Production of Genetically Engineered Extracellular Vesicles for Targeted Protein Delivery.

Extracellular vesicles (EVs) have emerged as promising carriers for the targeted delivery of therapeutic proteins to specific cells. Previously, we demonstrated that genetically engineered EVs can be used for targeted protein delivery. This protocol details the generation of mannose receptor (CD206)-targeted EVs using a modular plasmid system optimized for production in HEK293T cells. Three plasmids enable customizable EV budding, cargo loading, and surface modification for targeting to antigen-presenting cells (APCs). EVs are isolated via differential centrifugation and chromatography, characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), and validated through functional uptake assays in primary human activated dendritic cells. Our approach combines flexibility in engineering required EVs with robust, reproducible isolation and characterization workflows. Its modularity allows easy adaptation to alternative targets or cargoes, which can be validated immediately through in vitro testing. Key features • First detailed protocol for generating genetically engineered EVs with fusogenic VSV-G protein and CD206-specific targeting. • Enables rapid customization of EVs for diverse therapeutic cargo and cell-targeting applications. • Integrates gold-standard EV isolation with multi-modal characterization to ensure reliability. • A universal platform for customizable cell targeting: swapping VSV-G-linked llama nanobodies with diverse specificities.