RNA interference (RNAi) mediated by double- stranded small interfering RNA (siRNA) is a novel mechanism of sequence-specific, post-transcriptional gene silencing. There has been much research into the use of RNAi for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be efficacious therapeutically, effective RNAi targeting sequences and a suitable delivery system are required. In this study, we employed a polymerase chain reaction (PCR)-based siRNA expression strategy to rapidly screen for effective siRNA sequences. Two effective siRNAs sequences (designated as S2 and X1) which reduced the HBV RNA by >90% were identified. For delivering the siRNAs, they were cloned into a human foamy virus (HFV)-based vector to generate single siRNA expression vectors HFVU6-siS2, HFVU6-siX1 and a dual siRNA expression vector HFVU6-siSX. The results showed that these siRNA vectors effectively inhibited multiple HBV gene expression and viral DNA replication based on ELISA and quantitative PCR analysis. HFVU6-siSX which simultaneously expressed two siRNAs that targeted the S and X genes of HBV is the most potent inhibitor of HBV replication. In addition, the repression of HBV RNA and DNA was stable for up to 3 months post-transduction as determined by RT-PCR and Southern blotting. Collectively, the PCR-based siRNA expression strategy provides a rapid and easy approach for testing candidate anti-HBV siRNA sequences and for cloning selected siRNA expression cassettes into a vector. RNAi based on the HFV vector was able to achieve effective, long-term inhibition of HBV gene expression and viral DNA replication. The combination of the two techniques may provide a powerful tool in the treatment of viral infection.

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