Obuchi Wataru, Zargani-Piccardi Ayrton, Leandro Kevin, Rufino-Ramos David, Di Lanni Emilio, Frederick Dawn Madison, Maalouf Katia, Nieland Lisa, Xiao Tianhe, Repiton Pierre, Vaine Christine A, Kleinstiver Benjamin P, Bragg D Cristopher, Lee Hakho, Miller Miles A, Breakefield Xandra O, Breyne Koen
Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal.
J Extracell Vesicles. 2025 Jun;14(6):e70094. doi: 10.1002/jev2.70094.
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 affinity for mCherry. FLAG-tag-based immunocapture from cell conditioned media allowed selection of cargo-loaded E-NoMi-EVs, and tobacco etch virus (TEV) protease cleavage sites were used to remove the 3xFLAG-tag from the surface of E-NoMi-EVs after capture. For functional payload delivery to recipient cells, the vesicular stomatitis virus G (VSV-G) fusogenic protein was incorporated into E-NoMi-EVs to form fusogenic EV-based vectors (EVVs) and proved to be 10-fold more effective at cargo delivery than EVs generated by size-exclusion chromatography. Functional delivery of cargo with E-NoMi-EVVs was validated in two mouse brain models in vivo.
细胞外囊泡（EVs）是细胞间通讯的介质，通过在细胞之间传递核酸、脂质和蛋白质来实现。这一特性使生物工程改造的EVs成为有前景的治疗载体。然而，由于货物装载到EVs中的性质不均一，分离具有治疗性载荷的EVs仍然具有挑战性。在本研究中，通过对标志性的CD63跨膜蛋白进行工程改造，可以富集具有所需货物的EVs。E-NoMi是指在EV膜内侧带有mCherry且在EV膜外侧暴露有标签（3xFLAG）的工程化CD63。为了在生物发生过程中促进EVs的装载，将货物蛋白，如增强型绿色荧光蛋白（EGFP）、Cre重组酶和CRISPR-Cas核酸酶（SaCas9），与对mCherry具有高亲和力的纳米抗体（Nb）蛋白融合。基于FLAG标签从细胞条件培养基中进行免疫捕获，可选择装载有货物的E-NoMi-EVs，并且在捕获后使用烟草蚀纹病毒（TEV）蛋白酶切割位点从E-NoMi-EVs表面去除3xFLAG标签。为了将功能性货物递送至受体细胞，将水泡性口炎病毒G（VSV-G）融合蛋白整合到E-NoMi-EVs中以形成基于融合性EV的载体（EVVs），并证明其在货物递送方面比通过尺寸排阻色谱法产生的EVs有效10倍。在两种小鼠脑体内模型中验证了使用E-NoMi-EVVs进行货物的功能性递送。
