to create two double-stranded breaks per allele using the CRISPR / Cas9 system with the Cas9 protein. The sequence of the gRNA does not have to be completely identical to the target sequence, as long as it can bind to the target sequence. A polynucleotide encoding the gRNA (e.g., an expression vector) may be used to introduce the gRNA. The method for introducing gRNA and Cas9 into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. Note that step [1-1] may be performed using other site-specific nucleases, such as TALENs or ZFNs, instead of the CRISPR / Cas system.

[0074] [1-2] Introduction of Different Selectable Marker Genes into the Target Region of Each Allele To introduce a selectable marker gene, a selectable marker donor DNA is used. The "selectable marker donor DNA" contains a "selectable marker gene" between an "upstream homology arm sequence" and a "downstream homology arm sequence." The "upstream homology arm sequence" has a sequence capable of homologous recombination with an upstream sequence within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination) (e.g., if the target region is target region 1, chr16: 10,872,423-10,873,829 of the hg38 genomic sequence. For example, if the target region is target region 2, chr16: 10,873,026-10,873,829 of the hg38 genomic sequence). The upstream homology arm sequence does not need to be completely identical to the sequence adjacent to the upstream side of the target region, as long as homologous recombination is possible, but preferably has a sequence identity of 90% or more, and more preferably 95% or more. The "downstream homology arm sequence" has a sequence capable of homologous recombination with a downstream sequence within a target region present in one of two chromosomes (the chromosome to be subjected to homologous recombination) (for example, when the target region is target region 1, chr16: 10,925,166-10,926,506 of the hg38 genome sequence; for example, when the target region is target region 2, chr16: 10,925,155-10,925,966 of the hg38 genome sequence). The downstream homology arm sequence does not need to be completely identical to the sequence adjacent to the downstream side of the target region, as long as homologous recombination is possible, but preferably has a sequence identity of 90% or more, and more preferably 95% or more. The lengths of the upstream homology arm and downstream homology arm can be, for example, about 500 bp to 3 kbp, but can be appropriately designed by those skilled in the art, similar to the design of ordinary donor DNA. The "donor DNA for selection marker" contains a positive selection marker (e.g., a drug resistance gene) used in this step and a negative selection marker (e.g., a fluorescent protein) used in steps 2 and 3 described below. The selection marker can be selected appropriately depending on the type of cells used, etc. The selection marker may be one that serves as both a positive selection marker and a negative selection marker (a dual-purpose marker).An example of a dual-purpose marker is a fluorescent protein.

[0075] In this step, two types of donor DNA for selection markers are used. The two types of donor DNA for selection markers each have a positive selection marker gene that is distinguishable from one another. Furthermore, the two types of donor DNA for selection markers each have a negative selection marker gene that is distinguishable from one another. Examples of combinations of selection marker genes carried by the two types of donor DNA for selection markers include those described in "1. Method for knocking in SCD."

[0076] The donor DNA for a selection marker may be either linear or circular, but is preferably circular. Preferably, the donor DNA for a selection marker is a plasmid. The donor DNA for a selection marker may contain a spacer sequence between the above sequences.

[0077] The method for introducing the selectable marker donor DNA into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. It is preferable to simultaneously introduce gRNA, Cas9, and the selectable marker donor DNA into cells, and then simultaneously perform steps [1-1] and [1-2]. Homologous recombination (HDR) occurs between the selectable marker donor DNA having the "upstream homology arm sequence" and "downstream homology arm sequence" and the cell chromosome. Sequence replacement by homologous recombination can be performed using well-known conventional techniques. After homologous recombination, cells expressing two types of positive selectable markers are selected and used in step 2. In the selected cells, the target regions of the two alleles are replaced with different types of selectable marker genes. In other words, in the selected cells, the CIITA genes of the two alleles are knocked out, and instead, different types of selectable marker genes are knocked into each allele.

[0078] [Step 2] Deletion of selection marker genes from both alleles [2-1] Site-specific cleavage of DNA using the CRISPR / Cas system Using the CRISPR / Cas9 system, two types of gRNAs targeting each of the two types of positive selection marker genes used in step [1-2] are used, along with the Cas9 protein, to create a double-stranded break at one site per allele. The sequence of the gRNA does not need to be completely identical to the target sequence, as long as it can bind to the target sequence. A polynucleotide (e.g., an expression vector) encoding the gRNA may be used to introduce the gRNA. The method for introducing the gRNA and Cas9 into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. Note that step [2-1] may also be performed using other site-specific nucleases, such as TALENs or ZFNs, instead of the CRISPR / Cas system.

[0079] [2-2] Deletion of the selectable marker gene from both alleles To delete the selectable marker, a selectable marker deleting DNA is used. In the selectable marker deleting DNA, an "upstream homology arm sequence" and a "downstream homology arm sequence" are linked, with no other sequences present between the two homology arm sequences. The "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker deleting DNA are the same as the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker donor DNA used in step [1-2], respectively. Therefore, homologous recombination occurs between the "selectable marker deleting DNA" and the "allele containing the selectable marker gene," resulting in the removal of the selectable marker gene from the allele. Homologous recombination can be carried out using well-known conventional techniques. Note that, as long as homologous recombination occurs, the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker deleting DNA may be different from the "upstream homology arm sequence" and "downstream homology arm sequence" of the selectable marker donor DNA used in step [1-2], respectively.

[0080] The DNA for deleting the selection marker may be either linear or circular, but is preferably circular. Preferably, the DNA for deleting the selection marker is a plasmid.

[0081] The method for introducing the selectable marker deletion DNA into cells is not particularly limited, and known methods (e.g., lipofection) can be used without particular limitation. It is preferable to simultaneously introduce gRNA, Cas9, and SCD donor DNA into cells, and then simultaneously perform steps [2-1] and [2-2]. After homologous recombination, cells that do not express either of the two negative selection markers introduced in step [1-2] are selected. In the selected cells, the selectable marker gene is deleted from both alleles. In other words, in the selected cells, the CIITA gene is knocked out in both alleles. In other words, for example, if the target region is target region 2, the chromosomal region corresponding to chr16: 10,873,830-10,925,154 of the hg38 genome sequence (the region excluding the upstream homology arm and downstream homology arm from target region 2) is deleted in both alleles in the selected cells. Cells in which the "CIITA gene" has been deleted from both alleles have reduced expression of the gene compared to wild-type cells. "Wild-type cells" refer to cells that have not been subjected to steps 1 and 2 described above. Comparison of gene expression can be carried out using known gene expression analysis techniques (e.g., reverse transcription quantitative PCR). Cells in which the "CIITA gene" has been deleted from both alleles may lack expression of the gene. Cells in which the "CIITA gene" has been deleted from both alleles may lack expression of the protein encoded by the gene, or may lack expression of the protein encoded by the gene on the cell membrane.

[0082] In one aspect of the present invention, expression of an HLA class II molecule may be achieved by means other than the deletion (knockout) of the aforementioned CIITA gene, for example, by deleting the endogenous gene encoding RFXANK, which is one of the transcriptional regulators of the HLA class II molecule gene, or by deleting the endogenous gene for the HLA class II molecule.

