Despite growing successes associated with whole organ liver transplantation, surgical risks and complications contribute significantly to patient morbidity and mortality. Hepatocyte transplantation could be used to treat acute liver failure and liver-based metabolic diseases and would avoid surgical intervention and its associated risks. A potential alternative to the transplantation of primary hepatocytes would be the use of a clonal cell line. An hepatocyte cell line would provide the advantages of availability, uniformity and sterility and could be grown in unlimited quantity and at far less cost compared to isolated primary hepatocytes. This proposal will examine whether hepatocytes can be conditionally immortalized and engineered to be non-tumorigenic when transplanted in to recipients for the treatment of liver-based metabolic diseases and liver failure. To test this hypothesis eukaryotic expression vectors will be constructed which control expression of the SV40 large T antigen at the level of both transcription and translation. Vectors will contain inducible promoters and operator/repressor sequences. Primary hepatocytes will then be immortalized by transfection with these constructs and cells will be characterized at the permissive and non-permissive conditions for gene expression to determine their level of differentiated function. Regulation of gene transcription and translocation under the various conditions will be evaluated by measuring SV40 T antigen mRNA and protein production and cell growth will be assessed in vitro and following transplantation in immunodeficient mice. In order to provide one more way to protect against possible abnormal cell growth in recipients, conditionally immortalized cells will be transduced to express a suicide gene. Introduction of the Herpes Simplex Virus - thymidine kinase gene will provide a way of eliminating the transplanted cells if desired by treatment with gancyclovir. Conditionally immortalized hepatocytes will then be transplanted into Nagase aalbuminemic rats and rats with ammonium acetate inducible hepatic coma to assess the ability of these cells to correct deficiencies in liver function in vivo. In summary, these studies will determine whether conditionally immortalized hepatocyte cell lines can be engineered to be non-tumorigenic and safe for use in the treatment of metabolic and global liver deficiencies.
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