A platform for actively loading cargo RNA to elucidate limiting steps in EV‐mediated delivery

Abstract

Extracellular vesicles (EVs) mediate intercellular communication through transfer of RNA and protein between cells. Thus, understanding how cargo molecules are loaded and delivered by EVs is of central importance for elucidating the biological roles of EVs and developing EV-based therapeutics. While some motifs modulating the loading of biomolecular cargo into EVs have been elucidated, the general rules governing cargo loading and delivery remain poorly understood. To investigate how general biophysical properties impact loading and delivery of RNA by EVs, we developed a platform for actively loading engineered cargo RNAs into EVs. In our system, the MS2 bacteriophage coat protein was fused to EV-associated proteins, and the cognate MS2 stem loop was engineered into cargo RNAs. Using this Targeted and Modular EV Loading (TAMEL) approach, we identified a configuration that substantially enhanced cargo RNA loading (up to 6-fold) into EVs. When applied to vesicles expressing the vesicular stomatitis virus glycoprotein (VSVG) – gesicles – we observed a 40-fold enrichment in cargo RNA loading. While active loading of mRNA-length (>1.5 kb) cargo molecules was possible, active loading was much more efficient for smaller (~0.5 kb) RNA molecules. We next leveraged the TAMEL platform to elucidate the limiting steps in EV-mediated delivery of mRNA and protein to prostate cancer cells, as a model system. Overall, most cargo was rapidly degraded in recipient cells, despite high EV-loading efficiencies and substantial EV uptake by recipient cells. While gesicles were efficiently internalized via a VSVG-mediated mechanism, most cargo molecules were rapidly degraded. Thus, in this model system, inefficient endosomal fusion or escape likely represents a limiting barrier to EV-mediated transfer. Altogether, the TAMEL platform enabled a comparative analysis elucidating a key opportunity for enhancing EV-mediated delivery to prostate cancer cells, and this technology should be of general utility for investigations and applications of EV-mediated transfer in other systems.

Therapeutic and diagnostic applications of extracellular vesicles

“…Unfortunately, the EVs used in this study were not able to escape the endolysosomal degradation pathway and hence failed to functionally deliver the siRNA in contrast to anionic fusogenic liposomes that were equally loaded with chol-siRNA. Moreover, the endogenously present miRNAs were not able to silence their respective target proteins which is in accordance with recent reports describing that (1) even the most abundant miRNAs found in EVs are secreted at a (low) ratio of 1 molecule per 100 vesicles [46,177] and (2) internalized EVs are typically trafficked toward the lysosomes [140,178]. Although this particular combination of EVs and recipient cells did not lead to successful EV-mediated drug delivery [176], it does not invalidate the concept of EVs as drug carriers as their interaction with cells might be highly specific.…”

Selective release of circRNAs in platelet‐derived extracellular vesicles

“…Finally, to search for potential sorting determinants, we asked whether there may be a correlation between circRNA size and packaging into vesicles as hypothesised earlier for other classes of RNA [33]. CircRNA size appears to be plausible in this regard, considering that the available packaging volume within a vesicle might be limiting to accommodate RNP complexes of larger circRNA molecules.…”

Therapeutic biomaterials based on extracellular vesicles: classification of bio‐engineering and mimetic preparation routes

“…TAMEL is another genetic engineering tool that has recently been published [59] for the active cargo of RNA. This platform is a fusion between an engineered EV-loading protein and the RNA to be loaded.…”