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

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...

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Autores principales: Hung, Michelle E., Leonard, Joshua N.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Co-Action Publishing 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870355/
https://www.ncbi.nlm.nih.gov/pubmed/27189348
http://dx.doi.org/10.3402/jev.v5.31027
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author Hung, Michelle E.
Leonard, Joshua N.
author_facet Hung, Michelle E.
Leonard, Joshua N.
author_sort Hung, Michelle E.
collection PubMed
description 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.
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spelling pubmed-48703552016-05-25 A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery Hung, Michelle E. Leonard, Joshua N. J Extracell Vesicles Original Research Article 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. Co-Action Publishing 2016-05-13 /pmc/articles/PMC4870355/ /pubmed/27189348 http://dx.doi.org/10.3402/jev.v5.31027 Text en © 2016 Michelle E. Hung and Joshua N. Leonard http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Research Article
Hung, Michelle E.
Leonard, Joshua N.
A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery
title A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery
title_full A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery
title_fullStr A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery
title_full_unstemmed A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery
title_short A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery
title_sort platform for actively loading cargo rna to elucidate limiting steps in ev-mediated delivery
topic Original Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870355/
https://www.ncbi.nlm.nih.gov/pubmed/27189348
http://dx.doi.org/10.3402/jev.v5.31027
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