| Download | - View final version: Inducible HEK293 AAV packaging cell lines expressing Rep proteins (PDF, 3.3 MiB)
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| DOI | Resolve DOI: https://doi.org/10.1016/j.omtm.2023.07.002 |
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| Author | Search for: Jalšić, Lovro1; Search for: Lytvyn, Viktoria1; Search for: Elahi, Seyyed Mehdy1; Search for: Hrapovic, Sabahudin2; Search for: Nassoury, Nasha1; Search for: Chahal, Parminder Singh1; Search for: Gaillet, Bruno; Search for: Gilbert, Rénald1 |
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| Name affiliation | - National Research Council of Canada. Human Health Therapeutics
- National Research Council of Canada. Aquatic and Crop Resource Development
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| Funder | Search for: National Research Council Canada; Search for: Fonds de Recherche du Québec - Santé; Search for: Natural Sciences and Engineering Research Council of Canada |
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| Format | Text, Article |
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| Subject | AAV; packaging cell; HEK293; inducible expression; Rep; manufacturing; suspension; serum-free; transfection; gene therapy |
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| Abstract | Packaging or producer cell lines for scalable recombinant adeno-associated virus (rAAV) production have been notoriously difficult to create due in part to the cytostatic nature of the Rep proteins required for AAV production. The most difficult challenge being creating AAV packaging cell lines using HEK293 parental cells, currently the best mammalian platform for rAAV production due to the constitutive expression of E1A in HEK293 cells, a key REP transcription activator. Using suspension and serum-free media adapted HEK293SF carrying a gene expression regulation system induced by addition of cumate and coumermycin, we were able to create REP-expressing AAV packaging cells. This was achieved by carefully choosing two of the AAV Rep proteins (Rep 40 and 68), using two inducible promoters with different expression levels and integrating into the cells through lentiviral vector transduction. Three of our best clones produced rAAV titers comparable to titers obtained by standard triple plasmid transfection of their parental cells. These clones were stable for up to 7 weeks under continuous cultures condition. rAAV production from one clone was also validated at scale of 1 L in a wave bioreactor using serum-free suspension culture. |
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| Publication date | 2023-07-23 |
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| Publisher | Cell Press
American Society of Gene & Cell Therapy |
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| Language | English |
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| Peer reviewed | Yes |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | f8aa61c5-521a-4c64-9cd7-4e84775b6c53 |
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| Record created | 2023-11-15 |
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| Record modified | 2023-11-15 |
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