206 Engineering MRNA-Loaded Immunogenic Exosomes for Targeted Glioblastoma Immunotherapy

INTRODUCTION: Recent advancements in mRNA therapeutics have shown remarkable efficacy against pathogenic infections, prompting interest in their application for other diseases, particularly cancer. However, one major challenge is the precise delivery of genetic material to specific cells and tissues. Extracellular vesicles like exosomes are emerging as promising carriers due to their favorable immunogenic profile, long circulatory half-life, ability to cross biological barriers such as the blood-brain barrier, and potential for targeted delivery (Kim Lab Nature Biomedical Engineering 2019, 2023, Nature Communications 2023). METHODS: A microfluidic electroporation technique utilizing nano- and milli-second pulses was employed to efficiently load exosomes with interferon-gamma (IFN-γ) mRNA, resulting in the production of a substantial quantity of mRNA-loaded exosomes. To enable targeted delivery, CD64 molecules were overexpressed on the surface of these exosomes, acting as adaptors for docking targeting ligands, such as anti-CD71 and anti-programmed cell death-ligand 1 (PD-L1) antibodies. RESULTS: The engineered imExo successfully increased IFN-γ expression in GBM, which upregulated major histocompatibility complex I (MHC-I) in the tumor microenvironment, thereby enhancing the anti-tumor effect and increased the survival time of mice in both GBM models. Specifically, the treatment led to a significant increase in CD8 + T cells and MHC-I expression within the tumor microenvironment, indicating restored immunogenicity. Additionally, there was an observed increase in pro-inflammatory M1-polarized macrophages, which are recognized to promote tumor immunogenicity, in contrast to the immunosuppressive M0 and M2 macrophages CONCLUSIONS: This study presents a novel adaptive strategy for engineering mRNA-loaded exosomes with targeting functionality. Using CD64 to dock various targeting ligands onto the exosome surface proved effective in targeting cancer cells and generating strong antitumor activities. These findings suggest that these engineered exosomes have significant potential for application in cancer immunotherapy, offering a new avenue for the precise delivery of mRNA therapeutics to combat resistant tumors.