[0083] In the cells of the present invention, the expression of the α chain of the aforementioned specific HLA class I molecule (and thus the expression of functional HLA class I molecules having antigen-presenting ability) is suppressed, and therefore, the T cells (especially CD8 + T cells and / or CD4 + The rejection reaction due to attack from CD8 T cells is reduced compared to wild-type cells. The term "wild-type cells" refers to cells that have not been subjected to the above-mentioned process of knocking in SCD and / or SCT. The rejection reaction can be compared, for example, by comparing the rejection reaction of wild-type cells with that of wild-type cells. + T cells and / or CD4 + The proliferative activity of T cells can be used as an index for evaluation.

[0084] Furthermore, the cells of the present invention express single-chain fusion peptides (SCD and / or SCT) mimicking HLA-G or HLA-E, which function to suppress the activity (cytotoxicity) of NK cells and macrophages, and therefore, the rejection reaction due to attack by NK cells and / or macrophages in the transplant recipient is reduced compared to wild-type cells. "Wild-type cells" refer to cells that have not been subjected to the above-mentioned step of knocking in SCD and / or SCT. Rejection reactions can be compared, for example, using cytotoxicity by NK cells and / or macrophages as an index.

[0085] [Other HLA-associated factors] In one embodiment of the present invention, the cells of the present invention may be cells that carry the B2M gene. Here, the cells that carry the B2M gene may be cells that carry a wild-type B2M gene, or may be genetically modified cells that carry the B2M gene that a cell has before genetic modification. Furthermore, the cells that carry the B2M gene may be cells that express a protein encoded by the B2M gene.

[0086] [Uses of Genetically Modified Cells] The cells of the present invention, which have been subjected to the above-described genetic modification, can inhibit the expression of certain HLA class I molecules, thereby inhibiting T cells and the like (especially CD8 + T cells and CD4 +The expression of HLA gene-modified cells avoids attack by NK cells and the like, and the expression of SCD and / or SCT avoids attack by NK cells and the like. Cells with improved tissue compatibility with the recipient due to these characteristics can be used as transplant cells for regenerative medicine (particularly allogeneic transplantation). The cells of the present invention obtained by subjecting pluripotent stem cells such as iPS cells to the above-mentioned genetic modification can be differentiated and then transplanted depending on the purpose of transplantation, etc. Examples of differentiated cells include immune cells, cardiomyocytes, and nerve cells. Therefore, cells (e.g., differentiated cells) derived from HLA gene-modified cells that have been genetically modified according to the present invention also constitute one aspect of the present invention.

[0087] [Knock-in of Effector Cell Inhibitory Factors] Up to this point, we have described embodiments in which SCD and / or SCT, which are factors (effector cell inhibitory factors) that avoid recognition by NK cells and macrophages and suppress their attack, are used as the factors to be knocked in. However, in another embodiment of the present invention, other effector cell inhibitory factors can be used for knock-in in addition to SCD and / or SCT. In yet another embodiment of the present invention, other effector cell inhibitory factors can be used for knock-in instead of SCD and SCT. That is, in these embodiments, one or more effector cell inhibitory factors including SCD and SCT are knocked in. This embodiment includes the following HLA gene-modified cells 1 to 3. 1. An HLA gene-modified cell, characterized in that a polynucleotide encoding an effector cell inhibitory factor has been knocked in into a region containing the α-chain genes of HLA-F, HLA-G, and HLA-A, and further in that the α-chain genes of HLA-F, HLA-G, and HLA-A have been deleted in the cell before gene modification. 2. An HLA gene-modified cell, characterized in that a polynucleotide encoding an effector cell inhibitory factor has been knocked into a region containing the α-chain genes of HLA-C and HLA-B, and further, the α-chain genes of HLA-C and HLA-B have been deleted from the cell before gene modification. An HLA gene-modified cell, characterized in that the following polynucleotide (1) has been knocked into a region containing the α-chain genes of HLA-F, HLA-G, and HLA-A: (1) a polynucleotide encoding a first effector cell inhibitory factor; the following polynucleotide (2) has been knocked into a region containing the α-chain genes of HLA-C and HLA-B: (2) a polynucleotide encoding a second effector cell inhibitory factor that is the same as or different from the first effector cell inhibitory factor; and the α-chain genes of HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G have been deleted from the cell before genetic modification.

[0088] Examples of "effector cells" include NK cells and macrophages. Examples of "effector cell inhibitory factors" include known factors that can avoid recognition by effector cells and suppress their cytotoxicity. Specific examples include NK cell inhibitory factors (e.g., HLA-E, HLA-G, PD-L1, CD47, CD68, and ICAM-1) and macrophage inhibitory factors (e.g., HLA-E and HLA-G). An "effector cell inhibitory factor" may be a modified form of a naturally occurring factor. An example of a modified form is a fusion peptide comprising the α chain of HLA-G and β2-microglobulin (e.g., a single-chain dimeric fusion peptide, more specifically, SCD). Other examples of variants include fusion peptides comprising the α chain of HLA-E and β2 microglobulin, and fusion peptides (e.g., single-chain trimeric fusion peptides, more specifically, SCT) comprising the α chain of HLA-E, β2 microglobulin, and an HLA signal peptide (specifically, a signal peptide of an HLA class I molecule, for example, a signal peptide of HLA-A, HLA-B, HLA-C, or HLA-G). Knock-in of a polynucleotide encoding an effector cell inhibitory factor can be carried out using a homologous recombination method using a site-specific nuclease (e.g., the method described in eLife, 2017, 6, e27873; FEBS Letters, 591, 2017, 903-913; and WO 2021 / 206054), similar to the knock-in of polynucleotides encoding SCD and / or SCT described above. The region into which the polynucleotide encoding the effector cell inhibitory factor is knocked in is the same as the "region comprising the α-chain genes of HLA-F, HLA-G, and HLA-A" and / or the "region comprising the α-chain genes of HLA-C and HLA-B" described in relation to the knock-in of SCD and / or SCT. In another embodiment, polynucleotides encoding one or more effector cell inhibitory factors are knocked in only one allele of a target cell. In another embodiment, polynucleotides encoding one or more effector cell inhibitory factors are knocked in respectively into each allele of a target cell.In another aspect, one or more promoter sequences of the α-chain gene of HLA (e.g., HLA-F, HLA-G, HLA-A, HLA-B, or HLA-C) are knocked in together with the polynucleotide encoding the effector cell inhibitory factor. In another aspect, one or more promoter sequences whose expression is induced by IFNγ are knocked in together with the polynucleotide encoding the effector cell inhibitory factor. The promoter sequence is knocked in in a manner operably linked to the polynucleotide encoding the effector cell inhibitory factor. "Operably linked" means that the promoter sequence is positioned relative to the polynucleotide encoding the effector cell inhibitory factor in a manner that controls the expression of the polynucleotide.

