2018 Fiscal Year Research-status Report
Establishment of universal iPS cells for regenerative medicine applications through regulated HLA expression
| Project/Area Number |
17K07256
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| Research Institution | Kyoto University |
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Principal Investigator |
ウォルツェン クヌート 京都大学, iPS細胞研究所, 准教授 (50589489)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | Human leukocyte antigen / HLA / ゲノム編集 / iPS細胞 / CRISPR Cas9 / KRAB / B2M / allograft |
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| Outline of Annual Research Achievements |
Human leukocyte antigens (HLA) are highly polymorphic gene loci that encode cell-surface glycoproteins which are the strongest transplant antigens leading to allograft rejection. 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. To avoid the need for chronic dox treatment, we generated a tet-OFF system with higher sensitivity to dox than the original tet-ON system. Moreover, we prove that a single copy sgRNA targeted to the AAVS1 locus is sufficient to retain HLA-null status, even with cytokine stimulation.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Using the tet-ON CRISPRi iPS cell lines and B2M-null control iPS cells prepared in FY30, we performed a detailed analysis of the dynamics of KRAB-Cas9 expression as well as B2M repression and de-repression over time. We reproduced these results in a second iPS cell line known to have a different HLA haplotype. As chronic, high-level dox treatment is unfavorable for iPS cell growth, differentiation, and eventual in vivo application, we developed an AAVS1-targeted tet-OFF CRISPRi system. Using titration of dox concentrations we found the tet-OFF system to be significantly more sensitive to low-level dox treatment than tet-ON CRISPRi. We targeted the most effective gRNA in single-copy to the second AAVS1 allele, and re-analyzed for B2M knockdown. We found one gRNA copy sufficient to repress B2M expression, even when challenged with IFNg; treatment. Using qPCR we noted minimal pleiotropy on neighbouring gene expression. Additional CRISPRi gene targets have been selected, however held until empirical data on tet-ON and tet-OFF CRISPRi kinetics were established.
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| Strategy for Future Research Activity |
In FY31, we will focus on the differentiation of tet-ON or tet-OFF CRISPRi cell lines along three germ layers. 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 during differentiation. B2M-null and knockdown cells will be differentiated in order to begin cytotoxic T-lymphocyte assays. We will also confirm local epigenetic effects induced by CRISPRi in order to understand the phenomenon of pleiotropy and gene expression reversibility. Additional CRISPRi targets for reversible immune evasion will be considered for testing.
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| Causes of Carryover |
Only a small amount of additional funds will be carried over to FY31, which will be applied to cytotoxicity assays.
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