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Gouvernement du Canada / Government of Canada

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Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture

DOITrouver le DOI : https://doi.org/10.1038/sj.mt.6300383
AuteurRechercher : 1; Rechercher : 1; Rechercher : 1; Rechercher : 1; Rechercher : 1; Rechercher : 1; Rechercher : 1; Rechercher : 1
Affiliation
  1. Conseil national de recherches Canada. Institut de recherche en biotechnologie du CNRC
FormatTexte, Article
Sujettetracycline (Tet) switch; packaging cell lines; human immunodeficiency virus-1; vesicular stomatitis virus (VSV-G); glycoprotein; lentiviral vectors (LVs); gene transfer vehicle; biotechnology; cell line; cell; gene; pressure; transfection
Résumé
We have developed new packaging cell lines (293SFPacLV) that can produce lentiviral vectors (LVs) in serumfree suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2S-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 × 10⁷ transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 × 10⁷ TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SFPacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 × 10⁶ TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 × 10⁷ TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 × 10⁶ TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications.
Date de publication
Condition d’accès
  • available
  • unlimited
  • public
Dans
  • Molecular Therapy 16, nº 3 (8 janvier 2008) : 500–507.
Langueanglais
Publications évaluées par des pairsOui
Numéro du CNRC47809
NRCC 47809
Numéro NPARC8925854
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Identificateur de l’enregistrementbeae0de5-4fac-4c58-87e6-d5dc8796adbe
Enregistrement créé2009-04-23
Enregistrement modifié2020-04-15

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