Engineering Extracellular Vesicles As Targeted Delivery Vehicles

Extracellular vesicles (EVs) are nanoscale particles secreted by all cells that play an important role in intercellular communication by transporting proteins and nucleic acids between cells. These intrinsic properties, combined with the ability to engineer cargo loading and targeting moieties, make EVs a viable delivery platform for a wide range of biomolecules. Specifically, the nontoxic and nonimmunogenic nature of EVs yields a promising method for translatable delivery of engineered proteins and nucleic acids. One exciting prospect is the use of EVs to deliver Cas9 for in vivo gene editing, as delivery poses a challenging obstacle to such applications. Toward this end, we have validated a strategy for loading Cas9 protein into multiple EV populations and are investigating approaches for enhancing delivery. We created a fluorescence-based reporter system to evaluate functional delivery of Cas9 via EVs in different cell lines. To address the challenges of achieving EV-recipient cell fusion and recipient cell targeting, we developed a method for displaying single-chain variable fragments (scFvs) on the surface of EVs to promote high affinity interactions with target cells. We evaluated the extent to which scFv display impacts EV uptake in a target-specific fashion, including the use of cells that exhibit low basal EV uptake. Our method could be used to promote high affinity recipient cell targeting as well as to direct EVs to particular uptake pathways with the goal of increasing functional delivery of engineered cargo.