| Project/Area Number |
11670631
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurology
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| Research Institution | Kumamoto University |
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Principal Investigator |
UCHINO Makoto Department of Neurology, Kumamoto University Hospital Professor, 医学部・附属病院・神経内科, 教授 (20117336)
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| Co-Investigator(Kenkyū-buntansha) |
MAEDA Yasushi Department of Neurology, Kumamoto University Hospital physician, 医学部・附属病院・神経内科, 医員
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Gene therapy / gutless adenovirus / Cre / loxP system / recombinant adenovirus vector / skeletal muscle / repetitive transduction of therapeutic gene / coxsackie B and adenovirus receptor / 遺伝子治療 / loxPシステム / 新世代のアデノウイルスベクター / 免疫原性 / hCAR / 反復遺伝子導入 / 遺伝子導入効率 / ヘルパーウイルス依存型アデノウイルス |
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| Research Abstract |
We successfully produced and purified a new generation adenovirus vector (gutless adenovirus) aiming for gene therapy by using Cre/loxP system. This new generation adenovirus is helper-dependent adenovirus and lack virtually almost all of the wild adenoviral DNA.Instead of the deletion it was replaced to non-viral DNA longer than 30kb. After the repetitive propagations viral titer increased. Then propagated vectors could be condensed by CsCl buoyant density centrifugation to 1000-fold higher than that of crude lysate.
And we optimized the structure of packaging DNA and the structure of helper virus for a robust system capable of producing high-titer gutless adenovirus stocks with low helper contamination.
Furthermore, we constructed a gutless adenovirus containing the expression units of 16.5kb-long mouse full-length dystrophin and 2kb-long human Coxsackievirus and adenovirus receptor (CAR). By using this vector we could transduced the expression of dystrophin in tibialis anterior skeletal muscles of mdx-mouse in vivo.
As this vector is very promising for gene therapy, long-term gene expression and activation of immunity must be carefully studied for safe clinical application.
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