CasRx-Based KRASG12D Silencing for In Vivo Tumor Inhibition in Spontaneous Lung Adenocarcinoma
Lung adenocarcinoma with KRAS mutation represents a formidable challenge in cancer treatment due to its undruggable oncogenic mutation. Here, we explored the CasRx-editing approach on lung adenocarcinoma driven by the KRAS activation and Tp53 loss in vivo, a scenario closely resembling clinical conditions. We screened seven sgRNAs in cultured cells and identified that CasRx combined with sgRNA5 effectively suppressed murine KRASG12D transcript levels by 93.185 ± 4.758%, leading to significant attenuation of cell viability and proliferation. 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 targeted the classic 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 lung adenocarcinoma demonstrated an optimistic therapeutic outcome, characterized by reduced mutation burden, diminished tumor size, and seemingly improved overall survival. Remarkably, no obvious off-target events were detected in the treated tissues, highlighting the specificity and safety of CasRx + sgRNA5 therapy. This study establishes the feasibility of CasRx-based-KRASG12D editing for efficient in vivo tumor inhibition and provides insights into the potential role of the KRAS-associated protein network in the early development of lung cancer. These findings hold great promise for the development of novel targeted therapies for this aggressive malignancy. Overall design: To assess the expression levels of therapeutic vectors and Kras transcripts, elucidate the underlying mechanisms, and evaluate potential off-target effects of CRISPR/CasRx in the context of lung cancer therapy by targeting the driver KRASG12D, the research was performed on an in vivo spontaneous tumor model. We then performed gene expresion profiling analysis using data obtained from deep RNA_seq of lungs subjected to CasRx therapy for either two weeks of six weeks at the early stage of tumorigenesis. Additionally, we analyzed tumors that underwent CasRx treatment for a duration of six weeks. Comparative gene expression profiling analysis of RNA_seq data for tissues between experimental groups (AAV-CasRx + sgNC) and control groups (AAV-CasRx + sgRNA2 and/or AAV-CasRx + sgRNA5), considering diverse time points and experimental designs.