SUMMARY Despite recent advances in therapeutic strategies, the prognosis for patients with highly malignant brain tumors, glioblastomas (GBM) remains poor, with a median survival of 12-19 months. Immunotherapy has emerged as a promising approach for different cancer types. However, its efficacy in GBM has been limited primarily by overall systemic immune suppression and the immune-suppressive tumor micro-environment. Recently, we have shown CRISPR/Cas9 engineered self-targeting re-purposed cancer cells specifically home to tumor cells and release targeted ligands that induce tumor cell killing which translates into survival benefits in mouse models of primary and metastatic tumors. Based on our exciting studies, we have gene edited and subsequently engineered syngeneic immunosuppressive GBM to express bi-functional immunomodulatory and cytotoxic protein, interferon (IFN)β and granulocyte macrophage stimulating factor (GMCSF), which is known to induce both innate and adaptive immunity. Our preliminary data reveal that repurposed immunosuppressive GBM cells do not proliferate in vivo and elicit an active immunity which prevents tumor recurrence. These results although promising, have raised fundamental questions for our tumor cell based gene edited therapy strategy to be characterized and tested extensively in immunocompetent mouse tumor models that mimic clinical settings of immunosuppressive, resected and recurrent immune-profiled GBM tumors. In this proposal, we will first develop and extensively characterize a platform of gene edited and engineered syngeneic immunosuppressive and active GBM therapeutic tumor cells (ThTC) and assess them for their mechanism based direct killing of parental GBM cells and their ability to elicit active anti-tumor immunity in primary and recurrent mouse GBMs. Based on our previous findings that GBM tumor resection promotes the recruitment of CD4/CD8 T cells and local delivery of synthetic extracellular matrix (sECM) encapsulated immunomodulators has therapeutic efficacy, we will test sECM-ThTC for their therapeutic efficacy in resected GBM mouse tumor models. We hypothesize that ThTC will lead to specific killing of residual GBM cells in the tumor resection cavity of primary and recurrent GBMs and elicit active immunity. To ease clinical translation, we will ultimately CRISPR/Cas9 gene edit and subsequently engineer patient derived resected primary tumor cells (hTC) to express human IFN and GMCSF (hThTC). These hThTC will be tested in recurrent GBM models generated from glioma stem cell (GSC) lines in humanized mice. The integration of the safety kill switch, HSV-TK in ThTC will ensure safety in our approach and the incorporation of genetically engineered imaging markers into both ThTC and GBMs will allow us to follow fate and efficacy in vivo and thus to fine tune the proposed approaches. We anticipate that our findings will have a major contribution towards developing novel ThTC based therapies for GBM and are l...