Method for producing modified human induced pluripotent stem cells

Abstract

A method for producing modified human induced pluripotent stem cells (iPSCs) in which two or more target genes have been modified, said method comprising a transduction step for introducing CRISPR / Cas9 protein and multiple types of gRNAs for the aforesaid two or more target genes into human iPSCs.

Description

Technical Field

[0001] The present invention relates to a method for producing modified human induced pluripotent stem cells (iPSCs). More specifically, the present invention relates to a method for producing modified human iPSCs in which two or more target genes are modified, modified human iPSCs, differentiated cells derived from the modified human iPSCs, and a kit for modifying two or more target genes of human iPSCs.

Background Art

[0002] In the case of allogeneic transplantation in which donor cells are transplanted into a recipient (patient) who is another person, the transplanted cells are rejected due to an immune reaction.

[0003] The cell surface protein called the Major histocompatibility complex (MHC) or Human Leukocyte Antigen (HLA) plays the most important role in distinguishing self and non-self of cells.

[0004] HLAs are classified into class I and class II. Class I HLA proteins are expressed in most types of cells in the body. Class I HLA proteins form a heterodimer with β2-Microglobulin (B2M) and are expressed on the cell surface, and are responsible for the function of presenting peptides to CD8-positive cytotoxic T cells and inducing activation. The antigen peptides presented are endogenous, and many have a length of 8 to 10 amino acids.

[0005] Classified into HLA class I are mainly six genes: HLA-A gene, HLA-B gene, HLA-C gene, HLA-E gene, HLA-F gene, and HLA-G gene. In addition, a number of pseudogenes (such as HLA-H, HLA-J, HLA-K, HLA-L, HLA-P, HLA-T, HLA-U, HLA-V, HLA-W, HLA-X, HLA-Y, etc.) are also known. Among these, the HLA-A gene, HLA-B gene, and HLA-C gene have particularly high sequence diversity among individuals and play a major role in distinguishing self from non-self in transplantation immunity.

[0006] Class II HLA proteins are mainly expressed in immune cells such as macrophages, dendritic cells, activated T cells, and B cells. Class II HLA proteins are responsible for the function of forming a heterodimer of an α-chain and a β-chain and presenting peptides to CD4 helper T cells to induce activation. The antigenic peptides presented are exogenous, and many of them have a length of 15 - 24 amino acids.

[0007] Classified into HLA class II are HLA-DR (α-chain: HLA-DRA, β-chain: HLA-DRB), HLA-DQ (α-chain: HLA-DQA1, β-chain: HLA-DQB1), and HLA-DP (α-chain: HLA-DPA1 or HLA-DPA2, β-chain: HLA-DPB1 or HLA-DPB2). In addition, the existence of a number of pseudogenes (such as HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB) is also known.

[0008] Due to its sequence diversity, the HLA gene is involved in the recognition of self and non-self at the cellular level. In allogeneic transplantation, HLA matching is also very important for reducing immune rejection. For example, in hematopoietic stem cell transplantation, it is recommended to find a donor with antigenicity that matches as much as possible in both alleles of HLA-A, HLA-B, HLA-C, and HLA-DR, that is, a total of 8 loci of alleles, and perform transplantation.

[0009] Also, looking at the results of engraftment rates such as kidney transplantation, it has been reported that the higher the degree of HLA antigenicity match, the significantly higher the engraftment efficiency.

[0010] By the way, pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) have pluripotency to differentiate into various cell types. Therefore, cell therapy and regenerative medicine, which involve differentiating pluripotent stem cells into various cells and then transplanting them into patients, have attracted attention.

[0011] Actually, examples have been reported such as differentiating ESCs into nerve cells and transplanting them into patients with spinal cord injuries, and differentiating iPSCs into retinal pigment epithelial cells and transplanting them into patients with age-related macular degeneration. There are also reports that it is important to match the HLA type of the transplanted cells and the patient when transplanting such pluripotent stem cell-derived cells.

[0012] However, since pluripotent stem cells require a great deal of cost and time to establish, there is a problem that it is difficult to prepare cells with a matching HLA type for each patient. As one means of solving this problem, for example, Patent Document 1 describes cells in which the B2M gene required for cell surface presentation of class I HLA proteins has been deleted.

