1. ¹Groupe de Vecteurs de Génomique et Thérapie Génique, Institut de Recherche en Biotechnologie, Conseil National de Recherches Canada, Montréal, Quebec, Canada
2. ²Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Quebec, Canada
3. ³Groupe de Technologie des Cellules Animales, Institut de Recherche en Biotechnologie, Conseil National de Recherches Canada, Montréal, QC, Canada
4. ⁴Neuromuscular Research Group, Montreal Neurological Institute, Montreal, Quebec, Canada
5. ⁵Institut National de la Recherche Scientifique–Institut Armand-Frappier, Université du Québec, Laval, Quebec, Canada

Correspondence: Bernard Massie, Institut de recherche en biotechnologie, Conseil national de recherches Canada, 6100, Royalmount Avenue, Montréal, Quebec, Canada H4P 2R2. E-mail:bernard.massie@cnrc-nrc.gc.ca

Received 26 July 2007; Accepted 14 November 2007; Published online 8 January 2008.

Abstract

We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2^S-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 293SF-PacLV 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.