PROJECT SUMMARY/ABSTRACT Dr. Jessica Foster's career goal is to become a translational physician-scientist focused on immunotherapy for pediatric brain tumors. This proposal describes a five-year plan to facilitate her transition to independence through the acquisition of critical technical skills and scientific training in brain tumor modeling and evaluation of the tumor microenvironment with single cell RNA sequencing, integrated with comprehensive mentoring from a diverse team of faculty members. She will conduct the proposed studies under the proven mentorship of Dr. John Maris, an international leader in translational neuroblastoma research and immunogenomics. Additionally, her dedicated Advisory Committee is comprised of highly regarded physician-scientists with diverse expertise in immunotherapy and pediatric brain tumors. Finally, the collaborative research environment with unparalleled resources at the University of Pennsylvania and the Children's Hospital of Philadelphia provides an ideal setting to conduct these translational studies. Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain tumor that remains incurable despite decades of clinical trials, with a median survival of 11 months. This proposal seeks to use chimeric antigen receptor (CAR) T cell therapy, a form of immunotherapy, to target DIPG. Recently GD2 was identified as an immunotherapeutic target for DIPG, and lentiviral GD2-directed CAR T cells were able to successfully treat murine models of DIPG. However, a significant number of mice treated with CAR T cells died due to inflammation and herniation, prompting concerns for potential toxicity from this therapy, in particular in the pons. This proposal is building upon the applicant's experience utilizing mRNA for the creation of CAR T cells that are transient and can be titrated to effect to avoid toxicity, here using repeated local delivery of GD2- directed mRNA CAR T cells to effectively treat DIPG while still maintaining safety. Aim 1 will determine the effect of GD2-directed mRNA CAR T cells on tumor, microenvironment and normal tissue using both immunocompetent and xenograft models of DIPG. Aim 2 will use single cell RNA sequencing to investigate the role of microglia in DIPG development and test mRNA CAR T cells in combination with inhibition of microglia. Dr. Foster's ultimate goal is to create a clinical trial for patients with DIPG using mRNA CAR T cells directed against GD2, as well as generating a new treatment platform for all pediatric brain tumors. These efforts will provide an outstanding foundation for her career as a physician-scientist and the development of an independent translational research program.