CasRx-Based Kras G12D Silencing for In Vivo Tumor Inhibition in Spontaneous Lung Adenocarcinoma

Abstract KRAS mutation represents a formidable challenge in cancer treatment. Here, we explored the CasRx-editing approach on lung adenocarcinoma driven by KRAS activation and Tp53 loss in vivo, a scenario closely resembling clinical conditions. We screened seven sgRNAs in vitro and identified CasRx + sgRNA5 effectively suppressed Kras-G12D transcript levels, leading to attenuation of cell growth. Subsequently, we administered CasRx + sgRNA5 via in vivo Adeno-associated viruses (AAV) delivery at the early tumorigenesis stage, resulting in inhibition of tumor initiation and progression, ultimately enhancing the survival of the mice. Mechanistically, the treatment directly inhibited KRAS signal pathways and regulated a potential protein network comprising KRAS, DUSP8, NR4A1, DUSP1, and EGR1. Encouragingly, the CasRx + sgRNA5 treatment in mice bearing the induced adenocarcinoma demonstrated a positive therapeutic outcome, characterized by seemingly reduced mutation burden, diminished tumor size, and trend of improved overall survival. Remarkably, no obvious off-target events were detected, highlighting the specificity and safety of CasRx therapy. This study establishes the feasibility of CasRx-based-Kras-G12D editing for in vivo tumor inhibition and provides insights into the potential role of the KRAS-associated protein network in early development of lung cancer. These findings hold promise for development of CasRx therapies for this aggressive malignancy.