Method for producing low-antigenicity cell
阅读说明：本技术 低抗原性细胞的制造方法 (Method for producing low-antigenicity cell ) 是由 堀田秋津 徐淮耕 金子新 王博 于 2019-02-15 设计创作，主要内容包括：一种制造方法,所述制造方法是由供体细胞制造向受体进行同种异体移植时的排斥反应被降低的低抗原性细胞的方法,所述制造方法包括：分别确定上述供体细胞以及上述受体的人白细胞抗原(Human Leukocyte Antigen)(HLA)等位基因；特定不存在于上述受体中而存在于上述供体细胞中的HLA等位基因；以及,对已特定的上述HLA等位基因进行破坏或改变,得到包含没有表达对上述供体细胞特异的HLA蛋白质的细胞的细胞群,没有表达对上述供体细胞特异的HLA蛋白质的细胞为上述低抗原性细胞。(A method for producing, from a donor cell, a low-antigenicity cell having reduced rejection when allogeneic transplantation is performed on a recipient, comprising: determining the Human Leukocyte Antigen (HLA) alleles of said donor cell and said recipient cell, respectively; specifying an HLA allele that is not present in said recipient but is present in said donor cell; and disrupting or altering the specified HLA allele to obtain a cell population including cells that do not express an HLA protein specific for the donor cell, wherein the cells that do not express an HLA protein specific for the donor cell are the low-antigenicity cells.)
1. A method for producing low-antigenicity cells from donor cells, the rejection of which is reduced when the cells are transplanted into a recipient,
the manufacturing method comprises the following steps:
determining Human Leukocyte Antigen (HLA) alleles of said donor cell and said recipient, respectively;
(ii) an HLA allele that is not present in the recipient but is present in the donor cell; and
disrupting or altering the HLA allele that has been specified to give a population of cells comprising cells that do not express an HLA protein specific for the donor cell,
cells that do not express an HLA protein specific for the donor cell are the low antigenic cells.
2. The manufacturing method according to claim 1, wherein the HLA alleles determined in the determination of the HLA alleles of the donor cell and the recipient, respectively, include an HLA-a allele, an HLA-B allele, and an HLA-C allele.
3. The production method according to claim 1 or 2, further comprising, after obtaining a cell population including cells that do not express an HLA protein specific to the donor cell: recovering from the cell population cells that do not express an HLA protein specific for the donor cell,
the recycling comprises:
contacting an HLA protein expression inducing agent with the population of cells; and
recovering cells that do not express an HLA protein specific for the donor cell from the cell population using, as an indicator, the expression of an HLA protein specific for the donor cell in the cell population that has been contacted with the HLA protein expression-inducing agent.
4. The production method according to claim 3, 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.
5. The production method according to any one of claims 1 to 4, wherein the low-antigenic cells are pluripotent stem cells.
6. A kit for detecting low-antigenicity cells that have reduced rejection when allogeneic transplantation is performed on a recipient, comprising an HLA protein expression-inducing agent.
7. The kit according to claim 6, wherein the HLA protein expression-inducing agent is IFN- γ, IFN- α, IFN- β, IL-4, GM-CSF, TGF- α or TGF- β.
8. The kit of claim 6 or 7, wherein the low antigenic cells are pluripotent stem cells.
9. An HLA protein expression inducer consisting of IFN-gamma, IFN-alpha, IFN-beta, IL-4, GM-CSF, TGF-alpha or TGF-beta.
10. A cell, wherein at least one HLA allele is disrupted or altered and the cell is capable of expressing at least one HLA protein.
11. The cell of claim 10, wherein the HLA allele comprises an HLA-a allele, an HLA-B allele, or an HLA-C allele.
12. The cell of claim 10 or 11, which is a low antigenic cell with reduced rejection upon allograft transplantation to a recipient, the HLA allele that has been disrupted or altered being an HLA allele that is not present in the recipient.
13. The cell of any one of claims 10-12, which is a pluripotent stem cell.
14. The cell of any one of claims 10-13, wherein at least one of a class II major histocompatibility complex transactivator (CIITA) allele, a regulator X5(RFX5) allele, a regulator X-associated protein (RFXAP) allele, and a regulator X-associated ankyrin (RFXANK) allele is further disrupted or altered.
15. The cell of any one of claims 10-14, wherein the HLA-a allele and the HLA-B allele are disrupted, and the cell is capable of expressing at least one HLA-C protein.
16. The cell of claim 15, wherein the HLA-C protein is a protein encoded by one allele selected from the group consisting of an HLA-C01: 02 allele, an HLA-C02: 02 allele, an HLA-C03: 03 allele, an HLA-C03: 04 allele, an HLA-C04: 01 allele, an HLA-C05: 01 allele, an HLA-C06: 02 allele, an HLA-C07: 01 allele, an HLA-C07: 02 allele, an HLA-C08: 01 allele, an HLA-C12: 02 allele, and an HLA-C16: 01 allele.
17. A method for producing low-antigenicity cells from donor cells, the rejection of which is reduced when the cells are transplanted into a recipient, wherein,
the manufacturing method comprises the following steps:
disrupting or altering at least one allele of the donor cell comprising at least one of a CIITA allele, an RFX5 allele, an RFXAP allele, and an RFXANK allele,
cells following disruption or alteration of the CIITA allele, RFX5 allele, RFXAP allele or RFXANK allele are the low antigenic cells.
18. The cell of claim 17, wherein the low antigenic cell is a pluripotent stem cell.
19. A cell comprising a disruption or alteration in at least one allele of a CIITA allele, an RFX5 allele, an RFXAP allele, and an RFXANK allele.
20. The cell of claim 19, which is a pluripotent stem cell.
21. A gRNA having, as a target base sequence, a base sequence that maps to only one HLA haplotype when mapping base sequence data of genomic DNA of a whole HLA haplotype, and that does not map to base sequence data of whole genomic DNA other than HLA alleles.
22. The gRNA according to claim 21, wherein the target base sequence is composed of the base sequence of any one of SEQ ID Nos. 3, 4, 7, 45 to 52, and 72 to 2459, or is composed of a base sequence in which 1 or more bases are deleted, substituted, or added at the 5' -end of the base sequence of any one of SEQ ID Nos. 3, 4, 7, 45 to 52, and 72 to 2459.
23. The gRNA according to claim 21 or 22, wherein the target base sequence is composed of the base sequence of any one of SEQ ID Nos. 3, 4, 7, 45 to 52, or is composed of a base sequence in which 1 or more bases are deleted, substituted, or added at the 5' -end of the base sequence of any one of SEQ ID Nos. 3, 4, 7, 45 to 52.
24. A gRNA according to any one of claims 21-23, wherein the target base sequence maps to exon 2 or 3 of an HLA gene.
25. A gRNA having, as a target base sequence, a base sequence that maps to two or more HLA haplotypes that are targets when mapping base sequence data of genomic DNA of a whole HLA haplotype, and that does not map to base sequence data of whole genomic DNA other than HLA alleles.
26. The gRNA according to claim 25, wherein the target nucleotide sequence is composed of a nucleotide sequence of any one of SEQ ID Nos. 53 to 55 and 2460 to 8013, or a nucleotide sequence in which 1 or more nucleotides are deleted, substituted, or added at the 5' -end of the nucleotide sequence of any one of SEQ ID Nos. 53 to 55 and 2460 to 8013.
27. The gRNA according to claim 25 or 26, wherein the target base sequence is composed of the base sequence of any one of SEQ ID Nos. 53 to 55, or is composed of a base sequence in which 1 or more bases are deleted, substituted, or added at the 5' -end of the base sequence of any one of SEQ ID Nos. 53 to 55.
28. A gRNA according to any one of claims 25 to 27, wherein the target base sequence maps to exon 2 or 3 of an HLA gene.
29. A method for producing a target base sequence of a low-antigenicity cell having reduced rejection in allogeneic transplantation into a recipient by genome editing, comprising:
mapping candidate base sequences to the base sequence data of the genome DNA of the whole HLA haplotype;
mapping the candidate base sequences to base sequence data of whole genome DNA other than HLA alleles; and
the candidate base sequences, which are mapped to only one target HLA haplotype when the base sequence data of the genome DNA of the whole HLA haplotype is mapped and are not mapped when the base sequence data of the whole genome DNA other than HLA alleles is mapped, are specified as the target base sequences.
