| 研究実績の概要 |
The main cause of human genetic blood disorders is mutations in hematopoietic stem cells (HSCs). Allogeneic HSC transplantation is the first-line treatment for many genetic blood disorders. However, HSC transplantation is accessible to only up to 30% of patients. Thus, targeted genome repair in long-term HSCs, is considered to be a reliable curative treatment with the emergence of CRISPR/Cas9 gene editing technology. To develop effective targeted genome repair in long-term HSCs, we proposed the following two aims: 1) To develop ex vivo expansion of long-term HSCs for genome editing 2) Improvement the strategy of in vitro or in vivo HDR editing efficiency of expanded/edited long-term HSCs. Detailed experimental results are as follow. First, to improve HDR-mediated editing in LT-HSCs, we tested 1) GSK and mTOR inhibitors which are known to support the maintenance and self-renewal of HSCs. Treatment with two inhibitors, HDR-mediated editing improved from ~5% to 10-30% in vitro; from ~1% to ~10% in vivo 2) Modified single-strand DNA template to conjugate the template to Cas9, and it showed 5-20% in vitro; ~5% in vivo after serial transplantation. Further, we established and optimized the method to improve the edited cell engraftment by using beta-glucan. Our result showed that beta-glucan protects HSCs and improves their engraftment ability. These results showed that ex vivo expanded and HDR-edited cells are capable to repopulate HDR-edited multilineage cells after transplantation, demonstrating that precise genome editing of expanded long-term HSCs is feasible.
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