Glioblastoma (GBM) is a form of brain cancer with no cure and a five-year survival rate below 10%. Immunotherapies such as immune checkpoint blockade (ICB) have shown great promise in other solid tumors but have demonstrated no clinical benefits for GBM patients. In part due to a highly immunosuppressive tumor microenvironment that inhibits T cell activation and infiltration. There is consequently an urgent unmet need to engineer new immunotherapies for GBM. Retinoic acid-inducible gene I (RIG-I) is a promising target for cancer immunotherapy. RIG-I is an RNA sensing pattern recognition receptor in the cytosol that leads to downstream production of type I interferon, which can lead to CD8+ T cell infiltration into tumors. However, RNA delivery to the cytosol is a challenge because the negative charge of RNA prevents internalization. Therapeutics that activate RIG-I have been demonstrated to improve response to ICB in several preclinical models of solid tumors but have yet to be explored in GBM. This proposal aims to design and test a biomaterial therapy that locally delivers a RIG-I agonist, 5’-triphosphorylated double-stranded RNA (3pRNA), via a polymeric nanocarrier to elicit a pro-inflammatory response in mouse models of GBM. RIG-I is an unstudied target for treating GBM, and we will deliver a unique stem-loop 3pRNA (SLR) to activate the pathway. SLR will be delivered in pH responsive nanocarriers that destabilize the endosome and release SLR into the cytosol. An α-CD11b nanobody will be conjugated to the nanoparticle to drive uptake by antigen presenting cells, including resident microglia and infiltrating dendritic cells and macrophages. We propose two specific aims to 1) Engineer RANs for GBM immunotherapy and 2) determine their immunological consequences. We will test the aims using the following experiments: Aim 1) SLR will be covalently conjugated to polymeric nanoparticles, followed by click chemistry to attach an α-CD11b nanobody. We will verify RNA activity and uptake in vitro using interferon reporter cell lines and bone marrow-derived dendritic cells. The efficacy of our engineered nanoparticles will be tested in the GL261 and CT2A immunocompetent models of GBM using intracranial injection of the nanoparticles. We will determine if combination therapy with ICB leads to enhanced efficacy, as RIG-I activating nanoparticles will create a more immunogenic tumor microenvironment. Aim 2) Studies will determine the immunological mechanism of treatment. We will evaluate immune cell frequency and activation state in the tumor and brain. We will determine T cell dependence, if infiltrating T cells are necessary for efficacy, and if therapy creates memory T cells in the brain. We hypothesize that our polymer therapeutic will activate antigen presenting cells, leading to CD8+ T cell infiltration into the tumor. This project will improve the knowledge in the field of neuroimmunology through insight gained about the role of RIG-I activation in GB...