30. A method for producing a target base sequence of a low-antigenicity cell having reduced rejection in allogeneic transplantation into a recipient by genome editing, comprising:
mapping candidate base sequences to the base sequence data of the genome DNA of the whole HLA haplotype;
mapping the candidate base sequences to base sequence data of whole genome DNA other than HLA alleles; and
the candidate base sequences mapped to two or more target HLA haplotypes when mapping the base sequence data of the genome DNA of the whole HLA haplotype and not mapped when mapping the base sequence data of the whole genome DNA other than HLA alleles are specified as the target base sequences.
Technical Field
The present invention relates to a method for producing a cell with low antigenicity. More specifically, the present invention relates to a method for producing a low-antigenic cell, a kit for detecting a low-antigenic cell, a gRNA, and a method for specifying a target base sequence. The present application claims priority based on japanese patent application No. 2018-026421 filed in japan on 16/2/2018, the contents of which are incorporated herein by reference.
Background
In the case of allogeneic transplantation in which donor cells are transplanted into another person, i.e., a recipient (patient), the transplanted cells are rejected due to immune reaction.
The most important role to distinguish between self and non-self of cells is the cell surface proteins called HLA (human leukocyte Antigen) or Major Histocompatibility Complex (MHC).
HLA is classified into class I and class II. HLA class I proteins are expressed in most cell types in vivo. HLA class I proteins form heterodimers (heterodimers) with β 2-Microglobulin (B2M), are expressed on the cell surface, and have the function of presenting peptides to CD8 positive cytotoxic T cells and inducing activation. The antigen peptide presented is endogenous and mostly has a length of 8 to 10 amino acids.
Classified as HLA class I are 6 genes including HLA-A gene, HLA-B gene, HLA-C gene, HLA-E gene, HLA-F gene, and HLA-G gene. In addition, many pseudogenes are known (HLA-H, HLA-J, HLA-K, HLA-L, HLA-P, HLA-T, HLA-U, HLA-V, HLA-W, HLA-X, HLA-Y, etc.). Among these genes, the HLA-A gene, HLA-B gene and HLA-C gene have a large sequence diversity among individuals, and they play a major role in the recognition of self and non-self in transplantation immunity.
Class II HLA proteins are mainly expressed in immune cells such as macrophages, dendritic cells, activated T cells, and B cells. The α and β chains of HLA class II proteins form heterodimers and have the function of presenting peptides to CD4 helper T cells for induction activation. The antigen peptide to be presented is exogenous and mostly has a length of 15 to 24 amino acids.
Classified as HLA class II are HLA-DR (alpha chain: HLA-DRA, beta chain: HLA-DRB), HLA-DQ (alpha chain: HLA-DQA1, beta chain: HLA-DQB1), HLA-DP (alpha chain: HLA-DPA1 or HLA-DPA2, beta chain: HLA-DPB1 or HLA-DPB 2). In addition, it is known that many pseudogenes (HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB) are present.
HLA genes are involved in self and non-self recognition at the cellular level due to their sequence diversity. HLA matching is also important in order to mitigate immune rejection during allogeneic transplantation. For example, in hematopoietic stem cell transplantation, it is recommended to find out donors having the highest possible antigenicity among two alleles of HLA-A, HLA-B, HLA-C, HLA-DR, i.e., 8 total alleles, and transplant them.
In addition, it has been reported that the higher the degree of matching of HLA antigenicity, the higher the implantation efficiency, as seen from the survival rate of kidney transplantation and the like.
On the other hand, there is also an immune system induced by the absence of HLA antigens on the 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 to attack when cells in which HLA-E is not present are found. In addition, there is a receptor family called KIR (Killer cell Immunoglobulin-like receptor), which is classified into 2D and 3D according to the number of extracellular domains, and is classified into L (long) and S (short) according to the length of the intracellular domain. It is known that 2DL1 receptor recognizes HLA-C2, 2DL2 and 2DL3 receptor recognize HLA-C1, 2DL4 receptor recognizes HLA-G, 3DL1 recognizes HLA-Bw4, 3DL2 recognizes HLA-A3 or HLA-A11, and thereby the activity of NK cells is suppressed. The KIR has a polymorphism, and for example, almost all Japanese people (98% or more) have been reported to have 2DL1, 2DL3 and 3DL4, but 2DL2 is rare and is about 15%.
Further, pluripotent stem cells such as iPS cells and ES cells have multifunctionality capable of differentiating into various cell types, and therefore, cell therapy and/or regenerative medicine, in which pluripotent stem cells are transplanted to a patient after being differentiated into various cells, have attracted attention.
In fact, the following examples have been reported: examples of differentiation from ES cells into nerve cells and transplantation into bone marrow-injured patients; examples of differentiation from iPS cells into retinal pigment epithelial cells and transplantation to age-related macular degeneration patients, and the like. It has been reported that when the cells derived from pluripotent stem cells are transplanted, it is also important to match the transplanted cells with the HLA type of the patient.
However, the establishment of pluripotent stem cells requires a great deal of cost and time, and therefore has a problem that it is difficult to prepare HLA-type-identical cells for each patient. As a method for solving this problem, for example,
In recent years, by genome editing technology using CRISPR-Cas9 and guide rna (gRNA), a target base sequence of gRNA induces Double-stranded DNA cleavage (DSB) in genomic DNA depending on the target base sequence, thereby deleting a part of the genomic DNA, or a template DNA having a partially identical sequence near the Double-stranded DNA cleavage-inducing site is co-introduced with Cas9 and gRNA, thereby allowing insertion of an arbitrary DNA fragment.
However, DSB induction in genome editing techniques depends on the following base sequences: the gRNA to be identified and bound to the target sequence has a spacer base sequence of about 20 bases and a PAM sequence of about 3 to 5 bases. Therefore, if the nucleotide sequence of the gRNA does not match the nucleotide sequence of the target site, efficient DSB induction is difficult.
In addition, when a base sequence very similar to the base sequence of the target site, for example, a base sequence different from only 1 base, is present in the region other than the target site, it is known that there is a risk that DSB is erroneously induced in the region other than the target site, and an undesirable genomic sequence mutation is induced, that is, a so-called off-target mutation risk.
Disclosure of Invention
Problems to be solved by the invention
Since the cell described in
Therefore, it is considered that only an arbitrary HLA gene is selectively disrupted using the genome editing technique. However, there are many HLA genes, and many pseudogenes are included, and sequence identity between HLA genes is high. On the other hand, even if the same HLA gene is used, sequence diversity between individuals is large, and therefore, even if a target base sequence matching a certain HLA gene sequence is identified, it cannot be used in cells derived from other donors. Therefore, it is not easy to determine the appropriate target sequence for genome editing. In addition, in the conventional genome editing techniques, it takes a lot of time to search for a cell in which the target genome editing is performed.
The present invention aims to provide a technique for producing low-antigenicity cells in which rejection is reduced when a recipient is subjected to allogeneic transplantation.
Means for solving the problems
The present invention includes the following aspects.
[1] A method for producing, from donor cells, low-antigenicity cells in which rejection is reduced when allogeneic transplantation is performed on a recipient, comprising: determining the Human Leukocyte Antigen (HLA) alleles of said donor cell and said recipient cell, respectively; (ii) a HLA allele that is not present in said recipient but is present in said donor cell; and disrupting or altering the specified HLA allele to obtain a cell population including cells that do not express an HLA protein specific for the donor cell, wherein the cells that do not express an HLA protein specific for the donor cell are the low-antigenicity cells.
[2] The production method according to [1], wherein the HLA alleles specified in the specification of the HLA alleles of the donor cell and the recipient cell include HLA-A alleles, HLA-B alleles and HLA-C alleles.