Prior Art Documents

Patent Documents

[0013]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0014]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0015] Since the cells described in Patent Document 1 lack the B2M gene, class I HLA proteins are not presented on the cell surface. Therefore, it is considered that the immune reaction in the case of allogeneic transplantation is suppressed. However, if HLA proteins are not presented on the cell surface, the antigen-presenting ability of the cells is lost. When the cells lose their antigen-presenting ability, they cannot present antigens derived from viruses or tumors when infected with viruses or when tumorigenic, and as a result, there is a risk of assisting the growth of viruses and tumors. In addition, cells without HLA proteins presented on the cell surface are attacked by NK cells that recognize "missing self".

[0016] Therefore, it is conceivable to selectively disrupt only arbitrary HLA genes using genome editing technology. However, there are multiple HLA genes, and including many pseudogenes, the sequence homology is high among the HLA genes with each other. Thus, when there are multiple target genes, performing genome editing of each gene individually increases the time and labor required to produce the desired cells.

[0017] An object of the present invention is to provide a technique for producing modified human iPSCs in which two or more target genes are modified.

Means for Solving the Problems

[0018] The present invention includes the following aspects. [1] A method for producing modified induced pluripotent stem cells (iPSCs) in which two or more target genes are modified, the method comprising an introduction step of introducing CRISPR / Cas9 protein and multiple types of guide RNAs (gRNAs) for two or more target genes into human iPSCs. [2] The production method according to [1], wherein the human iPSCs are blood cell-derived iPSCs. [3] The production method according to [1] or [2], wherein the human iPSCs are homozygous for the HLA-A gene or the HLA-B gene. [4] The production method according to [3], wherein the human iPSCs include the A24:02 haplotype of the HLA-A gene and the B52:01 haplotype of the HLA-B gene. [5] The production method according to [3] or [4], wherein the human iPSCs are homozygous for the HLA-C gene. [6] The production method according to any one of [3] to [5], wherein the human iPSCs are homozygous for the HLA-DRB1 gene. [7] The production method according to any one of [1] to [6], wherein the introduction step is performed by electroporation. [8] The production method according to any one of [1] to [7], wherein the two or more target genes are genes selected from the group consisting of the HLA-A gene, the HLA-B gene, the HLA-C gene, the HLA-DR gene, and the Class II Major Histocompatibility Complex Transactivator (CIITA) gene. [9] The production method according to any one of [1] to [8], wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence in which one or several nucleotides are deleted, substituted, or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 2, or a nucleotide sequence in which one or several nucleotides are deleted, substituted, or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 2.

[10] The production method according to any one of [1] to [9], wherein in the introduction step, the CRISPR / Cas9 protein and the gRNA are introduced in the form of a complex.

[11] In the introduction step, the CRISPR / Cas9 protein and multiple types of the gRNA are introduced at a mass ratio of the mass of the CRISPR / Cas9 protein: the total mass of the multiple types of the gRNA = 5:1 to 1:10, the production method according to any one of [1] to

[10] .

[12] In the introduction step, the CRISPR / Cas9 protein is introduced at 1 to 50 μg per 1×10 5 cells to 3×10 6 cells of the human iPSC, the production method according to

[11] .

[13] The modification of the target gene is the disruption of the target gene, the production method according to any one of [1] to

[12] .

[14] A modified human iPSC or a differentiated cell derived from the modified human iPSC, having a disrupted HLA-A gene, a disrupted HLA-B gene, and a disrupted CIITA gene, wherein the disrupted HLA-A gene or the disrupted HLA-B gene is homozygous, the haplotype of the disrupted HLA-A gene includes A24:02, and the haplotype of the disrupted HLA-B gene includes B52:01.

[15] A modified human iPSC or a differentiated cell derived from the modified human iPSC, having a disrupted HLA-A gene, a disrupted HLA-B gene, and a disrupted CIITA gene, and the haplotype of the HLA-C gene includes C12:02.

[16] A kit for introducing into a human iPSC a CRISPR / Cas9 protein and multiple types of gRNAs for two or more target genes to modify the two or more target genes of the human iPSC.

[17] The kit according to

[16] , for performing the introduction by electroporation.

[18] The kit according to

[16] or

[17] , wherein the two or more target genes are genes selected from the group consisting of HLA-A gene, HLA-B gene, HLA-C gene, HLA-DR gene, and CIITA gene.

[19] The kit according to any one of

[16] to

[18] , wherein the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 1, and the gRNA consists of the nucleotide sequence set forth in SEQ ID NO: 2, or a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 2.

