A preclinical model of hepatocyte gene transfer: the in vivo, in situ perfused rat liver

Delivering retroviruses targeted to hepatocytes in vivo involves the inject ion of retroviruses directly into the portal vein. The aim of this work was to establish a clinically relevant system for retrovirus-mediated gene tra nsfer in a new model of in vivo, in situ perfused rat liver and to study th e transgene expression. At 24 h after partial hepatectomy, the liver was co mpletely excluded from the splanchnic circulation using an extracorporeal s hunt. Two independent normothermal, oxygenated perfusion systems were used. First, liver perfusion was carried out with a recirculating system (1 h). Culture supernatant containing retroviruses (1.5 x 10(8) ffu/ml, beta -gala ctosidase gene) was used as perfusate. Then the liver perfusion was maintai ned for more 30 min in a single liver passage system using culture medium w ithoutretro-viruses as perfusate. High hepatocyte transduction rates (up to 34.4%) were obtained. PCR analysis showed no provirus in extrahepatic orga ns. Viral titrations performed simultaneously (inflow and outflow liver lin es) showed that after 1 h of perfusion (up to 30 successive liver passages) retroviruses were still detected in the liver outflow perfusate (up to 2.0 x 10(7) ffu/ml). Washing the liver for 30 min dramatically decreased the l eakage of retroviruses in the outflow. In order to be of clinical use, the injection of retroviruses targeted to hepatocytes in vivo should be done wh ile the liver is completely excluded from the splanchnic circulation to avo id any extrahepatic retrovirus diffusion.