| Project/Area Number |
21K15872
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 52050:Embryonic medicine and pediatrics-related
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| Research Institution | Jichi Medical University |
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Principal Investigator |
Byambaa Suvd 自治医科大学, 医学部, 客員研究員 (90834193)
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| Project Period (FY) |
2021-04-01 – 2023-03-31
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| Project Status |
Completed (Fiscal Year 2022)
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| 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)
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| Keywords | CRISPR/Cas9 / Hematopoietic stem cells / HDR / Genome-editing / beta-glucan / mTOR inhibitor / GSK inhibitor / Engraftment / Hematopoietic stem cell / NHEJ / SCID |
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| 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.
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| 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.
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| 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.
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