[20] The kit according to any one of

[16] to

[19] , wherein the CRISPR / Cas9 protein and the plurality of types of the gRNA are used to be introduced into the human iPSC at a mass ratio of the mass of the CRISPR / Cas9 protein: the total mass of the plurality of types of the gRNA = 5:1 to 1:10.

[21] The CRISPR / Cas9 protein is used to be introduced at 1 to 50 μg per 1 × 10 5 cells to 3 × 10 6 cells of the human iPSC, and the kit is the one described in

[20] .

Advantages of the Invention

[0019] According to the present invention, it is possible to provide a technique for producing modified human iPSCs in which two or more target genes are modified.

Brief Description of the Drawings

[0020]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Modes for Carrying Out the Invention

[0021] [Method for Producing Modified Human iPSCs] In one embodiment, the present invention provides a method for producing modified human iPSCs in which two or more target genes are modified, the method including an introduction step of introducing CRISPR / Cas9 protein and a plurality of types of gRNAs for two or more target genes into human iPSCs.

[0022] In the production method of the present embodiment, the modification of the target gene may be the introduction of a mutation into the target gene or the disruption of the target gene, but it is preferably the disruption of the target gene. Also, the two or more target genes can be appropriately selected according to the purpose, and for example, they may be genes selected from the group consisting of HLA-A gene, HLA-B gene, HLA-C gene, HLA-DR gene, and Class II Major Histocompatibility Complex Transactivator (CIITA) gene.

[0023] The CIITA gene encodes a transcription factor that controls the transcriptional activation of HLA class II. Therefore, cells in which the CIITA gene is disrupted do not express HLA class II protein, and the rejection reaction when transplanted to a recipient is reduced.

[0024] The NCBI accession numbers of the human CIITA gene are NM_000246.3, NM_001286402.1, NM_001286403.1, etc.

[0025] In the production method of the present embodiment, the human iPSCs to be modified are preferably iPSCs derived from blood cells. Since iPSCs derived from blood cells can be obtained from cells in blood that can be collected with low invasiveness, they can be prepared relatively easily.

[0026] In the production method of the present embodiment, the human iPSC to be genetically modified preferably has a homozygous HLA-A gene or HLA-B gene. When both the HLA-A gene and the HLA-B gene are heterozygous, in order to disrupt these genes, it is necessary to modify a total of four genes, namely two types of HLA-A genes and two types of HLA-B genes, which requires a great deal of labor and production time. In contrast, by using human iPSCs with a homozygous HLA-A gene or HLA-B gene, the labor and production time required for gene modification can be significantly reduced. In particular, it is preferable that both the HLA-A gene and the HLA-B gene are homozygous because it can reduce the labor and production time required for gene modification.

[0027] The human iPSC with a homozygous HLA-A gene or HLA-B gene preferably has a haplotype of the HLA-A gene including A24:02 and a haplotype of the HLA-B gene including B52:01.

[0028] The above human iPSC preferably has a homozygous HLA-A gene and a haplotype of the HLA-A gene being A24:02. Also, it preferably has a homozygous HLA-B gene and a haplotype of the HLA-B gene being B52:01.

[0029] The above human iPSC is particularly preferably homozygous for both the HLA-A gene and the HLA-B gene, with the haplotype of the HLA-A gene being A24:02 and the haplotype of the HLA-B gene being B52:01.

[0030] Also, the above human iPSC preferably has a homozygous HLA-C gene. The remaining HLA-C gene can avoid the attack by NK cells. Also, having a homozygous HLA-C gene makes it easier to match the HLA type with the patient. When the HLA-C gene is heterozygous, it is preferable to disrupt one of the HLA-C genes.

[0031] Table 1 below shows the haplotypes in which the HLA-A gene, HLA-B gene, and HLA-C gene are all homozygous, in descending order of frequency in Japanese people. In Table 1, the frequencies of the HLA-C gene are shown in descending order.

[0032] [Table 1]

[0033] From the perspective of making it easier to match the HLA type with a Japanese patient, the human iPSC in which the HLA-A gene, HLA-B gene, and HLA-C gene are homozygous preferably has any one of the following haplotypes (i) to (iv), and particularly preferably has the haplotype of (i). (i) The haplotype of the HLA-A gene is A24:02, the haplotype of the HLA-B gene is B52:01, and the haplotype of the HLA-C gene is C12:02 (ii) The haplotype of the HLA-A gene is A24:02, the haplotype of the HLA-B gene is B54:01, and the haplotype of the HLA-C gene is C01:02 (iii) The haplotype of the HLA-A gene is A24:02, the haplotype of the HLA-B gene is B07:02, and the haplotype of the HLA-C gene is C07:02 (iv) The haplotype of the HLA-A gene is A33:03, the haplotype of the HLA-B gene is B44:03, and the haplotype of the HLA-C gene is C14:03

[0034] In the production method of the present embodiment, the human iPSC to be genetically modified preferably has a homozygous HLA-DRB1 gene. By having a homozygous HLA-DRB1 gene, it becomes easier to match the HLA type with the patient.

