| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2018: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2016: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
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| Outline of Final Research Achievements |
With the goal of establishing a strategy for safe and efficient genome editing therapy in a marmoset model of immunodeficiency, we identified the optimal combination of Cas protein and gRNA targeting near the mutation site in the IL2RG gene. The resultant CRISPR complex was delivered together with a donor AAV vector for homology-directed repair at the mutation site in CD34 hematopoietic progenitor cells. Gene repair via gene editing is expected to knock-in the Venus reporter gene precisely into the mutated exon 2 of the locus. The gene repair efficiency in the cell population was about 40%. When the efficiency was measured in more primitive colony-forming progenitors on methylcellulose, the efficacy was about 20% and 10% in human and marmoset cells, respectively. We also designed a novel negative selection reporter using the iCaspase 9 gene in order to avoid the integration of the donor AAV vector at undesired random chromosomal sites.
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