Development of Mesenchymal Stem Cell Membrane-Enveloped Nanovesicles for Enhanced Gene Delivery

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) hold great promise for therapeutic applications and regenerative medicine. EVs are nanoscale vesicles secreted by all known cell types, carrying diverse cargos including membrane-anchored proteins, soluble factors, multiple RNA species, and metabolites that regulate the physiology and behavior of recipient cells. While MSC-derived or engineered EVs can deliver therapeutic proteins and RNAs, EV-mediated DNA delivery remains challenging due to the lack of efficient mechanisms for sorting DNA sequences into vesicles. Previous work from our group and others demonstrated that adeno-associated virus (AAV)-containing EVs enable targeted nuclear delivery and sustained gene expression in vitro and in vivo. However, their production and isolation have been limited by low yield and time-intensive procedures. Here, we report the development of MSC membrane-enveloped nanovesicles generated by a size-defined extrusion method for efficient gene delivery. These vesicles, approximately 200 nm in diameter, mimic the properties of natural EVs while encapsulating recombinant AAV vectors carrying therapeutic gene sequences. Compared with conventional AAVs, the engineered MSC vesicles improved gene delivery efficiency and achieved significantly higher yields with reduced time and cost relative to naturally secreted EV-AAVs. In summary, we present a novel MSC-based membrane nanovesicle platform that combines the advantages of EV-mimicking structures with AAV-mediated gene transfer. This approach enhances delivery efficiency and production scalability, offering a promising strategy to advance gene therapy toward clinical translation.