Novel mutant-specific siRNAs induce selective mutant KRAS knockdown

Undergraduate: Anne Feng

Faculty Advisor: Chad Pecot
Department: Biology

---

The KRAS gene has been implicated as a driver mutation in nearly one third of all cancers. However, it remains an elusive target for molecular therapy and harder still to selectively knock down while sparing the wild-type allele. Thus, the purpose of this investigation was to determine the efficacy of novel custom mutant-specific (MS) siRNA sequences in targeting multiple mutant KRAS alleles over the wild-type. To do so, NIH 3T3 mouse fibroblasts that had been infected with wild-type, G12C, G12D, G12V, and G13D mutant human KRAS were reverse transfected with 3 control sequences, 2 sequences designed to be anti-sense to a single mutation each (G12C and G12D), and 12 novel MS siRNA sequences that target 3 mutations each. Levels of mRNA transcription were quantified using reverse transcription followed by real-time PCR. Current results indicate that the G12CD13D_1, G12CD13D_2, and G12CD13D_4 siRNA sequences exhibit higher knockdown of the mutant alleles while maintaining expression levels of WT KRAS that are comparable to the negative control siRNA. By contrast, neither of the single mutant siRNA sequences showed any particular affinity for their target mutants over either the other mutants or WT KRAS. As such, with their potential as an effective payload with mutant KRAS specificity, novel MS siRNAs present a promising avenue of pursuit for drugging the formerly "undruggable."