Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

Abstract

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors contalning genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liverspecific functions, including phenylalanine hydroxylase, a,-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.

A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.

“…However, this ex vivo approach is hindered by the fact that only 10-30% of the primary hepatocytes in culture can be infected by conventional retroviral vectors (5,18,19). To test whether a pseudotyped vector could mediate stable gene transfer into hepatocytes more efficiently than a vector containing the amphotropic envelope protein, we infected primary mouse hepatocytes with LSPONL(G) or LSPONL(A) and determined the amount of S antigen secreted into the culture medium.…”

“…Most importantly, the availability of murine retroviral packaging cell lines allows generation ofreplication-defective retroviral vectors that can deliver genes into target cells but cannot spread further (3,4). However, the titers ofretroviral vectors produced from these packaging cells are relatively low, and some human cells cannot be infected efficiently (5,6). Attempts to concentrate retroviral vectors by physical methods such as filtration or ultracentrifugation have generally resulted in massive loss of infectious virus, presumably due to instability of the retroviral envelope protein, which is essential for the interaction of virions with the cell-surface receptor and for their entry into the cell.…”

Pseudotyping of Moloney leukemia virus-based retroviral vectors with simian immunodeficiency virus envelope leads to targeted infection of human CD4+ lymphoid cells

“…In addition, some cell types, including hepatocytes and bone marrow cells, are inefficiently infected by A-MLVbased vectors, and this constitutes a limitation for gene therapy approaches to diseases affecting these cell types. 2 Thus, engineering of viral tropism is actively studied, because it could lead to significant improvements in the field.…”

Emerging Therapies in Hepatology: Liver-Directed Gene Transfer and Hepatocyte Transplantation

“…As mentioned before, HcTx is a prerequisite for liverdirected ex vivo gene therapy [Peng et al, 1988;Adams et al, 1992]. Familial hypercholesterolemia was the first metabolic disease, which was treated by this approach in humans.…”