367. Development of Lentiviral Vectors Carrying shRNA, Ribozyme and RNA Decoy for the Inhibition of HIV-1 Infection

Abstract

The replication-defective lentiviral vectors are able to transduce a wide array of quiescent cell types with sustained long-term expression of the genes within the vector backbone. These properties make them favorable vectors for anti-HIV-1 therapeutic gene delivery. The recently discovered small interfering RNA(siRNA) mediated RNA interference provides a powerful new tool for gene silencing in a sequence specific manner, which suggest the potential of siRNAs for gene therapy application. A stable expression system of siRNAs and the efficient delivery vehicle of lentiviral vectors would prove an excellent match as a powerful gene therapy tool. We have developed lentiviral vectors expressing U6 pol III promoter-driven short hairpin RNAs (shRNAs) targeting the rev and tat mRNAs of HIV-1. Utilizing these shRNAs we have achieved potent inhibition of HIV-1 replication in primary cells. Up to date, the majority of HIV-1 gene therapy protocols target a single therapeutic gene. Like any therapeutic approach for inhibition of HIV infection, gene therapy is also faced with the problem of development of viral variants that are resistant to therapeutic agents. We hypothesize that if a combination of therapeutic genes targeting different viral products and steps in the viral life cycle is used, the probability of viral escape mutants will be greatly reduced. Furthermore, there is the possibility of synergy between the different gene therapy agents. Previously, we created the pol III expressing cassettes expressing an anti-CCR5 ribozyme or a nucleolar localizing TAR decoy and cloned them into a self-inactivating lentiviral vector. Each of the constructs showed marked anti-HIV-1 activities. In this study, we combined the shRNAs with the ribozyme or the RNA decoy, or all three constructs into a single vector backbone. Each of these therapeutic RNAs targets a different gene and blocks HIV infection by a distinct mechanism. After transduction with the vectors and infection with HIV-1, a potential synergistic effect was apparent in CD34+ hematopoietic stem cells and primary T cells. We have also developed the vectors expressing only the various therapeutic genes but excluding the reporter gene and WPRE sequence to eliminate any elements that potentially interfere with the expression of the therapeutic genes or have adverse effects on target cells. Our long-term goal is to develop vectors suitable for human gene therapy.