EP1.14-25 Development of New Lung Cancer Therapies Based on Gene-Editing Technologies

Genome editing has enriched our understanding of mechanisms of the human pathology. Genome editing took a significant advance with the recent development of the CRISPR-Cas9 technology. CRISPR is an acronym for: Clustered Regularly Interspaced Short Palindromic Repeats and it is an adaptation of a prokaryotic functional system. It uses a single guide RNA to direct Cas9 activity to a specific part of the genome, therefore, this system can be used for gene editing and regulation. Cancer is a genetic disease where some DNA-damaged cells begin to divide without stopping and spread into surrounding tissues. Interestingly, in some tumors there is a dependency of a single oncogenic activity (oncogene addiction). This phenomenon indicates that mutations in key oncogenes (driver mutation) are able to drive carcinogenesis and maintain the tumor phenotype. Suggestively, if we can prevent or disrupt these mutations, we can difficult carcinogenesis or damage an established tumoral phenotype We seek out for using Crispr-Cas9 technology to target driver mutations and evaluate its therapeutic and preventive value. To develop a proof of concept, we focused on KRAS gene which represents the most frequently mutated family across all cancer types. About one-third of human lung adenocarcinomas, the most prevalent form of lung cancer, carry KRAS mutations. Most of these mutations are located on codon 12; the mutations KRAS c.34G>T (G12C) and KRAS c.35G>A (G12D) are the most frequent and important ones. We designed specific targeting strategies using HiFiCas9 nuclease which induced KRAS G12C and G12D edition while leaving KRAS WT untouched. Disruption of these KRAS mutations with the specific gRNA-guided-CRISPR-Cas9 decreases viability and proliferation of mutated cells such as H23 and A427. The generation of a transgenic mouse expressing Crispr-Cas9 designed to target these mutations will allow us to test the potential cancer-resistant phenotype. Crispr-Cas9 can be engineered to specifically target single nucleotide oncogenic mutations of KRAS. Edition of KRAS oncogenic mutations G12C and G12D led to a reduction in the viability of mutated lung adenocarcinoma cell lines.