Engineered Exosomes as Vehicles for Biologically Active Proteins

Abstract

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.

Post-translational add-ons mark the path in exosomal protein sorting

“…In addition, EVs represent a novel non-invasive and selective therapeutic tool to be developed for prognosis, diagnosis or treatment of diverse diseases, with very promising preliminary results [138,139,152,153]. PTMs confer specific properties to proteins, playing a relevant role in EVs protein sorting.…”

“…The size of these small particles make them suitable to carry proteins, lipids, RNA, DNA or small chemical drugs, protecting them from external factors in the blood stream [138,139]. They can be easily isolated in high amounts from diverse culture cells types.…”

“…The addition of a WW tag (a domain-containing PPxY motif) to a certain protein determines its incorporation into exosomes [139]. The l-domains of Ndfip1 recognize this particular mark, targeting this protein to exosomes [14].…”

Modularized Extracellular Vesicles: The Dawn of Prospective Personalized and Precision Medicine

“…In a recent report, late‐domain (L‐domain) proteins, which are involved in the recruitment of ALIX, could be used to load specific exogenous intracellular proteins with a WW tag for recognition by L‐domain proteins into EVs ( Figure 8 ). 116 Although ubiquitination and the ESCRT machinery have attracted the attention of many researchers and the underlying mechanisms have become increasingly clear, the packaging of proteins taking advantage of this feature still requires further investigation. We predict that utilizing the ESCRT machinery has the potential to be the major method of protein packaging.…”

Characteristics and roles of extracellular vesicles released by epidermal keratinocytes

“…On the other hand, oligodendrocyte‐derived EVs are preferentially internalized in microglia that do not seem to have antigen‐presenting capacity . In addition, to natural specific cell targeting by EVs, recently, EVs bearing engineered peptides on the surface have been generated in order to specifically target cells or tissues . However, up to date, there are no reports of any preferential cell targets of keratinocyte‐derived EVs.…”