[3] The production method according to [1] or [2], further comprising, after obtaining a cell population including cells that do not express an HLA protein specific to the donor cell: recovering cells that do not express an HLA protein specific to the donor cell from the cell population,
the above recovery comprises:
contacting an HLA protein expression-inducing agent with the cell population; and
and recovering cells that do not express an HLA protein specific to the donor cell from the cell population using, as an indicator, the expression of an HLA protein specific to the donor cell in the cell population contacted with the HLA protein expression inducer.
[4] The production method according to [3], 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.
[5] The production method according to any one of [1] to [4], wherein the low-antigenic cells are pluripotent stem cells.
[6] A kit for detecting low-antigenicity cells that have reduced rejection when allogeneic transplantation is performed on a recipient, comprising an HLA protein expression-inducing agent.
[7] The kit according to [6], wherein the HLA protein expression-inducing agent is IFN- γ, IFN- α, IFN- β, IL-4, GM-CSF, TGF- α or TGF- β.
[8] The kit according to [6] or [7], wherein the low-antigenic cells are pluripotent stem cells.
[9] An HLA protein expression inducer consisting of IFN-gamma, IFN-alpha, IFN-beta, IL-4, GM-CSF, TGF-alpha or TGF-beta.
[10] A cell, wherein at least one HLA allele is disrupted or altered and the cell is capable of expressing at least one HLA protein.
[11] The cell according to [10], wherein the HLA allele comprises an HLA-A allele, an HLA-B allele or an HLA-C allele.
[12] The cell according to [10] or [11], which is a low-antigenic cell having reduced rejection in allograft transplantation to a recipient, wherein the HLA allele that has been disrupted or changed is an HLA allele that is not present in the recipient.
[13] The cell according to any one of [10] to [12], which is a pluripotent stem cell.
[14] The cell of any one of [10] to [13], wherein at least one of a class II major histocompatibility complex transactivator (CIITA) allele, a regulator X5(RFX5) allele, a regulator X-associated protein (RFXAP) allele, and a regulator X-associated ankyrin (RFXANK) allele is further disrupted or altered.
[15] The cell according to any one of [10] to [14], wherein the HLA-A allele and HLA-B allele are disrupted, and the cell is capable of expressing at least one HLA-C protein.
[16] The cell according to [15], wherein the HLA-C protein is a protein encoded by one allele selected from the group consisting of HLA-C01: 02 allele, HLA-C02: 02 allele, HLA-C03: 03 allele, HLA-C03: 04 allele, HLA-C04: 01 allele, HLA-C05: 01 allele, HLA-C06: 02 allele, HLA-C07: 01 allele, HLA-C07: 02 allele, HLA-C08: 01 allele, HLA-C12: 02 allele and HLA-C16: 01 allele.
[17] A method for producing, from donor cells, low-antigenicity cells in which rejection is reduced when allogeneic transplantation is performed on a recipient, comprising: disrupting or altering at least one allele of the donor cell comprising a CIITA allele, an RFX5 allele, an RFXAP allele, and an RFXANK allele, wherein a cell after disrupting or altering the CIITA allele, the RFX5 allele, the RFXAP allele, or the RFXANK allele is a low antigenic cell.
[18] The cell according to claim 17, wherein the low-antigenic cell is a pluripotent stem cell.
[19] A cell comprising a disruption or alteration in at least one allele of a CIITA allele, an RFX5 allele, an RFXAP allele, and an RFXANK allele.
[20] The cell according to [19], which is a pluripotent stem cell.
[21] A gRNA having, as a target base sequence, a base sequence that maps (mapping) only to one target HLA haplotype when mapping base sequence data of genomic DNA of a whole HLA haplotype, and that does not map when mapping base sequence data of whole genomic DNA other than HLA alleles.
[22] The gRNA according to [21], wherein the target nucleotide sequence is composed of a nucleotide sequence of any one of SEQ ID Nos. 3, 4, 7, 45 to 52, and 72 to 2459, or a nucleotide sequence in which 1 or more nucleotides are deleted, substituted, or added at the 5' -end of the nucleotide sequence of any one of SEQ ID Nos. 3, 4, 7, 45 to 52, and 72 to 2459.
[23] The gRNA according to [21] or [22], wherein the target base sequence is composed of the base sequence of any one of SEQ ID Nos. 3, 4, 7, and 45 to 52, or is composed of a base sequence in which 1 or more bases are deleted, substituted, or added at the 5' -end of the base sequence of any one of SEQ ID Nos. 3, 4, 7, and 45 to 52.
[24] The gRNA according to any one of [21] to [23], wherein the target nucleotide sequence is mapped to
[25] A gRNA having, as a target base sequence, a base sequence that maps to two or more HLA haplotypes that are targets when mapping base sequence data of genomic DNA of a whole HLA haplotype, and that does not map when mapping base sequence data of whole genomic DNA other than HLA alleles.
[26] The gRNA according to [25], wherein the target nucleotide sequence is composed of the nucleotide sequence of any one of SEQ ID Nos. 53 to 55 and 2460 to 8013, or is composed of a nucleotide sequence in which 1 or more nucleotides are deleted, substituted, or added at the 5' -end of the nucleotide sequence of any one of SEQ ID Nos. 53 to 55 and 2460 to 8013.
[27] The gRNA according to [25] or [26], wherein the target nucleotide sequence is composed of a nucleotide sequence of any one of SEQ ID Nos. 53 to 55, or a nucleotide sequence in which 1 or more nucleotides are deleted, substituted, or added at the 5' -end of the nucleotide sequence of any one of SEQ ID Nos. 53 to 55.
[28] The gRNA according to any one of [25] to [27], wherein the target nucleotide sequence is mapped to
[29] A method for identifying a target base sequence for producing a low-antigenic cell with reduced rejection upon allogeneic transplantation to a recipient by genome editing, the method comprising:
mapping candidate base sequences to the base sequence data of the genome DNA of the whole HLA haplotype;
mapping the candidate base sequences on the base sequence data of the whole genome DNA other than the HLA allele; and
the candidate base sequences, which are mapped to only one target HLA haplotype when the base sequence data of the genomic DNA of the whole HLA haplotype is mapped and are not mapped when the base sequence data of the whole genomic DNA other than HLA alleles is mapped, are identified as the target base sequences.
[30] A method for identifying a target base sequence for producing a low-antigenic cell with reduced rejection upon allogeneic transplantation to a recipient by genome editing, the method comprising:
mapping candidate base sequences to the base sequence data of the genome DNA of the whole HLA haplotype;
mapping the candidate base sequences on the base sequence data of the whole genome DNA other than the HLA allele; and
the candidate base sequences mapped to two or more target HLA haplotypes when mapping the base sequence data of the genome DNA of the whole HLA haplotype and unmapped when mapping the base sequence data of the whole genome DNA other than HLA alleles are specified as the target base sequences.
Effects of the invention
The present invention can provide a technique for producing low-antigenicity cells in which rejection is reduced when a recipient is subjected to allogeneic transplantation.
Drawings
In fig. 1, (a) is a graph showing HLA alleles targeting sgrnas extracted in experimental example 1 by venn diagram; (b) the sgRNA identified in experiment example 1 is a diagram showing which site of HLA-A allele, HLA-B allele, and HLA-C allele is targeted.
Fig. 2 is a graph showing the results of the flow cytometer in experimental example 2.
Fig. 3 is a graph showing the results of the flow cytometer in experimental example 3.
In FIG. 4, (a) is a graph showing the results of the flow cytometer in Experimental example 4; (b) the results are shown in experimental example 4, which shows a time chart of IFN-. gamma.treatment of iPS cells and analysis of the expression level of HLA-A protein.
In fig. 5, (a) is a diagram showing a target site and a target base of a sgRNA in experimental example 6; (b) is a photograph showing the results of the T7 endonuclease I measurement in Experimental example 6.
In fig. 6, (a) and (b) are graphs showing the results of the flow cytometer in experimental example 7.
FIG. 7 is a graph showing the ratio of mutations in the nucleotide sequence of each clone in Experimental example 7.
In fig. 8, (a) and (b) are graphs showing the results of the flow cytometer in experimental example 8.
In fig. 9, (a) and (b) are graphs showing the results of the flow cytometer in experimental example 8.
