| Project/Area Number |
17590628
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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 |
Gastroenterology
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| Research Institution | The University of Tokyo (2007)
Gifu University (2005-2006) |
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Principal Investigator |
KIMURA Kiminori The University of Tokyo, Department of Immunotherapeutics,, Assistant professor (70397339)
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| Co-Investigator(Kenkyū-buntansha) |
MIYAGISHI Makoto The University of Tokyo, 21st Century COE Program, School of Medicine, Associate professor (30323538)
MORIWAKI Hisataka Gifu University school of Medicine, Dpearment of Gastroenterology, Professor (50174470)
NAGAKI Masahito Gifu University school of Medicine, Dpearment of Gastmenterology, Associate professor (30293559)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,580,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥180,000)
Fiscal Year 2007: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Hepatitis B virus / small interference RNA / antiviral drug / RNA technology / 創薬 / マウス / small interference RNA / RNA工学 / ウイルス複製 |
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| Research Abstract |
RNA interference (RNAi) is an evolutionarily conserved phenomenon in which gene expression is silenced by double-stranded RNA (dsRNA) in a sequence-specific manner. In case of mammalian cells, siRNA can be triggered by 21-nucleotide duplexes of short interfering RNA (siRNA). We examined effects of siRNA on hepatitis B virus (HBV) replication. We used HBV replicative hepatocyte cell lines (HBV-Met), in which hepatocytes were derived from the livers of HBV-transgenic mice that expressed the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met). These cell lines are well-differentiated, replicate HBV well. We designed 4 different siRNA expression vectors against HBV and determined whether siRNA system is effective for inhibition of HBV replication. First, transfection experiments demonstrated that the two of four siRNA expression vectors reduced the amount of HBV-pregenome RNA and resulted in reduction of the levels of replicative intermediates. W
… More
e also confirmed reduction of Hbe concentration in the culture media after siRNA expression vectors were transfected. Using Southern blot analysis, the level of open circular and single-stranded HBV-DNA, which is considered as an intracellular replicative intermediate, was clearly suppressed by siRNA vectors. As a next step, we tried to evaluate whether administration of these two siRNA vectors can reduce HBV RNA expression in the liver. For the purpose of in vivo experiment, we used HBV transgenic mice (107-5) which HBV envelope protein express in the hepatocytes. After 100 μg siRNA vectors were injected into HBV transgenic mice by hydrodynamic injection, we analyzed HBV RNA expression in the liver by Northern blot and real time PCR method. Nevertheless siRNA vectors were effective on HBV down-regulation in vitro system it did not work for in vivo experiment since reduction of HBV RNA expression in the liver was not clear. Thus, in this project we concluded that improvement of siRNA delivery system into the liver is necessary for therapeutic strategy. Less
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