High-fidelity Cas9-mediated targeting of KRAS driver mutations restrains lung cancer in preclinical models

Abstract Missense mutations in the 12 th codon of KRAS are key drivers of lung cancer, with glycine-to-cysteine (G12C) and glycine-to-aspartic acid (G12D) substitutions being among the most prevalent. These mutations are strongly associated with poor survival outcomes. Given the critical role of KRAS in lung cancer and other cancers, it remains as a major target for the development of new and complementary treatments. We have developed a CRISPR-High Fidelity (HiFi)-Cas9-based therapy strategy that can effectively and specifically target KRAS G12C and KRAS G12D mutants, avoiding KRAS WT off-targeting and affecting KRAS downstream pathways, thereby significantly reducing tumorgenicity. The delivery of HiFiCas9 components via ribonucleoprotein particles (RNPs) and adenovirus (AdV) effectively abrogates cell viability in KRAS -mutant Non-Small Cell Lung Cancer (NSCLC) preclinical models, including 2D and 3D cell cultures, cell-derived xenografts (CDX), and patient-derived xenograft organoids (PDXO). Our in vitro studies demonstrate that HiFiCas9-based therapy achieves superior KRAS inhibition compared to Sotorasib and effectively circumvents certain resistance mechanisms associated with Sotorasib treatment. Moreover, in vivo delivery using adenoviral particles significantly suppresses tumor growth in preclinical NSCLC models. Collectively, our findings establish HiFiCas9 as an effective therapeutic strategy with promising clinical applications, especially if in vivo delivery methods are further optimized.