[0089] [Method for Producing HLA Gene-Modified Cells] The present invention also relates to a method for producing the aforementioned HLA gene-modified cells. The method of the present invention comprises the step of introducing (knock-in) a polynucleotide encoding an effector cell inhibitory factor (e.g., SCD and SCT) into a region (target region) encoding the α chain of an HLA class I molecule of a target cell using gene modification techniques. Gene modification techniques that can be used include gene modification using site-specific nucleases (e.g., the homologous recombination method described herein). One embodiment of the method of the present invention comprises the step of knocking in a polynucleotide encoding an effector cell inhibitory factor into only one allele of the target cell. This embodiment may further comprise the step of introducing two types of selection markers into the target regions of the two alleles of the target cell during the knock-in process, thereby selecting cells in which the desired sequence has been introduced into only one allele. One embodiment of the method of the present invention (hereinafter also referred to as "one embodiment of the present invention") comprises the step of knocking in a polynucleotide encoding an effector cell inhibitory factor into each allele of the target cell. One aspect of the present invention comprises the step of knocking in one or more fusion peptides selected from a fusion peptide comprising the α chain of β2 microglobulin and HLA-G and a fusion peptide comprising the α chain of β2 microglobulin and HLA-E into one or more regions selected from a region comprising the α chain genes of HLA-F, HLA-G, and HLA-A, and a region comprising the α chain genes of HLA-C and HLA-B. Preferably, the fusion peptide comprising the α chain of β2 microglobulin and HLA-G is a single-chain dimer (SCD) comprising the α chain of β2 microglobulin and HLA-G. Furthermore, preferably, the fusion peptide comprising β2 microglobulin and the α chain of HLA-E is a single-chain trimeric fusion peptide comprising a signal peptide of HLA class I, β2 microglobulin, and the α chain of HLA-E, and more preferably, a single-chain trimeric fusion peptide (SCT) comprising a signal peptide of HLA-A, HLA-B, HLA-C, or HLA-G, β2 microglobulin, and the α chain of HLA-E.One aspect of the present invention comprises a step of knocking in a polynucleotide encoding a single-chain dimeric fusion peptide (Single Chain Dimer: SCD) comprising β2 microglobulin and the α chain of HLA-G into a region of a target cell comprising the α chain genes of HLA-F, HLA-G, and HLA-A. Another aspect of the present invention comprises a step of knocking in a polynucleotide encoding a single-chain trimeric fusion peptide comprising an HLA class I signal peptide, β2 microglobulin, and the α chain of HLA-E into a region of a target cell comprising the α chain genes of HLA-C and HLA-B, more preferably a step of knocking in a polynucleotide encoding a single-chain trimeric fusion peptide (Single Chain Trimer: SCT) comprising an HLA-A, HLA-B, HLA-C, or HLA-G signal peptide, β2 microglobulin, and the α chain of HLA-E into a region of a target cell comprising the α chain genes of HLA-C and HLA-B. One aspect of the present invention comprises a step of knocking in a polynucleotide encoding the SCD and a polynucleotide encoding the SCT into a region of a subject cell comprising the α-chain genes of HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G. In one aspect of the present invention, the region comprising the α-chain genes of HLA-F, HLA-G, and HLA-A is a chromosomal region corresponding to chr6:29,704,906-29,958,642 of the hg38 genome sequence. In another aspect of the present invention, the region comprising the α-chain genes of HLA-C and HLA-B is a chromosomal region corresponding to chr6:31,224,837-31,378,196 of the hg38 genome sequence. In one aspect of the present invention, the region containing the α-chain genes of HLA-C and HLA-B is a chromosomal region corresponding to chr6: 31,227,286-31,378,780 of the hg38 genome sequence. In one aspect of the present invention, the method further comprises the step of knocking out an HLA class II molecule gene in the target cell. In one aspect of the present invention, the HLA class II molecule gene is the CIITA gene. In one aspect of the present invention, the target cell is a human pluripotent stem cell. In one aspect of the present invention, the target cell is a human iPS cell or an ES cell.In one aspect of the present invention, the subject cells are human iPS cell line 253G4. In one aspect of the present invention, the subject cells are human iPS cell line 201B7. In one aspect of the present invention, the subject cells are cells differentiated from human pluripotent stem cells. In one aspect of the present invention, the human pluripotent stem cells are human iPS cells or ES cells. In one aspect of the present invention, the human pluripotent stem cells are human iPS cell line 253G4 or human iPS cells or ES cells. In one aspect of the present invention, the cells differentiated from human pluripotent stem cells are T cells or NK cells. In one aspect of the present invention, the polynucleotide encoding SCD comprises the nucleotide sequence shown in SEQ ID NO: 2 or 5. In one aspect of the present invention, the polynucleotide encoding SCT comprises the nucleotide sequence shown in SEQ ID NO: 7. In one aspect of the present invention, the step of knocking in the polynucleotide encoding SCD comprises a step of knocking in a promoter sequence of an HLA-A α-chain gene together with the polynucleotide encoding SCD, wherein the promoter sequence is operably linked to the sequence encoding SCD. In one aspect of the present invention, the promoter sequence of the HLA-A α chain gene comprises the nucleotide sequence shown in SEQ ID NO: 10. In one aspect of the present invention, the step of knocking in a polynucleotide encoding SCT comprises knocking in a promoter sequence of the HLA-B α chain gene together with the polynucleotide encoding SCT, wherein the promoter sequence is operably linked to a sequence encoding SCT. In one aspect of the present invention, the promoter sequence of the HLA-B α chain gene comprises the nucleotide sequence shown in SEQ ID NO: 11. In one aspect of the present invention, the production method does not comprise a step of deleting the B2M gene. In one aspect of the present invention, the production method does not comprise a step of deleting the B2M gene. In one aspect of the present invention, the production method does not comprise a step of reducing or deleting expression of the B2M gene. The step of reducing or deleting expression of the B2M gene includes, for example, a step of introducing a mutation that reduces or deletes expression of the B2M gene. In one aspect of the present invention, the production method does not comprise a step of reducing or deleting expression of a protein encoded by the B2M gene.In one embodiment of the present invention, the production method does not include a step of reducing or eliminating the function of a protein encoded by the B2M gene. The step of reducing or eliminating the function of a protein encoded by the B2M gene includes, for example, a step of introducing a mutation that reduces or eliminates the function of the protein encoded by the B2M gene. The production method of the present invention also includes any combination of two or more of the above-mentioned aspects. Furthermore, in addition to the above-mentioned aspects, the production method of the present invention also includes any combination of one or more of the production method features described in the section "Knock-in of polynucleotides encoding SCD and / or SCT" of this specification.

[0090] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0091] Example 1: Preparation of HLA gene-modified cells in which a region containing the α-chain genes of HLA-F, HLA-G, and HLA-A has been replaced (knocked in) with an SCD coding sequence HLA gene-modified cells were prepared according to the method described above in "1. Method for knocking in SCD." The "region containing the α-chain genes of HLA-F, HLA-G, and HLA-A" was a chromosomal region (target region 1) corresponding to chr6: 29,704,906-29,958,642 of the hg38 genome sequence. The CRISPR / Cas system was used as the site-specific nuclease.

[0092] The cells used for gene modification were human iPS cell line 253G4 (iPS cell stock derived from an HLA-homologous donor, Kyoto University iPS Cell Research Foundation, a public interest incorporated foundation) (diploid).

[0093] Step [1-1] A CRISPR / Cas9 system was used, using a guide RNA (gRNA) targeting the upstream region of the HLA-F α-chain gene (chr6: 29,707,002-29,707,024 in the hg38 genome sequence), a gRNA targeting the downstream region of the HLA-A α-chain gene (chr6: 29,956,465-29,956,487 in the hg38 genome sequence), and the Cas9 protein. The gRNA sequence was completely identical to the target sequence. An expression vector was used to introduce the gRNA. Lipofection was used to introduce the gRNA and Cas9 into cells.

[0094] Step [1-2] The upstream homology arm sequence was designed to target the upstream sequence (chr6:29,704,906-29,707,018 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The downstream homology arm sequence was designed to target the downstream sequence (chr6:29,956,471-29,958,642 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The following combinations of selectable marker genes contained in the two types of selectable marker donor DNA were used. The donor DNA for the selection marker was a plasmid. The donor DNA for the selection marker was introduced into the cells using the lipofection method. In this example, steps [1-1] and [1-2] were carried out simultaneously.

