Background: The prognosis of subjects with glioblastoma multiforme (GBM) after first recurrence is extremely poor with a median overall survival (OS) of approximately 90 - 160 days. New therapies are needed, thus we developed an adenoviral vector, Ad-RTS-IL-12 (AD), administered intratumorally under the control of the RheoSwitch Therapeutic System (RTS) expression platform. Gene expression and IL-12 protein production is tightly controlled by activator ligand veledimex (V) and in our mouse model of glioma we have consistently shown a dose-response and survival benefit compared to temozolamide, bevacizumab, and a PD-1 inhibitor. Methods: In a multicenter Phase I dose escalation trial, subjects with recurrent or progressive Grade III or IV glioma undergoing resection were injected intratumorally with AD 2x10¹¹viral particles and daily oral V for 15 doses, beginning on Day 0, prior to surgery. The study primary endpoint is safety and tolerability of AD+V with secondary endpoints including OS. Results: In the first cohort, consisting of 4 males and 3 females (ages 32-58), it was observed that V crossed the blood-brain barrier with 30±6% of the V plasma level in the resected tumor sample (17±6 ng/ml vs. 5±2 ng/ml). Peak serum IL-12 was observed at Day 3 (mean 23 pg/ml) and peak IFNg (mean 32 pg/ml) on Day 7. Peripheral blood samples showed an increase in CD8 T cells and an increase in the ratio of CD8/CD4 and CD8/FoxP3. AD+V was well tolerated with expected toxicities promptly reversing with discontinuance of V. Neurotoxicities were minimal and manageable. To date, 7/7 subjects are alive with 5 subjects having a follow-up of > 90 days and 3 > 160 days posttreatment. Conclusions: Intratumoral regulated IL-12 expression using AD+V in a salvage population with advanced and recurrent glioma has an acceptable and rapidly reversible safety profile. The intracranial administration of AD followed by oral V is clinically feasible and suggests possible benefit vs. historical control and provides a strong rational for dose-escalation as reviewed and approved by the independent Data Safety Monitoring Board. Clinical trial information: NCT02026271