Naturally Targeted Exosomal TLR7/8 Agonist for Immunotherapy of Medulloblastoma Despite aggressive and highly toxic treatment, nearly half of the children diagnosed with medulloblastoma will die from recurrent disease. Survivors are often left with disabling treatment-associated brain injury. More effective and less toxic therapies are needed to improve clinical outcomes for children affected by medulloblastoma. Sonic Hedgehog (SHH) medulloblastoma is enriched in tumor associated macrophages (TAMs) which express Toll-like receptor (TLR) 7 and 8. Reprogramming these TAMs could result in the loss of immunosuppressive signatures and acquisition of tumoricidal and proinflammatory signatures. We have shown that TLR7/8 agonist, resiquimod, encapsulated in polymeric nano-micelles significantly prolonged survival in G- Smo mice - genetically engineered mouse model (GEMM) of SHH medulloblastoma with intact blood brain barrier (BBB) and tumor microenvironment (TME). The free drug was not active in G-Smo mice medulloblastoma model. While micelles increase solubility and bioavailability of this agent to the brain capillaries, they do not target the BBB. Moreover, micelles are dynamic structures and release most of their cargo peripherally before reaching the brain, which can decrease therapeutic potential and increase side effects of this format. Hence, we propose a different formulation using monocyte/macrophage derived exosomes as natural targeted carriers of resiquimod for medulloblastoma. We discovered that exosomes secreted from macrophages are transported to the inflamed brain and carry therapeutic molecules across the intact BBB. Our preliminary data suggest that macrophage- derived exosomes accumulate in the brain of the G-Smo mice. Inspired by these discoveries we seek to develop exosome delivered resiquimod as a novel approach to medulloblastoma. Toward this goal we developed the exosome incorporated form of resiquimod (exo-Res) that was shown to polarize myeloid cells toward pro- inflammatory M1-like phenotype. We will test the hypotheses that (a) exo-Res exosomes in a G-Smo mouse model of medulloblastoma will effectively deliver the resiquimod to the tumor, (b) the delivered resiquimod will repolarize TAMs in medulloblastoma, (c) this novel targeted exo-Res therapeutic modality will inhibit the tumor growth and improve the clinically relevant outcomes compared to untargeted first generation nano-micelles, POx- Res PMs. Our Specific Aims will be: 1) Determine if exosomes improve the tumor distribution of resiquimod administered to medulloblastoma-bearing mice. SA2) Determine if exo-Res enhances the anti-tumor efficacy of resiquimod in medulloblastoma-bearing mice. If successful, this will lead to novel therapy that has potential to improve medulloblastoma treatment by replacing the current radiation and chemotherapy with the one that is less toxic and more effective.

Project Narrative We propose an entirely new way to treat medulloblastoma, the most common malignant pediatric brain tumor which if successful can replace the current radiation and chemotherapy with the one that is less toxic and more effective. We employ tiny extracellular vesicles called “exosomes” to carry an agonist of Toll-like receptor from the bloodstream into brain tumors to reprogram medulloblastoma tumor associated myeloid cells and enhance their tumoricidal properties. We use a genetically engineered mouse model of SHH subtype medulloblastoma to test the hypothesis that exosomes can effectively deliver drug to medulloblastoma, and this novel therapy will extend survival of tumor-bearing mice.