Gene therapy for HIV/AIDS: Harnessing RNA interference and the restriction factor TRIM5alpha to inhibit HIV-1 infection

Intracellular immunization against HIV through gene therapy offers promising therapeutic approaches. The recently discovered phenomenon of RNA interference and the species specific restriction factor TRIM5alpha were shown to inhibit HIV-1 infection with potential use for HIV gene therapy. In these studies, coreceptors CXCR4 and CCR5 essential for HIV entry were targeted using siRNAs. A monospecific short hairpin CXCR4 siRNA was initially designed and tested. Transfection of this shRNA into HIV-1 susceptible cells resulted in significant cell surface down regulation of CXCR4 with a concomitant viral resistance. To extend these findings to inhibit both T cell and macrophage tropic strains of HIV-1, a bispecific synthetic shRNA was designed targeting both CXCR4 and CCR5. Delivery of this construct into cells resulted in down regulation of both coreceptors which conferred resistance to HIV-1. For constitutive expression of these shRNAs, Pol-III promoter expression cassettes were designed and inserted into a third generation lentiviral vector, XHR. Upon transduction and subsequent analysis, stable down regulation of both CXCR4 and CCR5 was achieved. This down regulation conferred resistance to both X4 and R5-tropic strains of HIV-1 in cultured cells and CD34+ cell derived macrophages. Since the expression of shRNAs and the down regulation of normal cell surface markers may have detrimental effects, phenotypic and functional assays were performed on XHR transgenic macrophages which were found to be normal. Similarly, engineered expression of TRIM5alpharh by lentiviral vector transductions also restricted HIV-1 infections in cultured cells and CD34+ cell derived macrophages. Constitutive expression of TRIM5alpharh did not alter normal macrophage phenotypes or functionality. Targeting multiple stages of the viral life cycle is critical to avoid generating escape variants since HIV-1 is prone to high mutation rates. Accordingly, a Triple lentiviral vector containing a rev/tat shRNA, a TAR decoy, and a CCR5 ribozyme was used to generate transgenic thymocytes in a SCID-hu mouse model. Triple transgenic thymocytes were shown to be phenotypically normal and were resistant to HIV-1 infection. Together, these results showed the potential of these constructs to inhibit HIV-1 infection in a stem cell gene therapy setting.