We evaluated the ability of a SIV-specific ribozyme to inhibit viral replication in T cells and macrophages derived from transduced CD34+ hematopoietic cells. Rhesus CD34+ bone marrow cells were transduced with retroviral vectors containing either a hairpin ribozyme (Rz9456) that cleaves a conserved sequence in the 3' LTR of SIV and HIV-2 strains (M. Heusch, et al, Virology 1996, in press) and the selectable marker neo, or as control, a vector with neo alone. To evaluate expression of foreign genes in T cells derived from transduced hematopoietic progenitor cells, we established a culture system that supports the differentiation of rhesus and human CD34+ hematopoietic cells into mature T cells (Rosenzweig et al, in press). Following transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro T cell differentiation) or in the presence of cytokines (to support macrophage differentiation). Following expansion and selection with the neomycin analog G418, cells were challenged with lymphotropic and macrophagetropic strains of SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the SIV LTR ribozyme were highly resistant to challenge with SIVmac239. The SIV ribozyme resulted in over a 1000-fold inhibition of viral replication in comparison with T cells derived from CD34+ cells transduced with the control neo vector. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication when challenged with SIVmac316. No evidence for toxicity was observed in either T cells or macrophages containing the 9456 ribozyme. In conclusion, the SIV-specific ribozyme 9456 is highly effective at inhibiting SIV replication in both CD4+ T cells and macrophages derived from transduced CD34+ hematopoietic cells. The use of rhesus thymic stromal cultures to support T cell differentiation will facilitate in vitro evaluation of candidate genes for stem cell gene therapy for AIDS. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme will permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.
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