Abstract 471: CRISPR-Cas9 as a novel cancer gene therapy approach against pancreatic cancers

Abstract Pancreatic cancer (PC) is the third leading cause of cancer deaths in US with a 5-year survival rate of 13%. Most patients are diagnosed at advanced stages, and conventional treatments remain ineffective and highly toxic to these patients as both chemotherapy and radiation affect normal, healthy cells, leading to adverse side effects. We have adapted CRISPR-Cas9 as a cancer-specific killing strategy that spares normal cells. CRISPR-Cas9 is a molecular scissor that induces DNA double strand breaks (DSBs) at its targeted genomic locations. We use CRISPR-Cas9 to target the subset of somatic mutations that create protospacer adjacent motifs (PAMs). Using S. pyogenes Cas9 that recognizes a 5’-NGG-3’ PAM, we identified somatic single base substitutions (SBSs) that give rise to novel PAMs (e.g. NCG→NGG), and use the upstream sequence to design sgRNAs for cancer-targeting. We have discovered that ∼13% of SBSs in PC form novel PAMs, and these cancer-specific sgRNAs exhibited on-target activity and lack of off-target events. Notably, while examining WGS data from rapid autopsy samples, we found that 90% of PAMs were maintained among primary carcinomas and metastases. Using multi-target sgRNAs targeting 2-16 sites in the non-coding regions of human genome, we demonstrate a dose-response relationship in which >10 target sites are required to achieve >99% cell elimination. Moreover, we established that simultaneous induction of multiple DSBs by CRISPR-Cas9 causes extreme chromosomal instability that ultimately leads to cell death. We detected massive chromosomal aberrations, including dicentrics, ring chromosomes, and polyploidization. Surprisingly, CRISPR-Cas9-induced DSBs are significantly more cytotoxic in PCs than a comparable number of radiation-induced DSBs, in which the number of DSBs required to achieve equitoxic effects was ∼3 times higher for radiation than CRISPR-Cas9. Finally, we have developed a pipeline to screen for the most toxic sgRNAs in a given PC for selective killing. Using our PAM-based approach, we identified >100 Panc10.05-specific sgRNAs that have no off-target activity at 0- and 1-mismatch sites. We cloned them into lentiviral vectors and transduced in bulk, followed by NGS to discover sgRNAs that decreased in representation in the targeted cell line but not in the controls. We selected the top sgRNAs that have 100% variant allele frequency (VAF), cloned 4 of them into a multi-sgRNA expression vector, and transduced it into co-cultures consisting of two PC cell lines. Greater than 85% selective reduction of the targeted cell line was detected. Then, we selected nine top sgRNAs, transduced them as a pool into Panc10.05 cell line, and injected these cells into athymic, nude mice for tumor growth. We observed >90% tumor growth inhibition with these cancer-specific sgRNAs. Overall, our study highlights the therapeutic potential of CRISPR-Cas9 as an anti-cancer strategy and supports the genetic targeting of PCs. Citation Format: Selina Shiqing K. Teh, Akhil Kotwal, Kirsten Bowland, Alexis Bennett, Eitan Halper-Stromberg, Laura Morsberger, Sarah Wheelan, Nicholas J. Roberts, Chien-Fu Hung, Michael Goldstein, Ying S. Zou, James R. Eshleman. CRISPR-Cas9 as a novel cancer gene therapy approach against pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 471.