Rapid and Efficient Affinity Isolation of Engineered Extracellular Vesicles Encapsulating Functional Proteins via...

Extracellular vesicles (EVs) are mediators of intercellular communication and promising protein-delivery vehicles, but isolation of sufficiently pure, intact, engineered EVs remains challenging due to abundant cellular and serum-derived contaminants. Here, structure-guided engineering of the canonical EV tetraspanins, CD63 and CD9, is applied. Permissive sites for the internal insertion of the twin-Strep tag are identified within unstructured regions of their large extracellular loops, using flexible linkers. This design preserves scaffold functionality and enables efficient antibody-free affinity isolation of intact EVs using Strep technology. Fusing proteins of interest (POIs) to the C-terminus of these scaffolds, either directly or via the photocleavable protein PhoCl₂, enables efficient luminal POI loading with optional optogenetic control of POI release. Proteomic analysis confirms high purity of isolated EVs with more than 96% of contaminants removed, allowing sensitive detection of possibly novel EV-associated proteins. Additionally, this approach effectively removes hepatitis C virus, which has EV-like biophysical properties, and adeno-associated virus, demonstrating high specificity. Vesicular stomatitis virus glycoprotein coating of EVs facilitates endosomal escape, while PhoCl₂ cleavage enables POI release after EV uptake by target cells, including primary human hepatocytes and hepatocyte-like cells. This strategy provides a broadly applicable platform for producing intact, pure engineered EVs for basic and translational studies.