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...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Wiley
2016-05-01
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| Series: | Journal of Extracellular Vesicles |
| Subjects: | |
| Online Access: | http://www.journalofextracellularvesicles.net/index.php/jev/article/view/31027/pdf_63 |
| _version_ | 1818974504691433472 |
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| author | Michelle E. Hung Joshua N. Leonard |
| author_facet | Michelle E. Hung Joshua N. Leonard |
| author_sort | Michelle E. Hung |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-20T15:41:07Z |
| format | Article |
| id | doaj.art-c02a57ef1a124e688ad8bdea7f032fcc |
| institution | Directory Open Access Journal |
| issn | 2001-3078 |
| language | English |
| last_indexed | 2024-12-20T15:41:07Z |
| publishDate | 2016-05-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj.art-c02a57ef1a124e688ad8bdea7f032fcc2022-12-21T19:35:12ZengWileyJournal of Extracellular Vesicles2001-30782016-05-015011310.3402/jev.v5.3102731027A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated deliveryMichelle E. Hung0Joshua N. Leonard1 Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, USA Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USAExtracellular 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.http://www.journalofextracellularvesicles.net/index.php/jev/article/view/31027/pdf_63extracellular vesiclesexosomesmicrovesiclesactive loadingLamp2bVSVGMS2 coat protein dimerCD63 |
| spellingShingle | Michelle E. Hung Joshua N. Leonard A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery Journal of Extracellular Vesicles extracellular vesicles exosomes microvesicles active loading Lamp2b VSVG MS2 coat protein dimer CD63 |
| 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 | extracellular vesicles exosomes microvesicles active loading Lamp2b VSVG MS2 coat protein dimer CD63 |
| url | http://www.journalofextracellularvesicles.net/index.php/jev/article/view/31027/pdf_63 |
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