| Project/Area Number |
10470213
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | Jichi Medical School |
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Principal Investigator |
OZAWA Keiya Jichi Medical School, Faculty of Medicine, Professor, 医学部, 教授 (30137707)
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| Co-Investigator(Kenkyū-buntansha) |
URABE Masashi Jichi Med. Sch., Faculty of Medicine, Research Associate, 医学部, 助手 (40213516)
KUME Akihiro Jichi Med. Sch., Faculty of Medicine, Assistant Professor, 医学部, 講師 (10264293)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥10,300,000 (Direct Cost: ¥10,300,000)
Fiscal Year 1999: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1998: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Keywords | Gene theray / Gene transfer / Integration / AAV / AAVSI locus / Rep / ITR / TVI method |
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| Research Abstract |
Targeted integration of foreign DNA (TVI:targeted vector integration) is desirable for safe gene therapy. Adeno-associated virus (AAV) has the ability to integrate its genome into a defined locus, AAVS1 (19q13.3-qter). The inverted terminal repeat (ITR) at both ends of the AAV genome and Rep proteins are responsible for this site-specific integration. Therefore, two AAV components, Rep and ITR, are utilized to develop the TVI system. A mutant Rep protein that lacks cytotoxicity while retaining the ability to mediate AAVS1-specific integration is an attractive molecule for this system. We constructed plasmids containing the genes encoding mutant Rep proteins, in which all the charged amino acids in the N-terminal region were mutated to alanine. We found several mutants showed lower cytotoxicity, compared to the wild type Rep protein, without losing the ability of the site-specific integration. We are further screening a more suitable mutant Rep for the AAVS1-directed integration of genes. To analyze detailed mechanism of Rep-mediated integration, we amplified junctional regions between cellular and transgene sequences by using an Alu-PCR technique. The TVI system is valuable especially when dividing cells such as hematopoietic cells are transduced and long-term transgene expression is needed. We have showed that this TVI system could introduce a transgene into AAVS1 in K562 cells.
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