TOP-EV: Technology of protein loading through Extracellular Vesicle is a versatile platform for intracellular protein delivery

• Published in: Journal of controlled release
• Main Authors: Ilahibaks, Nazma F, Ardisasmita, Arif I, Xie, Songpu, Gunnarsson, Anders, Brealey, Joseph, Vader, Pieter, de Jong, Oliver G, de Jager, Saskia, Dekker, Niek, Peacock, Ben, Schiffelers, Raymond M, Sluijter, Joost P.G., Lei, Zhiyong
• Format: Journal Article
• Language: English
• Published: 01.01.2023
• Subjects: dimerization; drugs; genome; glycoproteins; liver; mutants; recombinases; therapeutics; Vesiculovirus
• ISSN: 0168-3659
• DOI: 10.1016/j.jconrel.2023.02.003

Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have limited delivery efficiency owning to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein loading through Extracellular Vesicles' (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. Our strategy promoted protein loading by integrating the vesicular stomatitis virus glycoprotein and rapamycin-induced heterodimerization of FKBP12 and T82L mutant FRB proteins in donor cells. The administration of TOP-EVs led to functional intracellular delivery of Cre recombinase, inducing Cre-mediated recombination in multiple cell types. Similarly, administration of Cas9 containing TOP-EV to Cas9 reporter cells led to highly efficient genome editing in the corresponding reporter cells. Furthermore, we showed that TOP-EVs can successfully mediate intracellular protein delivery in vivo in the liver. Taken together, TOP-EVs provides a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.