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

in Neoplasia (New York, N.Y.) by Sophie R Wu, Julianne Sharpe, Joshua Tolliver, Abigail J Groth, Reid Chen, María E Guerra García, Vennesa Valentine, Nerissa T Williams, Sheeba Jacob, Zachary J Reitman

TLDR

  • Researchers developed a novel mouse model for diffuse midline gliomas using CRISPR/Cas9 genetic engineering, which can be used to study tumorigenesis and potentially inform new treatment approaches.

Abstract

Diffuse midline gliomas (DMGs) are lethal brain tumors that arise in children and young adults, resulting in a median survival of less than two years. Genetically engineered mouse models (GEMMs) are critical to studying tumorigenesis and tumor-immune interactions, which may inform new treatment approaches. However, current midline glioma GEMM approaches are limited in their ability to multiplex perturbations and/or target specific cell lineages in the brain for genetic manipulation. Here, we combined the RCAS/tv-a avian retrovirus system and CRISPR/Cas9 genetic engineering to drive midline glioma formation in mice. CRISPR/Cas9-based disruption of Trp53, a tumor suppressor that is frequently disrupted in midline gliomas, along with the oncogene PDGF-B resulted in high grade tumor formation with moderate latency (median time to tumor formation of 12 weeks). We confirmed CRISPR-mediated Trp53 disruption using next-generation sequencing (NGS) and immunohistochemistry (IHC). Next, we disrupted multiple midline glioma tumor suppressor genes (Trp53, Pten, Atm, Cdkn2a) in individual mouse brains. These mini-pooled in vivo experiments generated primary midline gliomas with decreased tumor latency (median time to tumor formation of 3.6 weeks, P < 0.0001, log-rank test compared to single-plex gRNA). Quantification of gRNA barcodes and CRISPR editing events revealed that all tumors contained cells with various disruptions of all target genes and suggested a multiclonal origin for the tumors as well as stronger selection for Trp53 disruption compared to disruption of the other genes. This mouse modeling approach will streamline midline glioma research and enable complex experiments to understand tumor evolution and therapeutics.

Overview

  • The study focuses on developing a novel mouse model for diffuse midline gliomas (DMGs), which are aggressive and lethal brain tumors that affect children and young adults.
  • The researchers used a combination of the RCAS/tv-a avian retrovirus system and CRISPR/Cas9 genetic engineering to drive midline glioma formation in mice.
  • The primary objective of the study is to develop a reliable and versatile mouse model for DMGs, which can be used to study tumorigenesis and tumor-immune interactions, and potentially inform new treatment approaches.

Comparative Analysis & Findings

  • CRISPR/Cas9-based disruption of Trp53, a tumor suppressor frequently disrupted in midline gliomas, along with the oncogene PDGF-B resulted in high-grade tumor formation with moderate latency (median time to tumor formation of 12 weeks).
  • Disrupting multiple midline glioma tumor suppressor genes (Trp53, Pten, Atm, Cdkn2a) in individual mouse brains resulted in primary midline gliomas with decreased tumor latency (median time to tumor formation of 3.6 weeks, P < 0.0001, log-rank test compared to single-plex gRNA).
  • Quantification of gRNA barcodes and CRISPR editing events revealed that all tumors contained cells with various disruptions of all target genes, suggesting a multiclonal origin for the tumors and stronger selection for Trp53 disruption compared to disruption of the other genes.

Implications and Future Directions

  • This novel mouse model will streamline midline glioma research and enable complex experiments to understand tumor evolution and therapeutics.
  • This study opens up new avenues for understanding the role of specific genetic mutations in tumor development and progression, which can inform the development of targeted treatments.
  • Future studies can utilize this mouse model to investigate the effects of different genetic perturbations on tumor development and to identify potential biomarkers for early diagnosis and treatment of midline gliomas.
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