biostudies-literatureObuchi WNational Cancer InstituteNCI NIH HHSDaiichi Sankyo CompanyIonis PharmaceuticalsKayden-Lambert MGH Research Scholar AwardNational Science FoundationFriedreich's Ataxia Research Alliance Australiae70094https://www.ebi.ac.uk/biostudies/studies/S-EPMC12173531biostudies-literatureUnknown14(6)Extracellular vesicles (EVs) are mediators of intercellular communication through the transfer of nucleic acids, lipids and proteins between cells. This property makes bioengineered EVs promising therapeutic vectors. However, it remains challenging to isolate EVs with a therapeutic payload due to the heterogeneous nature of cargo loading into EVs. In this study, enrichment of EVs with a desired cargo was possible through engineering of the hallmark CD63 transmembrane protein. E-NoMi refers to engineered CD63 with mCherry on the inside of the EV membrane and a tag (3xFLAG) exposed on the outside of the EV membrane. To facilitate EV loading during biogenesis, cargo proteins, such as EGFP, Cre recombinase and the CRISPR-Cas nuclease (SaCas9), were fused to a nanobody (Nb) protein with a high Journal of extracellular vesiclesEngineering of CD63 Enables Selective Extracellular Vesicle Cargo Loading and Enhanced Payload Delivery.PMC12173531NIH DP2-CA259675NCI‐CA232103NIH R01‐GM138790NIH R01-GM138790NIH DP2‐CA281401NIH K22‐CA2802019‐01NCI-CA232103NIH DP2-CA281401NIH DP2‐CA259675NIH K22-CA2802019-01Nieland LVaine CADi Lanni ERepiton PLeandro KRufino-Ramos DLee HMiller MAXiao TKleinstiver BPBreyne KMaalouf KObuchi WBreakefield XOZargani-Piccardi ABragg DCFrederick DMfalseEngineering of CD63 Enables Selective Extracellular Vesicle Cargo Loading and Enhanced Payload Delivery.Extracellular vesicles (EVs) are mediators of intercellular communication through the transfer of nucleic acids, lipids and proteins between cells. This property makes bioengineered EVs promising therapeutic vectors. However, it remains challenging to isolate EVs with a therapeutic payload due to the heterogeneous nature of cargo loading into EVs. In this study, enrichment of EVs with a desired cargo was possible through engineering of the hallmark CD63 transmembrane protein. E-NoMi refers to engineered CD63 with mCherry on the inside of the EV membrane and a tag (3xFLAG) exposed on the outside of the EV membrane. To facilitate EV loading during biogenesis, cargo proteins, such as EGFP, Cre recombinase and the CRISPR-Cas nuclease (SaCas9), were fused to a nanobody (Nb) protein with a high 2025-01-01T00:00:00Z2025 Jun2026-05-29T17:27:44.282Z2026-04-08T05:34:15.878ZS-EPMC121735314052773310.1002/jev2.70094