A method for using the cell-penetrating peptides for loading plasmid DNA into secreted extracellular vesicles

The low bioavailability and high toxicity of pDNA-based therapeutics make their application in vivo challenging. Extracellular vesicles (EVs) have great potential to overcome those limitations, thus they are biocompatible native cargo carriers. Various strategies to load EVs with pDNA, such as electroporation, sonication, and co-incubation, have been previously investigated, but with questionable success. In this work, we report a unique method of loading EVs through transient transfection of donor cells with complexes formed between pDNA and CPPs. With this method, we achieve up to 10 4 fold higher expression levels of the luciferase reporter protein in recipient cells compared to the untreated cells. This indicates high transfection efficacy and bioavailability of delivered encapsulated nucleic acid. Also, the in vivo experimental data indicates that the use of pDNA-loaded-EV as the native delivery vehicle reduces the toxic effects associated with traditional nucleic acid (NA) delivery and treatment.