2000 Fiscal Year Final Research Report Summary
Gene Therapy for Sly disease using AAV vector.
| Project/Area Number |
11670790
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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 |
Pediatrics
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| Research Institution | The Jikei University School of Medicine |
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Principal Investigator |
OHASHI Touya Jikei Univ., Dept.of Pediatrics assi prof., 医学部, 講師 (60160595)
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| Co-Investigator(Kenkyū-buntansha) |
岩澤 京子 東京慈恵会医科大学, 医学部, 助手 (10301524)
IDA Hiroyuki Jikei Univ., Dept.of Pediatrics assi prof., 医学部, 講師 (90167255)
ETO Yoshikatsu Jikei Univ., Dept.of Pediatrics prof., 医学部, 教授 (50056909)
KOBAYASGI Hiroshi Jikei Univ., Dept.of Pediatrics senior investigator, 医学部, 助手 (90266619)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Gene Therapy / AAV / MPS VII / Sly disease |
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| Research Abstract |
We generated adeno-associate vector which express human beta-glucuronidase. Genzyme generously provided us AAV plasmid vector and helper virus. Using this system, full length human beta-glucuronidase cDNA was inserted to AAV expression plasmid, pNT C3CMV.Resultant plasmid was cotransfected with helper virus, p5rep-CMVcap to 293 cells. At the same time we infect temperature sensitive adenovirus, Ad5ts149 to 293 cells. After 48 hours culture at 39℃, the cells and medium was collected and recombinant virus was purified using sulfated sepharose column chromatography. We infected this purified virus to the skin fibroblasts cultivated from MPS VII mice. The activity of beta-glucuronidase increased in the infected fibroblasts. We also looked at transdution efficiency by histochemical staining of beta-glucuronidase activity in cells. More than 60% cells expressed human beta-glucuronidase. This observation suggest that our AAV vector efficiently infect Cosl cells in vitro. As an next step, we injected this virus to muscle of MPS VII mice. The enzymatic : activity increased in serum up to 20 % of normal serum. We are currently investigating the improvement of pathology of various organs from MPS VII.
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