[0095] [Step 2-1] Of the two types of positive selection marker genes, gRNA targeting the blasticidin resistance gene was used. The sequence of the gRNA was completely identical to the target sequence. An expression vector encoding the gRNA was used to introduce the gRNA. The lipofection method was used to introduce the gRNA and Cas9 into the cells.

[0096] [Step 2-2] The nucleotide sequence shown in SEQ ID NO: 5 was used as the SCD coding sequence. The donor DNA for SCD contained the promoter sequence of the HLA-A α chain gene shown in SEQ ID NO: 10. The donor DNA for SCD was a plasmid. The donor DNA for SCD was introduced into cells using the lipofection method. In this example, steps [2-1] and [2-2] were carried out simultaneously.

[0097] [Step 3-1] Of the two types of positive selection marker genes, gRNA targeting the puromycin resistance gene was used. The sequence of the gRNA was completely identical to the target sequence. An expression vector encoding the gRNA was used to introduce the gRNA. The lipofection method was used to introduce the gRNA and Cas9 into the cells.

[0098] [Step 3-2] The DNA for deleting the selection marker was a plasmid. The DNA for deleting the selection marker was introduced into the cells using the lipofection method. In this example, steps [3-1] and [3-2] were carried out simultaneously.

[0099] [Expression Analysis of SCD Coding Sequence in Genetically Modified Cells] Expression of the SCD coding sequence in the genetically modified cells prepared in the above Examples was analyzed using the expression of the HLA-G α-chain gene contained in the SCD coding sequence as an index. Using a 6-well plate, 3 × 10 cells before genetic modification (human iPS cell line 253G4) or three genetically modified cell lines (AL-UDC (ver. 0.1)) were added to 1.5 mL of Stemfit AK02N medium (Ajinomoto Co., Inc.) containing 10 μM Rho-associated kinase inhibitor (Y-27632). 5The cells were seeded at a cell density of 1000 cells / mL and cultured at 37°C in 5% CO2. The next day and the day after, the medium was replaced with 2 mL of Stemfit AK02N medium supplemented with 50 ng / mL interferon gamma (IFNγ), and the culture was continued. IFNγ was used to stimulate the promoter of the HLA-A α-chain gene and express the SCD coding sequence. After 3 days of culture, the supernatant was removed with an aspirator and the cells were washed twice with 2 mL of DPBS(-) (D-PBS(-) without Ca and Mg, liquid, Nacalai). 0.5x TrypLE (TrypLE TMSelect Enzyme (1X), no phenol red, Gibco) / DPBS was added and incubated at 37°C, 5% CO2 for approximately 3 minutes. After removing the supernatant with an aspirator, the cells were suspended in 1 mL of DPBS and centrifuged at 300g for 3-5 minutes. The cells were again suspended in 1 mL of DPBS, dispensed in 250 μL aliquots, and centrifuged at 300g for 3-5 minutes. The supernatant was removed, and RNA was extracted using the RNeasy Mini Kit (250) (QIAGEN). Using the PrimerScript RT reagent Kit with gDNA Eraser (TaKaRa), reverse transcription was performed in a 10 μL reaction system using 200 ng of RNA as a template. After the reaction, the reaction mixture was diluted with 40 μL of ultrapure water, aliquoted into 5 μL aliquots, and 45 μL of ultrapure water was added (diluted cDNA). For each sample, 3.3 μL of DEPC treated water, 7.5 μL of TB Green® PremixEx Taq™ II (Tli RNase H Plus) (TaKaRa), 1.2 μL of 2.5 μM each primer mix (a mixture of three primers (Primer 1, Primer 2, and Primer 3) that amplify the ORF region of the HLA-G α-chain gene), and 3 μL of diluted cDNA were mixed and added to a 96-well plate. The expression level of the HLA-G α-chain gene was quantified by qPCR using a program consisting of 40 cycles of 95°C for 30 seconds, 95°C for 5 seconds, and 60°C for 30 seconds, followed by 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds. Regarding the expression of the HLA-G α-chain gene, the relative expression level in the genetically modified cells (AL-UDC (ver. 0.1)) is shown in Figure 1, where the expression level in the cells before genetic modification (iPS253G4) is set to 1. Unlike the cells before genetic modification (iPS253G4), the genetically modified cells (AL-UDC (ver. 0.1)) showed a significant increase in expression level before the addition of IFNγ (IFNγ - ), compared with after the addition of IFNγ (IFNγ + ) The expression level of the HLA-G α-chain gene increased. This result indicates that the SCD coding sequence had been knocked in so as to be expressible in the genetically modified cells prepared in the examples.

[0100] Example 2: Preparation of HLA gene-modified cells in which the region containing the α-chain genes of HLA-F, HLA-G, and HLA-A has been replaced (knocked in) with an SCD coding sequence, and the region containing the α-chain genes of HLA-C and HLA-B has been replaced (knocked in) with an SCT coding sequence. HLA gene-modified cells were prepared by carrying out the above-mentioned "1. Method for knocking in SCD" followed by "2. Method for knocking in SCT."

[0101] The cells used for gene modification were human iPS cell line 201B7 (iPS cell stock derived from an HLA-homologous donor, Kyoto University iPS Cell Research Foundation, a public interest incorporated foundation) (diploid).

[0102] Example 2-1: Knock-in of SCD According to the method described above in "1. Method for knocking-in of SCD," an SCD coding sequence was knocked into a "region containing the α-chain genes of HLA-F, HLA-G, and HLA-A" of a cell. The "region containing the α-chain genes of HLA-F, HLA-G, and HLA-A" was a chromosomal region (target region 1) corresponding to chr6: 29,704,906-29,958,642 of the hg38 genome sequence. In addition, the CRISPR / Cas system was used as the site-specific nuclease.

[0103] Step [1-1] A CRISPR / Cas9 system was used, using a guide RNA (gRNA) targeting the upstream region of the HLA-F α-chain gene (chr6: 29,707,002-29,707,024 of the hg38 genome sequence), a gRNA targeting the downstream region of the HLA-A α-chain gene (chr6: 29,956,465-29,956,487 of the hg38 genome sequence), and the Cas9 protein. The sequence of the gRNA was completely identical to the target sequence. An expression vector was used to introduce the gRNA. Lipofection was used to introduce the gRNA and Cas9 into cells.

[0104] Step [1-2] The upstream homology arm sequence was designed to target the upstream sequence (chr6:29,704,906-29,707,018 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The downstream homology arm sequence was designed to target the downstream sequence (chr6:29,956,471-29,958,642 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The following combinations of selectable marker genes contained in the two types of selectable marker donor DNA were used. The donor DNA for the selection marker was a plasmid. The donor DNA for the selection marker was introduced into the cells using the lipofection method. In this example, steps [1-1] and [1-2] were carried out simultaneously.

[0105] [Step 2-1] Of the two types of positive selection marker genes, gRNA targeting the blasticidin resistance gene was used. The sequence of the gRNA was completely identical to the target sequence. An expression vector encoding the gRNA was used to introduce the gRNA. The lipofection method was used to introduce the gRNA and Cas9 into the cells.