[0035] In the production method of the present embodiment, it is preferable to perform the introduction step of introducing CRISPR / Cas9 protein and gRNA into human iPSCs by the electroporation method. As will be described later in the examples, by using the electroporation method, modified human iPSCs in which two or more target genes have been modified can be produced with high efficiency.

[0036] Examples of the CRISPR / Cas9 protein include those derived from Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, Geobacillus stearothermophilus, etc. As the CRISPR / Cas9 protein, commercially available products such as the product name "Recombinant Cas9 Protein GMP grade" (Takara Bio Inc.) may be used.

[0037] Also, it is preferable that the gRNA is a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2. In this case, the human iPSCs to be the target for gene modification are homozygous for the HLA-A gene and the HLA-B gene, the haplotype of the HLA-A gene is A24:02, and the haplotype of the HLA-B gene is B52:01. Further, instead of the gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1, a gRNA consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 1 can also be used. Similarly, instead of the gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2, a gRNA consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 2 can also be used. Here, "one or several" may be 1 to 5, may be 1 to 4, may be 1 to 3, or may be 1 or 2.

[0038] The gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 is a common single-guide RNA (sgRNA) for both the HLA-A gene and the HLA-B gene. The sgRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 can modify both the HLA-A gene with a haplotype of A24:02 and the HLA-B gene with a haplotype of B52:01. Further, the gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2 is an sgRNA for the CIITA gene.

[0039] In the production method of the present embodiment, in the introduction step of introducing the CRISPR / Cas9 protein and the gRNA into human iPSCs, the CRISPR / Cas9 protein, the gRNA, and the human iPSCs may be mixed in any order.

[0040] The gRNA may be mixed after mixing the CRISPR / Cas9 protein and the human iPSCs, the CRISPR / Cas9 protein may be mixed after mixing the gRNA and the human iPSCs, or the human iPSCs may be mixed after mixing the CRISPR / Cas9 protein and the gRNA. Among these, it is preferable to mix this mixture and the human iPSCs after mixing the CRISPR / Cas9 protein and the gRNA. Thereby, it is considered that the complex Cas9 protein-gRNA complex (RNP complex) of the CRISPR / Cas9 protein and the gRNA is formed and introduced into human iPSCs in the form of the complex. As will be described later in the examples, modified human iPSCs can be produced with high efficiency by introducing the CRISPR / Cas9 protein and the gRNA in the form of a complex.

[0041] In the introduction step of introducing the CRISPR / Cas9 protein and the gRNA into human iPSCs, it is preferable to introduce the CRISPR / Cas9 protein and a plurality of types of gRNAs at a mass ratio of CRISPR / Cas9 protein mass: total mass of a plurality of types of gRNAs = 5:1 to 1:10.

[0042] The mass ratio of the CRISPR / Cas9 protein to the total mass of multiple types of gRNAs (mass of CRISPR / Cas9 protein: total mass of multiple types of gRNAs) is preferably from 3:1 to 1:10, more preferably from 3:1 to 1:5, and particularly preferably from 2:1 to 1:5. Also, each of the multiple types of gRNAs is preferably introduced in equal mass amounts.

[0043] As will be described later in the examples, when the mass ratio of the CRISPR / Cas9 protein to the gRNA is within the above range, modified human iPSCs can be produced with high efficiency.

[0044] Also, in the introduction step of introducing the CRISPR / Cas9 protein and the gRNA into human iPSCs, the introduction amount of the CRISPR / Cas9 protein is preferably 1 to 50 μg per 1×10 5 to 3×10 6 human iPSCs.

