EXTH-55. DELIVERY OF CAS9/SGRNA RIBONUCLEOPROTEIN CONJUGATE VIA VIRUS-LIKE PARTICLES FOR EFFICIENT GENE EDITING OF TRANSCRIPTION FACTOR BRACHYURY IN GLIOBLASTOMA

Brachyury, a transcription factor that is a critical driver during normal embryonic development, is highly expressed in glioblastoma (GBM) but rarely expressed in normal adult tissues. Recent evidence suggests that brachyury plays a crucial role in regulating GBM cell proliferation and stemness, implying the potential of brachyury as a therapeutic target. However, like other transcription factors, brachyury is not readily inhibited pharmacologically due to the absence of ligand-accessible small-molecule binding pockets, and a direct inhibitor of brachyury has not been identified. Our team has recently developed a novel virus-like particle (VLP)-based system by fusing aptamer-binding protein to the lentiviral nucleocapsid protein within the group-specific antigen, which allows for efficient packaging of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) for gene editing. Our data suggests that our VLP-mediated Cas9/gRNA RNP allows for transient expression of Cas9 in patient-derived GBM cells while maintaining effective gene editing efficiency (∼40%). Our strategy results in 90% knockdown of brachyury in vitro with subsequent inhibition of GBM cell proliferation and stemness as well as enhanced susceptibility of tumor cells to radiation via increased cleavage of caspase-3. In addition, our in vivo results using a xenograft mouse model further demonstrates that transduction with VLP-packaged Cas9/brachyury gRNA inhibits GBM progression. Our findings demonstrate that our novel VLP-based system allows for efficient delivery of Cas9/gRNA RNP-based brachyury gene editing technology and represents a novel treatment strategy for GBM.