FIG. 10, (a) is a diagram showing the base mutation pattern of each clone recovered in Experimental example 8; (b) is the nucleotide sequence of the clone of the representative example recovered in Experimental example 8.
Fig. 11 is a graph showing the results of the flow cytometer in experimental example 9.
FIG. 12, (a) is a diagram showing the base mutation pattern of each clone recovered in Experimental example 9; (b) is the nucleotide sequence of the clone of the representative example recovered in Experimental example 9.
FIG. 13 shows the base mutation pattern of each clone collected in Experimental example 10. (c) Is the nucleotide sequence of the clone of the representative example recovered in Experimental example 10.
FIG. 14, (a) is a view showing the base mutation pattern of each clone recovered in Experimental example 11; (b) is the nucleotide sequence of the clone of the representative example recovered in Experimental example 11.
FIG. 15, (a) is a graph showing the results of the flow cytometer in Experimental example 11; (b) is a figure showing the base mutation pattern of each clone recovered in Experimental example 11; (c) is the nucleotide sequence of the clone of the representative example recovered in Experimental example 11.
Fig. 16, (a) is a diagram showing a target site of sgRNA in experimental example 12; (b) and (c) is a graph showing the results of the flow cytometer in experimental example 12.
FIG. 17, (a) is a diagram showing the base mutation pattern of the clone recovered in Experimental example 12; (b) is the nucleotide sequence of the clone recovered in Experimental example 12.
FIG. 18, (a) is a graph showing the results of the flow cytometer in Experimental example 13; (b) and (d) is a figure showing the base mutation pattern of each clone recovered in Experimental example 13; (c) and (e) nucleotide sequences of clones representing the example recovered in Experimental example 13.
In fig. 19, (a) and (b) are graphs showing the results of the flow cytometer in experimental example 14; (c) is a figure showing the base mutation pattern of each clone recovered in Experimental example 14; (d) is the nucleotide sequence of the clone of the representative example recovered in Experimental example 14.
Fig. 20, (a) is a diagram showing a target site of sgRNA in experimental example 15; (b) and (c) is a graph showing the results of the flow cytometer in experimental example 15.
FIG. 21, (a) and (b) are schematic diagrams illustrating homologous recombination in Experimental example 16; (c) is a graph showing the results of the flow cytometer in experimental example 15.
In FIG. 22, (a) and (b) are graphs showing the results of analysis of the nucleotide sequence in Experimental example 16.
In fig. 23, (a) and (b) are graphs showing the results of the flow cytometer in experimental example 17; (c) is a graph showing the results of analysis of the nucleotide sequence in Experimental example 17.
FIG. 24(a) is a graph showing the results of the flow cytometer in Experimental example 18; (b) is a graph showing the results of nucleotide sequence analysis in Experimental example 18.
In fig. 25, (a) to (e) are graphs showing the results of the flow cytometer in experimental example 19.
In fig. 26, (a) to (f) are graphs showing the results of the flow cytometer in experimental example 20.
In fig. 27, (a) to (e) are graphs showing the results of the flow cytometer in experimental example 21.
Fig. 28 is a schematic diagram showing the procedure of experimental example 22.
In fig. 29, (a) to (e) are graphs showing the results of the flow cytometer in experimental example 22.
FIG. 30 is a schematic diagram showing the procedure of Experimental example 23.
FIG. 31 is a graph showing the results of Experimental example 23.
FIG. 32 is a graph showing the results of Experimental example 24.
FIG. 33 is a graph showing the results of Experimental example 25.
In fig. 34, (a) and (b) are graphs showing the results of the flow cytometer in experimental example 26; (c) is a graph showing the results of experimental example 26.
In fig. 35, (a) to (g) are graphs showing the results of the flow cytometer in experimental example 27.
FIG. 36 is a graph showing the results of Experimental example 28.
In fig. 37, (a) to (f) are phase contrast micrographs showing the results of experimental example 29.
In fig. 38, (a) to (f) are phase contrast micrographs showing the results of experimental example 30.
In fig. 39, (a) and (b) are graphs showing the results of the flow cytometer in experimental example 31; (c) is a graph showing the results of experimental example 31.
In fig. 40, (a) and (b) are graphs showing the results of the flow cytometer in experimental example 32; (c) is a graph showing the results of experimental example 32.
In FIG. 41, (a) is a diagram illustrating an experiment schedule in Experimental example 33; (b) is a photograph showing the results of fluorescence of EB-derived blood cell-like cells in Experimental example 33; (c) is a graph showing the survival rate of EB-derived hemocyte-like cells in Experimental example 33.
FIG. 42, (a) is a graph showing the results of flow cytometer analysis in Experimental example 34; (b) is a graph showing the results of experimental example 34.
FIG. 43, (a) is a graph showing the results of flow cytometer analysis in Experimental example 35; (b) is a graph showing the results of experimental example 35.
In fig. 44, (a) and (b) are graphs showing the results of flow cytometer analysis in experimental example 36.
In FIG. 45, (a) is a graph illustrating an experiment schedule in Experimental example 37; (b) is a photograph showing the results of fluorescence of EB-derived blood cell-like cells in Experimental example 37; (c) is a graph showing the relative survival rate of EB-derived hemocyte-like cells in Experimental example 37.
FIG. 46 is a graph showing the results of Experimental example 38.
FIG. 47(a) is a table showing the HLA haplotype of the residual cells 585A1-C7 used in Experimental example 39 and the HLA haplotype of the residual cells 585A1-C7 after the CIITA gene was knocked out, prepared in Experimental example 39; (b) is a figure showing the base mutation pattern of each clone obtained in Experimental example 39; (c) the nucleotide sequence of the clone obtained in Experimental example 39 was used.
In fig. 48, (a) to (d) are graphs showing the results of the flow cytometer in experimental example 40.
In fig. 49, (a) to (c) are graphs showing the results of the flow cytometer in experimental example 41.
In fig. 50, (a) to (c) are diagrams for explaining the steps of experimental example 42.
FIG. 51 is a graph showing the results of the flow cytometer in Experimental example 42 (a); (b) is a diagram showing the base mutation pattern of each subclone obtained in Experimental example 42.
In FIG. 52, (a) and (b) are graphs showing the base mutation patterns of each subclone obtained in Experimental example 42.
FIG. 53 is a table showing allele frequencies of HLC-C alleles in various human races.
FIG. 54 is a view showing the base mutation pattern of each subclone obtained in Experimental example 43.
FIG. 55 is a diagram showing the base mutation pattern of each subclone obtained in Experimental example 44.
In fig. 56, (a) and (b) are graphs showing the results of the flow cytometer in experimental example 45.
Fig. 57 is a graph showing the results of the flow cytometer in experimental example 46.
FIG. 58 is a view showing the base mutation pattern of each subclone obtained in Experimental example 47.
FIG. 59 is a diagram showing the base mutation pattern of each subclone obtained in Experimental example 48.
FIG. 60 is a view showing the base mutation pattern of each subclone obtained in Experimental example 49.
Detailed Description
[ method for producing Low-antigen cells with reduced rejection ]
(embodiment 1)
The manufacturing method of
As described below in examples, according to the manufacturing method of
In the present specification, an HLA locus is sometimes referred to as "HLA allele", and the antigenic diversity of HLA proteins presented on the cell surface is sometimes referred to as "HLA type". Hereinafter, each step will be explained.
(Process (a))
In this step, HLA alleles of the donor cell and the recipient cell are determined. The donor cell may be a human cell or a non-human animal cell. The donor cell may be a pluripotent stem cell or a differentiated cell. In the present specification, pluripotent stem cells refer to embryonic stem cells (ES cells), artificial pluripotent stem cells (iPS cells), and the like.
The low-antigen cells produced by the production method of
In addition, the recipient is preferably an animal of the same species as the low antigenic cells. The recipient may be a patient who is actually scheduled to be transplanted, or may be a hypothetical recipient having an HLA allele which may become a recipient in the future.
