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

1. OBJECTIVE Extracellular Vesicles (EVs) are emerging drug delivery tools with key roles in physiological and pathological processes. Red Blood Cells (RBCs) are an attractive source due to accessibility, safety, non-immunogenicity, and lack of DNA. Our goal is to produce RBC-derived Extracellular Vesicle (RBCEVs) as RNA carriers, readily translatable to clinical use. 2. 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 achieved via hypotonic dialysis with synthetic, codon-optimized, and modified mRNA for enhanced stability in murine models. RBCEVs were characterized, per MISEV2023 guidelines, and loading efficiency assessed by qRT-PCR. RBCEVs were tested in mice for pharmacokinetics (PK) and biodistribution studies. Finally, blood toxic metabolite levels were assessed to evaluate post-treatment therapeutic efficacy. 3. RESULTS RBCs were efficiently loaded with mRNA encoding the GAMT enzyme, producing RBCEVs that retained the cargo while preserving integrity, as soft extrusion is non-disruptive. RBCEVs showed high homogeneity and great biological features. PK and biodistribution analyses revealed extended half-life, efficient liver targeting, and minimal spleen uptake. Preliminary data suggest that EV-mediated delivery reduced GAA levels. 4. DISCUSSION RBCEV mimetics show efficient RNA loading and delivery, making them a safe, efficient, and scalable platform for RNA-based therapies, e.g. for metabolic disorders.