Unlocking extracellular vesicle-mediated efficient DNA delivery for long-lasting transgene expression.

Achieving efficient DNA delivery for sustained transgene expression while minimizing immune response remains a significant challenge in gene therapy. Here, we conduct an in-depth characterization of red blood cell-derived extracellular vesicles (RBCEVs) and evaluate their potential as DNA delivery vehicles targeting specific cell types. We aim to achieve stable, prolonged transgene expression, minimizing the need for frequent treatments. Proteomic analysis shows that RBCEVs closely resemble red blood cells in protein content with over 90% of lumen proteins composed of hemoglobin family proteins. Biodistribution of RBCEVs in non-human primates indicates predominant liver accumulation, highlighting the liver's potential as a biofactory organ for RBCEV-mediated gene therapies. In mice, systemic administration of RBCEVs loaded with luciferase plasmids achieves stable, long-lasting luciferase expression, maintained throughout a 13-months monitoring period. RBCEV-mediated delivery of therapeutic human factor IX plasmids produces durable factor IX protein levels in bloodstream. RBCEVs can also co-deliver two plasmids simultaneously. Incorporating heavy- and light-chain antibody constructs into RBCEVs enables sustained expression of Herceptin antibodies in circulation. Further utilization of cell-specific promoters enhances transgene expression and concomitantly reduces undesired immune responses. These results highlight RBCEVs as a safe, scalable, and versatile DNA delivery platform for achieving sustained transgene expression in gene therapy.