PROJECT SUMMARY/ABSTRACT Antitumor immune responses require a functional repertoire of innate and adaptive immune cells. Glioblastoma (GBM), however, harbors a profoundly immunosuppressed microenvironment, particularly its T cell ignorance caused by bone marrow sequestration; T cell exhaustion caused by immune checkpoint molecules on the surface of T cells that suppress T cell function; and impaired memory T-cell responses. Unfortunately, efforts to target the immunosuppressed GBM microenvironment with systemic immunotherapies have not produced meaningful impact in clinical trials. Localized viral treatments have also been investigated for GBM and, while these viruses elicit an anti-tumoral immune response, these treatments have also failed to impact survival in clinical trials. To address these limitations, we have investigated intratumoral delivery of a replicating retrovirus expressing RLI, which encodes an interleukin-15 fusion protein that enhances CD8+ and CD4+ naïve and memory T-cell proliferation, as a therapeutic strategy free of the toxicities of systemic treatments targeting the tumor microenvironment. We demonstrated that replicating retroviral delivery of RLI prolonged survival of immunocompetent mice with intracranial gliomas using multiple different models. Here, we will build upon our data by investigating our central hypothesis that intratumoral RLI immunomodulatory gene therapy can be potentiated by adding other immunomodulatory strategies, incorporating immunogenic cell death, or targeting resistance mechanisms. We will investigate our hypothesis through four specific aims: (1) Potentiate RLI immunomodulatory gene therapy by enhancing T-cell mobilization, co-stimulation, and memory; (2) Determine if targeting checkpoint pathways potentiates retroviral RLI immunomodulatory gene therapy; (3) Enhance RLI immunomodulatory gene therapy by incorporating immunogenic cell death; and (4) Identify and target glioblastoma-expressed proteins that counteract retroviral RLI immunomodulatory gene therapy. Our pursuit of these aims will utilize novel technologies developed by our lab such as our binary retroviral system to deliver a large payload of immunomodulatory genes and our retroviral compact Cas13d RNA-targeting CRISPR to target resistance mechanisms. We will combine these innovative approaches with cutting-edge technologies such as CyTOF to characterize the effects of RLI-based retroviral therapies on the full cohort of innate and adaptive immune responses; customized CRISPRi libraries; paired immunodeficient and immunocompetent mice strains to isolate immunologic resistance mechanisms; and single cell sequencing to profile T-cell subsets altered by these therapies. These studies will develop our novel localized RLI retroviral immunotherapy in a manner that addresses the spectrum of mechanisms creating local and systemic immunodeficiency in GBM by accounting for T-cell ignorance and exhaustion, and identifying and targeting tumor cell and immune cell-driven resistance mechanisms before they evolve. In doing so, we will validate our hypothesis regarding the impact of RLI-based immunomodulatory gene therapy on GBM, a novel strategy with significant translational potential.

PROJECT NARRATIVE Considerable preclinical and clinical evidence has emerged in recent years suggesting that the poor prognosis faced by glioma patients is in part due to the lack of an effective antitumor T-cell response. Here, we will investigate a localized and comprehensive approach of replicating retroviral delivery of RLI, an IL-15 fusion protein, and other immunomodulatory genes as a therapeutic strategy for glioblastoma. Direct stimulation of an anti-tumor T-cell mediated immune response through retroviral delivery of these genes will reverse the T-cell poor nature of glioblastoma without systemic toxicity.