BTK gene repair by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector

Research Project

Project/Area Number	26860811
Research Category	Grant-in-Aid for Young Scientists (B)
Allocation Type	Multi-year Fund
Research Field	Pediatrics
Research Institution	Kyushu University
Principal Investigator	YAMAMOTO Hiroyuki 九州大学, 環境発達医学研究センター, 学術研究員 (00710170)
Project Period (FY)	2014-04-01 – 2016-03-31
Project Status	Completed (Fiscal Year 2015)
Budget Amount *help	¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000) Fiscal Year 2015: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000) Fiscal Year 2014: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Keywords	遺伝子変異修復 / アデノ・アデノ随伴ウイルスハイブリッドベクター / BTK遺伝子 / CD34陽性細胞 / CD34陽性造血幹細胞
Outline of Final Research Achievements	We constructed a new helper-dependent adenovirus/adeno-associated virus hybrid vector (HD-Ad.AAV hybrid vector) that is composed of the genomic sequence containing BTK exons 2-19, and GFP/puromycin resistant gene. Using this HD-Ad.AAV hybrid vector and CRISPR-Cas9, we found that the targeting in the BTK gene occurred in 10 among 24 hygromycin-resistant BFU-E colonies differentiated from the cord blood CD34+ cells.　The estimated targeting efficiency was 1.8 x10-5 per cell. Our study shows the possibility to perform gene repair of BTK gene by homologous recombination in hematopoietic stem cells using this HD-Ad.AAV hybrid vector and CRISPR-Cas9 system.

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