PROJECT SUMMARY High-grade gliomas are the most common primary brain tumors in adults that continue to have poor median 5-year survival rates despite maximal safe resection and chemoradiation. Over the last 20 years, the standard treatment for high-grade gliomas has not changed drastically; therefore, attention has shifted to developing virotherapies to improve outcomes from this lethal disease. Viral based gene therapies are preferred biological agents that selectively deliver transgenes to tumor cells and are not permissible in normal cells. The first clinical candidate retroviral replicating vector (RRV) (DB107, vocimagene amiretrorepvec) delivers a transgene, cytosine deaminase, to convert the non-toxic prodrug 5-fluorocytosine (5-FC) to an intracellular chemotherapeutic, 5-fluorouracil. Recently, phase III randomized clinical trials (Toca 5, NCT02414165) using the first clinical candidate DB107 (formerly named Toca511) demonstrated an improvement in survival in a select subgroup of patients with a novel biomarker, DGM7 compared to patients receiving standard of care alone (SOC) (18.3 vs. 12.0 months, HR: 0.5, p<0.0167). Since DGM7 is highly predictive of a positive response to retroviral gene therapy, we are conducting a Phase II, biomarker-driven trial to a priori identify “responders” to DB107+5- FC. Additionally, we have previously shown that DB107+5-FC suppresses myeloid derived suppressor cells and recruits tumor infiltrating CD8+ T-cells. Therefore, we hypothesize that DB107+5-FC will not only improve outcomes in patients with recurrent high-grade gliomas, but will also induce a robust anti-tumor immune response. To non-invasively characterize the immune response, we plan to employ an multiplex immuno-ELISA in treated patients before and after exposure to DB107+5-FC. Additionally, we will validate these in vivo immuno- ELISA signatures with single-nuclei RNA-seq of HGG ex vivo. The fundamental goal of this study is to 1) prospectively validate DGM7 as a novel biomarker to identify patients who will respond to DB107 and 2) comprehensively characterize the anti-tumor immune response after viral-based gene therapy through serum- based ELISA and transcriptomic approaches. The proposed clinical trial may not only validate clinical DGM7 as a prognostic marker for DB107 in recurrent high-grade gliomas but also set the foundation for the clinical translation of biomarker-driven clinical trials in neuro-oncology.