Development and application of lentiviral packaging cell systems

Lentiviral vectors based on the human immunodeficiency virus type 1 (HIV-1) can deliver exogenous genes to both dividing and nondividing cells and subsequently establish a stable provirus in these target cells which can allow long-term expression of a transferred gene.; CHAPTER 1 of this dissertation describes a stable lentiviral packaging cell line which is devoid of HIV-1 tat, vif, vpr, vpu, and nef. To avoid the cytotoxicity associated with constitutive expression of HIV-1 Protease and the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by the ecdysone inducible system. Using this cell line, we have generated concentrated pseudotyped vector virus with titers in the range of 108 infectious units per milliliter (IU/ml) which can efficiently transduce dividing and growth-arrested cells in vitro.; Liver regeneration through hepatocyte transplantation could be adapted for use as an ex vivo gene therapy procedure where functional hepatocytes are genetically modified by transduction with a lentiviral vector harboring a therapeutic gene and transplanted back into a dysfunctional liver. In CHAPTER 2 we have described the production of pseudotyped HIV-1 vectors capable of infecting primary rat hepatocytes and quiescent rat fetal liver stem cells. Our studies suggest that transduced liver stem cells can potentially be used as vehicles for gene therapy and liver repopulation with the ultimate goal of reversing chronic and acute liver failure.; The introduction of highly active antiretroviral therapy (HAART) for the treatment of HIV-1 infection and AIDS has caused a sharp decline in patient morbidity and mortality. However, drug toxicity, side effects and the emergence of drug-resistant viral strains have hampered the ability of the current drug regimens to eradicate viral infection. These challenges make clear the urgent need for new anti-HIV-1 chemotherapeutic compounds. Our laboratory has previously generated a lentiviral vector producing cell line which mimics all of the steps of the viral life cycle in the absence of replication-competent HIV-1. CHAPTER 3 discusses the novel application of a lentiviral vector system as a cell-based assay for the discovery of anti-HIV-1 drugs and tests its feasibility by investigating the antiviral effects of five well-characterized compounds.