Identification of Novel Scaffold Proteins for Improved Endogenous Engineering of Extracellular Vesicles

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. In this study 244 candidate scaffold proteins were studied, resulting in the identification of 24 proteins with conserved EV-sorting abilities across five types of producer cells. TSPAN2 and TSPAN3 were detected as the lead candidates for cargo loading, outperforming the well-known CD63 scaffold. EVs from human embryonic kidney epithelial (HEK-293T) cells, either wild type or overexpressing TSPAN2, TSPAN3 or CD63, were analyzed with label-free top-down proteomics to understand the effect of EV-engineering on protein signatures. The proteomics findings demonstrated that TSPAN2/TSPAN3-based engineering gives rise to EV subpopulations distinct from CD63. The discovery of these novel scaffolds provides a new platform for EV-based therapies.