HLA alleles can be identified by conventional methods such as PCR-rSSO (PCR reverse Sequence Specific oligonucleotide), PCR-SSP (Sequence Specific primer), PCR-SBT (Sequence based typing), and new-generation sequencing methods, and can be performed using commercially available HLA typing kits. In addition, HLAreport, HLA-PRG, HLA-genotyper, PHLAT, Optitype, neXtype, Athlates, HLAfforestoes, SOAP-HLA, HLAminer, seq2HLA, GATK HLA Caller, and the like are known as software for HLA typing from sequence data of a new generation sequencer such as the Whole Genome Sequence (WGS), exome sequence (WES), RNA-seq, and the like.
The HLA allele specified in the present step is preferably an HLA allele having a high association with rejection reaction at the time of cell transplantation, and preferably contains an HLA-A allele, an HLA-B allele and an HLA-C allele.
The HLA allele specified in the present step may include HLA-DR allele, HLA-DQ allele, HLA-DP allele, or other HLA alleles.
(Process (b))
In this step, an HLA allele that is not present in the recipient but present in the donor cell (hereinafter, may be referred to as "donor-specific HLA allele") is specified. Donor-specific HLA alleles are specified by comparing the HLA alleles of the donor cells and the HLA alleles of the recipient.
The HLA allele is a nucleotide sequence encoded on a genomic DNA and encodes an HLA protein. Serological classification of HLA proteins is referred to as HLA antigenic type. Specific examples of HLA antigen types include a method of analyzing reactivity with a serum or an antibody recognizing a specific HLA antigen type; and a method of comparing the DNA of HLA allele or the nucleotide sequence of RNA transcribed from HLA allele with a known correspondence table or IPD-IMGT database (https:// www.ebi.ac.uk/IPD/IMGT/HLA /).
In the case of the HLA antigen type, usually, the distinction of the 1st region (2-digit symbol) of HLA allele symbols (http:// HLA. alloles. org/nomenclature/naming. html) is sometimes referred to, but in the present specification, even when the HLA antigen type is serologically the same, it is judged that different HLA alleles are present when the amino acid sequence is different (non-synonymous substitution).
Specifically, whether the HLA alleles are identical or different is determined by the difference between the 1st region and the 2nd region (4-digit symbol) of the HLA allele symbols. In addition, HLA alleles matching the 1st region and the 2nd region of the HLA allele symbols are judged to be the same HLA allele. In addition, when it is necessary to distinguish the nucleotide sequence of the genome, such as when the target sequence for genome editing is specified, the distinction between the 3 rd region (nucleotide substitution inside the translation region without amino acid mutation) and the 4 th region (nucleotide substitution outside the translation region) may be considered.
(step (c))
When cells having HLA proteins of an HLA antigen type that are not present in a recipient are transplanted allogeneic to the recipient, rejection occurs and the transplanted cells are rejected. Therefore, in this step, the donor-specific HLA allele is disrupted or altered to obtain a cell population including cells that do not express the HLA protein specific to the donor cell (hereinafter, may be referred to as "donor-specific HLA protein").
Here, the cells that do not express the donor-specific HLA protein are cells in which the expression of the specific HLA protein is negative due to the destruction of the HLA allele of the donor cell; or cells that change from a particular HLA type to another HLA type due to a change in the HLA allele of the donor cell.
The cells obtained by this step, which do not express the donor-specific HLA protein, are low-antigenic cells. In the absence of donor-specific HLA proteins, this step is not required.
It is preferable that the B2M gene is not disrupted in the low-antigenicity cell produced by the production method of
Genome editing
In the examples described below, disruption or alteration of the HLA allele of the donor cell can be performed by, for example, genome editing. Disruption of HLA alleles can be performed by specifically cleaving HLA alleles and inducing double-stranded DNA cleavage (DSB). When a base is deleted or added during the repair of DSB and a frame shift mutation occurs, HLA alleles are disrupted and HLA proteins are no longer expressed. In the present specification, the disruption of an HLA allele may be referred to as HLA allele knock-out.
In addition, DSB is induced by specifically cleaving HLA alleles in the presence of donor DNA, thereby enabling homologous recombination to be induced during repair of DSB to alter HLA alleles. Specifically, for example, as described below in examples, the HLA-a 02:07 allele can be changed to HLA-a 01:01 allele, and the like.
As the donor DNA, a DNA that has sequence identity with regions around the cleavage position of double-stranded DNA including genomic DNA and encodes a desired HLA allele can be used. The donor DNA may be a single-stranded DNA or a double-stranded DNA. The donor DNA may be a DNA having a single nucleotide sequence, or a mixture of DNAs having a plurality of nucleotide sequences.
Herein, having sequence identity means: the nucleotide sequence of the donor DNA is 90% or more identical to that of a region including the double-strand cleavage site of the target genomic DNA. The donor DNA preferably has a nucleotide sequence of 95% or more, more preferably 99% or more, in a region including the double-strand cleavage site of the genomic DNA.
The donor DNA may be a single-stranded DNA having about 50 to 5000 bases, or a double-stranded DNA having about 50 to 5000 bases. When the donor DNA is a single-stranded DNA, the donor DNA may have sequence identity with any of the double strands of the genomic DNA.
Sequence specific DNA cutter
In general, sequence-specific DNA cleaving enzymes used for inducing DSBs to perform genome editing are roughly classified into RNA-inducible nucleases and artificial nucleases. The sequence-specific DNA cleaving enzyme may be an RNA-inducible nuclease or an artificial nuclease.
The sequence-specific DNA cleaving enzyme is not particularly limited as long as it specifically cleaves the genomic DNA to a double strand by the target sequence. The length of the target sequence recognized by the sequence-specific DNA cleaving enzyme may be, for example, about 10 to 60 bases.
The DNA cleaving enzyme specific to the sequence may be, for example, a DNA cleaving enzyme in which a plurality of nicking enzymes are combined. Here, nicking enzyme refers to an enzyme that forms a nick on one strand of double-stranded DNA. For example, a double-stranded cut can be formed by nicking both strands of a double-stranded DNA at close positions on the genomic DNA.
An RNA-inducible nuclease is an enzyme that binds a target sequence to a short-chain RNA as a guide and recruits a nuclease having 2 DNA cleavage domains (nuclease domains), thereby inducing sequence-specific cleavage. As RNA-inducible nucleases, CRISPR-Cas family proteins can be cited. CRISPR-Cas family proteins are broadly classified into
Examples of CRISPR-Cas family proteins include Cas9, Cpf1, CasX, CasY, Cas12, Cas13, and
Examples of Cas9 include Cas9 derived from Streptococcus pyogenes, staphylococcus aureus, Streptococcus thermophilus (Streptococcus thermophilus), bacillus stearothermophilus (Geobacillus stearothermophilus), and the like. Examples of Cpf1 include Cpf1 derived from the genera Aminococcus (Acidaminococcus), Lachnospira (Lachnospira), Chlamydomonas (Chlamydomonas), and Francisella neogericina (Francisella-Novicida).
An artificial nuclease is an artificial restriction enzyme having a DNA binding domain designed and created so as to specifically bind to a target sequence, and a nuclease domain (e.g., a DNA cleavage domain of fokl as a restriction enzyme). Examples of the artificial nuclease include, but are not limited to, Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), megabase Meganuclease (Meganuclease), and the like.
Introduction of DNA cleavage enzyme specific for sequence
As a sequence-specific DNA cleaving enzyme to be introduced into the donor cell, for example, in the case of using CRISPR-Cas, the target base sequence is determined by the gRNA. Details regarding grnas are described later. The gRNA may be introduced into a donor cell in the form of RNA, or may be introduced into a donor cell in the form of an expression vector, and expressed intracellularly.
Examples of a method for producing a gRNA in the form of RNA include: constructs comprising a nucleic acid fragment encoding a gRNA and a promoter such as T7 added upstream thereof, a method of synthesis by in vitro transcription reaction, a method of chemical synthesis, and the like. In the case of chemically synthesizing a gRNA, chemically modified RNA can be used.
Examples of expression vectors include: a plasmid vector or a viral vector for transcribing a gRNA from Pol III promoter such as H1 promoter or U6 promoter. When the gRNA is expressed using an expression vector, the gRNA may be expressed all the time or may be expressed under the control of an expression-inducible promoter.
