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

Abstract

HIV/AIDS continues to be a major public health problem worldwide with millions of people currently infected and new infections continuing to rise. As no effective vaccines are currently available for prevention, new and innovative therapies need to be developed. Current therapies such as highly active antiretroviral therapies (HAART) which includes protease and reverse transcriptase inhibitors have shown to be efficacious but also possess certain drawbacks. Intracellular immunization against HIV through gene therapy approaches offers a promising alternative approach to current therapies. Based on this concept, a number of strategies that showed varied efficacies in vitro have been tested. These include antisense RNA, RNA decoys, ribozymes, transdominant proteins, and bacterial toxins. The recently discovered phenomenon of RNA interference (RNAi) offers another novel approach, which appears to be even more potent in targeted gene silencing and therefore can be potentially harnessed for HIV gene therapy. With the recent advances in knowledge of HIV evolution and species specific restriction, the intracellular protein, TRIM5α, has been shown to be responsible for restricting retroviral infections between species. By harnessing this evolutionarily selected mechanism, its potential for inhibiting HIV infections can be exploited in gene therapy applications. Here I describe the use of siRNAs and the rhesus macaque isoform of TRIM5α for inhibiting HIV-1 infections in a gene therapy setting. The siRNAs used target the critical coreceptors CXCR4 and CCR5 required for HIV attachment and entry. A monospecific short hairpin form CXCR4 siRNA was initially designed to down regulate CXCR4 alone and confer resistance to T cell tropic NL4-3 HIV-1. Transfecting this shRNA into HIV-1 susceptible cells resulted in significant cell surface down regulation with a concomitant resistance to HIV-1 infection. 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. Again, transfecting this bispecific construct into cultured cells resulted in down regulation of both coreceptors which conferred resistance to HIV-1. For constitutive expression of these shRNAs, Pol-III expression cassettes were designed and inserted into a third generation lentiviral vector (XHR) for transduction of target cells. Upon transduction and subsequent analysis, stable down regulation of both CXCR4 and CCR5 was achieved. This down regulation of both coreceptors conferred resistance to both X 4 and R5-tropic strains of HIV-1 in both cultured cell lines and CD34+ derived macrophages. As the expression of shRNAs and the down regulation of normal cell surface markers may have detrimental effects on normal cell physiology, phenotypic and functional assays were performed on XHR transgenic macrophages. Normal levels of the cell surface markers, CD14, CD4, and MHCII were observed. Upon LPS stimulation, similar levels of B7.1 upregulation and IL-1 and TNF-α secretion were also found. The ability to phagocytose foreign material was also observed at normal levels. Engineered expression of TRIM5αrh by lentiviral vector transductions also restricted productive infections of both tropisms of HIV-1 in both cultured cell lines and CD34+ cell derived macrophages. Constitutive expression of TRIM5αrh did not alter normal macrophage phenotypes or functionality. Targeting multiple stages of the viral life cycle is critical to avoid generating escape variants as HIV-1 is prone to a high mutation rate. 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. Transgenic thymocytes were shown to be phenotypically normal displaying all cell subsets (CD4+, CD8+, CD4+/CD8+) and expressing the normal T cell markers CD45RA, CCR7, CXCR4, and CD28. Triple transgenic thymocytes were also resistant to T cell tropic HIV-1 infection. Together, these results show the potential of these constructs to inhibit HIV-1 infection in a stem cell gene therapy setting.