The Development of Resistance of Human Immunodeficiency Virus to RNA Interference Therapies: Understanding Mechanism and Developing Strategies to Overcome

Human immunodeficiency virus (HIV) possesses a prolific ability to mutate and adapt to an ever-changing environment. This intrinsic capacity for mutation not only allows HIV to evade the immune response, but also allows the virus to develop resistance to antiretroviral therapies. As an approach that targets RNA sequence rather than protein structure, RNA interference (RNAi) offers the potential for faster drug development and fewer side effects for treating HIV infection. However, the very sequence-specificity that gives RNAi-based therapies these advantages also makes the therapy susceptible to HIV escape, and the development of resistance to RNAi has been extensively documented. The work presented in this dissertation systematically analyzes how therapy delivery limitations, properties of RNAi targets, combinatorial approaches and existing HIV diversity can affect therapy efficacy and the development of resistance to RNAi./nWe have demonstrated that HIV can escape RNAi by indirect mechanisms of resistance to RNAi. When HIV was exposed to a RNAi therapy targeting the highly conserved trans-activation response (TAR) hairpin of the long terminal repeat (LTR), we failed to isolate any viral clones with mutations in the target site. Instead, we identified many mutations in the U3 region of the LTR that served to tune viral gene expression and overwhelm the RNAi pathway./nOne method to combat resistance is to use combinations of siRNAs in a manner similar to the existing highly active antiretroviral therapy (HAART). Combinations inhibit the virus at multiple loci, making it highly unlikely that a variant resistant to all components of the combination will emerge. While combinatorial RNAi therapy may delay the onset of resistance, our results indicate that the distribution or compartmentalization of the combination within cellular subpopulations may not be a critical factor in determining therapy efficacy. While we isolated several viral clones with mutations with the RNAi targeted regions, extensive sequence analysis indicated that these mutations were not fixed. We again identified mutations in the U3 region of the LTR, many of which were fixed and unique to virus that was exposed to a RNAi selective pressure. When compared to HIV that was propagated in the absence of a RNAi selective pressure, a significantly higher number of mutations in the U3 region correlated with the degree of sequence conservation of the RNAi target site. Taken together, these data suggest that high degrees of sequence conservation at the RNAi target site could divert selective pressure to the U3 region of the LTR./nFinally, we have explored how existing global sequence diversity of HIV can affect a sequence-specific therapy such as RNAi. We identified two regions of the HIV genome that could potentially serve as targets for a global RNAi therapy and we developed a cell culture system that could serve as the foundation of any long-term studies of the evolution of different HIV subtypes in response to a RNAi therapy./nIn summary, RNAi is a promising therapy for HIV; however, a number of challenges concerning viral escape remain. We have developed a number of systems to study HIV evolution in response to RNAi therapies, and our findings emphasize that one must look "outside the target" when searching for resistance as this may represent a more general mechanism by which viruses adapt to selective pressure and escape antiviral therapy.

Advisor: Schaffer, David V