Creating cancer-specific genotoxicity by targeting Cas9 nickase to focal gene amplifications

Focal gene amplification is a hallmark of many types of cancer that drives tumorigenesis, such as MYCN-amplified neuroblastoma. Gene amplification affords an opportunity for therapeutic exploitation if DNA damage can be site-specifically targeted to amplified loci, since the damage will accumulate specifically in cancer cells. DNA nicks (single strand breaks, SSBs) can create single ended double strand breaks (seDSBs) within a proliferating cell if encountered by a DNA replication fork, where large numbers of seDSBs can lead to cell death. SpCas9 nickase can be employed to selectively create large numbers of SSBs within the genome of proliferating cancer cells by targeting highly amplified loci resulting in a toxic level of seDSBs. MYCN-amplified neuroblastoma provides an ideal cancer to test the efficacy of Cas9 nickase as a cancer-selective genotoxic agent when targeting focal gene amplification. MYCN-amplified neuroblastoma is observed in ~20% of diagnosed cases and has an overall survival rate of ~50%. Thus, it represents a target with important unmet medical need. Based on our preliminary data, Cas9 nickase shows promise as a selective therapeutic agent for MYCN-amplified neuroblastoma cells in vitro and in vivo. We propose to improve the properties of Cas9 nickase for selective killing of MYCN-amplified neuroblastoma cells and demonstrate its therapeutic potential in vivo in an orthotopic xenograft model of MYCN-amplified neuroblastoma. Evaluation of the efficacy and safety of new anti-cancer therapeutics delivered systemically by nanoparticles are most appropriately evaluated in an in vivo mouse model of neuroblastoma, where the impact of therapeutic treatment on tumor metastasis, which is a critical component in high risk MYCNamplified neuroblastoma, can be assessed, which is not possible in cell culture/organoid systems. Tumor plasticity and evolution in response to genotoxic damage is best approximated with in vivo orthotopic/disseminated tumors that recapitulate the aggressive, highly vascular and invasive character of high risk MYCN-amplified neuroblastoma. In Aim 1, we will optimize the efficiency of neuroblastoma cell killing by Cas9 nickase through improvements to the nickase, sgRNA and choice of target site. In Aim 2, we will optimize our lipid nanoparticle carrier formulation for delivery of Cas9 nickase mRNA to an orthotopic xenograft model of neuroblastoma and evaluate the efficacy of tumor reduction by Cas9 nickase. In Aim 3, we will evaluate small molecule inhibitors of DNA damage response pathways for their ability to enhance Cas9 nickase toxicity. We have already demonstrated that Cas9 nickase toxicity is enhanced by a CHK1 inhibitor, which blocks a DNA damage response pathway that responds to cellular replication stress. We will then evaluate the efficacy of tumor reduction in vivo by Cas9 nickase in conjunction with promising inhibitors of DNA damage response pathways. At the conclusion of this study, we will have a therapeutic lead for the treatment of refractory MYCN-amplified neuroblastoma that can undergo further optimization, and efficacy and safety assessments in vitro and in vivo in the context of assembling a pre-IND package for FDA interactions. Project Number: 1R01CA311503-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: SCOT WOLFE | Institution: UNIV OF MASSACHUSETTS MED SCH WORCESTER, WORCESTER, MA | Award Amount: $622,611 | Activity Code: R01 | Study Section: Drug Discovery and Molecular Pharmacology C Study Section[DMPC] View on NIH RePORTER: https://reporter.nih.gov/project-details/11343314