802. Silencing of HIV-1 with RNA Interference: A Multiple shRNA Approach

Abstract

Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). The double-stranded RNA can be expressed in the cell as a short hairpin RNA (shRNA) with a stem of 21–23 base pairs. Previously, we have shown that stable expression of a shRNA targeting the Nef gene of HIV-1 results in strong inhibition of HIV-1 replication. However, the application of this single shRNA was not sufficient to maintain inhibition. One of the hallmarks of RNAi, its sequence specificity, presented a way out for the virus, as single nucleotide substitutions in the target region abolished the inhibition. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs (ter Brake and Berkhout 2005). Therefore, we screened 86 different shRNAs targeting highly conserved regions. We identified multiple shRNAs that act as potent inhibitors of virus replication (Figure 1). We show, for the first time, that the expression of three different shRNAs from a single lentiviral vector results in similar levels of inhibition per shRNA as compared to single shRNA vectors. Thus, their combined expression results in an additive effect on inhibition of virus production. Moreover, when we expressed multiple shRNAs in a cell line that is susceptible for HIV-1 replication, viral escape was prevented. These results confirm that RNAi has great potential as antiviral gene therapy approach and supports our efforts to develop this strategy for the treatment of HIV-1 infected individuals. Brake, O. ter., and Berkhout, B. (Journal of RNAi and Gene Silencing 2005, 1(2): 56–65). Figure 1 86 shRNA expression plasmids were co-transfected with the HIV-1 molecularLAI and virus production measured. Figure 2Position of effective shRNA targets in the HIV-1 genome.