This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The introduction of inhibitory genes into hematopoietic stem cells offers the potential for long-lived immune reconstitution with progeny cells resistant to HIV-1 replication. However, successful development of stem cell gene therapy for AIDS is likely to require preclinical testing in the rhesus macaque model. We examined the ability of a HIV-1-based lentiviral vector (VRX494) encoding a 937 bp antisense HIV-1 envelope sequence to inhibit viral replication. Because HIV-1 does not replicate in rhesus macaques, chimeric SIV/ HIV-1 viruses with the HIV-1 envelope were used to determine the efficacy of VRX494. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the efficacy of the VRX494 in CD4+ T cells derived from transduced CD34+ cells, rhesus CD34+ bone marrow cells were transduced with VRX494 and then cultured on rhesus fetal thymus stromal cells to induce T cell differentiation. Transduction conditions for CD34+ cells were optimized so as to yield relatively high levels of transduction efficiency (greater than 50 per cent), with minimal effective multiplicity of infection. Purified CD4+GFP+ T cells derived from VRX494-transduced cells strongly inhibited SHIV HXBc2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of T cell clones derived from transduced CD34+ cells revealed a subset of cells with multiple proviral copies per cell highlighting the importance of optimizing transduction conditions to minimize the possibility of multiple integration events per cell. These results indicate that a lentiviral vector expressing an HIV-1 antisense sequence strongly inhibits HIV-1 and SHIV replication and that the SHIV macaque model should serve as a useful preclinical model to evaluate stem cell gene therapy for AIDS.
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