Identification of Novel Extracellular Vesicles Scaffold Proteins for Versatile Cargo Engineering

ABSTRACT Extracellular vesicles (EVs) are promising drug delivery platforms that have been engineered to carry various drug modalities. Key strategies for generating those therapeutic EVs involve the direct fusion of protein of interest (POI) to EVs scaffold proteins with inherently high EVs-sorting ability. In this work, we identified raftlin (RFTN1) and poliovirus receptor (PVR) as novel EVs scaffold proteins for loading EVs lumen and surface cargos respectively. Truncation studies revealed that the N-terminus 15 residues from RFTN1 (RFTN1-N15) were sufficient for EVs engineering, as demonstrated by distinct cargos including gene editing tools, cytosolic enzymes as well as type II transmembrane proteins. On the other hand, PVR efficiently displayed secreted proteins including antibodies and serum albumins on EVs surface. Critically, RFTN1 and PVR-engineered EVs demonstrated consistent and efficient cargo delivery in vivo . In summary, the discovery of RFTN1 and PVR can potentially benefit EVs engineering for both fundamental research and clinical translation in the future.