Improvements in genome editing technologies for treating a marmoset model of human inherited blood disorder
| Project/Area Number |
16K08642
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Research Field |
Human genetics
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| Research Institution | Saitama Medical University |
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Principal Investigator |
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| 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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| Keywords | 遺伝子治療 / ゲノム編集 / 免疫不全症 / ウイルスベクター / 疾患モデルマーモセット |
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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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| Academic Significance and Societal Importance of the Research Achievements |
疾患モデルマーモセットの原因遺伝子であるIL2RG遺伝子エクソン3のDNA変異部位の近辺を標的とする、効率の良いCRISPRと標的配列の同定と、AAVベクターのデザインに成功した。そして実際にin vitroで、ヒトとマーモセットでの造血幹前駆細胞での遺伝子修復に成功した。まだ治療レベルから低く数倍の効率の改善が望まれるが、一度達成されれば世界初の霊長類を用いたゲノム編集治療の治療効果ならびに安全性の解析を行うことが可能となる。
また、修復用ドナーDNAのランダムな染色体部位への組込みを防ぐためのネガティブ選択法も、方法論の確率まであと一歩の所に到達している。
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Report
(4 results)
Research Products
(4 results)