Next, Cas9 is prepared. Cas9 can be introduced into donor cells in the form of an expression vector expressed from Pol II promoter, or in the form of a purified protein. Examples of the expression vector include a transposon vector, a virus vector, an Episomal vector (Episomal vector), and a plasmid vector.
Introduction of the gRNA and Cas9 into the donor cell may be added only to the medium of the donor cell when the gRNA and Cas9 are in the form of a viral vector. Examples of the viral vector include adeno-associated virus, adenovirus, retrovirus, lentivirus, sendai virus, and baculovirus.
When the gRNA and Cas9 are transposon vectors, episomal vectors, plasmid vectors, or the like, or when the gRNA is RNA and Cas9 is a purified protein, they can be introduced into donor cells by transfection reagents or electroporation.
Examples of transfection reagents that can be used include Lipofectamine 2000, Lipofectamine3000, CRISPRMAX, RNAMAX (all from THERMO FISHER SCIENTIFIC),
The electroporation can be carried out by using a device such as NEPA21(Neppagene Co., Ltd.), Neon (THERMO FIHERSCIIENTIFIC Co., Ltd.), 4D-Nucleofector (Lonza Co., Ltd.).
(Process (d))
In the step (c), a cell population containing low-antigenicity cells that do not express donor-specific HLA proteins can be obtained. However, the cell population obtained in step (c) may include not only the target cells (low-antigen cells) in which the HLA allele is disrupted or modified, but also cells in which the HLA allele is not disrupted or modified, cells in which the HLA allele is not completely disrupted or modified, and the like. Therefore, after the step (c), the step (d) of collecting cells that do not express the donor-specific HLA protein (low-antigen cells) may be further performed.
The step (d) comprises a step of bringing an HLA protein expression-inducing agent (HLA protein expression-inducing agent) into contact with a cell population containing cells having low antigenicity; and collecting cells that do not express the donor-specific HLA protein from the cell population using, as an index, the expression of the donor-specific HLA protein in the cell population contacted with the HLA protein expression-inducing agent. The step of bringing the HLA protein expression inducer into contact with the cell population may be performed, for example, by adding the HLA protein expression inducer to a culture medium of the cell population.
In this step, the cells that do not express the donor-specific HLA protein may be recovered from the cell population by detecting the presence of the expressed HLA protein and recovering the cells that do not express the donor-specific HLA protein based on the presence or absence of the expressed HLA protein, using the expression of the donor-specific HLA protein as an indicator.
Examples of the HLA protein expression inducer include cytokines such as IFN-. gamma., IFN-. alpha., IFN-. beta., IL-4, GM-CSF, TGF-. alpha., and TGF-. beta. These expression inducers may be used singly or in combination of two or more.
The cytokine is preferably a human-derived cytokine when used in human cells. The NCBI accession numbers of human IFN-. gamma.proteins are NP-000610.2, etc., the NCBI accession numbers of human IFN-. alpha.proteins are NP-076918.1, NP-000596.2, NP-066546.1, etc., the NCBI accession numbers of human IFN-. beta.proteins are NP-002167.1, etc., the NCBI accession numbers of human IL-4 proteins are NP-000580.1, NP-758858.1, NP-001341919.1, etc., the NCBI accession numbers of human GM-CSF proteins are NP-000749.2, etc., the NCBI accession numbers of human TGF-. alpha.proteins are NP-001093161.1, NP-003227.1, NP-001295088.1, NP-001295087.1, etc., and the NCBI accession numbers of human TGF-. beta.proteins are NP-000651.3, XP-011525544.1, etc.
The cytokine may have a mutation with respect to the amino acid sequence described in each of the aforementioned accession numbers, as long as it has an activity of inducing the expression of an HLA protein. More specifically, the amino acid sequence may have an amino acid sequence obtained by deleting, substituting or adding 1 or more amino acids from the amino acid sequence described in each of the above-mentioned accession numbers. Here, the 1 or more amino acids may be, for example, 1 to 10 amino acids, for example, 1 to 5 amino acids, for example, 1 to 3 amino acids. The cytokine may have an amino acid sequence from which a signal peptide is removed.
It is known that pluripotent stem cells do not express HLA proteins on the cell surface in a generally undifferentiated state, and it is difficult to detect HLA proteins. Differentiation induction is required for presentation of HLA proteins. However, the differentiation induction process is generally complicated and time-consuming. In addition, the cells after the primary differentiation induction do not spontaneously return to an undifferentiated state, and therefore, even if a differentiated cell population negative for HLA proteins is recovered, it is no longer a pluripotent stem cell.
In view of the above, the present inventors have found that the expression of an HLA protein can be induced while maintaining the pluripotency of pluripotent stem cells by allowing the HLA protein expression-inducing agent to act on the pluripotent stem cells.
This enables the efficient detection and recovery of pluripotent stem cells (low-antigen cells) in which the target HLA allele has been disrupted or altered by inducing the expression of HLA proteins in the cell population obtained in step (d).
For example, after the expression of HLA protein in cells is induced, the cells are stained with an anti-HLA protein antibody, whereby HLA-expressing cells or non-expressing cells can be distinguished by flow cytometry and recovered by sorting.
Alternatively, the anti-HLA protein antibody may be adsorbed on a magnetic bead, and the magnetic bead-bound cell may be collected by bringing the anti-HLA protein antibody into contact with the HLA protein-expressing cell and using a magnetic force.
Alternatively, only target low-antigen cells may be collected by labeling cells expressing a specific HLA protein on a culture dish with an anti-HLA protein antibody or the like, and removing unnecessary target extracellular cells by aspiration, laser irradiation, or the like.
Alternatively, only target low-antigen cells may be recovered by mixing T cells recognizing HLA proteins other than the target into HLA protein-expressing cells on a culture dish, and attacking only the cells expressing HLA proteins other than the target and removing the cells.
The production method according to
The CIITA gene encodes a transcription factor that controls transcriptional activation of HLA class II together with the RFX5 gene, RFXAP gene, and RFXANK gene. Thus, cells disrupted in at least one of the CIITA allele, RFX5 allele, RFXAP allele and RFXANK allele do not express HLA class II protein, and rejection of the cells by allograft to a recipient is further reduced.
The NCBI accession numbers of human CIITA gene are NM _000246.3, NM _001286402.1, NM _001286403.1 and the like, the NCBI accession numbers of human RFX5 gene are NM _000449.3, NM _001025603.1 and the like, the NCBI accession numbers of human RFXAP gene are NM _000538.3 and the like, and the NCBI accession numbers of human RFXANK gene are NM _001278727.1, NM _001278728.1, NM _003721.3, NM _134440.2 and the like.
(embodiment 2)
The production method of
As described above, it is known that the expression of the CIITA gene, the RFX5 gene, the RFXAP gene, and the RFXANK gene is essential for the expression of HLA class II proteins. Thus, cells disrupted in at least one of the CIITA allele, RFX5 allele, RFXAP allele, and RFXANK allele do not express HLA class II protein and rejection is reduced upon allograft transplantation to a recipient.
In the production method of
In addition, the recipient is preferably an animal of the same species as the low antigenic cells. The recipient may be a patient who is actually planned to be transplanted, or may be a hypothetical recipient having an HLA allele that is expected to become a recipient in the future.
In addition, as described above, for cells in which the B2M allele was disrupted, the HLA class i protein was not expressed even when the HLA allele was wild type. The production method according to
Disruption of the CIITA allele or the B2M allele of the donor cell can be performed, for example, by genome editing. Genome editing is the same as described above.
[ kit for detecting Low-antigen cells ]
In one embodiment, the present invention provides a kit for detecting low-antigenicity cells in which rejection is reduced when a recipient is subjected to allograft transplantation, the kit comprising an HLA protein expression-inducing agent. The kit of the present embodiment can detect and recover low-antigenic cells. Therefore, the kit of the present embodiment may also be referred to as a kit for recovering low-antigenic cells.
