Development of a highly efficient gene targeting strategy using adenovirus-mediated delivery of donor DNA and artificial nucleases

2014 Fiscal Year Final Research Report

• Project/Area Number: 24590405
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Human genetics
• Research Institution: Saitama Medical University
• Principal Investigator: MITANI Kohnosuke 埼玉医科大学, 医学部, 教授 (10270901)
• Co-Investigator(Renkei-kenkyūsha): ISHII Naoto 東北大学, 医学研究科(研究院), 教授 (60291267)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Keywords: 遺伝子ターゲッティング / iPS細胞 / 遺伝子治療 / TALEN / アデノウイルスベクター / 遺伝子修復 / CRISPR / ゲノム編集

Outline of Final Research Achievements

Several strategies are known to enhance gene targeting in mammalian cells. Expression of sequence-specific nucleases, such as TALENs and CRISPRs, introduces DNA double-strand breaks at the target chromosomal sites, thus stimulating homology-mediated DNA repair. As an alternative strategy, delivery of donor DNA by a helper-dependent adenoviral vectors (HDAdV), also reportedly enhances gene targeting efficiencies. In this study, we combined TALEN/CRISPR expression and HDAdV-mediated delivery of donor DNA for gene targeting at the HPRT and the IL2RG loci in human induced pluripotent stem cells. At both loci, the combination of the nucleases and donor HDAdV enhanced the targeting efficiencies by ~2 folds. Delivery of both the nuclease and the donor DNA by a single HDAdV further enhanced the targeting by additional ~2 folds. These results suggest that adenovirus-mediated gene targeting technologies can be further improved in a variety of cells.

Free Research Field

遺伝子治療学