EXTH-37. TARGETING THE IDH1R132H MUTATION IN GLIOMAS BY CRISPR-CAS PRECISION BASE EDITING

Abstract

Abstract Gliomas are the most frequent malignant primary brain tumors and continue to lack curative therapies. To aid the development of novel therapeutic approaches, a better understanding of glioma-specific molecular patterns is essential. We therefore investigate the role of the canonical point mutation in the isocitrate dehydrogenase 1 gene (IDH1R132H). Its early occurrence in oncogenesis and ubiquitous expression suggests a causative driver function; however, current literature is inconclusive on the impact of IDH1R132H, requiring innovative approaches to further elucidate the functional consequences and therapeutic potential of IDH1R132H. METHODS Clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas precision base editing systems enable the alteration of a specific base pair without suffering from off-target editing associated with previous, double strand brake-inducing systems and thus making them an ideal tool to revert the point mutation in IDH1R132H gliomas. RESULTS By selecting a CRISPR-Cas precision base editing system, the IDH1R132H site was edited with editing efficiencies of up to 50%. Primary patient derived cell lines and organoids were corrected in their IDH1R132H locus. Phenotypical changes, such as a change in IDH1R132H protein levels, 2-hydroxyglutarate concentration as well as proliferation rates were observed upon the reversal of the point mutation. Furthermore, the precision base editing system was packaged into a dual-AAV-vector split intein system and showed successful in vitro gene editing. In conclusion, this precise genetic intervention provides a methodology to create accurate patient derived models to analyze the impact of IDH1R132H on glioma biology and provides a framework for in vivo gene therapy.