[0106] [Step 2-2] The upstream homology arm sequence was designed to target the upstream sequence (chr6:29,704,906-29,707,018 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The downstream homology arm sequence was designed to target the downstream sequence (chr6:29,956,471-29,958,642 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The nucleotide sequence set forth in SEQ ID NO:5 was used as the SCD coding sequence. The donor DNA for SCD contained the promoter sequence of the HLA-A α-chain gene set forth in SEQ ID NO:10. The donor DNA for SCD was a plasmid. The donor DNA for SCD was introduced into cells using the lipofection method. In this example, steps [2-1] and [2-2] were carried out simultaneously.

[0107] [Step 3-1] Of the two types of positive selection marker genes, gRNA targeting the puromycin resistance gene was used. The sequence of the gRNA was completely identical to the target sequence. An expression vector encoding the gRNA was used to introduce the gRNA. The lipofection method was used to introduce the gRNA and Cas9 into the cells.

[0108] [Step 3-2] The DNA for deleting the selection marker was a plasmid. The DNA for deleting the selection marker was introduced into the cells using the lipofection method. In this example, steps [3-1] and [3-2] were carried out simultaneously. The modified cells obtained in step [3-2] were used for knock-in of SCT (Example 2-2).

[0109] [Example 2-2: Knock-in of SCT] According to the method described above in "2. Method for knocking-in of SCT," an SCT coding sequence was knocked into the "region containing the α-chain genes of HLA-C and HLA-B" of the cells prepared in Example 2-1. The "region containing the α-chain genes of HLA-C and HLA-B" was a chromosomal region (target region 2) corresponding to chr6: 31,227,286-31,378,780 of the hg38 genome sequence. In addition, the CRISPR / Cas system was used as the site-specific nuclease.

[0110] Step [1-1] A CRISPR / Cas9 system was used, using a guide RNA (gRNA) targeting the upstream region of the HLA-C α-chain gene (chr6: 31,228,800-31,228,822 in the hg38 genome sequence), a gRNA targeting the downstream region of the HLA-B α-chain gene (chr6: 31,377,258-31,377,277 in the hg38 genome sequence), and the Cas9 protein. The gRNA sequence was completely identical to the target sequence. An expression vector was used to introduce the gRNA. Lipofection was used to introduce the gRNA and Cas9 into cells.

[0111] Step [1-2] The upstream homology arm sequence was designed to target the upstream sequence (chr6: 31,227,286-31,228,805 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The downstream homology arm sequence was designed to target the downstream sequence (chr6: 31,377,261-31,378,780 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The following combinations of selectable marker genes contained in the two types of selectable marker donor DNA were used. The donor DNA for the selection marker was a plasmid. The donor DNA for the selection marker was introduced into the cells using the lipofection method. In this example, steps [1-1] and [1-2] were carried out simultaneously.

[0112] [Step 2-1] Of the two types of positive selection marker genes, gRNA targeting the blasticidin resistance gene was used. The sequence of the gRNA was completely identical to the target sequence. An expression vector encoding the gRNA was used to introduce the gRNA. The lipofection method was used to introduce the gRNA and Cas9 into the cells.

[0113] [Step 2-2] The upstream homology arm sequence was designed to target the upstream sequence (chr6: 31,227,286-31,228,805 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The downstream homology arm sequence was designed to target the downstream sequence (chr6: 31,377,261-31,378,780 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The nucleotide sequence set forth in SEQ ID NO: 7 was used as the SCT coding sequence. The donor DNA for SCD contained the promoter sequence of the HLA-B α-chain gene set forth in SEQ ID NO: 11. The donor DNA for SCD was a plasmid. The donor DNA for SCD was introduced into cells using the lipofection method. In this example, steps [2-1] and [2-2] were carried out simultaneously.

[0114] [Step 3-1] Of the two types of positive selection marker genes, gRNA targeting the puromycin resistance gene was used. The sequence of the gRNA was completely identical to the target sequence. An expression vector encoding the gRNA was used to introduce the gRNA. The lipofection method was used to introduce the gRNA and Cas9 into the cells.

[0115] [Step 3-2] The DNA for deleting the selection marker was a plasmid. The DNA for deleting the selection marker was introduced into the cells using the lipofection method. In this example, steps [3-1] and [3-2] were carried out simultaneously.

[0116] In the genetically modified cells obtained in this Example, deletion of the HLA-F, HLA-G, HLA-A, HLA-C, and HLA-B genes and introduction of polynucleotide sequences encoding SCD and SCT were confirmed by PCR based on the presence or absence of bands indicating amplification of the target region. In the genetically modified cells obtained in [Example 2-1: SCD knock-in], deletion of the HLA-F, HLA-G, and HLA-A genes and introduction of the polynucleotide sequence encoding SCD were confirmed by PCR. In the genetically modified cells obtained in [Example 2-2: SCT knock-in], deletion of the HLA-B and HLA-C genes and introduction of the polynucleotide sequence encoding SCT were confirmed by PCR.

[0117] Example 3: Expression analysis of genetically modified cells prepared in Example 2 The expression of HLA-A, HLA-B, HLA-C, SCD, and SCT in the genetically modified cells prepared in Example 2 (hereinafter referred to as "genetically modified cells") was analyzed.

[0118] Example 3-1: Analysis of HLA-A, HLA-B, and HLA-C Expression The cell surface expression levels of HLA-A, HLA-B, and HLA-C in genetically modified cells were analyzed using flow cytometry. A suspension of genetically modified cells or control cells (human iPS cell line 201B7 before genetic modification) was mixed with a primary antibody that recognizes HLA-A, HLA-B, and HLA-C (company name: BioLegend, catalog number 311415, clone number W6 / 32) and incubated on ice for 30 minutes. After the incubation, the cells were washed with a washing solution (PBS solution containing 2% BSA), and a sample containing only cells bound to the primary antibody was collected. Next, each sample was reacted with a fluorescently labeled antibody (secondary antibody) that recognized the primary antibody and subjected to a flow cytometer (Thermo Fisher Science, Attune) to analyze the cell surface expression levels of HLA-A, HLA-B, and HLA-C. The results are shown in the "HLA-A, B, C" column in the upper part of Figure 2. Figure 2 includes the results (panels) of a control cell sample (201B7) and three genetically modified cell samples (AL-UDC v0.2#1-#3). Each panel contains two experimental results: IC (gray) and IFN-g(+) (black). The IC (isotype control) indicates the results obtained using a fluorescently labeled primary antibody instead of the primary antibody. The IC was used to measure the proportion of nonspecific binding of the primary antibody. "IFN-g(+)" indicates the results obtained when IFNγ was added. For this sample, cells were seeded at 13,000 cells / well and cultured in AK03N medium (Ajinomoto Healthy Supply Co., Ltd.) for 5 days. Then, they were stimulated with 50 ng / mL of IFN-γ and cultured for an additional 2 days before expression analysis. IFN-γ is a substance that induces the expression of HLA-A, HLA-B, and HLA-C. The horizontal axis of the figure represents fluorescence intensity. The vertical axis (Percent of Max) represents the percentage of fluorescence intensity relative to the fluorescence intensity of the most numerous cells in the sample. The percentages in the figure represent the proportion of cells expressing each protein (positive cells). In the control cell sample (201B7), the addition of IFN-γ significantly shifted the histogram to the right (toward higher fluorescence intensity).This indicates that the addition of IFNγ increased the expression levels of HLA-A, HLA-B, and HLA-C. On the other hand, in the genetically modified cell samples (AL-UDC v0.2#1-#3), the degree of shift to the right side of the histogram due to the addition of IFNγ was small. This indicates that the expression levels of HLA-A, HLA-B, and HLA-C did not increase even with the addition of IFNγ. This result indicates that the "knock-in of SCD into the region containing the α-chain gene of HLA-A" and the "knock-in of SCT into the region containing the α-chain genes of HLA-C and HLA-B" in the genetically modified cells resulted in the loss of expression of HLA-A, HLA-B, and HLA-C.

