Abstract Intracellular delivery of protein and RNA therapeutics represents a major challenge. Here, we developed highly potent engineered extracellular vesicles (EVs) by incorporating essential bio-inspired attributes required for effective delivery. These comprise engineered mini-intein proteins with self-cleavage activity for active cargo loading and release, and fusogenic VSV-G protein to activate productive endosomal escape. Combining these components allowed high efficiency recombination and genome editing in vitro following EV-mediated delivery of Cre recombinase and Cas9/sgRNA RNP cargoes, respectively. In vivo , single dose EV-mediated Cre delivery to the brains of Cre-LoxP R26-LSL-tdTomato reporter mice resulted in greater than 40% and 30% recombined cells in hippocampus and cortex respectively. In addition, we demonstrate therapeutic potential of this platform by showing inhibition of LPS-induced systemic inflammation via delivery of a super-repressor of NF-ĸB activity. Our data establish these engineered EVs as a novel platform for effective delivery of multimodal therapeutic cargoes, including for efficient genome editing.

Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors