PROJECT SUMMARY Glioblastoma (GBM), one of the most fatal of cancers, remains impervious to treatment with the current standard of care (SOC) therapies. Even immunotherapies like immune checkpoint inhibitors (ICI) have failed for GBM despite their success with other cancers. One critical reason for this failure is that the GBM tumor microenvironment (TME) is highly immunosuppressive. To change the GBM microenvironment, we and others have been utilizing direct intra-tumoral administration of gene therapies that express powerful immune-activators, both preclinically and clinically. In fact, we have concluded a phase 1 clinical trial of intratumoral administration of a regulatable interleukin 12 (IL12) immunogene therapy in subjects with recurrent GBM (rGBM)1 (NCT02026271). In this trial, post-injection tumors showed increased inflammatory infiltrates including increased numbers of CD8+ T cells and elevated levels of tumor IFNγ. However, this also was coupled with increases in tumor PD-L1 and PD-1 positive cells, characteristic of chronic exhaustion and immune escape. Therefore, the trial suggested that addition of anti-PD-1 immune checkpoint signaling should be beneficial to IL12 immunogene therapy efficacy. Based on this, we finished accrual to two multi-institutional clinical trials of intratumoral IL12 immunogene therapy (to increase infiltration of IFNγ producing, CD8+ cytotoxic T cells) coupled with neoadjuvant immune checkpoint inhibition (ICI) (NCT03636477 and NCT04006119). These trials have shown that the combination of these two immunotherapies is well tolerated in humans, but unexpectedly there was no improvement in efficacy by adding ICI. Based on this clinical result, we propose to explore two questions using preclinical models of GBM: 1- Does the timing of ICI administration with respect to IL12 immunostimulation change the therapeutic response? 2- Does GBM immune-evasion from IL12 depend on more than single PD-1 signaling? To provide answers to these two questions, we plan to pursue the following aims: Aim 1- Compare efficacy, tolerability, and immune effects of neo-adjuvant vs. adjuvant anti-PD1 therapy in mouse models of GBM treated with intratumoral IL12 immunogene therapy, and Aim 2- Test the efficacy, tolerability, and immune effects of silencing the immunoevasive noncoding RNA, INCR1, when combined with intratumoral IL12 immunogene therapy. The impact from these studies will be to significantly inform whether IL12 (or other cytokines) immunogene therapy can be improved by changing the timing of ICI administration or by targeting a different pathway that regulates multiple immunoevasive signals, leading us to the next design of clinical trials using cytokine immunogene therapy.