Engineered exosomes as carriers of biologically active proteins

A critical component for devising drug delivery systems is to package and protect molecules with the ability to release them specifically in a temporal and spatial manner. To date a number of systems have been developed as drug delivery vehicles; however, many have limitations in regard to immunogenicity, packaging efficiency and stability. Recent interest in vectors has shifted to naturally occurring nanovesicles called exosomes which can package proteins, lipids, DNA and various forms of RNA for delivery to recipient cells. These small vesicles are released from nearly every cell type and are known to play a role in cellular communication pathways through the delivery of the aforementioned cargo. However, mechanisms for loading functional molecules into exosomes are relatively unexplored. Here I report the use of the evolutionarily conserved late-domain (L-domain) pathway as a mechanism for loading exogenous proteins into exosomes. I demonstrate that labelling of a target protein, Cre recombinase, with a WW-tag leads to recognition by the L-domain protein Ndfip1, resulting in ubiquitination and loading into exosomes. My results show that Ndfip1 expression acts as a molecular switch for exosomal packaging of WW-Cre that can be suppressed using the exosome inhibitor GW4869. When taken up by floxed reporter cells, exosomes containing WW-Cre were capable of inducing DNA recombination indicating functional delivery of the protein to recipient cells. Using this functional in vitro assay I was able to identify differences in the communication potential between distinct cell types as well as the functionality of exosomes after storage. Engineered exosomes were administered to the brain of transgenic reporter mice using the nasal route to test for intracellular protein delivery in vivo. This resulted in the transport of engineered exosomes predominantly to recipient neurons in a number of brain regions. The ability to engineer exosomes to deliver biologically active protei