| Project/Area Number |
05557033
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
内科学一般
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| Research Institution | NAGOYA UNIVERSITY |
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Principal Investigator |
SEO Hisao RES.INT.ENVIRON MED.PROFESSOR, 環境医学研究所, 教授 (40135380)
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| Co-Investigator(Kenkyū-buntansha) |
SATOH Yuichiro BASICRES.LAB.TORAY INDUST.INC.CHIEF INVESTIGATOR, 創薬第二研究室, 主任研究員
KAKUMU Shinichi SCHOOL OF MED.LECTURER, 医学部, 講師 (10115545)
YOSHIDA Jun SCHOOL OF MED.PROFESSOR, 医学部, 教授 (40158449)
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1993: ¥4,300,000 (Direct Cost: ¥4,300,000)
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| Keywords | HEPATITIS B / GENE THERAPY / LIPOSOME / INTERFERON / ASIARO-GLYCOPROTEIN / ウイルス性肝炎 / 遺伝子療法 / アシアロ糖蛋白 / 肝細胞培養 / リポゾーム |
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| Research Abstract |
1. INHIBITION OF HEPATITIS beta VIRUS REPLICATION BY INTRODUCTION OF INTERFERON-BETA EXPRESSING PLASMID
In developing gene therapy to treat the hepatitis B,we introduced plasmid expression human interferon-beta to a hepatoma cell line producing the hepatitis B virus. The cell line was established by transfecting a human hepatoma cell line, HepG2 using pHBV-3 containing the HBV genome. A liposome-mediated introduction of the interferon-beta expressing plasmid resulted in the secretion of interferon-beta into the medium at constnt levels for at least 6 days (30-40 IU/ml). However, exogenously added human interferon-beta (1,000 IU/ml) deteriorated rapidly and became undetectable after 3 days. Viral replication was assessed by determining the levels of the hepatitis B antigen in the culture medium. Introduction of the plasmid proved to markedly inhibit viral replication, while exogenously added human interferon-b caused almost no inhibition. It is thsu indicated that continuous productiono
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finterferon-beta by its gene delivery inhibits hepatitis beta viral replication much more efficiently than does an exogenous administration of linterferon-beta.
2. HEPATOCYTE SPECIFIC GENE DELIVERY
To establish gene therapy for viral hepatitis by introducing interferon-beta expressing plasmid, it is mandatory to develop hepatocyte specific gene delivery. First we evaluated the usefulness of gene delivery via asialoglycoprotein receptor which is specifically expressed on the plasma membrane of the hepatocyte. Asialofetuin or asialo-alpha1-acid glycoprotein was conjugated with poly-L-lysine which binds with plasmid DNA due to its positive charge. A firefly luciferase-expressing plasmid was incubated with each of the conjugate and introduced into two forms of primary cultured rat hepatocytes or HepG2 cells. Efficiency of the gene transfer was assessed by the determination of luciferase activity. An inhibitor of the lysozomal enzyme chloroquine was required for efficient gene transfer. As a ligand for the receptor, asialo-alpha1-acid glycoprotein was more efficient than asialofetuin. Rapidly growing cells (HepG2 cells and monolayr cultured rat hepatocytes) were readily transfected while non-grwing rat hepatocytes in shperoid culture could not be transfected easily, suggesting that cell division in required for gene transfer. The transfected cell population was less than 10%. To increse the efficiency of transfection, luciferase reporter gene was introduced into replication defective adenovirus vector. The recombinat virus infected almost 100% of the hepatocytes either growing or not growing. When injected to the tail-vein fo the mouse, luciferase activity was detected in liver, kidney, lung and muscles, while the activity in liver was 10 times more than that in the rest of organs. It is thus demonstrated that adenovirus vector could be used for hepatocyte specific gene delivery when coupled with promoter region which define hepatocyte specific expression. Less
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