Glycoengineering of extracellular vesicles enhances cellular uptake and cargo delivery to target cells

ABSTRACT The glycocalyx is a major regulator of membrane recognition, yet its specific influence on extracellular vesicles (EVs) cellular uptake remains poorly defined. We established a genetic glycoengineering platform to systematically investigate how the major glycan classes on small EVs (sEVs) modulate cell interactions and functional cargo delivery. Using an isogenic panel of HEK293F lines lacking distinct glycan biosynthetic pathways, we find that removing glycosaminoglycans (ΔGAG-sEVs) yields a strong increase in cellular uptake and delivery of diverse cargos, including DNA oligonucleotides, siRNA, proteins, and plasmid DNA. Glycan-modified recipient cells show that sEV-cell communication and internalization is jointly governed by glycan features on both membranes. ΔGAG-sEVs strongly improve gene delivery and expression in recipient cells and in a physiologically relevant human airway epithelial model. These findings establish glycan structures as tunable regulators of sEV uptake and position ΔGAG-sEVs as potent vehicles for improved drug delivery and gene therapy.