[0119] Example 3-2: Expression analysis of SCD and SCT The cell surface expression levels of SCD and SCT in genetically modified cells were analyzed using flow cytometry. Expression analysis was performed according to the same procedure as in Example 3-1, except that an anti-HLA-G antibody (abcam, catalog number: ab24384, clone number: MEM-G / 9) was used as the primary antibody recognizing SCD (single-chain fusion peptide mimicking HLA-G), and an anti-HLA-E antibody (abcam, catalog number: ab272335, clone number: 3D12) was used as the primary antibody recognizing SCT (single-chain fusion peptide mimicking HLA-E). The results for SCD are shown in the "SCD" column in the lower part of Figure 2. The results for SCT are shown in the "SCT" column in the middle of Figure 2. For SCD, the control cell sample (201B7) did not undergo a histogram shift even with the addition of IFNγ. On the other hand, in the genetically modified cell samples (AL-UDC v0.2#1-#3), the addition of IFNγ significantly shifted the histogram to the right (toward increasing fluorescence intensity). This indicates that in genetically modified cells in which HLA-G expression (due to the gene possessed by the pre-modification cells) was lost due to "knock-in of SCD into the region containing the HLA-G α-chain gene," the promoter sequence (SEQ ID NO: 10) of the HLA-A α-chain gene knocked in using SCD donor DNA was stimulated by the addition of IFNγ, resulting in increased expression of the downstream SCD (single-chain fusion peptide mimicking HLA-G). Therefore, this result indicates that SCD is knocked in and expressed in the genetically modified cells. For SCT, the addition of IFNγ significantly shifted the histogram to the right (toward increasing fluorescence intensity) for both the control cell sample (201B7) and the genetically modified cell samples (AL-UDC v0.2#1-#3). This indicates that the addition of IFNγ stimulated the promoter sequence (sequence number 11) of the HLA-B α-chain gene knocked in by the SCT donor DNA, increasing the expression level of the downstream SCT.Although the HLA-E α-chain gene remains in the genetically modified cells, the leader sequence of the HLA-E α-chain gene is not translated due to the knockout of the HLA-A, HLA-B, and HLA-C α-chain genes, and therefore the HLA-E α-chain is not expressed on the cell surface. Therefore, this result indicates that SCT is knocked in and expressed in the genetically modified cells.

[0120] Example 3-3: T cell responsiveness to genetically modified cells T cells recognize self / non-self based on the HLA class I molecules (HLA-A, HLA-B, and HLA-C) expressed by target cells. T cells that recognize target cells as non-self are activated, divide, and proliferate, strengthening cellular immunity (attacking the target cells). Utilizing this property of T cells, the responsiveness of T cells to genetically modified cells was evaluated using the proliferation of T cells when co-cultured with genetically modified cells as an indicator. The proliferation of T cells was evaluated using the fluorescence intensity of the fluorescent dye CytelRed as an indicator. When T cells that have incorporated this fluorescent dye divide, the fluorescence intensity of the divided T cells decreases. Before the start of co-culture, T cells (human T lymphocytes; LONZA, catalog number 2W-300) were stained with the fluorescent dye CytelRed (AAT Bioquest, catalog number 22255). T cell samples at the time of staining (when awake) were subjected to flow cytometry to measure the percentage of viable cells. The criteria for viable cells were negative for the 7-AAD antibody (7-AAD(-)), which binds to dead cells, and positive for anti-CD8 antibody (CD8(+)) (CD8 is an indicator of cytotoxic T cells). In addition to the genetically modified cells, human iPS cell line 201B7 (the parent line of the genetically modified cells; hereafter referred to as "WT cells") and human iPS cell line 201B7 lacking the B2M gene (hereafter referred to as "B2MKO cells") were used as evaluation cells. The evaluation cells and T cells were co-cultured for 10 days. After co-culture, the cell samples were subjected to flow cytometry to measure fluorescence intensity, and the results were compared with those obtained when awake. The results are shown in Figure 3. Figure 3 shows the results (panels) of T cell samples at the start of culture (Day 0), co-culture samples with genetically modified cells (AL-UDC v0.2), co-culture samples with wild-type cells (201B7), co-culture samples with B2MKO cells (B2MKO), a positive control (PC), and a negative control (NC). The horizontal axis of the figure represents CytelRed fluorescence intensity. The vertical axis (Count) represents cell number. The percentage in the figure represents the proportion of divided T cells. For PC, T cells stimulated with 5 μg / mL anti-CD3 antibody (OKT3) (Invitrogen, catalog number 16-0037-85) were cultured alone for 10 days and evaluated.Anti-CD3 antibodies bind to the T cell surface antigen CD3, activating and promoting T cell proliferation. A decrease in CytelRed fluorescence intensity (i.e., T cell proliferation) occurred in the PC, confirming the validity of this test system. In the NC, T cells cultured alone for 10 days without stimulation with the anti-CD3 antibody were evaluated. No decrease in CytelRed fluorescence intensity occurred in the NC, indicating no T cell proliferation. In the WT cell co-culture group, a cell population with lower fluorescence intensity was observed compared to Day 0 of culture. This indicates that T cells recognized WT cells expressing HLA class I molecules (HLA-A, HLA-B, and HLA-C) as non-self, resulting in activation and proliferation. In the B2MKO cell co-culture group, fluorescence intensity remained unchanged compared to T cells on Day 0 of culture. This indicates that the T cells did not recognize B2MKO cells (in which the expression of HLA class I molecules is suppressed due to a deletion of the gene encoding B2M, a component of HLA class I molecules) as non-self and did not activate (proliferate). The genetically modified cell group also had a fluorescence intensity comparable to that of T cells on day 0 of culture. This indicates that the T cells did not recognize the genetically modified cells (in which the expression of HLA class I molecules is suppressed by "knocking in SCD into the region containing the α-chain genes of HLA-A" and "knocking in SCT into the region containing the α-chain genes of HLA-C and HLA-B") as non-self and did not activate (proliferate). The results of this example support the idea that the HLA gene-modified cells of the present invention have the property of avoiding attack by T cells.

