Background: The KRAS oncogene is a key driver of human malignancies, but targeting the KRAS protein with conventional small-molecule inhibitors remains challenging due to its lack of accessible drug-binding pockets. Antisense oligonucleotides (ASOs) offer an approach for suppressing KRAS production by directly targeting RNA transcripts rather than the protein product. While current KRAS-targeting ASOs largely rely on RNase H-dependent gapmer designs for mRNA degradation, exploring alternative steric-blocking mechanisms represents an unexploited strategy.
Methods & Results: We designed and evaluated a panel of fully 2′-O-methyl (2′-OMe) phosphorothioate (PS)-modified RNA ASOs targeting KRAS pre-mRNA/mRNA. ASOs 1–8 were complementary to different regions of exon-1, while ASOs 9–15 were specific to exon-2. Initial screens identified ASO-6 and ASO-9 as the most effective candidates for reducing full-length KRAS transcript levels. Further analysis revealed that this reduction was partly driven by exon skipping, where ASO-6 induced exon-1 skipping and ASO-9 mediated exon-2 skipping. Follow-up dose-response experiments confirmed that ASO-9 suppressed KRAS mRNA more effectively than ASO-6 at the transcript level. Finally, Western blot analysis verified these results at the protein level, confirming that ASO-9 achieved a stronger downregulation of KRAS expression than ASO-6.
Conclusion: These findings demonstrate that fully 2′-OMe PS-modified RNA ASOs can effectively suppress KRAS expression. Consequently, ASO-9 was selected as the lead sequence for subsequent downstream evaluation.