Naturally Targeted Exosomal TLR7/8 Agonist for Immunotherapy of Medulloblastoma

天然靶向的外泌体TLR 7/8激动剂用于髓母细胞瘤的免疫治疗 尽管积极和高毒性的治疗，近一半的儿童诊断为髓母细胞瘤将 死于复发性疾病。幸存者通常会留下与治疗相关的致残性脑损伤。更 需要有效和毒性较小的治疗方法来改善儿童的临床结果， 髓母细胞瘤Sonic Hedgehog（SHH）髓母细胞瘤富含肿瘤相关巨噬细胞 （TAM），其表达Toll样受体（TLR）7和8。重新编程这些TAM可能会导致 免疫抑制特征和获得杀肿瘤和促炎特征。我们已经表明 TLR 7/8激动剂，瑞喹莫特，包封在聚合物纳米胶束显着延长生存在G- Smo小鼠-具有完整血脑屏障的SHH髓母细胞瘤基因工程小鼠模型（GEMM） (BBB)肿瘤微环境（TME）游离药物在G-Smo小鼠髓母细胞瘤模型中没有活性。 虽然胶束增加了这种药剂对脑毛细血管的溶解度和生物利用度，但它们不靶向脑毛细血管。 BBB.此外，胶束是动态结构，并且在到达之前将其大部分货物释放到外围 大脑，这可能会降低治疗潜力并增加这种形式的副作用。因此，我们建议 使用单核细胞/巨噬细胞衍生的外来体作为瑞喹莫特的天然靶向载体的不同制剂 神经管母细胞瘤我们发现，巨噬细胞分泌的外泌体被运送到发炎的 并携带治疗分子穿过完整的BBB。我们的初步数据显示巨噬细胞- 衍生的外泌体在G-Smo小鼠的脑中积累。在这些发现的启发下， 外泌体递送瑞喹莫特作为髓母细胞瘤的新方法。为了实现这一目标，我们开发了 外泌体掺入形式的瑞喹莫特（exo-Res），其显示出使髓样细胞向前 炎性M1样表型。我们将测试以下假设：（a）G-Smo小鼠中的exo-Res外泌体 成神经管细胞瘤模型将有效地将瑞喹莫特递送至肿瘤，（B）递送的瑞喹莫特将 （c）这种新型靶向exo-Res治疗方式将抑制肿瘤 与非靶向的第一代纳米胶束相比，POx- Res PMs.我们的具体目标将是：1）确定外泌体是否改善瑞喹莫特的肿瘤分布 给药于携带髓母细胞瘤的小鼠。SA 2）确定exo-Res是否增强了抗肿瘤功效 resiquimod在髓母细胞瘤小鼠中的作用。如果成功，这将导致新的治疗方法，有潜力， 改善髓母细胞瘤的治疗，取代目前的放疗和化疗， 毒性更小，更有效。

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.