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  • Title: Stringent testing identifies highly potent and escape-proof anti-HIV short hairpin RNAs.
    Author: von Eije KJ, ter Brake O, Berkhout B.
    Journal: J Gene Med; 2009 Jun; 11(6):459-67. PubMed ID: 19384894.
    Abstract:
    BACKGROUND: RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences. METHODS: In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone and also infected shRNA-expressing T cell lines with HIV-1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA-expressing T cells upon challenge with increasing dosages of HIV-1, and measured the dose required to result in massive virus-induced syncytia formation in this 2-week assay. RESULTS: Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co-transfection assays. The use of increased dosages of HIV-1 selected the same highly potent shRNAs as the laborious and extended escape study. CONCLUSIONS: These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs.
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