| Project/Area Number |
14370307
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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 Kaiya Jichi Medical School, Faculty of Medicine, Professor, 医学部, 教授 (30137707)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUSHITA Takashi Jichi Med.Sch., Faculty of Medicine, Research Associate, 医学部, 助手 (20343444)
OKADA Takashi Jichi Med.Sch., Faculty of Medicine, Research Associate, 医学部, 助手 (00326828)
MIZUKAMI Hiroaki Jichi Med.Sch., Faculty of Medicine, Assistant Professor, 医学部, 講師 (20311938)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥14,200,000 (Direct Cost: ¥14,200,000)
Fiscal Year 2003: ¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 2002: ¥7,700,000 (Direct Cost: ¥7,700,000)
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| Keywords | AAV vector / tissue specificity / protein-supplement gene therapy / targeted vector integration / Rep / ITR / adenoviral vector / adaptor molecule / エリスロポエチン / 凝固因子 / 免疫反応 / 遺伝子治療 / 遺伝子導入 / 血清型 / AAVS1領域 / TVI法 |
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| Research Abstract |
AAV (adeno-associated virus) vectors are considered to be promising gene-delivery vehicles for gene therapy, because they are derived from non-pathogenic virus and efficiently transduce non-dividing cells such as muscles, hepatocytes and neurons. Recently, 5 distinct serotypes have been characterized and developed as AAV vectors. In this study, we investigated the level of transgene expression by comparing different serotypes as well as promoters, aiming at designing optimal AAV vector structure for protein-supplement gene therapy. As a result, the combination of serotype 1 and CMV promoter was most efficient for muscle-mediated expression, and the combination of serotype 5 and CAG promoter was the best for liver-mediated expression. No pathological change was observed at the vector injection sites. Another project is the development of TVI (targeted vector integration) technology using the two AAV-derived components [ITR (inverted terminal repeat) sequence and Rep proteins]. With this method, a gene of interest will be integrated site-specifically into the AAVS 1 locus on chromosome 19 (19q13.3-qter). Since Rep proteins are cytotoxic, inducible Rep-expression adenoviral vector system was constructed using a Cre-loxP switching element. The feasibility of this system was demonstrated by model experiments using plasmid transfection. In addition, we have developed an adaptor molecule (CAR-SCF fusion protein) that bridges adenoviral fibers and the c-Kit receptor to alter adenoviral tropism to immature hematopoietic cells, since these cells do not express the coxsackie-adenoviral receptor (CAR). The addition of CAR-SCF in the culture medium enhanced adenoviral vector-mediated gene transfer into MO7e cells. The TVI technology will also be applied to the genetic manipulation of human ES cells in the future.
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