2021 Fiscal Year Research-status Report
CRISPR/Cas9-mediated genome-editing of long-term hematopoietic stem cells
| Project/Area Number |
21K15872
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| Research Institution | Jichi Medical University |
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Principal Investigator |
スブド ビャンバー 自治医科大学, 医学部, 助教 (90834193)
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| Project Period (FY) |
2021-04-01 – 2023-03-31
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| Keywords | CRISPR/Cas9 / Hematopoietic stem cell / Genome-editing / NHEJ / HDR / SCID |
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| Outline of Annual Research Achievements |
First-line treatment for the most of genetic blood disorders is allogenic hematopoietic stem cell (HSC) transplantation, which only available for ~30% of patients. Gene therapy and targeted genetic correction in HSCs could possibly cure the diseases as alternates, and the preliminary results of genome-editing in HSCs are considered promising with the emergence of CRISPR/Cas9. We aimed to (1) expand long-term repopulating HSCs (LT-HSCs) to achieve more genome-editing with higher repopulation ability and (2) develop a method for homologous-direct repair (HDR) in HSCs.
First, we enriched mouse LT-HSCs by sorting Lineage marker negative (Lin-), cKit+, Sca1+, CD150+, and CD34- cells from bone marrow, and expanded using PVA-containing media. LT-HSCs were expanded to XXX times in YYY days. Second, we optimized HDR-based genome-editing in expanded LT-HSCs. For the optimization, we chose Ptprc (CD45) as the target. Only a single amino acid polymorphism causes CD45.1 and CD45.2, which are detectable with selective antibodies. After the optimization of the delivery method of Cas9 ribonucleoprotein (RNP) and single stranded DNA repair template to LT-HSCs, we achieved ~10% of conversion from CD45.2 to CD45.1. We then transplanted the genome edited HSCs. After the engraftment, we observed only ~1% of donor HSC-derived peripheral blood cells was CD45.1.
The current study demonstrated that precise genome editing of expanded LT-HSCs is feasible, however, further improvement is necessary for the realization of this method to cure genetic blood disorders.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
I hypothesized that (1) quiescence of long-term HSCs (rarely proliferate) limits the HDR activity in long-term HSCs, hence, (2) re-activation of the cell cycle in long-term HSCs may enhance HDR and increase the number of repaired HSCs, simultaneously. I have the following 2 aims. Specific aim 1) To develop HDR-editing in long-term HSCs by CRISPR/Cas9. Specific aim 2)To develop reactivation of the cell cycle in long-term HSCs to enhance HDR and increase the number of repaired HSCs. The followings are sub-aims for each specific aim.
1-1.Ex vivo expansion of long-term HSCs: KSL CD150+ CD34- HSCs expanded 50 times after 2 weeks of culture PVA-based Dr.Yamazaki’s medium and expanded mouse long-term HSCs were engraftable in NOG mice.
1-2. Comparison of HDR vs. NHEJ editing efficiency in long-term HSCs: 5 to 40% HDR-mediated editing and 20-50% NHEJ-mediated editing in vitro
2-1. Re-activation of the cell cycle in long-term HSCs: Treatment of mTOR and GSK-3 inhibitor media can keep long-term HSCs in more quiescence stage compared with no treated cells. Moreover, the treatment improved the HDR-mediated genome-editing 2-fold in long-term HSCs. ssODN-AltR (IDT) modified ssODN improved HDR efficiency from ~5% to 10-20%.
2-2. To analyze engraftment of genome-edited long-term HSCs after transplantation:
We are middle of this experiment. Preliminary results showed that edited long-term HSCs are engraftable after transplantation, the result showed 0.1-1% HDR-edited CD45.1 cells, and 15-30% NHEJ~edited cells in vivo.
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| Strategy for Future Research Activity |
In future we are planning to work more on specific aim two.
We are planning to do following experiments:
1) Optimization of in vitro HDR-mediated genome-editing of Expanded / Edited / Engraftable (3E) HSCs. We are successfully expanded the long-term HSCs, and we could see efficient genome-editing in long-term HSCs. However we need to work more on making engraftable HSCs.
2) Need to further improve in vivo HDR editing efficiency and transplantation setting. We are planning to set up the C57BL/6J (CD45.2) to C57BL/6J-GFP mouse transplantation setting. Further, we are planning to compare GSK and mTOR treated or none treated+edited HSCs transplantation.
3) Disease modeling and therapeutical approach is essential for the future studay. Thus we want to set up the experimental design for X-SCID long-term HSCs editing in vitro and in vivo.
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| Causes of Carryover |
Due to COVID-19 situation, could not attend to the planned meetings. And will attend next fiscal year.
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Research Products
(3 results)
