Using exogenous sequences to express RNA interference (RNAi) activators has potential for the treatment of chronic viral infections. However, availability of a variety of suitable of promoter elements is important to optimize transcription control of silencing sequences and facilitate multitargeting. Recent demonstration that tRNA miR genes occur naturally has prompted investigating the incorporation these tRNA Pol III promoters into exogenous RNAi-activating cassettes. We have assessed efficacy of Pol III tRNA(Lys3) short hairpin RNA (shRNA) sequences that target hepatitis B virus (HBV). These cassettes achieved good silencing at low concentrations, and efficacy compared favorably to that of equivalent U6, H1 and CMV expression cassettes. HBV replication in cell culture was inhibited and northern blot hybridization analysis confirmed processing of the tRNA(Lys3) transcripts to form intended antiviral guide sequences. Importantly effects were observed without evidence of disruption of endogenous miR function. Analysis in a murine hydrodynamic injection model of HBV replication confirmed that the tRNA(Lys3) expression cassettes are also effective in vivo. Usefulness of tRNA(Lys3) antiviral expression cassettes expands the repertoire of promoters available for RNAi-mediated HBV silencing and advances the application of expressed sequences for therapeutic gene silencing.
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