TY  - JOUR
TI  - Production of Genetically Engineered Extracellular Vesicles for Targeted Protein Delivery
AU  - Ovchinnikova, Leyla A.
AU  - Evtushenko, Evgeniy G.
AU  - Bagrov, Dmitriy V.
AU  - Goncharov, Anton O.
AU  - Gabibov, Alexander G.
AU  - Lomakin, Yakov A.
VL  - 15
IS  - 21
PY  - 2025
DA  - 2025/11/05
SP  - e5494
C1  - Bio-protocol 2025;15:e5494
DO  - 10.21769/BioProtoc.5494
UR  - https://doi.org/10.21769/BioProtoc.5494
AB  - 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.
KW  - EVs
KW  - Engineered EVs
KW  - Protein delivery
KW  - NTA
KW  - VSV-G
KW  - Target delivery
KW  - Antigen-presenting cells
KW  - CD206
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False