Engineered Exosomes as Vehicles for Biologically Active Proteins

• Published in: Molecular therapy Vol. 25; no. 6; pp. 1269 - 1278
• Main Authors: Sterzenbach, Ulrich, Putz, Ulrich, Low, Ley-Hian, Silke, John, Tan, Seong-Seng, Howitt, Jason
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.06.2017; Elsevier Limited; American Society of Gene & Cell Therapy
• Subjects: Animals; Biological activity; Blood-brain barrier; Blood-Brain Barrier - metabolism; Brain - drug effects; Brain - metabolism; Brain research; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Line; Cerebellum; Cortex (olfactory); Cre recombinase; Deoxyribonucleic acid; DNA; drug delivery; Drug Delivery Systems; Drug development; ESCRT; Exosomes; Exosomes - metabolism; Exports; extracellular vesicles; Extracellular Vesicles - metabolism; Gene Expression; Genetic Engineering - methods; Immunoglobulins; Integrases - metabolism; intraluminal vesicles; L-domain; Localization; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Nasal Absorption; nasal delivery; Neostriatum; Olfactory bulb; Original; Packaging; Permeability; Protein Transport; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombination; therapy; Transgenic mice; ubiquitin; Ubiquitination; United States > US; L-domain; blood-brain barrier; intraluminal vesicles; extracellular vesicles; ESCRT; therapy; drug delivery; nasal delivery; ubiquitin
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2017.03.030

Exosomes represent an attractive vehicle for the delivery of biomolecules. However, mechanisms for loading functional molecules into exosomes are relatively unexplored. Here we report the use of the evolutionarily conserved late-domain (L-domain) pathway as a mechanism for loading exogenous proteins into exosomes. We demonstrate that labeling of a target protein, Cre recombinase, with a WW tag leads to recognition by the L-domain-containing protein Ndfip1, resulting in ubiquitination and loading into exosomes. Our 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. 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, including the olfactory bulb, cortex, striatum, hippocampus, and cerebellum. The ability to engineer exosomes to deliver biologically active proteins across the blood-brain barrier represents an important step for the development of therapeutics to treat brain diseases. [Display omitted] Exosomes are nanovesicles capable of intercellular communication. As such, they represent a highly attractive delivery vehicle. Sterzenbach et al. investigated a mechanism for loading exosomes with exogenous proteins through the sequestration of an evolutionary conserved pathway. Using this method, they loaded exosomes with Cre recombinase and, through nasal administration, have identified the functional delivery of exosomes over large distances into the brain.