Extracellular vesicles (EVs) are gaining ground as next-generation drug delivery modalities. Genetic fusion of the protein of interest to a scaffold protein with high EV-sorting ability represents a robust cargo loading strategy. To address the paucity of such scaffold proteins we conducted a large-scale comparative study involving 244 candidate proteins. Their EV-sorting potential was evaluated using a simple but reliable assay that can distinguish intravesicular cargo proteins from surface and non-vesicular proteins. Notably, 24 proteins with conserved EV-sorting abilities across five types of producer cells were identified. Most of these are first to be reported including TSPAN2 and TSPAN3, which emerged as lead candidates, outperforming the well-known CD63 scaffold. Importantly, these engineered EVs show promise as delivery vehicles as demonstrated byin vitroandin vivointernalization studies with luminal cargo proteins as well as surface display of functional domains. The discovery of these novel scaffolds provides a new platform for EV-based engineering.
