This IP/CP application proposes to use recombinant SV40-derived vectors (rSV40) to deliver combination genetic therapy against HIV/SIV. This approach is based on three principles: 1. better inhibition of HIV/SIV can be achieved using combinations of anti-lentiviral transgenes that attack different stages of the lentiviral replicative cycle; 3. transduction of bone marrow progenitor cells may provide a reservoir of stem cells capable of producing differentiated progeny that are resistant to lentivirus infection. We will apply these principles, using rSV40 vectors to transduce the same cells consecutively with 3 transgenes chosen to inhibit complementary stages of retrovirus replication: (a) MIP-1beta-KDEL, to sequester CCR5 co-receptor for HIV/SIV in the endoplasmic reticulum and impede HIV/SIV entry into the cell; (b) RT#3, single chain antibody vs. HIV-1 reverse transcriptase (RT), to block RT early in infection; and (c) RevM10, dominant negative mutant on the nuclear export protein, Rev, to prevent unspliced lentivirus transcripts from exiting the nucleus. A multi- institutional group of collaborators will assay transduction, toxicity, and inhibition of HIV/SIV in vitro in T lymphocytes and CD34+ cells, and in vivo in two different animal models of AIDS. We present significant preliminary data to support our contention that: these transgenes- and designated alternates-will inhibit HIV/SIV in this setting; rSV40 vectors deliver them effectively to CD34+ cells, T cell lines and primary human peripheral blood mononuclear cells (pbmc); rSV40 vectors will stably transduce these cells with multiple transgenes, with >98% efficiency; and the proposed rSV40 transduction will protect susceptible cells from challenge with HIV-1. Project #1, directed by David Strayer, in collaboration with Roger Pomerantz, will devise and optimize strategies for inhibiting HIV-1 in cultured cell lines and pbmc, using combinations of rSV40 vectors; to minimize toxicity, maximize expression, and provide high levels of protection from HIV-1. Projects #2 and #3 will study will combination gene delivery to human and simian CD34+ cells ex vivo, and consequent protection of differentiated progeny of these cells in vitro and following reimplantation in vivo. Project #2, directed by Harris Goldstein, will evaluate combination rSV40 transduction of human CD34+ cells, test effects of such combination transduction on maturation of these cells in SCID-hu mice, and measure protection of these animals from challenge with HIV-1. Project #3, directed by Paul Johnson, will test ex vivo rSV40 transduction of rhesus CD34+ cells, assess protection of differentiated progeny from challenge with SIV or SHIV(RT) challenge in vitro and in vivo, and identify any side effects of the treatment. Project #3 will also test whether the high transduction efficiency of rSV40 vectors can be exploited to deliver genetic therapy directly into the bone marrow in rhesus macaque monkeys. These projects are supported by a Vector Cor, which will produce and characterize rSV40 vectors for use in these projects, and test for anti- SV40 antibody in treated animal sera, and by an Administrative Core, which will assure smooth functioning of the individual grant component units. Thus, we propose an consortium IP/CP study to evaluate rSV40-derived combination anti-lentiviral gene therapy in relevant experimental systems, in vitro and in vivo, and to translate these findings into clinical studies.