[0121] Example 3-4: T cell cytotoxicity against genetically modified cells In this example, the cytotoxicity of T cells against genetically modified cells was evaluated using an impedance assay. The impedance assay utilizes the property of live cells to adhere to, spread, and proliferate on an electrode. When live cells on the electrode proliferate and the cell number increases, it becomes difficult for current to flow across the electrode, and the electrical resistance value (impedance) of the electrode increases. On the other hand, when live cells are killed by T cell injury and the number of live cells present on the electrode decreases, the impedance decreases. This assay indirectly evaluates the cytotoxicity of T cells against the cells being evaluated, using the change in impedance as an indicator. Primary T cells (LONZA, catalog number 2W-300) were cultured at 6 x 10 cells in wells coated with 5 μg / mL anti-CD3 antibody (Invitrogen, catalog number 16-0037-85), a T cell activator. 4Activated T cells (effector cells) were produced by seeding the cells at 12,500 cells / well and culturing them for 7 days with Optimizer medium (ThermoFisher) containing 200 U / mL IL-2 (PeproTech, catalog number 200-02). Genetically modified cells, WT cells, and B2MKO cells were used as target cells, as in Example 3-1. The day before co-culture, target cells (12,500 cells / well) were seeded onto wells equipped with electrodes, and the activated T cells were added the following day and co-cultured for 3 days. Impedance values ​​were measured over time using a real-time cell analyzer (xCELLigence). Cell Index (an index of cell number) was calculated from the measured values. The assay was performed with the effector (E) cell to target (T) cell ratio (E / T ratio) set at 10 and 5. The results are shown in Figure 4. At both E / T ratios, the cell index of WT cells (201B7) increased less over time than that of the genetically modified cells (AL-UDC v0.2) and B2MKO cells. This indicates that T cells recognized WT cells expressing HLA class I molecules (HLA-A, HLA-B, and HLA-C) as non-self and attacked (damaged) them, resulting in the death of some WT cells and the suppression of their proliferation. In contrast, the genetically modified cells and B2MKO cells in which expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) was suppressed showed attenuated damage from T cells and proliferated. The results of this example support the idea that the HLA gene-modified cells of the present invention have the property of avoiding attack by T cells.

[0122] Example 3-5: NK cell cytotoxicity against genetically modified cells In this example, the cytotoxicity of NK cells against genetically modified cells was evaluated using an impedance assay. Regarding the relationship between NK cells and HLA class I molecules on target cells, when HLA-G expressed on target cells binds to inhibitory receptors expressed on NK cells (e.g., KIR2DL4 and LILRB1), the cytotoxicity of NK cells is suppressed. Furthermore, when HLA-E expressed on target cells binds to inhibitory receptors expressed on NK cells (e.g., CD94 / NKG2A and CD94 / NKG2B), the cytotoxicity of NK cells is suppressed. Primary NK cells (LONZA, catalog number 2W-501) were cultured in flasks for 6 days using an Expansion Kit (R&D Systems, catalog number CDK015) and used as effector cells. As in Example 3-1, genetically modified cells, WT cells, and B2MKO cells were used as target cells. The day before co-culture, target cells (12,500 cells / well) were seeded onto wells equipped with electrodes. The following day, the NK cells were added and co-cultured for three days. Impedance values ​​were measured over time using a real-time cell analyzer (xCELLigence). Cytolysis (%) (an index of target cell lysis due to NK cell damage) was calculated from the measured values. The assay was performed with the effector (E) cell to target (T) cell ratio (E / T ratio) set to 1.25, 2.5, and 5. The results are shown in Figure 5. At all E / T ratios, cytolysis (%) of B2MKO cells was higher than that of WT cells (201B7). This indicates that NK cell-mediated cytolysis of B2MKO cells, in which expression of HLA-E and HLA-G is suppressed due to deletion of the B2M coding gene, was greater than that of WT cells. On the other hand, at E / T ratios of 2.5 and 5, the cytolysis rate (%) of the genetically modified cells (AL-UDC v0.2) was lower than that of B2MKO cells.This indicates that the expression of SCD (single-chain fusion peptide mimicking HLA-G) and SCT (single-chain fusion peptide mimicking HLA-E) suppressed cytotoxicity by NK cells. The results of this example support the idea that the HLA gene-modified cells of the present invention have the property of avoiding attack by NK cells.

[0123] Example 4: Preparation of HLA gene-modified cells in which the region containing the α-chain genes of HLA-F, HLA-G, and HLA-A has been replaced (knocked in) with an SCD coding sequence, the region containing the α-chain genes of HLA-C and HLA-B has been replaced (knocked in) with an SCT coding sequence, and the region containing the CIITA gene has been deleted (knocked out) The gene-modified cells prepared in Example 2 above were subjected to "4. Method for knocking out the CIITA gene" to prepare CIITA gene-deleted cells.

[0124] [Knockout of the CIITA gene] The "region containing the CIITA gene" in cells was knocked out according to the method described above in "4. Method for knocking out the CIITA gene." The "region containing the CIITA gene" was a chromosomal region (target region 2) corresponding to chr16: 10,873,026-10,925,966 of the hg38 genome sequence. In addition, the CRISPR / Cas system was used as a site-specific nuclease.

[0125] Step [1-1] A CRISPR / Cas9 system was used, using a guide RNA (gRNA) targeting the upstream region of the CIITA gene (chr16: 10,873,824-10,873,846 in the hg38 genome sequence), a gRNA targeting the downstream region of the CIITA gene (chr16: 10,925,149-10,925,171 in the hg38 genome sequence), and the Cas9 protein. The gRNA sequence was completely identical to the target sequence. An expression vector was used to introduce the gRNA. Lipofection was used to introduce the gRNA and Cas9 into cells.

[0126] Step [1-2] The upstream homology arm sequence was designed to target the upstream sequence (chr16: 10,873,026-10,873,829 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The downstream homology arm sequence was designed to target the downstream sequence (chr16: 10,925,155-10,925,966 of the hg38 genome sequence) within the target region present in one of the two chromosomes (the chromosome to be subjected to homologous recombination). The following combinations of selectable marker genes contained in the two types of selectable marker donor DNA were used. The donor DNA for the selection marker was a plasmid. The donor DNA for the selection marker was introduced into the cells using the lipofection method. In this example, steps [1-1] and [1-2] were carried out simultaneously.

[0127] [Step 2-1] Two types of gRNA were used, each targeting one of the two types of positive selection marker genes used in step [1-2]. The gRNA sequence was completely identical to the target sequence. An expression vector encoding the gRNA was used to introduce the gRNA. The lipofection method was used to introduce the gRNA and Cas9 into the cells.

[0128] [Step 2-2] The "upstream homology arm sequence" and "downstream homology arm sequence" of the DNA for deleting the selection marker were the same as the "upstream homology arm sequence" and "downstream homology arm sequence" of the donor DNA for the selection marker used in step [1-2]. A plasmid carrying the DNA for deleting the selection marker was used to introduce the DNA for deleting the selection marker. The lipofection method was used to introduce the DNA for deleting the selection marker into the cells. In this example, steps [2-1] and [2-2] were carried out simultaneously.

[0129] The deletion of the CIITA gene in the genetically modified cells obtained in this example was confirmed by the absence of a band indicating amplification of the target region by PCR. In the genetically modified cells obtained in this example, the expression of HLA class II molecules is suppressed due to the deletion of the CIITA gene in addition to the deletion of the HLA class I gene and the introduction of SCD and SCT in the genetically modified cells prepared in Example 2. The suppression of HLA class II molecule expression is confirmed by culturing cells differentiated from the genetically modified cells obtained in this example (e.g., cells differentiated into mesenchymal stem cells, or cells differentiated into antigen-presenting cells such as B cells, monocytes, macrophages, dendritic cells, and Langerhans cells) for a period sufficient for the differentiated cells to mature, and then evaluating mRNA expression by qPCR or HLA class II protein expression by flow cytometry. To evaluate HLA class II protein expression by flow cytometry, an antibody that recognizes HLA class II (e.g., an antibody against HLA-DR / DQ / DP) is used. The expression of HLA class II molecules can also be evaluated under the conditions of whether or not induction by IFNγ is present.

[0130] The present invention can be used in the field of cell-based regenerative medicine.

