Rapid and Efficient Affinity Isolation of Engineered Extracellular Vesicles Encapsulating Functional Proteins via Internal Twin‐Strep Tetraspanin Tagging

ABSTRACT 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 PhoCl 2 , 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 PhoCl 2 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.