Elucidating Key Proteins in the Internalization Pathway of Small Extracellular Vesicles

Abstract

The delivery of oligonucleotide therapeutics, such as silencing RNAs (siRNA), into the cytoplasm of cells is the central plight of their use as a therapeutic. Our lab demonstrated that small extracellular vesicles (sEVs) are highly efficient delivery vehicles, capable of delivering 3-30% of the RNAs they contain into cells, and, importantly, traverse the blood–brain barrier, highlighting their promise as RNA carriers and making them a potent candidate for drug delivery in neurodegenerative diseases. The stark therapeutic potential of sEVs is contrasted heavily by the fact that the mechanism surrounding sEV internalization is relatively understudied. Our electron microscopy results reveal that sEVs can enter the cell through direct fusion with the cell membrane through a series of intermediates that deliver the sEV contents directly to the cytoplasm. Typical fusion mechanisms involve recruiting fusion proteins to decrease the free energy needed for a complete fusion event. Using mass spectrometry and gene ontology term analysis, the retromer complex was identified as a significantly enriched complex surrounding our sEVs as they enter into cells. Functional knock-down of core retromer subunits abolishes fusion-dependent lipid mixing and prevents siRNA-mediated gene silencing, implicating retromer as a previously unrecognized facilitator of vesicle fusion and cargo release.. Elucidating the mechanism of sEV internalization will fill the knowledge gap of a foundational pathway in cell biology, facilitating their use as therapeutic vehicles for various drugs and increasing our understanding of their use in neurodegenerative diseases.