2019 Fiscal Year Annual Research 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 |
We aimed to generate ‘Universal’ iPS cells which bear no intrinsic HLA signature. Our strategy used doxycycline(dox)-regulated epigentic repression (CRISPRi) of B2M to reversibly “cloak” (hide) cells from the immune system. We generated a new Tet-OFF system which is 10,000-fold more sensitive to dox than Tet-ON. Just 0.1 ng / mL dox is sufficient for HLA recovery. Moreover, we proved that a single-copy sgRNA at the AAVS1 locus is sufficient to repress HLA, even in the presence cytokine (IFN-gamma) stimulation. We evaluated Tet-OFF iPS cell differentiation. We found that endoderm and ectoderm maintained the CRISPRi system, but mesoderm showed a loss of expression. We hypothesized that mesodermal cells would be unable to maintain B2M repression, and would become HLA-ABC+. We differentiated Tet-OFF iPS cells to CD45+ hematopoietic cells and found they were HLA-ABC negative. Although the B2M promoter is not normally methylated in iPS cells or CD45+ cells, we found that, unlike de-repressed (+ dox) iPS cells, the B2M promoter of CD45+ cells was highly methylated. In conclusion, we created a novel CRISPRi system which is reversible and sensitive, avoiding the need for chronic, high-dosage dox treatment, making it suitable for in vivo applications. We made a new finding that epigenetic changes caused by dCas9-KRAB may persist through differentiation, and suggest a potentially valuable approach to permanently regulate gene expression without genetic mutation. We submitted our findings to be considered for publication as an article in Nature Biomedical Engineering.
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Research Products
(5 results)
