Abstract LT02: CRISPR-Cas9 as a Novel Cancer Gene Therapy Approach against Pancreatic Cancers

Somatic mutations are desirable targets for selective cancer elimination, yet most are found within noncoding regions. We have adapted CRISPR-Cas9 as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks (DSBs). We hypothesized that somatic 5’-NGG-3’ PAMs arising from base substitutions (e.g. NCG→NGG) could serve as CRISPR-Cas9 targets for selective cancer elimination through the induction of multiple DSBs. Whole genome sequencing (WGS) of paired tumor-normal (T-N) samples from three PC patients were put through PAMfinder (software for somatic PAM discovery). On average, 417 somatic PAMs per PC case were found (>99% of them in non-exons) and 97% of the selected subset were Sanger confirmed. We then analysed T-N samples from ICGC which revealed medians of 455 somatic PAMs per tumor in two PC cohorts (N=174 combined), 2790 in lung (N=29), and 3236 in esophageal (N=388) cancer cohorts. We performed γH2AX staining on PC cell lines transduced with 4-9 sgRNAs designed from our PAM discovery approach, and observed more foci with increased number of sgRNAs in the target cell line but not in the non-target cell line. Deep sequencing and WGS on clones transduced with cancer-specific sgRNAs also demonstrated on-target activity in the target cell line but not in either the patient’s normal cells or an irrelevant cancer cell line. No off-target events were detected. To test selective toxicity in vitro and in vivo, we transduced 4-9 sgRNAs into PC co-cultures and found 69-99% selective cell death of three target cell lines through flow cytometry. Finally, using mouse xenograft models, 92% weight reduction (N=10, P=0.0004) of tumors transduced with a pool of 9 cancer-specific sgRNAs was achieved compared to tumors transduced with non-targeting sgRNAs (N=8). Citation Format: Selina Shiqing K. Teh, Kirsten Bowland, Alexis Bennett, Eitan Halper-Stromberg, Akhil Kotwal, Hong Liang, Sarah Wheelan, Chien-Fu Hung, Michael Goldstein, Nicholas J. Roberts, and James R. Eshleman. CRISPR-Cas9 as a Novel Cancer Gene Therapy Approach against Pancreatic Cancers [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr LT02.