Retroviral gene transfer into rat liver cells, in vitro and in vivo.

Abstract

Gene therapy is being considered in the treatment of several acquired and inherited diseases and represents a novel approach for treatment of disease based on direct modification of gene expression in somatic cells. Current approaches to gene therapy include adenoviral and retroviral gene transfer methods. Recombinant retroviruses are attractive because they can stably transform a wide variety of cell types in many species. Alternatively, adenoviral gene transfer offers transient expression at high levels. Many acquired and inherited diseases are present in liver tissue and are well suited for treatment with gene therapy. Presently two paradigms gene therapy are <italic>ex vivo</italic> and <italic>in vivo</italic>. In <italic> ex vivo</italic> gene therapy cells from liver tissue are isolated, grown, and genetically transduced <italic>in vitro</italic> followed by transplantation into the animal. Parameters of the hepatocyte cultures and retroviral particle were studied to define optimal conditions for gene transfer. Studies to retroviral infection of primary rat hepatocytes demonstrate that hepatocyte proliferation is required but not sufficient for retroviral infection. The infected cells were shown to be hepatocytes by colocalization of retroviral gene product and hepatocyte specific enzymatic activity; furthermore, the cultures expressed hepatocyte specific mRNAs throughout 6 days in culture. Rat cultures could be retrovirally infected by two types of recombinant retroviruses, amphotropic and GALV, but not by ecotropic viruses. Saturation studies indicated that the amphotropic receptors on rat hepatocytes were not saturated. To study whether overexpression of the retroviral receptor in cells increases retroviral mediated gene transfer, the GALV receptor gene was cloned into an adenoviral vector and overexpressed in primary hepatocytes. Northern analysis showed that GALV mRNA was increased 25 fold in the adenovirally infected cultures. Subsequent infection with GALV retroviruses demonstrated a three fold increase in retroviral gene transfer. To assess whether <italic>in vivo</italic> retroviral gene transfer could be accomplished via the biliary tract, rat bile ducts were ligated to induce proliferation of the cells in the periportal region. Cells retrovirally transduced were demonstrated to cytokeratin 19 negative, indicating that these cells may be the putative liver stem cell.