Abstract 5156: Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of midline glioma

Abstract Diffuse midline gliomas (DMGs) are fatal pediatric brain tumors that commonly arise in the brainstem and other midline brain structures, with a median overall survival of less than two years. Genetically engineered mouse models (GEMMs) are key to studying tumorigenesis and tumor-immune interactions, which may inform the development of urgently-needed treatment approaches. However, current midline glioma GEMM approaches are limited in their ability to model multiple gene perturbations and to target specific cell lineages in the brain. Here, we establish a platform that combines the RCAS/tv-a avian retrovirus system with CRISPR/Cas9 gene editing to drive midline glioma formation in mice. To express endonuclease Cas9 and the avian retrovirus receptor TVA in mouse neural stem cells, we generated mice of genotype Nestin-TVA; Nestin-Cre; LoxP-STOP-LoxP-Cas9-EGFP (NNC). RCAS retrovirus plasmids were cloned containing guide RNAs (gRNAs) targeting various DMG-relevant genes for perturbation, including Trp53, Cdkn2a, Atm, Pten, and a non-targeting control (Cntl). Two types of plasmids were generated for each gene target, one with a platelet-derived growth factor beta ligand (PDGFB) oncogene driver and another with a blue fluorescent protein (BFP) marker.To confirm if the combined system could generate high grade midline gliomas in mice, the Trp53 tumor suppressor that is frequently disrupted in midline gliomas was targeted for CRISPR/Cas9 based disruption. We observed high grade tumors with moderate latency (median time to tumor formation of 12.57 weeks). Immunohistochemistry (IHC) and next-generation sequencing (NGS) confirmed tumor features that model human DMG, expression of Cas9 and PDGFB, and CRISPR-mediated loss of p53. Next, we disrupted multiple tumor suppressor genes (Trp53, Atm, Cdkn2a, Pten) in a mini-pooled in vivo experiment, generating midline gliomas with decreased tumor latency (median time to tumor formation 3.6 weeks, P < 0.0001, log-rank test compared to a single-plex gRNA). Quantification of gRNA barcodes and CRISPR editing events using NGS revealed that all tumors contained cells with disruption of all target genes, suggesting a multiclonal origin for the tumors and stronger selection for Trp53 disruption compared to other genes. This mouse modeling approach may streamline future GEMM DMG research by enabling complex experiments in which new genes of interest can be rapidly perturbed by CRISPR/Cas9. Sophisticated experiments such as pooled in vivo genetic experiments may allow for screens to identify genetic determinants of tumorigenesis or treatment response. Citation Format: Sophie R. Wu, Julianne Sharpe, Joshua Tolliver, Abigail J. Groth, Reid Chen, María E. Guerra Garcia, Vennesa Valentine, Nerissa T. Williams, Sheeba Jacob, Zachary J. Reitman. Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of midline glioma [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 5156.