| Co-Investigator(Kenkyū-buntansha) |
KANEDA Yasufumi Institute for Molecular and Cellular Biology, Osaka University, Associate Profes, 細胞生体工学センター, 助教授 (10177537)
NISHIDA Makoto Osaka University Hospital, Medical Staff, 医学部・付属病院, 医員
NOZAKI Shuichi Osaka University Medical School, Assistant Professor, 医学部, 助手 (30252646)
YAMASHITA Shizuya Osaka University Medical School, Assistant Professor, 医学部, 助手 (60243242)
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| Research Abstract |
To investigate the efficiency of gene transfer, we constructed a liposome with the Sendai-virus(HVJ), which has the ability for cell fusion, and the nuclear protein (HMG-l) to make DNA to transfer efficiently into the nucleus. Then the reporter gene (BETA-galaczosidase) was introduced to the liposome, and the construct was injected to the livers of rats and mice via portal vein or directly, resulting in the expression of the reporter gene in 5 to 10% of the hepatocytes for I or 2 weeks. Next, human low density lipoprotein (LDL) receptor gene was introduced to the liposome and transferred to rat liver in vivo using this method. It was found that the human LDL receptor mRNA was expressed in the hepatocytes, and approximately 20% reduction of serum total cholesterol level was observed in vivo. However, immunohistochemical examinations revealed that the expression of the LDL receptor protein was not adequate in the hepatocytes. Thus we exchanged the expression vector from pMy3 to pCL1, whi
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ch is smaller in size (7.5kb) than pMy3 and has chicken BETA-actin promoter, and it was observed that the efficiency of gene transfer was improved. Then we transferred the human LDL receptor gene to Watanabe heritable hyperlipidemic (WHHL) rabbits using this procedure, and found that the human LDL receptor gene was expressed in the hepatocytes and that serum cholesterol levels were decreased about 20% before the gene transfer. However, control rabbits to which only the HVJ-liposome was transferred also showed the similar reduction of serum cholesterol levels, thus we are investigating the mechanism of the expression of the LDL receptor gene in these rabbits by Northern blotting, RNase protection assay and immunohistochemical studies, and examining the mechanism of the reduction of serum cholesterol level by the HVJ-Iiposome. We are also developing the efficient transfer of retrovirus, which has been used for ex vivo gene therapy in the United States, using the HVJ-liposome method. For the future application of the gene therapy to the human, we are going to investigate the transfer of the human LDL receptor gene to the LDL receptor knock out mice and WHHL rabbits. Less
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