In vivo generation of CAR macrophages via the enucleated mesenchymal stem cell delivery system for glioblastoma therapy

Glioblastoma multiforme (GBM) is one of the most aggressive intracranial tumors for which there is no effective treatment. Chimeric antigen receptor macrophage (CAR-M) therapies have demonstrated impressive therapeutic efficacy in solid tumors; however, the cost and rigor associated with manufacturing engineered macrophages ex vivo can be prohibitive. Here, we utilized enucleated mesenchymal stem cells (MSCs) as vehicles for the targeted delivery of CAR-encoding plasmid to reprogram glioma-associated microglia/macrophages (GAM), thereby achieving CAR-M preparation in vivo. Specifically, we observed that the enucleated cells retained the key organelle function and membrane integrity, and actively homed to glioma tissue. Interestingly, enucleated MSCs underwent intrinsic apoptosis due to the absence of the nucleus, which subsequently triggered macrophage-specific endocytosis, thereby achieving precise delivery of CAR-plasmids to GAM. Compared with lipid nanoparticles, this strategy specifically generated sufficient numbers of CAR-M in glioma situ to achieve GBM therapy. Moreover, this process altered the immune cell profiles within the tumor by increasing cytotoxic T cells and M1-like macrophages with antitumor activity. When combined with CD47-blocking therapies, tumor growth was completely suppressed in the GBM orthotopic mouse model, resulting in a 90-d survival rate of 83%. Collectively, our strategy provides a viable platform technology for CAR-M generation in vivo, which is expected to provide an approach for GBM therapy.