| Project/Area Number |
17591112
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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 | Jichi Medical University |
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Principal Investigator |
MATSUSHITA Takashi Jichi Medical University, School of Medicine, Assistant Professor, 医学部, 助手 (20343444)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2006: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Adeno-associated virus / Gene Therapy / AAV / アデノ随伴ウイルス(AAV) / FVIII / 血友病A |
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| Research Abstract |
I constructed AAV-human FVIII BDD (B domain deletion) vector plasmids with p6 promoter of parvovirus B19. Expression level of human FVIII from these vector plasmids are comparable to that were expressed by FVIII Heavy chain/Light chain-Dual vector system plasmids which FVIII was droved by CAG promoters in vitro. AAV1 and AAV9 vectors carrying FVIII BDD gene with p6 promoter were produced by Adenovirus-Free system because these two serotypes were attractive for muscle tissue. These vectors were injected into tibialis anterior muscles of C57BL/6 mice(5 weeks) and blood samples were taken every two weeks. Human FVIII antigen in mice blood was measured by ELISA and neutralizing antibodies were measured by Bethesda method. Unfortunately insufficient expression levels of FVIII were observed in mice which received both 3x10e11 and 6x10e10 g.c. of AAV1-p6 FVIIIBDD vectors and the amount of neutralizing antibodies was gradually increased since second week. To prevent production of neutralizing antibodies, a specific sequence for microRNA which worked in only hematopoietic cells was inserted in 3'-untranslated region of FVIIIBDD gene (based on Nat.Med. 12(5),p585-91,2006). AAV1 or AAV9 vectors of p6FVIIIBDD including this sequence were injected into tibialis anterior muscles of C57BL/6 mice(5 weeks) and blood samples were taken every two weeks. Human FVIII antigen in mice blood was not detected. It would be that this sequence was sensitive for microRNA even if in muscle tissue. The neutralizing antibodies measured by Bethesda method were also not detected because it would be that FVIII antigen was not expressed in muscle tissue.
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