Scalable production and purification of engineered ARRDC1-mediated microvesicles in a HEK293 suspension cell system
Abstract Engineering of human ARRDC1-mediated microvesicles (ARMMs) as non-viral vehicles for delivery of gene therapies bears the potential to enable novel therapeutic paradigms. We evaluated two scalable strategies to generate ARMMs loaded with protein cargo, by transient transfection or stable ce...
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Nature Portfolio
2025-03-01
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| Online Access: | https://doi.org/10.1038/s41598-025-87674-5 |
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| author | Kristin Luther Ali Navaei Leah Gens Carson Semple Pearl Moharil Ilaria Passalacqua Komal Vyas Qiyu Wang Shu-Lin Liu Lucy Sun Senthil Ramaswamy Davide Zocco Joseph F. Nabhan |
| author_facet | Kristin Luther Ali Navaei Leah Gens Carson Semple Pearl Moharil Ilaria Passalacqua Komal Vyas Qiyu Wang Shu-Lin Liu Lucy Sun Senthil Ramaswamy Davide Zocco Joseph F. Nabhan |
| author_sort | Kristin Luther |
| collection | DOAJ |
| description | Abstract Engineering of human ARRDC1-mediated microvesicles (ARMMs) as non-viral vehicles for delivery of gene therapies bears the potential to enable novel therapeutic paradigms. We evaluated two scalable strategies to generate ARMMs loaded with protein cargo, by transient transfection or stable cell line-based production. The upstream ARMMs production processes utilized a suspension-adapted HEK293-derived line, termed 5B8. 5B8 cells yielded robust production of ARMMs after transient transfection with the ARMMs loading construct or using a stable cell line containing a transgene that encodes the ARMMs loading cassette, in shake flasks or a stirred tank bioreactor, respectively. ARMMs were purified by ultracentrifugation (small scale) or a combination of TFF and AEX (scalable production). Both purification methods produced comparable ARMMs, in terms of size and payload incorporation. Single particle analysis showed approximately 50% were payload-containing ARMMs. Additionally, an in vivo study was conducted in mice to investigate the half-life and biodistribution of ARMMs administered intravenously. ARMMs showed rapid biodistribution predominantly to the spleen and liver and, to a lesser extent, kidneys, and lungs. The half-life of ARMMs in plasma was 6 ± 0.4 min. Altogether, this work advances knowledge on scale-up of engineered cell-derived vesicles for future in vivo delivery of therapeutic molecules. |
| format | Article |
| id | doaj-art-ea5a622874f9481cae55c39c8b84d0db |
| institution | DOAJ |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-ea5a622874f9481cae55c39c8b84d0db2025-08-20T02:59:35ZengNature PortfolioScientific Reports2045-23222025-03-0115111710.1038/s41598-025-87674-5Scalable production and purification of engineered ARRDC1-mediated microvesicles in a HEK293 suspension cell systemKristin Luther0Ali Navaei1Leah Gens2Carson Semple3Pearl Moharil4Ilaria Passalacqua5Komal Vyas6Qiyu Wang7Shu-Lin Liu8Lucy Sun9Senthil Ramaswamy10Davide Zocco11Joseph F. Nabhan12Vesigen TherapeuticsLonza Cell & Gene Technologies, Lonza Walkersville Inc.Vesigen TherapeuticsVesigen TherapeuticsVesigen TherapeuticsLonza SienaVesigen TherapeuticsVesigen TherapeuticsVesigen TherapeuticsVesigen TherapeuticsLonza Cell & Gene Technologies, Lonza Walkersville Inc.Lonza SienaVesigen TherapeuticsAbstract Engineering of human ARRDC1-mediated microvesicles (ARMMs) as non-viral vehicles for delivery of gene therapies bears the potential to enable novel therapeutic paradigms. We evaluated two scalable strategies to generate ARMMs loaded with protein cargo, by transient transfection or stable cell line-based production. The upstream ARMMs production processes utilized a suspension-adapted HEK293-derived line, termed 5B8. 5B8 cells yielded robust production of ARMMs after transient transfection with the ARMMs loading construct or using a stable cell line containing a transgene that encodes the ARMMs loading cassette, in shake flasks or a stirred tank bioreactor, respectively. ARMMs were purified by ultracentrifugation (small scale) or a combination of TFF and AEX (scalable production). Both purification methods produced comparable ARMMs, in terms of size and payload incorporation. Single particle analysis showed approximately 50% were payload-containing ARMMs. Additionally, an in vivo study was conducted in mice to investigate the half-life and biodistribution of ARMMs administered intravenously. ARMMs showed rapid biodistribution predominantly to the spleen and liver and, to a lesser extent, kidneys, and lungs. The half-life of ARMMs in plasma was 6 ± 0.4 min. Altogether, this work advances knowledge on scale-up of engineered cell-derived vesicles for future in vivo delivery of therapeutic molecules.https://doi.org/10.1038/s41598-025-87674-5Extracellular vesiclesARRDC1-mediated microvesiclesARMMs5B8Transient transfectionStable producer cell line |
| spellingShingle | Kristin Luther Ali Navaei Leah Gens Carson Semple Pearl Moharil Ilaria Passalacqua Komal Vyas Qiyu Wang Shu-Lin Liu Lucy Sun Senthil Ramaswamy Davide Zocco Joseph F. Nabhan Scalable production and purification of engineered ARRDC1-mediated microvesicles in a HEK293 suspension cell system Scientific Reports Extracellular vesicles ARRDC1-mediated microvesicles ARMMs 5B8 Transient transfection Stable producer cell line |
| title | Scalable production and purification of engineered ARRDC1-mediated microvesicles in a HEK293 suspension cell system |
| title_full | Scalable production and purification of engineered ARRDC1-mediated microvesicles in a HEK293 suspension cell system |
| title_fullStr | Scalable production and purification of engineered ARRDC1-mediated microvesicles in a HEK293 suspension cell system |
| title_full_unstemmed | Scalable production and purification of engineered ARRDC1-mediated microvesicles in a HEK293 suspension cell system |
| title_short | Scalable production and purification of engineered ARRDC1-mediated microvesicles in a HEK293 suspension cell system |
| title_sort | scalable production and purification of engineered arrdc1 mediated microvesicles in a hek293 suspension cell system |
| topic | Extracellular vesicles ARRDC1-mediated microvesicles ARMMs 5B8 Transient transfection Stable producer cell line |
| url | https://doi.org/10.1038/s41598-025-87674-5 |
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