Inverted chimeric RNAi molecules synergistically co-target MYC and KRAS

Mutant KRAS has been implicated in driving a quarter of all cancer types. Although inhibition of the KRASG12C mutant protein has shown promise in the clinic, there is still a need for therapies that overcome clinical resistance and target non-KRASG12C mutations. Mutant KRAS activates downstream MYC, which is also a challenging-to-drug oncogene. We have developed a novel “inverted” RNAi molecule in which the passenger strand of the MYC-targeting siRNA is fused to the guide strand of the KRAS-targeting siRNA. The chimeric molecule simultaneously inhibits KRAS and MYC, showing marked improvements in efficacy beyond the individual siRNA components. This effect is mediated by 5'-dT overhangs following endosomal metabolism. The synergistic RNAi activity led to a >10-40-fold improvement in inhibiting cancer cell viability in vitro. When conjugated to an epidermal growth factor receptor (EGFR)-targeting ligand, the chimeric siRNA was co-delivered to and internalized by tumor cells. Notably, as compared with individual KRAS or MYC siRNAs, the chimeric design resulted in significantly improved metabolic stability in tumors, enhanced silencing of both oncogenes, and reduced tumor progression in lung cancer models. This inverted chimeric design establishes proof-of-concept for ligand-directed, dual-silencing of KRAS and MYC in cancer and constitutes a new molecular strategy for co-targeting any two genes of interest, which has broad implications. Overall design: To understand the transcriptional effects of our siRNAs, we transfected A427 cells with MYC-targeting siRNA, KRAS-targeting siRNA, or MYC/KRAS co-targeting siRNA at 20nM for 24 hours.