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, researchers at Northwestern University developed a platform for actively loading engineered cargo RNAs into EVs. In their 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, the researchers 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 – they 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. They 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.
Evaluation of RNA loading into EVs via the TAMEL platform
(a) This cartoon summarizes the concept of facilitating active loading of cargo RNA into EVs via our TAMEL platform. A TAMEL EV-loading protein comprises an EV-enriched protein (EEP, blue) fused to an RNA-binding domain (RBD, green), which localizes to EVs. Actively loaded RNA (green) contains a motif that binds to the RBD, resulting in enhanced loading into EVs relative to passively loaded RNA (orange). (b) RNA cargo design impacts active loading. The “fold enrichment of cargo mRNA +/−MS2” is defined as the ratio of cargo RNA/GAPDH mRNA in EVs derived from cells expressing Lamp2b–MS2–HA divided by the same RNA ratio in EVs derived from cells expressing Lamp2b–HA. All experiments were performed in biological triplicates. (c) Cartoon illustrating the 3′ RNA fragment analysis technique. Cargo RNA is first reverse transcribed using an oligo dT primer, and amplicons corresponding to the RNA 5′ or 3′ ends (the latter is located ~500 bases upstream of the polyA site) are then quantified by qPCR using the primer pairs indicated. Note that the amplicon near the RNA 3′ end will be present in cDNA derived from both full-length RNA and 3′ RNA fragments. (d) Analysis of 3′ RNA fragment loading into EVs. Cargo RNA levels were quantified as depicted in panel c and normalized to GAPDH. Passive loading: cells transfected with Lamp2b–HA; active loading: cells transfected with Lamp2b–MS2–HA. (e) Full-length RNA and 3′ fragment RNA levels in EVs were quantified following incubation at 37°C.