ENGINEERING MRNA ENCAPSULATED EXTRACELLULAR VESICLES FOR IN VIVO CHIMERIC ANTIGEN RECEPTOR MACROPHAGE THERAPY FOR GLIOBLASTOMA - PROJECT SUMMARY GLIOBLASTOMA (GBM) IS THE MOST COMMON AND LETHAL PRIMARY CENTRAL NERVOUS SYSTEM CANCER IN ADULTS, WHERE TREATMENTS ARE INEFFECTIVE AND OFTEN DEBILITATING. UNLIKE MANY OTHER PERIPHERAL SOLID TUMORS, GBM IS HIGHLY RESISTANT TO CANCER IMMUNOTHERAPY. THE ABSENCE OF IMMUNE INFILTRATES IN GBM RESULTS IN AN IMMUNOLOGICALLY "COLD" TUMOR AND INTRINSIC IMMUNE RESISTANCE MEDIATED BY GBM CELLS IS AN ADDITIONAL FACTOR CONTRIBUTING TO THE FAILURE OF IMMUNOTHERAPY IN GBM THUS FAR. CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELL THERAPY HAS RECENTLY SHOWN REMARKABLE SUCCESS IN THE TREATMENT OF HEMATOLOGIC CANCERS, AND THERE HAVE BEEN ATTEMPTS MADE TO EXPLORE CAR T CELL STRATEGIES AGAINST GBM. HOWEVER, CAR T CELLS FOR GBM FACE CHALLENGES INCLUDING INTRATUMOR HETEROGENEITY, DYNAMIC EXPRESSION OF TARGET RECEPTORS, AND OFTEN THE INABILITY OF T CELLS TO TRAFFIC TO TUMORS TO MEDIATE THE DESIRED ANTITUMOR EFFECT. IN CONTRAST TO THE LACK OF T CELL INFILTRATES, MANY SOLID TUMORS, ESPECIALLY GBM, ARE ABUNDANT IN IMMUNE SUPPRESSIVE MYELOID CELLS INCLUDING MACROPHAGES. THEREFORE, CONVERTING THESE IMMUNE SUPPRESSIVE CELLS INTO TUMORICIDAL PHENOTYPE REPRESENTS A PROMISING STRATEGY FOR CELL-BASED THERAPY. THERE IS NOW STRONG INTEREST IN GENERATING CAR MACROPHAGES IN WHICH AUTOLOGOUS MACROPHAGES ARE TRANSDUCED WITH CARS DELIVERED BY VIRAL VECTORS EX VIVO TO ENHANCE THEIR EFFECTOR FUNCTIONS. HOWEVER, EX VIVO PREPARATION OF CAR MACROPHAGES IS COMPLEX, TIME CONSUMING, AND DUE TO THE NON-DIVIDING NATURE OF MACROPHAGES, IS OFTEN INEFFICIENT. WE PROPOSE AN INNOVATIVE STRATEGY THAT REPRESENTS A REVOLUTIONARY WAY TO PRODUCE CAR MACROPHAGES IN VIVO AND OFFERS A PROMISING NEW APPROACH FOR CELL THERAPY AGAINST GBM. THIS WILL BE THE FIRST STUDY TO EVALUATE THE FEASIBILITY OF PRODUCING CAR MACROPHAGES IN VIVO USING AN MRNA DELIVERY PLATFORM AND TO ASSESS THE ANTITUMOR EFFICACY OF CAR MACROPHAGES FOR GBM IMMUNOTHERAPY. WE HYPOTHESIZE THAT GENERATING CAR MACROPHAGES TARGETING THE EPIDERMAL GROWTH FACTOR RECEPTOR VARIANT III (EGFRVIII) IN VIVO USING MRNA-LOADED EXOSOMES WILL HAVE SIGNIFICANT ACTIVITY AGAINST GBM. EGFRVIII IS A MUTANT PROTEIN THAT IS EXPRESSED ON THE SURFACE OF 50% OF GBM CELLS. OUR PREVIOUS STUDY SHOWED THAT WE CAN EFFICIENTLY PRODUCE MRNA-LOADED EXOSOMES TO RESTORE PROTEIN EXPRESSION IN SOLID TUMORS IN VIVO. FURTHERMORE, OUR PRELIMINARY EXPERIMENTS SHOWED THAT THE EXOSOMES LOADED WITH EGFRVIII CAR MRNA CAN PRODUCE CAR MACROPHAGES IN VIVO WITH ENHANCED EFFECTOR FUNCTIONS. OUR CURRENT STUDY WILL TEST THE OVERALL HYPOTHESIS THROUGH THE FOLLOWING SPECIFIC AIMS. IN AIM 1, WE WILL EVALUATE THE DYNAMICS AND TOXICITY OF CAR MACROPHAGE PRODUCTION IN VIVO USING CAR MRNA EXOSOMES. IN AIM 2, WE WILL EVALUATE TRANSCRIPTOMIC AND FUNCTIONAL PROFILES OF IN VIVO GENERATED CAR MACROPHAGES. FINALLY, IN AIM 3, WE WILL ASSESS THE ANTITUMOR EFFECT OF IN VIVO GENERATED CAR MACROPHAGES AS A MONOTHERAPY OR IN COMBINATION WITH OTHER MYELOID-DIRECTED THERAPY AGAINST BOTH MURINE AND HUMAN EGFRVIII EXPRESSING GBMS. IF SUCCESSFUL, OUR PROPOSED RESEARCH CAN OVERCOME A MAJOR TECHNICAL HURDLE THAT HAS LIMITED CELL THERAPY IN GBM AND GREATLY EXPAND ITS UTILITY FOR CANCER TREATMENT.