ADENOVIRAL VECTOR TRANSDUCTION OF QUIESCENT PRIMARY HUMAN HEPATOCYTES

The hepatocyte is a potential target for gene therapy using adenoviral vectors because the cell does not replicate in the mature organ, and transduction efficiency of retroviral Vectors is low. fn this study, w e tested the efficiency of adenoviral vector-mediated gene transfer an d the persistence of transgene expression in freshly isolated as well as in quiescent, terminally differentiated, primary, human hepatocytes cultured under serum-free conditions for at least 1 1/2 months. Human hepatocytes were transduced with an adenoviral vector bearing a nucle ar targeted beta-galactosidase gene (Av1LacZ4) 48 hr after isolation o r during quiescent periods at 15 and 30 days in culture. Transgene exp ression was evaluated 2, 6, 15 and 26 days after transduction. In fres hly isolated hepatocytes, transduction efficiency was enhanced to 41, 65, 92 and 99% with a multiplicity of infection of 2, 5, 10 and 50, re spectively. Human hepatocytes continued to express beta-galactosidase while retaining the ability to express cytokeratins and albumin for at least 4 weeks after transduction. No apparent cytopathic Or cytotoxic effects were noted with a multiplicity of infection of 10 or less, as determined by morphologic evaluation and cell viability assays. To ou r knowledge, this is the first demonstration of sustained transgene ex pression in quiescent primary human hepatocytes after adenoviral vecto r-mediated gene transfer. This in vitro model system would be useful f or testing the safety and efficacy of adenovirus-based gene therapy Ve ctors for hemophilia and other genetic disorders.