Combination of KRAS ASO and RIG-I agonist in extracellular vesicles transforms the tumor microenvironment towards effective treatment of KRAS-dependent cancers.

Rationale: Mutations in the KRAS gene drive many cancers, yet targeting KRAS mutants remains a challenge. Here, we address this hurdle by utilizing a nucleic acid-based therapeutic strategy delivered via extracellular vesicles (EVs) to simultaneously inhibit KRAS mutants and activate the RIG-I pathway, aiming to enhance anti-tumor immunity. Methods: Antisense oligonucleotides against KRAS mutants (KRAS ASOs) and RIG-I agonist immunomodulatory RNA (immRNA) were loaded into EVs and administered to KRAS-mutant cancer models. The therapeutic effects were assessed in colorectal and non-small cell lung cancer (NSCLC) tumor models, as well as patient-derived pancreatic cancer organoids. Immune responses were evaluated by analyzing tumor microenvironment's changes, dendritic cell activation, and T cell memory formation. The treatment efficacy was evaluated based on the tumor development and overall survival. Results: The KRAS-ASO and immRNA combination treatment induced immunogenic tumor cell death and upregulated interferons in KRAS-dependent cancers. In a colorectal tumor model, the therapy shifted the tumor microenvironment to an immunogenic state, activated dendritic cells in sentinel lymph nodes, and promoted memory T cell formation. In an aggressive NSCLC model, the treatment resulted in a strong anti-tumor activity and extended survival without any adverse effects. Validation in patient-derived pancreatic cancer organoids confirmed the clinical translation potential of this approach. Conclusions: EV-mediated delivery of ASOs and immRNA effectively inhibits KRAS mutants and activates RIG-I, leading to a robust anti-tumor immune response. This strategy holds promise for effectively treating KRAS-driven cancers and improving clinical outcomes.