Claims

1. An HLA gene modified cell, in which the following polynucleotides (1) and (2) have been knocked into a region containing the α-chain genes of HLA-A, HLA-B, HLA-C, HLA-F and HLA-G: (1) a polynucleotide encoding a single-chain dimeric fusion peptide (Single Chain Dimer: SCD) containing β2 microglobulin and the α-chain of HLA-G; (2) a polynucleotide encoding a single-chain trimer fusion peptide (Single Chain Trimer: SCT) containing the signal peptide of HLA-A, HLA-B, HLA-C or HLA-G, β2 microglobulin and the α-chain of HLA-E.

2. The cell according to claim 1, in which expression of the α-chain genes of HLA-A, HLA-B, HLA-C, HLA-F and HLA-G is reduced compared to wild-type cells.

3. The cell according to claim 1, in which the α-chain genes of HLA-A, HLA-B, HLA-C, HLA-F and HLA-G have been deleted from the cell before genetic modification.

4. The cell according to claim 1, in which (1) a polynucleotide encoding SCD has been knocked into a region containing the α-chain genes of HLA-F, HLA-G, and HLA-A, and (2) a polynucleotide encoding SCT has been knocked into a region containing the α-chain genes of HLA-C and HLA-B.

5. The cell according to claim 1, in which a polynucleotide encoding (2) SCT has been knocked into a region containing the α-chain genes of HLA-F, HLA-G, and HLA-A, and a polynucleotide encoding (1) SCD has been knocked into a region containing the α-chain genes of HLA-C and HLA-B.

6. An HLA gene modified cell, in which the following polynucleotide (1) has been knocked into a region containing the α-chain genes of HLA-F, HLA-G and HLA-A: (1) a polynucleotide encoding a single-chain dimer fusion peptide (Single Chain Dimer: SCD) containing β2-microglobulin and the α-chain of HLA-G.

7. The cell according to claim 6, in which expression of the α-chain genes of HLA-F, HLA-G and HLA-A is reduced compared to that of a wild-type cell.

8. The cell according to claim 6, wherein the HLA-F, HLA-G and HLA-A α-chain genes of the cell before genetic modification have been deleted.

9. An HLA gene modified cell, in which the following polynucleotide (2) has been knocked into a region containing the α-chain genes of HLA-C and HLA-B: (2) A polynucleotide encoding a single chain trimer (SCT) comprising a signal peptide of HLA-A, HLA-B, HLA-C or HLA-G, β2 microglobulin, and the α-chain of HLA-E.

10. The cell according to claim 9, in which expression of HLA-C and HLA-B α-chain genes is reduced compared to wild-type cells.

11. The cell according to claim 9, wherein the HLA-C and HLA-B α-chain genes of the cell prior to genetic modification have been deleted.

12. The cell according to claim 1 or 6, wherein the region containing the α-chain genes of HLA-F, HLA-G and HLA-A is a chromosomal region corresponding to the chromosomal region of chr6: 29,704,906-29,958,642 of the hg38 genome sequence.

13. The cell according to claim 1 or 9, wherein the region containing the α-chain genes of HLA-C and HLA-B is a chromosomal region corresponding to the chromosomal region of chr6: 31,224,837-31,378,196 of the hg38 genome sequence.

14. The cell according to claim 1 or 9, wherein the region containing the α-chain genes of HLA-C and HLA-B is a chromosomal region corresponding to the chromosomal region of chr6: 31,227,286-31,378,780 of the hg38 genome sequence.

15. A cell described in claim 1, 6 or 9, in which (1) a polynucleotide encoding SCD and / or (2) a polynucleotide encoding SCT is knocked in to only one allele of the target cell.

16. The cell of claim 1, 6 or 9, further comprising a modification that reduces expression of an HLA class II molecule gene compared to a wild-type cell.

17. The cell of claim 1, 6 or 9, further comprising a modification that deletes an HLA class II molecule gene.

18. The cell of claim 16, wherein the HLA class II molecule gene is the CIITA gene.

19. The cell of claim 17, wherein the HLA class II molecule gene is the CIITA gene.

20. The cell of claim 1, 6 or 9, which is a human pluripotent stem cell.

21. The cell of claim 1, 6 or 9, which is a human iPS cell or ES cell.

22. The cell described in claim 1, 6 or 9, which is a human iPS cell strain 253G4 or a human iPS cell strain 201B7.

23. The cell according to claim 1, 6 or 9, which is a cell differentiated from a human pluripotent stem cell.

24. The cell described in claim 23, wherein the human pluripotent stem cell is a human iPS cell or an ES cell.

25. The cell described in claim 23, wherein the human pluripotent stem cell is a human iPS cell 253G4 strain, a human iPS cell, or an ES cell.

26. The cell according to claim 23, wherein the cell differentiated from a human pluripotent stem cell is an immune cell, a tissue stem cell or a somatic cell.

27. The cell of claim 26, wherein the immune cell is a T cell or a NK cell.

28. The cell of claim 1 or 6, wherein the SCD comprises an amino acid sequence set forth in SEQ ID NO:1 or 4, or an amino acid sequence having 80% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or 4.

29. The cell of claim 1 or 9, wherein the SCT comprises an amino acid sequence set forth in SEQ ID NO:6 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

6.

30. A cell described in claim 1 or 6, wherein the polynucleotide of (1) comprises a nucleotide sequence shown in SEQ ID NO: 2 or 5, or a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2 or 5.

31. The cell described in claim 1 or 9, wherein the polynucleotide of (2) comprises a nucleotide sequence set forth in SEQ ID NO:7 or a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO:

7.

32. The cell of claim 1 or 6, wherein the SCD further comprises a linker sequence between β2 microglobulin and the α chain of HLA-G.

33. The cell of claim 1 or 9, wherein the SCT further comprises a linker sequence between the signal peptide and β2 microglobulin.

34. The cell of claim 1 or 9, wherein the SCT further comprises a linker sequence between the β2 microglobulin and the α chain of HLA-E.

35. The cell of claim 32, wherein the linker sequence comprises a peptide consisting of glycine and serine.

36. The cell of claim 33, wherein the linker sequence comprises a peptide consisting of glycine and serine.

37. The cell of claim 34, wherein the linker sequence comprises a peptide consisting of glycine and serine.

38. The cell of claim 32, wherein the linker sequence comprises the amino acid sequence shown in SEQ ID NO:

3.

39. The cell of claim 33, wherein the linker sequence comprises the amino acid sequence shown in SEQ ID NO:

8.

40. The cell of claim 34, wherein the linker sequence comprises the amino acid sequence set forth in SEQ ID NO:

9.

41. The cell described in claim 1 or 6, in which a promoter sequence of the HLA-A α chain gene is knocked in together with the polynucleotide of (1), and the promoter sequence is operably linked to a sequence encoding SCD.

42. The cell described in claim 41, wherein the promoter sequence of the HLA-A α chain gene comprises the nucleotide sequence shown in SEQ ID NO:10 or a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:

10.

43. The cell described in claim 1 or 9, in which a promoter sequence of the HLA-B α chain gene is knocked in together with the polynucleotide of (2), and the promoter sequence is operably linked to a sequence encoding SCT.

44. The cell of claim 43, wherein the promoter sequence of the HLA-B α-chain gene comprises the nucleotide sequence shown in SEQ ID NO:11 or a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:

11.

45. The cell of claim 1, 6 or 9, which is induced by a T cell and / or NK cell-mediated rejection response attenuated compared to wild-type cells.

46. ​​T cells are CD8 + T cells and / or CD4 + 46. ​​The cell of claim 45, which is a T cell.

47. A cell derived from the HLA gene modified cell of claim 1, 6 or 9.

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