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.

• extracellular vesicles
• exosomes
• microvesicles
• active loading
• Lamp2b
• VSVG
• MS2 coat protein dimer
• CD63

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Acknowledgements

The authors acknowledge Charlene Wilke for her assistance with transmission electron microscopy. TEM was performed at the Northwestern University Biological Imaging Facility on a JEOL 3200 FETEM supported by the NU Office for Research and NCRR 1S10RR025092. The authors thank Dr. Arabela Grigorescu for assistance with NanoSight analysis. NanoSight analysis was performed at the Northwestern University Keck Biophysics Facility. Traditional sequencing services were performed at the Northwestern University Genomics Core Facility. BCA assays were performed at the Northwestern University High Throughput Analysis Lab, which is supported by the Chicago Biomedical Consortium and The Searle Funds at The Chicago Community Trust. Flow cytometry was performed at the Northwestern University Flow Cytometry Facility, which is supported by a Cancer Center Support Grant (NCI CA060553). The authors acknowledge support from a 3M Non-tenured Faculty Award, the Lynn Sage Breast Cancer Foundation and the Northwestern University Prostate Cancer Specialized Program of Research Excellence (SPORE) through NIH award P50 CA090386 (to JNL). This work was also supported by the National Science Foundation's Graduate Research Fellowship Program (NSF GRFP) award DGE-0824162 (to MEH).

Notes

To access the supplementary material to this article, please see Supplementary files under ‘Article Tools’.