Abstract BACKGROUND GD2-directed Chimeric Antigen Receptor (CAR) T cell therapy is a promising immunotherapeutic modality for diffuse midline glioma (DMG). We developed mRNA CAR T cells as a safe alternative to virally transduced CAR T cells and have shown that intra-tumoral (IT) infusions of mRNA GD2-directed CAR T cells result in significant tumor regression with improved toxicity in murine DMG models. For clinical translation, we sought to determine the optimal route and trafficking patterns of CAR T cells administered into the cerebrospinal fluid (CSF). METHODS NSG mice engrafted with DMG xenograft SU-DIPG13P* in the pons were treated with 5x106 mRNA CAR T cells either into the lateral ventricle (LV) or into the cisterna magna (CM), with brains harvested after 24 hours for analysis using confocal microscopy. RESULTS Both LV and CM treated mice showed higher GD2-directed CAR T cell migration to tumors compared to CD19-directed controls, with higher accumulation of GD2-directed CAR T cells in the parenchyma for LV treated mice compared to CM (p<0.001). To evaluate efficacy of delivery in the CSF, NSG mice engrafted with DMG xenograft 7316-6349 in the pons were treated with 5x106 mRNA CAR T cells IT, LV, or CM twice a week for 3 weeks totaling 6 doses. Mice were imaged weekly to monitor bioluminescent tumor signal, showing that mice treated with GD2-directed CAR T cells IT had significantly decreased tumor burdens compared to LV and CM GD2 CAR treated groups (p < 0.05 and p < 0.01 respectively), as well as compared to CD19 controls (p < 0.01). CONCLUSIONS Despite local infiltration, mRNA CAR T cells delivered into the CSF did not fully reduce tumor burden, and thus, future work is aimed at evaluating potential routes of priming tumors to attract subsequent cellular therapy doses delivered intra-ventricularly.