800. Complete Suppression of CCR5 Expression and Inhibition of HIV-1 Infection by Transfected and Lentiviral Vector Expressed shRNAs

Abstract

SiRNAs have been shown to be highly potent and sequence specific in targeted gene silencing with great potential for use as therapeutics in combating HIV infection. By targeting the critical coreceptor CCR5, infection can be inhibited at the level of viral entry. The chemokine receptor, CCR5, has been shown to be fully dispensable for normal homeostasis and is, therefore, an excellent target for gene therapy applications. Several reports have so far described the use of CCR5 siRNAs showing varied levels of gene knockdown. In the present study, a new generation of anti-CCR5 siRNAs was designed with the goal of achieving complete silencing. Based on rational target sequence identification criteria, new and more effective CCR5 siRNAs were identified. Complete knockdown of CCR5 expression as determined by FACS and quantitative real time PCR was observed. To achieve stable and constitutive knockdown of CCR5, siRNA coding sequences were cloned individually and in tandem into a lentiviral vector. Vector transduced cells showed similar impressive levels of CCR5 knockdown. When challenged with R5-tropic HIV|[ndash]|1, the siRNA transduced cells exhibited resistance to infection. These results are the first to demonstrate complete constitutive knockdown of CCR5 expression by the use of siRNAs after years of efforts in several laboratories.