In the kit of the present embodiment, the HLA protein expression inducer includes cytokines such as IFN-. gamma., IFN-. alpha., IFN-. beta., IL-4, GM-CSF, TGF-. alpha., TGF-. beta.and the like, as described above. The kit of the present embodiment may contain 1 kind of these expression-inducing agents alone, or 2 or more kinds thereof.
In the kit of the present embodiment, the low-antigenic cells may be pluripotent stem cells. As described below in examples, the kit of the present embodiment can induce expression of HLA protein while maintaining the pluripotency of low-antigen cells, even when the low-antigen cells are pluripotent stem cells.
[ cells ]
(embodiment 1)
The cell of
Pluripotent stem cells generally have poor expression of HLA proteins. Here, the expression weak means that: for example, when a flow cytometry analysis is performed by staining cells with an anti-HLA protein antibody, the expression of HLA protein cannot be clearly detected.
However, in the examples described below, if an HLA protein expression-inducing agent is brought into contact with a pluripotent stem cell, the expression of an HLA protein is enhanced. In the cell according to
In the examples described below, for example, for cells in which the B2M allele is disrupted, the HLA class i protein is not expressed even if the HLA allele is wild-type. The cell of
In the cell of
The cells of
The cells according to
The low-antigenic cells according to
For the cell of
(embodiment 2)
The cell of
The cell of
The cells according to
The low-antigenic cells according to
(use of Low-antigenic cells)
The cells according to
When the low-antigen cells are pluripotent stem cells, for example, the cells may be transplanted into a patient after differentiation into nerve cells, liver cells, islet cells, cardiac muscle cells, kidney cells, hematopoietic stem cells, cytotoxic T cells, or the like.
Alternatively, the gene may be transferred into low-antigen cells and then transplanted into a patient. Examples thereof include: after differentiation of pluripotent stem cells into T cells, Chimeric Antigen Receptor (CAR) is genetically introduced, and the cells are transplanted as CAR-T cells into cancer patients and the like.
[ method for specifying target nucleotide sequence ]
(embodiment 1)
In one embodiment, the present invention provides a method for producing a low-antigenic cell with reduced rejection in allograft transplantation to a recipient by genome editing, which is a specific method for a target base sequence, comprising mapping candidate base sequences to base sequence data of genomic DNA of a whole HLA haplotype; mapping the candidate base sequences on the base sequence data of the whole genome DNA other than the HLA allele; and identifying the candidate base sequence as a target base sequence, the candidate base sequence being mapped to only one target HLA haplotype when mapping the base sequence data of the genomic DNA of the whole HLA haplotype and not mapped when mapping the base sequence data of the whole genomic DNA other than HLA alleles.
The target base sequence of the gRNA according to
As described above, the HLA gene has a plurality of pseudogenes, and the sequence identity of HLA genes with each other is high, and the sequence diversity between individuals is large. Therefore, it is not easy to determine an appropriate target base sequence for genome editing using a sequence-specific DNA cleaving enzyme.
The target nucleotide sequence of the DNA cleaving enzyme specific to the sequence specific to the HLA gene can be specified, for example, as follows. First, base sequence data of genomic DNA of the whole HLA haplotype (base sequence data of all known HLA genes) is obtained. Base sequence data for all HLA genes can be obtained from, for example, the IPD-IMGT/HLA database (https:// www.ebi.ac.uk/IPD/IMGT/HLA /). In this database, base sequences of 12544 HLAI genes and 4622 HLAII genes were registered by 7 months in 2017.
Then, the target nucleotide sequence of the DNA cleaving enzyme specific to the sequence used is extracted from the nucleotide sequence of each HLA allele. For example, when CRISPR-Cas is used as a sequence-specific DNA cleaving enzyme, a nucleotide sequence that includes a PAM sequence of about 3 to 5 bases (for example, "NGG" in the case of Cas9 derived from streptococcus pyogenes, and "TTTN" in the case of Cpf1 derived from aminoacidococcus) and that can be used as a target nucleotide sequence of a gRNA is extracted as a candidate nucleotide sequence.
In the method of the present embodiment, the target base sequence includes a PAM sequence. The length of the target base sequence including the PAM sequence differs depending on the sequence-specific DNA cleaving enzyme. For example, when the sequence-specific DNA cleaving enzyme is Cas9 derived from Streptococcus pyogenes, the length of the target nucleotide sequence including the PAM sequence is preferably 19 to 33 nucleotides, and more preferably 20 to 24 nucleotides. When the sequence-specific DNA cleaving enzyme is Cpf1 derived from the genus Aminococcus, the length of the target nucleotide sequence including the PAM sequence is preferably 20 to 34 nucleotides, and more preferably about 24 nucleotides.
Next, candidate base sequences including the PAM sequence are mapped to the base sequence data of the genomic DNA of the whole HLA haplotype.
Mapping is an operation of specifying a position having high sequence identity of a query nucleotide sequence (herein, a candidate nucleotide sequence) on a reference nucleotide sequence (herein, nucleotide sequence data of genomic DNA of a whole HLA haplotype). The sequence identity between the query nucleotide sequence and the reference nucleotide sequence is preferably 90% or more, more preferably 95% or more, still more preferably 99% or more, and particularly preferably 100%.
Here, the sequence identity of the query nucleotide sequence to the reference nucleotide sequence can be determined, for example, as follows. First, the reference base sequence and the query base sequence are compared. Then, the number of bases of the matched bases in the reference base sequence and the query base sequence is calculated, and the sequence identity can be obtained by the following formula (1).
Number of bases having identical (%) sequence identity/total number of bases in query base sequence × 100(1)
In an embodiment, as described below, the mapping may be performed using, for example, the Bowtie program (http:// Bowtie-bio. The mapping can be performed using a sequence identity (sequence identity) search program other than the Bowtie program, such as BWA, BLAST, BLAT, SOAP, Novolalign, TopHat, and the like.
Next, the candidate base sequences are mapped to the base sequence data of the whole genomic DNA other than the HLA allele. As the base sequence data of the whole genome DNA, a reference human genome sequence (Hg19) or the like can be used. In addition, a sequence identity search program other than the Bowtie program or the Bowtie program can be used for mapping.
Next, a candidate base sequence to which only one target HLA haplotype is mapped when mapping the base sequence data of the genomic DNA of the whole HLA haplotype and which is not mapped when mapping the base sequence data of the whole genomic DNA other than the HLA allele is specified as the target base sequence. Here, the target HLA haplotype is an HLA haplotype of a subject to be destroyed or altered.
In examples, as described below, a gRNA having a target base sequence specified by the method of embodiment 1(a gRNA of
The HLA gene is composed of 8 exons in many cases, but the target base sequence is preferably a protein coding region of the HLA gene. The target base sequence is preferably targeted to
Alternatively, when the target nucleotide sequence is set at 2 positions, the nucleotide sequences between the target nucleotide sequences can be significantly deleted. For use, 2 or more target nucleotide sequences may be designed to include a gene region encoding a protein of an HLA gene.
In addition, it is preferable that the target base sequence is not targeted to a genomic region other than the HLA gene or mitochondrial DNA. Even when a few base mismatches are allowable, it is preferable to select a target base sequence having a small number of target sites other than the HLA gene.
(embodiment 2)
In one embodiment, the present invention provides a method, which is a specific method of a target base sequence, for producing low-antigenic cells with reduced rejection upon allogeneic transplantation to a recipient by genome editing, comprising: mapping candidate base sequences to the base sequence data of the genome DNA of the whole HLA haplotype; mapping the candidate base sequence to base sequence data of a whole genome DNA other than HLA alleles; and identifying the candidate base sequence, which is mapped to two or more target HLA haplotypes when mapping the base sequence data of the genome DNA of the whole HLA haplotype and is not mapped to when mapping the base sequence data of the whole genome DNA other than HLA alleles, as the target base sequence.
The method of
The method according to
By the method of this embodiment, a target base sequence of a gRNA according to
[gRNA]
(embodiment 1)
In one embodiment, the present invention provides a gRNA having, as a target base sequence, a base sequence that maps to only one target HLA haplotype when mapping base sequence data of genomic DNA of a full HLA haplotype, and that does not map to base sequence data of full genomic DNA other than HLA alleles.