[0045] For example, the amount of the CRISPR / Cas9 protein introduced into 3×10 5 human iPSCs may be 1 μg or more, 2 μg or more, 3 μg or more, or 4 μg or more. Also, the amount of the CRISPR / Cas9 protein introduced into 3×10 5 human iPSCs is preferably 10 μg or less, more preferably 9 μg or less, still more preferably 7 μg or less, and particularly preferably 5 μg or less. The above upper and lower limits can be arbitrarily combined. Alternatively, the amount of the CRISPR / Cas9 protein introduced into 1.5×10 6 human iPSCs may be 5 μg or more, 10 μg or more, 15 μg or more, or 20 μg or more. Also, the amount of the CRISPR / Cas9 protein introduced into 1.5×10 6The amount of CRISPR / Cas9 protein introduced into individual human iPSCs is preferably 50 μg or less, more preferably 40 μg or less, still more preferably 30 μg or less, and particularly preferably 25 μg or less. The above upper and lower limits can be arbitrarily combined. As will be described later in the examples, when the CRISPR / Cas9 protein is present in excess relative to the gRNA, the knockout efficiency tends to decrease.

[0046] In the production method of the present embodiment, the two or more target genes are not limited to genes selected from the group consisting of HLA-A gene, HLA-B gene, HLA-C gene, HLA-DR gene, and CIITA gene. According to the production method of the present embodiment, even when modifying any two or more target genes other than these genes, modified human iPSCs can be produced with high efficiency.

[0047] [Modified human iPSCs and differentiated cells derived from the modified human iPSCs] In one embodiment, the present invention provides a modified human iPSC or a differentiated cell derived from the modified human iPSC, which has a disrupted HLA-A gene, a disrupted HLA-B gene, and a disrupted CIITA gene, wherein the disrupted HLA-A gene or the disrupted HLA-B gene is homozygous, the haplotype of the disrupted HLA-A gene includes A24:02, and the haplotype of the disrupted HLA-B gene includes B52:01.

[0048] It is preferable that the above human iPSC or differentiated cell is homozygous for the HLA-A gene and the haplotype of the HLA-A gene is A24:02. Also, it is preferable that the HLA-B gene is homozygous and the haplotype of the HLA-B gene is B52:01.

[0049] It is particularly preferable that the above human iPSC or differentiated cell is homozygous for both the HLA-A gene and the HLA-B gene, the haplotype of the HLA-A gene is A24:02, and the haplotype of the HLA-B gene is B52:01.

[0050] In another embodiment, the present invention provides a modified human iPSC or a differentiated cell derived from the modified human iPSC, which has a disrupted HLA-A gene, a disrupted HLA-B gene, and a disrupted CIITA gene, and the haplotype of the HLA-C gene includes C12:02.

[0051] The modified human iPSC and the differentiated cell derived from the iPSC of the present embodiment have a reduced rejection reaction when allografted to a recipient. Also, the differentiated cell derived from the modified human iPSC of the present embodiment also has a reduced rejection reaction when allografted to a recipient. Therefore, these cells can be suitably used for cell therapy and regenerative medicine, in which pluripotent stem cells are differentiated into various cells and then transplanted into a patient.

[0052] In addition, humans in whom both the HLA-A gene and the HLA-B gene are homozygous, the haplotype of the HLA-A gene is A24:02, and the haplotype of the HLA-B gene is B52:01 are frequent in Japanese people. Therefore, when producing the modified human iPSC of the present embodiment, it is relatively easy to obtain a human iPSC to be modified. Also, as described above, when the HLA-A gene or the HLA-B gene is homozygous, or both the HLA-A gene and the HLA-B gene are homozygous, the labor and production time required for gene modification can be significantly reduced.

[0053] Also, as described above, the human iPSC to be modified is preferably an iPSC derived from blood cells. Since iPSCs derived from blood cells can be obtained from cells in blood that can be collected with low invasiveness, they can be prepared relatively easily.

[0054] In the modified human iPSCs or differentiated cells of the present embodiment, "the haplotype of the disrupted HLA-A gene includes A24:02, and the haplotype of the disrupted HLA-B gene includes B52:01" means that although the HLA-A gene and the HLA-B gene are disrupted in a state where they cannot be expressed, the haplotype of the HLA-A gene includes A24:02, and the haplotype of the HLA-B gene includes B52:01.

[0055] It is preferable that the HLA-C gene remains in the modified human iPSCs or differentiated cells of the present embodiment. As described above, by the remaining HLA-C gene, the attack by NK cells can be avoided. The haplotype of the HLA-C gene preferably includes C12:02. Also, it is preferable that the HLA-C gene is homozygous. By the HLA-C gene being homozygous, it becomes easier to match the HLA type with the patient. When the HLA-C gene is heterozygous, it is preferable to disrupt either one of the HLA-C genes.

