Effective drug delivery remains a challenge in many areas of medicine. Extracellular vesicles (EVs) are nanocarriers of therapeutic cargoes including proteins, nucleic acids, and/or small molecules, with their natural biogenesis conferring limited immunogenicity, enhanced stability and biocompatibility relative to synthetic nanoparticles.
The current COVID-19 pandemic underlines the limitations of traditionally-derived vaccines and presents a pressing need for novel therapies. We customised EVs with EVPS, a protein engineering technology aimed at targeting host cells susceptible to SARS-CoV-2 infection. By grafting EVs with viral coat proteins, viral tropism is transferred to the EVs, enabling delivery of customised cargo into host target cells.
We synthesised a construct encoding a fusion protein comprised of: a) an addressing domain (SARS-CoV-2 Spike) for the intended recipient cell, b) an exosome-anchoring domain (a VSVg fragment), and, c) an intravesicular ‘cargo’ domain (in this case fluorescent proteins). The construct was cloned into a mammalian-expression vector and subsequently introduced into HEK-293T cells. Correct construction was validated by Sanger sequencing, expression by immunoblotting and localisation by high-resolution confocal microscopy.
In cells transfected with empty vector, fluorescence was seen throughout the cytoplasm and nucleus. In contrast, cells expressing SARS-CoV-2 EVPS demonstrated punctate and reticular distribution in discrete regions of the cytosol and no signal in the nucleus. This pattern of SARS-CoV-2 EVPS expression is consistent with localisation in the endocytic compartments that are sites of exosome biogenesis.
These findings demonstrate that EVPS is a valid system for successful generation of SARS-CoV-2 EVPS, serving as a potentially novel and highly targeted therapeutic strategy for treating COVID-19, especially when the EVs are loaded with antiviral compounds. With customisation of constructs with proteins specific for different target cell types of interest, EVPS offers a flexible and safer approach to concentrate therapeutic agents to desired target cell populations when compared to conventional therapeutic approaches.