Abstract 1826: CRISPR mediated KRAS mutant gene editing in pancreatic and colorectal cancer therapy

Background: KRASG12V and KRASG12D mutations are among the most commonly observed mutations in pancreatic (30% and 51%) and colorectal (27% and 45%) adenocarcinomas and have been associated with poor prognosis. Activating mutations in KRAS play potent roles in cancer initiation, propagation, and maintenance. Although they represent important therapeutic targets, no effective treatment for direct inhibition of KRASG12V and KRASG12D mutants have been reported. We have developed a new potent strategy combing the potency of CRISPR for gene editing with a tumor selective nanocarrier, to impair cancer cell proliferation by directly targeting G12V and G12D mutations of the KRAS oncogene. Methods: ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with CRISPR/Cas9. ADGN nanoparticles containing Cas9mRNA and gRNA targeting either KRASG12D or KRASG12V were evaluated on pancreas (PANC1, PK-45H) and colorectal (LS513, SW480, SW403) cancer cells harboring KRASG12D or KRASG12V mutations. KRAS mutant indel frequency was evaluated by T7E1 method and compared to KRASWT (HT29, HS-69) cells. Cell proliferation was determined by flow cytometry. In-vivo efficacy of IV-administered ADGN/mRNACas9/gRNA complexes was evaluated in PANC1-LUC (KRASG12D) and SW403 (KRASG12V) mouse xenografts. The phosphorylation of ERK and AKT was measured by western blot, KRAS mutation level in the tumor and variation of housekeeping genes were estimated by deep sequencing. Results: We have developed highly specific gRNAs targeting KRASG12V and KRASG12D mutants complexed with ADGN-nanoparticles. These complexes efficiently and selectively silenced KRASG12V and KRASG12D in colorectal and pancreatic cancer cells in vitro and in vivo. Silencing KRAS mutations reduced the cell proliferation by 70% and the phosphorylation of the downstream effector pathways ERK and AKT. ADGN-nanoparticles containing CRISPR/gRNA targeting KRASG12D abolished PANC1 tumor growth and induced KRASG12D gene editing in the tumor with an indel frequency of 76%. ADGN-nanoparticles containing CRISPR/gRNA targeting KRASG12V abolished SW403 tumor growth and induced KRASG12V gene editing in the tumor with an indel frequency of 82%. In contrast, no effect on tumor growth were observed with ADGN-nanoparticles containing non-specific gRNA or gRNA targeting other KRAS mutations. ADGN/mRNACas9/gRNA targeting KRASG12D or KRASG12V abolished pancreatic and colorectal tumor growth respectively in vivo, without inducing any significant toxicity, emergence of off target effects or other KRAS mutations. Conclusions: ADGN-nanoparticle containing mRNACas9/gRNA targeting KRASG12D or KRASG12V were effective in selectively targeting mutated KRAS both in vitro and in vivo. Our study provides a proof-of-concept that ADGN/CRISPR can be applied to target driver mutations of cancers in vivo and permanently disrupt the oncogenic alleles, leading to inhibition of tumor growth. We proposed a new strategy for targeting KRAS oncogenes that can be useful in treatment of pancreatic and colorectal cancers. Citation Format: Gilles Divita, Elodie Czuba, Melanie Guidetti, Audrey Gunenberger, Leslie Tempremant, Veronique Josserand, Neil Desai. CRISPR mediated KRAS mutant gene editing in pancreatic and colorectal cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1826.