[0056] [Kit] In one embodiment, the present invention provides a kit for introducing into human iPSCs a CRISPR / Cas9 protein and a plurality of types of gRNAs for two or more target genes to modify the two or more target genes of the human iPSCs. With the kit of the present embodiment, the method for producing the modified human iPSCs described above can be preferably implemented.

[0057] In the kit of the present embodiment, the CRISPR / Cas9 protein is the same as that described above.

[0058] In the kit of the present embodiment, the two or more target genes can be appropriately selected according to the purpose. For example, they may be genes selected from the group consisting of the HLA-A gene, the HLA-B gene, the HLA-C gene, the HLA-DR gene, and the CIITA gene. In this case, human iPSCs with reduced rejection reaction when allografted to a recipient can be produced.

[0059] In the kit of this embodiment, the gRNA may be a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2. In this case, the human iPSC to be modified for the gene has homozygous HLA-A gene and HLA-B gene, the haplotype of the HLA-A gene is A24:02, and the haplotype of the HLA-B gene is B52:01. Further, instead of the gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1, a gRNA consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 1 can also be used. Similarly, instead of the gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2, a gRNA consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 2 can also be used. Here, "one or several" may be 1 to 5, may be 1 to 4, may be 1 to 3, or may be 1 or 2.

[0060] As described above, the gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 is a common sgRNA for both the HLA-A gene and the HLA-B gene. The sgRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 can modify both the HLA-A gene with a haplotype of A24:02 and the HLA-B gene with a haplotype of B52:01. Also, the gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2 is an sgRNA for the CIITA gene.

[0061] The kit of this embodiment is preferably for introducing the CRISPR / Cas9 protein and a plurality of types of gRNAs into human iPSCs by the electroporation method.

[0062] Also, the kit of this embodiment is preferably used to introduce the CRISPR / Cas9 protein and the plurality of types of the gRNAs into human iPSCs at a mass ratio of CRISPR / Cas9 protein mass: total mass of the plurality of types of gRNAs = 5:1 to 1:10.

[0063] The mass ratio of the CRISPR / Cas9 protein to the total mass of multiple types of gRNAs (mass of CRISPR / Cas9 protein: total mass of multiple types of gRNAs) is preferably 3:1 to 1:10, more preferably 3:1 to 1:5, and particularly preferably 2:1 to 1:5. Further, each of the multiple types of gRNAs is preferably introduced in the same mass.

[0064] As described later in the examples, when the mass ratio of the CRISPR / Cas9 protein to the gRNA is within the above range, modified human iPSCs can be produced with high efficiency.

[0065] In addition, the kit of this embodiment is preferably used such that there is 1 to 50 μg of CRISPR / Cas9 protein per 1×10 5 to 3×10 6 human iPSCs.

[0066] For example, the amount of CRISPR / Cas9 protein introduced into 3×10 5 human iPSCs may be 1 μg or more, 2 μg or more, 3 μg or more, or 4 μg or more. Also, the amount of CRISPR / Cas9 protein introduced into 3×10 5 human iPSCs is preferably 10 μg or less, more preferably 9 μg or less, still more preferably 7 μg or less, and particularly preferably 5 μg or less. The above upper and lower limits can be arbitrarily combined. Alternatively, the amount of CRISPR / Cas9 protein introduced into 1.5×10 6 human iPSCs may be 5 μg or more, 10 μg or more, 15 μg or more, or 20 μg or more. Also, 1.5×10 6The amount of CRISPR / Cas9 protein introduced into individual human iPSCs is preferably 50 μg or less, more preferably 40 μg or less, still more preferably 30 μg or less, and particularly preferably 25 μg or less. The above upper and lower limits can be arbitrarily combined. As will be described later in the examples, when the CRISPR / Cas9 protein is present in excess relative to the gRNA, the knockout efficiency tends to decrease.

[0067] In the kit of this embodiment, the two or more target genes are not limited to the genes selected from the group consisting of the HLA-A gene, HLA-B gene, HLA-C gene, HLA-DR gene, and CIITA gene. According to the kit of this embodiment, even when modifying any two or more target genes other than these genes, modified human iPSCs can be produced with high efficiency.

Examples

[0068] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples.

[0069] Using the CRISPR / Cas9 genome editing technology, modified human iPSCs with the HLA-A gene, HLA-B gene, and CIITA gene of human iPSCs deleted were produced. Here, the conditions for genome editing were examined.

