Multi-SubType Escape Reducing (MuSTER) RNAi Trigger Retains Potent Anti-Viral Activity Despite Emerging Mutations in the Target Site
Ulrike Jung, Daniela Castanotto, Marc Jung, John J. Rossi. Department of Molecular and Cell Biology, Beckman Research Institute at City of Hope Medical Center, Duarte, CA; Department of Cancer Biology, Beckman Research Institute at City of Hope Medical Center, Duarte, CA
The method of RNAi is a powerful and a very appealing approach in therapy for diseases. However, conventional si/shRNA designs are sometimes hampered by their very specificity when used against non-conserved or mutating targets such as viruses.
In this work, we were able to overcome this shortfall of si/shRNAs by a new design approach actively using non-canonical paring/non-Watson Crick pairings (nWCp). Our strategy employed targeting the ambiguous sequence of an artificial HIV strain covering mutations from strains of different subtypes and allowed nWCp with an shRNA guide sequence. One resulting shRNA (MuSTER 5, Fig. 1) was comparable or better than a conventional highly potent anti-HIV shRNA. And - contrary to traditional designs - it was able to retain its activity against several mutant viral strains while target specific RNAi could be proven as underlying mechanism.
Fig. 1.: MuSTER shRNAs inhibit a broad range of HIV-1 subtypes and outperform conventional shRNAs in packaging and challenge tests. A p24 assay from five different HIV strains 48 hours post co-transfection with conventional or MuSTER shRNA plasmids, 2 independent experiments. Values normalized to empty construct = 100, mean and average deviation. B p24 assay from CEM NKR CCR5 Tcells stably expressing MuSTER cassettes after sorting, challenged with pool of HIV strains from A (Pool, day 12 post challenge) or HIV BAL III quasispecies (BAL, day 15 post challenge).
In summary, this novel strategy allows the selection of potent multi-subtype, escape reducing (MuSTER) RNAi triggers, which is of paramount importance in the treatment of highly mutagenic infectious diseases, such as HIV and Hepatitis C, whose current therapies are hampered by escape mechanisms. Furthermore, our results also show that the parameters used for prediction of target sites for naturally occurring microRNAs as well as off-target effects of artificial shRNA/siRNAs need to be revised to include nWCp.
Keywords: RNAi and shRNA; Infectious Diseases; Clinical Gene Therapy
Session: Poster Session: Hematologic & Immunologic Diseases II (5:15 PM-7:15 PM)
Date/Time: Friday, May 23, 2014 - 5:15 PM
Room: Hall A and B South