Brain tumors are the most lethal form of pediatric cancers, especially high-grade gliomas (WHO grade IV), which have a 5-year survival rate of 17%. Developments in immunotherapy could provide new opportunities for the treatment of these patients. Effector T cells are capable of eliminating malignant cells, however, the response often gets suppressed by the tumor in one way or another. Immunotherapies are designed to relieve this suppression and has been successful in several cancer types. In adult and pediatric glioma immunotherapy has been unable to improve survival, likely because these tumors use different mechanisms of suppression that are not properly targeted by the current therapies. One barrier to success of immunotherapy is the baseline infiltration of T cells. Glioma vasculature is structurally and functionally impaired and does not properly support the infiltration of T cells. In fact, the vasculature can express markers that directly suppress or eliminate T cells. In this application my overall goal is to identify all suppressive mechanisms of the glioma vasculature and reverse the suppression with vasculature targeted therapies. I will utilize our single cell RNA sequencing dataset of endothelial cells, derived from human and murine glioma. The cells will be clustered with bioinformatic tools, to identify functionally distinct subsets. Furthermore, underlying regulators/pathways will be identified through a CRISPR-CAS9 knock-out screen of transcription factors. The genes underlying T cell suppression will then be targeted using glioma vasculature-targeted adeno-associated viral (AAV) vectors in murine glioma models, and AAVs will be combined with immunotherapies. The ultimate goal of my research is to translate findings to the clinic and utilize the AAV-based targeting of the vasculature to enhance efficacy of immunotherapy, and survival of glioma patients.