[0070] As the human iPSCs, the Ff-I14s04 strain at passage 14 was used. The Ff-I14s04 strain is homozygous for the HLA-A gene, HLA-B gene, and HLA-C gene. Also, the haplotype of the HLA-A gene is A24:02, the haplotype of the HLA-B gene is B52:01, and the haplotype of the HLA-C gene is C12:02.

[0071] By the electroporation method, CRISPR / Cas9 protein and two types of gRNAs were introduced into the Ff-I14s04 strain. Electroporation was performed using a 4D-Nucleofector TMIt was carried out using [Roche Diagnostics Japan Ltd.].

[0072] As the gRNA, HLA-A24-ex2g1 (SEQ ID NO: 1), which is a common sgRNA for both the HLA-A gene and the HLA-B gene, and CIITA-ex3g5 (SEQ ID NO: 2), which is an sgRNA for the CIITA gene, were used.

[0073] In addition, as the containers used for electroporation, 20 μL strips and 100 μL cuvettes were used. Also, the mass ratio of the CRISPR / Cas9 protein to the gRNA was varied.

[0074] First, the Ff-I14s04 strain was detached from the culture container. Subsequently, each cell was centrifuged at room temperature at 120 × g for 5 minutes, and then the supernatant was removed.

[0075] Subsequently, when using the 20 μL strip, the cells were suspended in 20 μL of P3 Primary Cell Solution ([Roche Diagnostics Japan Ltd.]) and supplement ([Roche Diagnostics Japan Ltd.]). When using the 20 μL strip, 3 × 10 5 cells were used per reaction.

[0076] Also, when using the 100 μL cuvette, the cells were suspended in 100 μL of P3 Primary Cell Solution ([Roche Diagnostics Japan Ltd.]) and supplement ([Roche Diagnostics Japan Ltd.]). When using the 100 μL cuvette, 1.5 × 10 6 cells were used per reaction.

[0077] Subsequently, in the combinations of Test Groups I to VI shown in Table 2 below, the CRISPR / Cas9 protein and the gRNA were mixed respectively to form a Cas9 protein-gRNA complex (RNP complex), and then mixed with the cell suspension (n = 2). In Test Groups I to V, 20 μL strips were used, and in Test Group VI, 100 μL cuvettes were used.

[0078] [Table 2]

[0079] Subsequently, each 20 μL strip or 100 μL cuvette was set in a 4D-Nucleofector TM system (Lonza Group Ltd.), program CA-137 was selected, and electroporation was performed.

[0080] Subsequently, when using a 20 μL strip, approximately 80 μL of the medium supplemented with Y-27632 (CAS No.: 129830-38-2) from Ajinomoto Health Supply Co., Ltd. was taken from the well in the incubator and added to the cuvette, and the total amount of cells inside the cuvette was seeded into one well of a 6-well plate.

[0081] Also, when using a 100 μL cuvette, 20 μL was aspirated from the cuvette and seeded into one well of a 6-well plate (n = 4).

[0082] Subsequently, the day when the cells after electroporation were seeded was designated as day 0, and the first medium change was performed on day 2. Subsequently, for cell state stabilization, subculture was carried out once. Subculture was performed with an additional amount for one well for flow cytometry analysis. Subsequently, the cells were cryopreserved, and the cells in the well for analysis were analyzed by flow cytometry in a bulk state.

[0083] Specifically, 50 ng / mL of IFN-γ was added to the iPSC medium and treated for 48 hours to induce the expression of HLA protein. After that, staining was performed with an anti-HLA-A2 antibody (product number: 740082, BD), and the expression of HLA-A2 protein was examined by flow cytometry analysis.

[0084] Figures 1A to 1D are graphs showing the results of flow cytometry analysis of each cell electroporated with tubes 1 to 11 in Table 1 above. In Figures 1A to 1D, the vertical axis represents the number of cells, and the horizontal axis represents the staining intensity with anti-HLA-A2 antibody. Also, "negative control" shows the results of the Ff-I14s04 strain not subjected to genome editing, and "unstained" shows typical results of cells not stained with anti-HLA-A2 antibody.

[0085] In addition, Table 3 below shows the ratio of the mass of Cas9 protein to the total mass of gRNA and the average value of the knockout efficiency of HLA-A2 protein in each test group. Here, the negative rate of HLA-A2 protein was used as the knockout efficiency.

