CRISPR/Cas9-mediated genome-editing of long-term hematopoietic stem cells

Research Project

Project/Area Number	21K15872
Research Category	Grant-in-Aid for Early-Career Scientists
Allocation Type	Multi-year Fund
Review Section	Basic Section 52050:Embryonic medicine and pediatrics-related
Research Institution	Jichi Medical University
Principal Investigator	Byambaa Suvd 自治医科大学, 医学部, 客員研究員 (90834193)
Project Period (FY)	2021-04-01 – 2023-03-31
Project Status	Completed (Fiscal Year 2022)
Budget Amount *help	¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000) Fiscal Year 2022: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000) Fiscal Year 2021: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Keywords	CRISPR/Cas9 / Hematopoietic stem cells / HDR / Genome-editing / beta-glucan / mTOR inhibitor / GSK inhibitor / Engraftment / Hematopoietic stem cell / NHEJ / SCID
Outline of Research at the Start	In the proposed study, I will develop HDR-edited long-term HSC for autologous transplantation to cure X-SCID in the mice. In the future, this approach can be easily converted to the genome-editing of human true HSC and translate to the large animal study.
Outline of Final Research Achievements	To improve in vitro HDR editing efficiency of expanded/edited long-term HSCs, we tested first, 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. Second, 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.
Academic Significance and Societal Importance of the Research Achievements	Genome-edited HSCs engraftment capability is an important point for successful HSCs transplantation. This work shows the ex vivo treatment of beta-glucan before the genome-editing process has protective effects for genome-edited HSCs to survive in the recipient body after transplantation.