Abstract:

Extracellular vesicles (EV) are submicron membrane vesicles released by most cells with a fundamental role in cell-to-cell communication. Much interest is flourishing towards their exploitation in regenerative medicine and diagnostics. However, the fulfilment of the EV promise is hampered by severe limitations in their isolation, characterization and manufacturing. A particularly arduous task is to move the isolation of specific EV subpopulations beyond the analytical scale and towards scalable processes. In this scenario, our project will leverage on DNAdirected reversible immunocapturing (rDDI), a new technology developed within FET-OPEN project “INDEX”. rDDI relies on the reversible EV isolation mediated by immunoaffinity followed by intact vesicles recovery upon enzymatic cleavage of a DNA linker used to anchor antibodies on solid supports. Despite unprecedented efficiency in the recovery of highly pure EVs, limitations inherent to antibodies (high costs, batch-to-batch variation and limited versatility of chemical manipulation) substantially impair the scalability of rDDI for any operating scale exceeding the analytical one. MARVEL targets a paradigm shift from antibodies to peptides as an alternative class of affinity ligands for EV capturing by introducing membrane-sensing peptides (MSP) as novel ligands for the size-selective capturing of small EV, unbiased by differential surface protein expression. MARVEL mission is to combine and implement rDDI and MSP technologies, towards the first and best performing ever affinity-based technology for scalable and reversible small EV ( <200nm ) isolation. The modularity in scaling-up of the novel protocols and kits will be demonstrated on medium/large sample volumes in relevant environments for therapeutic and diagnostics use of EVs and specifically: 1) In the manufacturing of GMP-grade EVs as a medicinal product for cardiac repair; 2) In urine-based liquid biopsy for bladder cancer diagnostics.