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US-20260125655-A1 - METHOD FOR PRODUCING LOW-ANTIGENIC CELL

US20260125655A1US 20260125655 A1US20260125655 A1US 20260125655A1US-20260125655-A1

Abstract

A method for producing, from a donor cell, a low-antigenic cell in which a rejection reaction is reduced in a case where the cell is allogeneically transplanted into a recipient, the method including: determining human leukocyte antigen (HLA) alleles for the donor cell and the recipient, respectively; specifying an HLA allele that is present in the donor cell but is not present in the recipient; and disrupting or modifying the specified HLA allele to obtain a cell population including a cell not expressing an HLA protein specific to the donor cell, in which the cell not expressing the HLA protein specific to the donor cell is the low-antigenic cell.

Inventors

  • Akitsu Hotta
  • Huaigeng Xu
  • Shin Kaneko
  • Bo Wang

Assignees

  • KYOTO UNIVERSITY

Dates

Publication Date
20260507
Application Date
20250922
Priority Date
20180216

Claims (14)

  1. 1 .- 30 . (canceled)
  2. 31 . A method for preventing attack of a cell by natural killer (NK) cells, the method comprising disrupting an HLA-A allele and an HLA-B allele of the cell by genome editing such that the cell is not attacked by NK cells, wherein the cell is an iPSC that can express an endogenous HLA-C protein.
  3. 32 . The method of claim 31 , wherein the HLA-C protein is a protein encoded by one kind of allele selected from the group consisting of an HLA-C*01:02 allele, an HLA-C*02:02 allele, an HLA-C*03:03 allele, an HLA-C*03:04 allele, an HLA-C*04:01 allele, an HLA-C*05:01 allele, an HLA-C*06:02 allele, an HLA-C*07:01 allele, an HLA-C*07:02 allele, an HLA-C*08:01 allele, an HLA-C*12:02 allele, and an HLA-C*16:01 allele.
  4. 33 . The method of claim 31 , wherein the genome editing is performed by introducing a sequence-specific DNA-cleaving enzyme into the iPSC.
  5. 34 . The method of claim 33 , wherein the sequence-specific DNA-cleaving enzyme is CRISPR-Cas and the genome editing is performed by introducing a gRNA and the CRISPR-Cas into the iPSC.
  6. 35 . A method for producing a low-antigenic cell in which a rejection reaction is reduced in a case where the cell is allogeneically transplanted into a recipient, the method comprising: (i) disrupting an HLA-A allele and an HLA-B allele of the cell by genome editing, wherein the cell is an iPSC that can express an endogenous HLA-C protein; (ii) applying a HLA protein expression-inducing agent to the iPSC and inducing expression of an HLA protein; and (iii) recovering the iPSC in which the HLA-A allele and the HLA-B allele are disrupted.
  7. 36 . The method of claim 35 , wherein the HLA-C protein is a protein encoded by one kind of allele selected from the group consisting of an HLA-C*01:02 allele, an HLA-C*02:02 allele, an HLA-C*03:03 allele, an HLA-C*03:04 allele, an HLA-C*04:01 allele, an HLA-C*05:01 allele, an HLA-C*06:02 allele, an HLA-C*07:01 allele, an HLA-C*07:02 allele, an HLA-C*08:01 allele, an HLA-C*12:02 allele, and an HLA-C*16:01 allele.
  8. 37 . The method of claim 35 , wherein the HLA protein expression-inducing agent is interferon (IFN)-γ, IFN-α, IFN-β, interleukin (IL)-4, granulocyte macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF)-α, or TGF-β.
  9. 38 . The method of claim 35 , further comprising differentiating the iPSC in which HLA-A allele and an HLA-B allele are disrupted.
  10. 39 . The method of claim 35 , wherein the genome editing is performed by introducing a sequence-specific DNA-cleaving enzyme into the iPSC.
  11. 40 . The method of claim 39 , wherein the sequence-specific DNA-cleaving enzyme is CRISPR-Cas and the genome editing is performed by introducing a gRNA and the CRISPR-Cas into the iPSC.
  12. 41 . An induced pluripotent stem cell in which an HLA-A allele and an HLA-B allele are disrupted and at least one kind of endogenous HLA-C protein can be expressed, wherein the HLA-C protein is a protein encoded by one kind of allele selected from the group consisting of an HLA-C*01:02 allele, an HLA-C*02:02 allele, an HLA-C*03:03 allele, an HLA-C*03:04 allele, an HLA-C*04:01 allele, an HLA-C*05:01 allele, an HLA-C*06:02 allele, an HLA-C*07:01 allele, an HLA-C*07:02 allele, an HLA-C*08:01 allele, an HLA-C*12:02 allele, and an HLA-C*16:01 allele.
  13. 42 . The induced pluripotent stem cell of claim 41 , wherein at least one allele of a class II major histocompatibility complex transactivator (CIITA) allele, a regulatory factor X5 (RFX5) allele, a regulatory factor X associated protein (RFXAP) allele, or a regulatory factor X associated ankyrin containing protein (RFXANK) allele is further disrupted or modified.
