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, which include the isolation and culture of hepatocytes, followed by their transduction with a retrovirus. Subsequently, autologous hepatocytes are transplanted. The number of hepatocytes that can be transduced by retroviruses bearing the therapeutic gene is one of the limiting steps that can impair the success of this strategy.

We presently describe an experimental approach that leads to improved transduction efficiency in mouse and human hepatocytes in vitro. By using a recombinant retrovirus bearing the Escherichia coli β-galactosidase gene, we show that addition of growth factors to the cells, namely human hepatocyte growth factor (HGF), allows marked increase in the transduction efficiency in mouse (up to 80%) and human (40%) hepatocytes. Familial hypercholesterolemia (FH) is due to mutation in the low-density lipoprotein (LDL) receptor gene and results in a deficiency in LDL receptors.

Transduction of the human LDL receptor cDNA under the transcriptional control of the L-type pyruvate kinase promoter-activator into mouse hepatocytes led to an elevated tissue-specific expression of the human protein. These results suggest that the ex vivo approach remains a promising alternative for hepatic gene therapy.

Overview summary

One approach to the treatment of inherited metabolic diseases involving the liver is represented by gene transfer into hepatocytes. We describe an efficient method for retroviral-mediated gene transfer ex vivo into murine and human hepatocytes. By use of a recombinant vector that encodes the human low-density lipoprotein (LDL) receptor and transduces murine hepatocytes, we demonstrate high expression, with a tissue-specific promoter, of the human LDL receptor protein.