EFFICIENT RETROVIRAL-MEDIATED GENE-TRANSFER INTO PRIMARY CULTURE OF MURINE AND HUMAN HEPATOCYTES - EXPRESSION OF THE LDL RECEPTOR

The ex vivo approach to hepatic gene therapy involves several steps, w hich include the isolation and culture of hepatocytes, followed by the ir transduction with a retrovirus. Subsequently, autologous hepatocyte s are transplanted. The number of hepatocytes that can be transduced b y retroviruses bearing the therapeutic gene is one of the limiting ste ps that can impair the success of this strategy. We presently describe an experimental approach that leads to improved transduction efficien cy in mouse and human hepatocytes irt vitro. By using a recombinant re trovirus bearing the Escherichia coli beta-galactosidase gene, we show that addition of growth factors to the cells, namely human hepatocyte growth factor (HGF), allows marked increase in the transduction effic iency in mouse (up to 80%) and human (40%) hepatocytes. Familial hyper cholesterolemia (FH) is due to mutation in the low-density lipoprotein (LDL) receptor gene and results in a deficiency in LDL receptors. Tra nsduction of the human LDL receptor cDNA under the transcriptional con trol of the L-type pyruvate kinase promoter-activator into mouse hepat ocytes led to an elevated tissue-specific expression of the human prot ein. These results suggest that the ex vivo approach remains a promisi ng alternative for hepatic gene therapy.