Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors
To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-02-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/12/4/619 |
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| author | Seyyed Mehdy Elahi Nazila Nazemi-Moghaddam Claire Guilbault Mélanie Simoneau Rénald Gilbert |
| author_facet | Seyyed Mehdy Elahi Nazila Nazemi-Moghaddam Claire Guilbault Mélanie Simoneau Rénald Gilbert |
| author_sort | Seyyed Mehdy Elahi |
| collection | DOAJ |
| description | To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene. |
| first_indexed | 2024-03-11T09:01:14Z |
| format | Article |
| id | doaj.art-c41cd24baee147d1848ba9f1f160f61a |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T09:01:14Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-c41cd24baee147d1848ba9f1f160f61a2023-11-16T19:44:53ZengMDPI AGCells2073-44092023-02-0112461910.3390/cells12040619Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus VectorsSeyyed Mehdy Elahi0Nazila Nazemi-Moghaddam1Claire Guilbault2Mélanie Simoneau3Rénald Gilbert4Department of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montreal, QC H4P 2R2, CanadaDepartment of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montreal, QC H4P 2R2, CanadaDepartment of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montreal, QC H4P 2R2, CanadaDepartment of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montreal, QC H4P 2R2, CanadaDepartment of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montreal, QC H4P 2R2, CanadaTo increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.https://www.mdpi.com/2073-4409/12/4/619protease-deleted adenovirussingle-cycle adenovirus vectorstable cell linesuspension culture |
| spellingShingle | Seyyed Mehdy Elahi Nazila Nazemi-Moghaddam Claire Guilbault Mélanie Simoneau Rénald Gilbert Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors Cells protease-deleted adenovirus single-cycle adenovirus vector stable cell line suspension culture |
| title | Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors |
| title_full | Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors |
| title_fullStr | Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors |
| title_full_unstemmed | Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors |
| title_short | Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors |
| title_sort | complementary cell lines for protease gene deleted single cycle adenovirus vectors |
| topic | protease-deleted adenovirus single-cycle adenovirus vector stable cell line suspension culture |
| url | https://www.mdpi.com/2073-4409/12/4/619 |
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