Red Blood Cells-derived Extracellular Vesicles as innovative and promising RNA delivery system

Introduction Extracellular Vesicles (EVs) are emerging as versatile drug delivery systems with key roles in many biochemical processes. Red Blood Cells (RBCs) represent an appealing EV source due to their abundance, safety, lack of immunogenicity, and absence of DNA. Our aim is to produce RBC-derived Extracellular Vesicle (RBCEVs) as RNA carriers, suitable for clinical translation. Materials and Methods We developed mRNA-loaded RBCEVs from pre-loaded RBCs using our patented soft extrusion method, mimicking the natural splenic passage. Cargo encapsulation was performed through hypotonic dialysis with synthetic, codon-optimized, and modified mRNA for enhanced stability in murine models. RBCEVs were characterized according to MISEV2023 guidelines, and loading efficiency assessed by qRT-PCR. In vivo studies in mice assessed pharmacokinetics (PK), biodistribution, hepatic protein translation, and circulating toxic metabolite levels to evaluate therapeutic potential. Results RBCs were successfully loaded with mRNA encoding the GAMT enzyme, producing RBCEVs that retained the cargo while preserving integrity, consistent with the non-disruptive nature of soft extrusion. The resulting RBCEVs displayed high homogeneity and favorable biological properties. PK and biodistribution analyses revealed prolonged circulation, efficient liver targeting, and minimal spleen accumulation. Preliminary data in KO mice indicate that a single RBCEV administration can induce hepatic protein expression and reduce GAA levels up to 7 days. Moreover, further studies on therapeutic effect with repeated administration are still ongoing. Conclusions RBCEVs mimetics enable efficient RNA loading and delivery, offering a safe, scalable, and clinically translatable platform for RNA-based therapies, e.g. for metabolic disorders.