The gRNA according to
In the present embodiment, the gRNA may be a complex of CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA), or may be a single synthetic gRNA (sgrna) in which tracrRNA and crRNA are combined. The gRNA of any structure can specifically induce DSB by a target base sequence.
The target nucleotide sequence is preferably a sequence specified by the above-mentioned method. As described above, the base sequence of interest in the present specification is a base sequence including a PAM sequence. Therefore, a base sequence obtained by removing the PAM sequence from the target base sequence is used as the spacer base sequence of the crRNA or sgRNA.
When the gRNA of the present embodiment is an sgRNA, the base sequence of the sgRNA may be as follows.
First, a base sequence obtained by removing the PAM sequence from the target base sequence is used as a spacer base sequence. Next, a nucleotide sequence to which a Scaffold sequence is ligated is designed at the 3' -end of the spacer nucleotide sequence. The base sequence of SEQ ID NO. 39 can be used as the Scaffold sequence, but the present invention is not limited thereto.
The base sequence of SEQ ID NO. 39 may function as a Scaffold sequence, or may be a base sequence obtained by deleting, substituting or adding 1 or more bases from the base sequence of SEQ ID NO. 39. Here, the 1 or more bases may be, for example, 1 to 10 bases, for example, 1 to 5 bases, for example, 1 to 3 bases.
The designed sgRNA can be prepared by chemical synthesis or the like. The sgRNA may be directly introduced into a donor cell as RNA preparation, or may be prepared as DNA, incorporated into an expression vector, and introduced into a donor cell in the form of an expression vector to express the sgRNA in the cell.
When the sgRNA is expressed in a cell, a polymerase III promoter such as a U6 promoter or an H1 promoter can be used. In this case, the base sequence of the 5' end of the sgRNA may be changed to G or GG in order to improve the transcription efficiency. Even if the base sequence of the 5' end of the sgRNA is changed to G or GG, the cleavage activity of Cas9 is hardly affected.
For example, when the base sequence of sgRNA obtained by removing the PAM sequence from the target base sequence is "5 '-NNNNNNNNNNNNNNNNNNNN-3'" (SEQ ID NO: 62), the base sequence of sgRNA to which the target base sequence is specifically cleaved may be "5 '-NNNNNNNNNNNNNNGUUUAGAGCAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAACUAUGAAAGAAGUGGCACCGAGUCGGUGCUUUUUUUUUUUUUUU-3'" (SEQ ID NO: 63).
The gRNA of the present embodiment may be a complex of crRNA and tracrRNA. The crRNA and tracrRNA may be introduced directly into the donor cell, or may be introduced into the donor cell in the form of an expression vector and expressed intracellularly. When crRNA and tracrRNA are expressed in cells, a polymerase III promoter such as U6 promoter or H1 promoter may be used. In this case, the base sequence of the 5' -end of the crRNA or tracrRNA may be changed to G or GG in order to improve the transcription efficiency. Even if the base sequence of the 5' end of the crRNA or tracrRNA is changed to G or GG, there is little effect on the cleavage activity of
When the gRNA of the present embodiment is a complex of crRNA and tracrRNA, the base sequences of the crRNA and the tracrRNA may be the following base sequences.
First, a base sequence obtained by removing the PAM sequence from the target base sequence is used as a spacer base sequence. Next, a nucleotide sequence to which a Scaffold sequence is ligated is designed at the 3' -end of the spacer nucleotide sequence, and a nucleotide sequence of crRNA is prepared. For example, when the nucleotide sequence obtained by removing the PAM sequence from the target nucleotide sequence is "5 '-NNNNNNNNNNNNNNNNNNNN-3'" (SEQ ID NO: 62), the nucleotide sequence of the crRNA may be "5 '-NNNNNNNNNNNNNNNNGUUUAGAGCUUGUUG-3'" (SEQ ID NO: 64). The base sequence of tracrRNA may be, for example, "5'-CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'" (SEQ ID NO: 65).
The base sequence of the crRNA may function as the crRNA, and may have a mutation with respect to the base sequence of SEQ ID NO. 64. More specifically, the nucleotide sequence may be a nucleotide sequence obtained by deleting, substituting or adding 1 or more nucleotides from the nucleotide sequence of SEQ ID NO. 64. Here, the 1 or more bases may be, for example, 1 to 10 bases, for example, 1 to 5 bases, for example, 1 to 3 bases.
The base sequence of tracrRNA may function as tracrRNA, and may be a base sequence obtained by deleting, substituting, or adding 1 or more bases from the base sequence of seq id No. 65. Here, the 1 or more bases may be, for example, 1 to 10 bases, for example, 1 to 5 bases, for example, 1 to 3 bases.
The designed crRNA and tracrRNA can be prepared by chemical synthesis or the like. The crRNA and tracrRNA may be prepared as RNA, directly introduced into a donor cell, or prepared as DNA, incorporated into an expression vector, and introduced into a donor cell in the form of an expression vector to be expressed in the cell.
Specific examples of the gRNA according to
Among them, preferred is a gRNA having the base sequence of any one of SEQ ID Nos. 3, 4, 7, and 45 to 52 as a target base sequence, or a gRNA having a base sequence in which 1 or more bases are deleted, substituted, or added at the 5' -end of the base sequence of any one of SEQ ID Nos. 3, 4, 7, and 45 to 52 as a target base sequence. In examples, as described below, the gRNA having these base sequences as the target base sequence can destroy or alter HLA alleles specifically for HLA haplotypes, thereby producing low-antigenic cells with reduced rejection when allogeneic transplantation is performed on a recipient.
(embodiment 2)
In one embodiment, the present invention provides a gRNA having, as a target base sequence, a base sequence that is mapped to two or more target HLA haplotypes when mapping base sequence data of genomic DNA of a full HLA haplotype and is not mapped to base sequence data of full genomic DNA other than HLA alleles.
Specific examples of the gRNA according to
Among them, a gRNA having the base sequence of any one of SEQ ID Nos. 53 to 55 as a target base sequence, or a gRNA having a base sequence in which 1 or more bases are deleted, substituted or added at the 5' -end of the base sequence of any one of SEQ ID Nos. 53 to 55 as a target base sequence, is preferable.
As described above, in order to improve the transcription efficiency from the polymerase III promoter, the base sequence of the 5' -end of the gRNA may be changed to G or GG.
For example, when focusing on the HLA-A allele, 2 genes, i.e., a paternal HLA-A gene and a maternal HLA-A gene, are present in the HLA-A allele, and the HLA-A genes may be different from each other or the same. When it is desired to maintain the antigen-presenting ability of HLA-A, it is necessary to cleave only one HLA-A gene to induce knockdown and leave the other HLA-A gene. Alternatively, when a complete knock-out is to be induced, both HLA-A genes need to be cleaved.
In general, when 2 genes are targeted for CRISPR-grnas, 2 grnas are usually designed to induce DNA cleavage, respectively. Therefore, it is considered that grnas specific to individual HLA genes are used when knocking out a plurality of HLA genes.
On the other hand, in the examples described below, the inventors of the present invention, utilizing the feature that HLA genes have high DNA sequence identity to each other, searched for grnas that can induce multiple HLA genes (for example, both a paternal-derived sequence and a maternal-derived sequence of an HLA-a gene, or an HLA-a gene and an HLA-B gene) by cleavage with 1 gRNA, and identified multiple such gRNA sequences.
In examples, as described below, by using the gRNA according to
[ other embodiments ]
In one embodiment, the present invention provides an HLA protein expression-inducing agent for inducing expression of an HLA protein while maintaining pluripotency of pluripotent stem cells, which comprises IFN-. gamma., IFN-. alpha., IFN-. beta., IL-4, GM-CSF, TGF-. alpha.or TGF-. beta.as an active ingredient.
In one embodiment, the present invention provides a method for inducing expression of an HLA protein while maintaining pluripotency of a pluripotent stem cell, comprising the step of adding IFN-. gamma., IFN-. alpha., IFN-. beta., IL-4, GM-CSF, TGF-. alpha.or TGF-. beta.to a culture medium for the pluripotent stem cell.
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