Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.
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 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) 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 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) 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.
Broussau
Sophie
S
Groupe de Vecteurs de Génomique et Thérapie Génique, Institut de Recherche en Biotechnologie, Conseil National de Recherches Canada, Montréal, Quebec, Canada.
Jabbour
Nadine
N
Lachapelle
Guillaume
G
Durocher
Yves
Y
Tom
Rosanne
R
Transfiguracion
Julia
J
Gilbert
Rénald
R
Massie
Bernard
B
D019149:Bioreactors / Q000821:virology; D018929:Cell Culture Techniques; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D015685:Fusion Proteins, gag-pol / Q000235:genetics; D015757:Genes, rev / Q000235:genetics; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics
D016895:Culture Media, Serum-Free; D015685:Fusion Proteins, gag-pol