Silencing of HIV-1 by AgoshRNA molecules.

RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by the expression of a short hairpin RNA (shRNA). shRNA molecules enter the RNAi pathway at the Dicer processing step. Recent studies indicated that the cellular microRNA miR-451 is not recognized by Dicer, but that it is processed instead by the Argonaute 2 (Ago2) protein. Subsequently, Dicer-independent shRNAs were described that rely on Ago2 for processing, as well as the subsequent silencing step. We called these AgoshRNA molecules because they depend on Ago2 both for maturation and activation. Processing of an AgoshRNA yields only a single active RNA strand, thus reducing the chance of adverse off-target effects induced by the passenger strand of regular shRNAs. In this study, we converted several anti-HIV-1 shRNAs into AgoshRNAs. Seven of the 21 designed AgoshRNAs were potent anti-HIV molecules, although their RNAi activity is generally somewhat reduced compared with the matching shRNAs. The AgoshRNA candidates revealed no cellular toxicity. This may relate to the absence of passenger strand expression, which was verified for these AgoshRNA candidates. Furthermore, we demonstrate that a toxic shRNA can be converted into a non-toxic AgoshRNA.

The impact of HIV-1 genetic diversity on the efficacy of a combinatorial RNAi-based gene therapy.

A hurdle for human immunodeficiency virus (HIV-1) therapy is the genomic diversity of circulating viruses and the possibility that drug-resistant virus variants are selected. Although RNA interference (RNAi) is a powerful tool to stably inhibit HIV-1 replication by the expression of antiviral short hairpin RNAs (shRNAs) in transduced T cells, this approach is also vulnerable to pre-existing genetic variation and the development of viral resistance through mutation. To prevent viral escape, we proposed to combine multiple shRNAs against important regions of the HIV-1 RNA genome, which should ideally be conserved in all HIV-1 subtypes. The vulnerability of RNAi therapy to viral escape has been studied for a single subtype B strain, but it is unclear whether the antiviral shRNAs can inhibit diverse virus isolates and subtypes, including drug-resistant variants that could be present in treated patients. To determine the breadth of the RNAi gene therapy approach, we studied the susceptibility of HIV-1 subtypes A-E and drug-resistant variants. In addition, we monitored the evolution of HIV-1 escape variants. We demonstrate that the combinatorial RNAi therapy is highly effective against most isolates, supporting the future testing of this gene therapy in appropriate in vivo models.