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Abstract
Inhibition of HIV-1 by Hybrid tRNA-shRNA Constructs
Kim D.1, Soifer H.1, Scherer L.1, Rossi J.1
1Beckman Research Institute of the City of Hope, Duarte, United States of America
Introduction: Highly active antiretroviral therapy (HAART) for HIV-infected individuals can lead to problems of drug-resistance and toxicity. In the search for alternative and complementary treatment strategies, RNA Interference (RNAi) has emerged as a promising approach to inhibiting HIV infection. RNA intermediates in the replication cycle of HIV can be targeted for the development of RNAi-based therapeutics. Successful RNAi-based strategies against HIV face several challenges, among them efficient intracellular functioning and long-term expression of the RNAi-triggering constructs. Short hairpin RNAs (shRNAs) can be delivered into human cells to trigger the RNAi pathway and target HIV proviral transcripts through Dicer-processed small interfering RNAs (siRNAs).
Methods: We have constructed several hybrid tRNA-shRNAs targeting a site in both the tat and rev transcripts to test various parameters affecting their efficacy of HIV-1 inhibition. Our constructs are expressed using a dual U6-tRNA promoter system, which yields higher levels of small RNA therapeutic transcripts relative to the tRNA promoter alone.
Results: We report that tRNA-shRNA expression cassettes cotransfected directly into human cells with HIV proviral DNA downregulate HIV expression in a dual luciferase assay.
Conclusions: Our results indicate the potential utility of our constructs for future therapeutic applications. A Phase I clinical trial is in the works for later this year at the City of Hope that will gauge the efficacy of shRNAs targeting HIV tat and rev transcripts in AIDS-related lymphoma patients undergoing bone-marrow transplantation. Extensive research has been done in our lab and at the City of Hope on RNAi-based HIV inhibition and stem cell therapy, respectively, and this work aims to develop safe and effective alternatives to HAART for HIV-infected individuals.
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