  14. 43 . The induced pluripotent stem cell of claim 41 , wherein the cell expresses at least one class I HLA protein selected from the group consisting of an HLA-C protein, and HLA-E protein and an HLA-G protein; and wherein the expression of the class I HLA protein is stimulated by an HLA protein expression-inducing agent chosen from interferon (IFN)-γ, INF-α, INF-β, interleukin (IL)-4, granulocyte macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF)-α, and TGF-β.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 16/969,222, filed Aug. 12, 2020, which is a U.S. National Stage of International Application No. PCT/JP2019/005524, filed Feb. 15, 2019, which claims the benefit of Japanese Patent Application No. 2018-026421, filed Feb. 16, 2018, the entire contents of each which are fully incorporated herein by reference. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY The present application is being filed along with a sequence listing in electronic format. The sequence listing is provided as a file entitled “70815A_SeqListing.xml, created Sep. 18, 2025, which is 7,074,572 bytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. TECHNICAL FIELD The present invention relates to a method for producing a low-antigenic cell. More specifically, the present invention relates to a method for producing a low-antigenic cell, a kit for detecting a low-antigenic cell, a cell, a gRNA, and a method for specifying a target base sequence. Priority is claimed on Japanese Patent Application No. 2018-026421, filed Feb. 16, 2018, the content of which is incorporated herein by reference. BACKGROUND ART In a case of allogeneic transplantation in which donor cells are transplanted into a recipient (patient) who is the other person, transplanted cells are rejected due to an immune reaction. A protein that plays the most important role in distinguishing between self cells and non-self cells is a cell surface protein called a human leukocyte antigen (HLA) or a major histocompatibility complex (MHC). HLA is classified into class I and class II. Class I HLA proteins are expressed in most cell types in a body. The class I HLA protein has a function of forming a heterodimer with β2-Microglobulin (B2M) to be expressed on a cell surface, and presenting a peptide with respect to CD8-positive cytotoxic T cells to induce activation. The antigenic peptides presented are endogenous and have a length of 8 to 10 amino acids in many cases. Genes classified into class I HLA are mainly six genes of an HLA-A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, and an HLA-G gene. In addition, many pseudogenes (HLA-H, HLA-J, HLA-K, HLA-L, HLA-P, HLA-T, HLA-U, HLA-V, HLA-W, HLA-X, HLA-Y, and the like) are also known. Among the genes, three genes of the HLA-A gene, HLA-B gene, and HLA-C gene have particularly great sequence diversity among individuals and play a major role in identifying self cells and non-self cells in transplantation immunity. Class II HLA proteins are mainly expressed in immune cells such as macrophages, dendritic cells, activated T cells and B cells, and the like. The class II HLA protein has a function of forming a heterodimer with an α chain and a β chain and presenting a peptide with respect to CD4 helper T cells to induce activation. The antigenic peptides presented are exogenous and have a length of 15 to 24 amino acids in many cases. Genes classified into class II HLA are HLA-DR (α chain: HLA-DRA, β chain: HLA-DRB), HLA-DQ (α chain: HLA-DQA1, β chain: HLA-DQB1), HLA-DP (α chain: HLA-DPA1 or HLA-DPA2, β chain: HLA-DPB1 or HLA-DPB2). In addition, many pseudogenes (HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB) are also known to exist. HLA genes have sequence diversity and thus are involved in recognition of self and non-self cells at the cellular level. HLA matching is also particularly important for reducing immune rejection during allogeneic transplantation. For example, in transplantation of hematopoietic stem cells, it is recommended to find and transplant donors in which antigenicities in both alleles of HLA-A, HLA-B, HLA-C, and HLA-DR, that is, alleles at a total of 8 loci, are as consistent as possible. In addition, in consideration of results of engraftment rates of kidney transplantation and the like, it has been reported that, as the degree of consistency of HLA antigenicity becomes high, engraftment efficiency becomes significantly high. There is also an immune system induced by the absence of HLA antigens on a cell surface. NK cells express multiple inhibitory receptors. For example, it is known that a complex receptor of CD94 and NKG2A, which recognizes HLA-E and inhibits the action of NK cells, induces activation of NK cells when a cell without HLA-E is found, and attacks the cells. In addition, there is also a receptor family called Killer cell Immunoglobulin-like Receptor (KIR), which is classified into 2D and 3D according to the number of extracellular domains, and is also divided into L (long) and S (short) depending on lengths of intracellular domain. It is known that a 2DL1 receptor recognizes HLA-C2, 2DL2 and 2DL3 receptors recognize HLA-C1, a 2DL4 receptor recognizes HLA-G, a 3DL1 recognizes HLA-Bw4, and a 3DL2 recognizes HLA-A3 or HLA-A11 to inhibit activity of NK cells. There are polymorphisms in KIR, and for example, almos