Project #1 of this IP/CP application proposes exploit the high transduction efficiency of recombinant SV40-derived gene therapy vectors, to test combination genetic therapies to inhibit HIV-1 infection in vitro in cultured cell lines and primary peripheral blood mononuclear cells (pbmc). rSV40 vectors are replication-defective viruses derived from Tag-deleted simian virus-40 genomes that integrate very rapidly in to cellular genomes, and infect almost all cell types. They have very high production titers (up to 10/11 infectious units (IU)/ml) and transduction efficiencies (>98%). These vectors deliver several different anti-lentiviral transgenes, or in sequence, to cultured cell lines, human pbmc and human and simian CD34+ cells. These inhibit HIV-1 in transduced cells. Our overall hypotheses for this project are: 1. SV40 can deliver multiple transgenes to the same cells to inhibit lentivirus replication with high efficiency 2. Simultaneous expression of combinations of transgenes, each targeting a different phase of the lentivirus replicative cycle, will provide enhanced protection from lentiviral challenge This project proposes to develop and evaluate rSV40 delivery of combinations of anti-lentiviral transgenes, each targeting a different phase of the HIV-1 replicative cycle: RT#3, single chain antibody vs. HIV-1 reverse transcriptase (RT); Rev410, dominant negative mutant Rev protein; and MIP-1beta-KDEL, a ligand for the CCR5 co-receptor,, targeted to the endoplasmic reticulum. Combinations of these transgenes, delivered in sequence by their respective rSV40 vectors, will be studied for levels and duration of expression in transduced cell lines and pbmc, cytotoxicity and effects on cell survival. Protection conferred by these transgenes, administered in the proposed combinations, will be measured using both cell lines and normal pbmc as targets, and using and T- tropic strains of HIV-1, including at least one clinical isolate of HIV-1. In the event that combinations of these transgenes prove either ineffective or toxic, alternative transgenes are on hand that will be used in their steads. Toxicity and efficacy data from Projects #2 and #3 will be collated with data from this project to determine the need (if any) for revising transduction protocols and/or transgenes used. Finally, as new and promising genetic therapies for lentiviral infection are reported and become available, rSV40 vectors incorporating these will be developed and studied in the same fashion. Therefore, in this project we propose to take advantage of the strengths of the rSV40 vector system and, working in tandem with investigators in Projects #2 and #3, will develop combinations gene therapy to inhibit HIV-1.
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