| 研究実績の概要 |
Human leukocyte antigens (HLA) are highly polymorphic gene loci that encode cell-surface glycoproteins which are the strongest transplant antigens leading to T-cell activation, antibody production, and allograft rejection. Chronic immunosuppressive regimens are often used to avoid transplant rejection, but are associated with drug toxicity and an increased risk for opportunistic infections and malignancies. Although personalised iPS cells hold great promise as cellular therapies by autologous transplantation, on-demand generation or banking of personal iPS cells to treat all patients is neither technically nor economically feasible. On the other hand, ‘Universal’ iPS cells, which bear no intrinsic HLA signature, would avoid immune rejection and have unrestricted application. To generate such ‘Universal’ iPS cells, we initially aimed to target the gene encoding B2M, which is required for HLA-A,B,C presentation on the cell surface. We used two strategies, either CRISPR-Cas9 nuclease for B2M knockout by random indel formation or drug-regulated KRAB-dCas9 for reversible B2M knockdown by epigentic repression (CRISPRi). Using the CRISPRi strategy, we aim to “cloak” (hide) cells from the immune system, with the option to reactivate gene expression and “reveal” transplanted cells to the host immune system as a safety net in the prevention of graft malfunction or tumorigenesis. We have identified Cas9 guide RNAs (gRNAs) which achieve >95% B2M knockdown by qPCR and FACS, and are currently verifying these results in a second iPS cell line.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
We prepared control iPS cell lines, where the common HLA cI subunit B2M was targeted for knockout using conventional CRISPR-Cas9 nuclease and a gRNA computationally evaluated for low off-target cleavage potential. Clones were selected where both B2M alleles were verified to be mutated by the same indel. These B2M-null iPS cell lines were used to establish conditions for flow cytometry analysis of cell surface expression of HLA class I heavy chains (antibody W6/32, binding HLA-A, -B, and -C) on undifferentiated iPS cells. For reversible B2M knockdown, we used an iPS cell line containing a tet-ON dCas9-KRAB transgene inserted at the AAVS1 (PPP1R12C) safe-harbor locus by gene targeting. We selected and tested 6 gRNAs targeting the transcription start site of B2M, and found 2 gRNAs that achieved > 95 % knockdown, even when B2M expression was boosted by IFN-g treatment. The specificity of KRAB-dCas9 for B2M knockdown, has been verified by qPCR for B2M, TAP1, Nanog, and GAPDH. Construction of a tet-OFF KRAB-dCas9 system for AAVS1 targeting has been completed.
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| 今後の研究の推進方策 |
In FY30, we will perform a detailed analysis of dox application and withdrawal to verify the dynamics of KRAB-Cas9 expression as well as B2M repression and de-repression over time. We will target the new tet-OFF system to the AAVS1 locus and similarly verify the activity of B2M gRNA activity and repression dynamics. These results will be reproduced in a second iPS cell line known to have a different HLA haplotype. The most effective gRNA will be targeted in single-copy to the second AAVS1 allele, and re-analyzed for B2M knockdown. B2M-null and knockdown cells will be differentiated in order to begin cytotoxic T-lymphocyte assays. Additional effort will be made to confirm local and global gene pleiotropy induced by CRISPRi. Additional CRISPRi targets for reversible immune evasion will be considered.
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| 次年度使用額が生じた理由 |
Screening of functional gRNAs for B2M knockout and knockdown proceeded more quickly than initially expected, reducing cell culture and antibody costs for FY29. We are therefore able to move more rapidly to functional assays in FY30. These assays are resource intensive, requiring iPS cell differentiation and co-culture with cytotoxic T-lymphocytes. Funds carried-over will also be used for genetic and epigenetic analyses to confirm local and global gene pleiotropy induced by CRISPRi.
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