An effective gene-knockdown using multiple shRNA-expressing adenovirus vectors

Viral vectors expressing short hairpin RNA (shRNA) are attractive for efficient and tissue-specific RNA interference (RNAi) delivery. We and others previously reported that recombinant adenovirus (Ad) vector-mediated RNAi has great potential for a variety of applications in molecular biology studies and gene therapy. In the present study, we have developed an efficient Ad vector-mediated RNAi system, in which an Ad vector carries four shRNA-expression cassettes (Ad-multi-shRNA vector), a simple and effective strategy for enhancing the RNAi response per Ad vector particle. The data demonstrated that the Ad-multi-shRNA vectors showed an enhanced RNAi effect compared to conventional Ad vectors containing a single shRNA-expression cassette. An application of the Ad-multi-shRNA vector carrying four same shRNA-sequences against the RET finger protein, an oncogene known to desensitize cells to oxidative stress and cisplatin, resulted in an enhanced cytotoxic effect of cisplatin, demonstrating the advantages of the Ad-multi-shRNA vector for silencing target genes. Furthermore, an Ad-multi-shRNA carrying four different shRNA-sequences efficiently silenced the multiple target genes simultaneously. These data suggest the potential usefulness of the Ad-multi-shRNA vector not only in basic research but also in clinical gene therapy.