title: Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free suspension culture.
abstract: E1-deleted adenovirus vectors (AdV) are important gene transfer vehicles for gene therapy and vaccination. Amplification of AdV must take place in cells that express the adenovirus E1A and E1B genes. Sequence homology between AdV and the E1 genes integrated within the complementing cells should be minimal to reduce the odds of generating replication-competent adenovirus (RCA). The present study describes the establishment of AdV complementing cells constructed by stable transfection of the minimal E1A and E1B genes into human lung carcinoma (A549). Because some transgene products can be cytotoxic, the cells were engineered to stably express the repressor of the cumate-switch (CymR) to silence transgene transcription during vector growth. For regulatory compliance and to facilitate the scale-up, the resulting complementing cells (SF-BMAdR) were adapted to serum-free suspension culture. The best clone of SF-BMAdR produced AdV carrying an innocuous transgene to the same level as 293 cells, but titers were better for AdV carrying transgene for a cytotoxic product. Elevated titers were maintained for at least two months in suspension culture in the absence of selective agent and the cells did not produce RCA. Because of their advantageous properties, SF-BMAdR cells should become an important tool for developing large-scale production processes of AdV for research and clinical applications.
authors.affiliation: Department of Antibodies & Bioprocessing, National Research Council, Building Montreal, Montreal, QC, Canada; Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada. Electronic address: renald.gilbert@cnrc-nrc.gc.ca.
authors.forename: Rénald
authors.initials: R
authors.lastname: Gilbert
authors.affiliation: Department of Antibodies & Bioprocessing, National Research Council, Building Montreal, Montreal, QC, Canada.
authors.forename: Claire
authors.initials: C
authors.lastname: Guilbault
authors.affiliation: Department of Antibodies & Bioprocessing, National Research Council, Building Montreal, Montreal, QC, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Gagnon
authors.affiliation: Department of Downstream Processing and Analytics, National Research Council, Building Montreal, Montreal, QC, Canada.
authors.forename: Alice
authors.initials: A
authors.lastname: Bernier
authors.affiliation: Department of Antibodies & Bioprocessing, National Research Council, Building Montreal, Montreal, QC, Canada.
authors.forename: Lucie
authors.initials: L
authors.lastname: Bourget
authors.affiliation: Department of Antibodies & Bioprocessing, National Research Council, Building Montreal, Montreal, QC, Canada.
authors.forename: Seyyed Mehdy
authors.initials: SM
authors.lastname: Elahi
authors.affiliation: Department of Antibodies & Bioprocessing, National Research Council, Building Montreal, Montreal, QC, Canada; Department of Bioengineering, McGill University, Montreal, QC, Canada.
authors.forename: Amine
authors.initials: A
authors.lastname: Kamen
authors.affiliation: Department of Antibodies & Bioprocessing, National Research Council, Building Montreal, Montreal, QC, Canada; Département de Microbiologie et Immunologie, Université de Montréal, Montréal, QC, Canada.
authors.forename: Bernard
authors.initials: B
authors.lastname: Massie
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics / Q000254:growth & development*; D001709:Biotechnology / Q000379:methods; D002460:Cell Line; D016895:Culture Media, Serum-Free; D004847:Epithelial Cells / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D006377:T-Lymphocytes, Helper-Inducer / Q000502:physiology; D013678:Technology, Pharmaceutical / Q000379:methods*; D019562:Viral Load; D014776:Virus Cultivation / Q000379:methods
chemical_list: D016895:Culture Media, Serum-Free
keywords: Adenovirus vectors; Cell line; Replication-competent adenovirus; Serum-free medium; Suspension culture