Targeted KRAS suppression in colorectal cancer via in vivo self-assembled siRNAs encapsulated in endogenous small extracellular vesicles.

KRAS is a prevalent oncogenic driver whose therapeutic targeting has remained challenging beyond the G12C mutation. Here, we developed a gene circuit platform that enables the in vivo self-assembly of small extracellular vesicles (sEVs) encapsulating KRAS-specific siRNAs for broad targeting of KRAS mutants. The system is based on intravenous injection of a synthetic gene circuit engineered to co-express KRAS-targeting siRNAs (directed against conserved regions or specific mutations such as G12D) and the colorectal cancer (CRC)-targeting peptide TCP-1 in hepatocytes. Upon hepatic absorption, the circuit drives the production of sEVs that package the siRNAs and display TCP-1 on sEV surface for tumor-specific delivery. In orthotopic models of KRASG12D-, KRASG12V-, and KRASG13D-driven CRC, the platform mediated efficient tumor targeting and significantly suppressed tumor growth, achieving complete regression in some cases. Tandem circuits co-expressing two siRNAs exhibited synergistic and superior efficacy compared to single-siRNA circuits or the small-molecule inhibitor MRTX1133. Mechanistic studies confirmed downregulation of KRAS expression and suppression of downstream ERK and AKT phosphorylation. Comprehensive safety evaluation revealed minimal off-target effects and no detectable systemic toxicity, highlighting the favorable safety profile of the platform. Collectively, this study establishes a robust and targeted siRNA delivery system that effectively overcomes the historical limitations of KRAS targeting, providing a promising therapeutic strategy for a broad spectrum of KRAS-driven cancers.