"Engineered red blood cell extracellular vesicles as a therapeutic strategy for the treatment of renal diseases" in Meeting abstracts of the 3° EVIta symposium

The use of engineered extracellular vesicles (EVs) for clinical purposes represents a novel strategy in regenerative medicine. However, cellular EV sources are limited in terms of availability and safety. Red blood cell-derived extracellular vesicles (RBC-EVs) could be used for large-scale production of engineered EVs with therapeutic potential and for autologous therapy. In literature, different approaches have been described to exploit RBC-EVs as a drug delivery system. We are currently evaluating different protocols of RBC-EVs engineering that would allow the loading of therapeutic RNAs and peptides. RBC-EVs have been isolated from healthy donors using ultracentrifugation and tangential flow filtration, in order to scale up EV production. RBC-EVs are characterized by nanoparticle tracking analysis, transmission electron microscopy (TEM), and super-resolution microscopy, in order to verify their number, dimension, purity, and marker expression. TEM analysis showed intact RBC-EVs with a diameter of around 50 nm. RBC-EVs express the tetraspanins CD9, CD63, CD81, and the RBC marker CD47, as evaluated by super-resolution microscopy. EV loading is performed using cholesterol-modified miRNAs and a peptide targeting KIM-1, specifically expressed in injured proximal tubular cells, to obtain specific cell targeting and increase the delivery efficiency. Engineered RBC-EVs will be tested on in vitro models of renal cell carcinoma using renal cancer stem cells, and of acute and chronic kidney damage using a temperature-sensitive human renal proximal tubule cell line. We aim to obtain an easy scalable protocol for RBC-EVs isolation and engineering that would allow the broadening of the therapeutic applications of extracellular vesicles in regenerative medicine.