OGATA Yoshiki TERUMO Corporation R & D Center Researcher, 研究開発センター, 研究員
UCHIYAMA Hideki TERUMO Corporation R & D Center Research Specialist, 研究開発センター, 専門研究員
SHIMADA Takashi Nippon Medical School Department of Biochemistry and Molecular Biology Professor, 教授 (20125074)
ENDO Fumio Kumamoto University School of Medicine Department of Hospital Lecturer, 医学部・附属病院, 講師 (00176801)
YAMAMOTO Tetsuro Kumamoto University Graduate School of Medical Sciences Division of Molecular Pa, 医学部, 教授 (60112405)
内山 秀樹 テルモ(株)研究開発センター, 専門研究員
犬童 康弘 熊本大学, 医学部・附属病院, 助手 (40244131)
|Budget Amount *help
¥17,400,000 (Direct Cost: ¥17,400,000)
Fiscal Year 1996: ¥7,300,000 (Direct Cost: ¥7,300,000)
Fiscal Year 1995: ¥10,100,000 (Direct Cost: ¥10,100,000)
1) non-viral gene transfer
The issue of liposome is generally of too low efficiency to be used as vector. We developed several new liposome vectors, which were modified on surface of multilameral vesicle (TRX series #9,13,20) in order to obtain higher transfection efficiency of transgenes. For evaluating efficiency of the TRX,Lipofectin and Transfectin, which are commercialty avalable and popular in many laboratories, were used as references. Three different TRX liposomes, which havor beta galactosidase, human OTC gene or the responsible gene for Wilson disease (Wdeu) under CAG promotor, were tested in cultured cells including Cos1, HepG2, and established liver cells from LEC rat (animal model of Wilson disease). Transfection efficiency of TRX vectors (#9,13 and 20) was all 5-10 times higher than those of Lipofectin and Transfectin, and farthermore, the espession was increased in the presence of 20% of serum in TRX vectors, but was reduced in Lipofectin. This is an advantage of TRX vector when in vitro study is designed.
TRX vector harboring human OTC gene and Wilson gene was injected into OTC deficient mice and LEC rat through tail vein, respectively. Expression of the transgene was found only in kupffer cells but not in hepatocyte. However, when injected those in liver tissue directly, some expression was observed even in hepatocyte. Thus, some more modification of surface of TRX are necessary, and, if achieved, availability of TRX in vitro study will be much progressed.
2) Virus vector
Adenovirus vector Adex CAG hOTC,which harbor human OTC gene under CAG promoter was injected into OTC deficient mice, and normalization of OTC activity was evident for 60 days after the injection. AAV vector harboring the same gene is now studying.
3) Though the development of vector is not enough to apply for human therapy, we learned more about the issue for transfection of gene, based on which further study should be continued.