[0086]

Table 3

[0087] As a result, under conditions other than Test Group IV, very high knockout efficiencies of about 80% or more were obtained in all cases. In Test Group IV (tubes 7 and 8), the knockout efficiency was lower compared to other test groups. The reason for this was considered to be that there was more Cas9 protein than the added gRNA. From this, it was considered that the presence of a large amount of free Cas9 protein during RNP complex formation might reduce the knockout efficiency. From this result, it was considered that the balance of the mixing amount of gRNA and Cas9 protein is important for the knockout efficiency. Also, from the comparison between the case of using a 20 μL strip and the case of using a 100 μL cuvette, it became clear that the shape of the cuvette does not affect the knockout efficiency.

[0088] Also, when comparing the results of tubes 11-1 to 11-4 in Figures 1C to 1D, when an aliquot of the required amount (20 μL) was taken from the cuvette (100 μL) after electroporation, no variation in knockout efficiency was observed in the cell population for each aliquot taken.

[0089] From the above results, it became clear that in order to increase or decrease the knockout efficiency, it is effective to adjust the balance of the mixing amounts of gRNA and Cas9 protein. Also, it became clear that when there is excess Cas9 with respect to the optimized balance, the knockout efficiency decreases. Further, since the shape of the cuvette does not affect the knockout efficiency, it became clear that any of them can be used as appropriate. [Industrial Applicability]

[0090] According to the present invention, it is possible to provide a technique for producing modified human iPSCs in which two or more target genes are modified.

Claims

1. comprising an introduction step of introducing a CRISPR / Cas9 protein and two types of gRNAs for two or more target genes into human induced pluripotent stem cells (iPSCs), wherein the two or more target genes are HLA-A gene, HLA-B gene and CIITA gene, and the gRNAs are a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 1, and a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2, or a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 2 ; A method for producing modified human iPSCs in which the HLA-A gene, HLA-B gene and CIITA gene are disrupted.

2. The production method according to claim 1, wherein the human iPSCs are blood cell-derived iPSCs.

3. The production method according to claim 1 or 2, wherein the human iPSCs are homozygous for the HLA-A gene or the HLA-B gene.

4. The production method according to claim 3, wherein the haplotype of the HLA-A gene of the human iPSCs includes A24:02, and the haplotype of the HLA-B gene includes B52:

01.

5. The production method according to claim 3 or 4, wherein the human iPSCs are homozygous for the HLA-C gene.

6. The production method according to any one of claims 3 to 5, wherein the human iPSCs are homozygous for the HLA-DRB1 gene.

7. The production method according to any one of claims 1 to 6, wherein the introduction step is performed by electroporation.

8. The production method according to any one of claims 1 to 7, wherein in the introduction step, the CRISPR / Cas9 protein and the gRNAs are introduced in the form of a complex.

9. The production method according to any one of claims 1 to 8, wherein in the introduction step, the CRISPR / Cas9 protein and a plurality of types of the gRNAs are introduced at a mass ratio of the mass of the CRISPR / Cas9 protein: the total mass of the plurality of types of the gRNAs = 5:1 to 1:

10.

10. In the introduction step, the CRISPR / Cas9 protein is introduced at 1 to 50 μg per 1 × 10 ５ to 3 × 10 ６ human iPSCs. The manufacturing method according to claim 9.

11. comprising a CRISPR / Cas9 protein and two types of gRNAs for two or more target genes, wherein the two or more target genes are HLA-A gene, HLA-B gene and CIITA gene, and the gRNAs are A gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 1, and a gRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2, or a nucleotide sequence in which one or several nucleotides are deleted, substituted or added at the 5'-end of the nucleotide sequence set forth in SEQ ID NO: 2 is a kit for introducing into human iPSCs to disrupt the HLA-A gene, HLA-B gene and CIITA gene of the human iPSCs.

12. The kit according to claim 11, for performing the introduction by electroporation.

13. The kit according to claim 11 or 12, wherein the CRISPR / Cas9 protein and the plurality of types of the gRNAs are used to introduce them into the human iPSCs at a mass ratio of the mass of the CRISPR / Cas9 protein: the total mass of the plurality of types of the gRNAs = 5:1 to 1:

10.

14. The CRISPR / Cas9 protein was added to 1×10 of the human iPSCs. ５ pieces ~ 3×10 ６ The kit according to any one of claims 11 to 13, which is used to introduce 1 to 50 µg per individual.

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