title: Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells.
abstract: Recombinant adeno-associated virus (rAAV) has emerged in recent years as a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broadening the usage of rAAV vectors remains the limited capacity of available production systems to provide sufficient rAAV quantities for preclinical and clinical trials. The impracticality of expanding commonly used adherent cell lines represents a limitation to large-scale production. This paper describes successful productions of rAAV type 2 using suspension-growing human embryonic kidney (HEK293) cells in serum-free medium. The developed process, based on triple transfection employing polyethylenimine (PEI) as DNA transporter, allowed for a serum-free production of AAV, yielding viral vector titer up to 4.5x10(11) infectious viral particles (IVP) in a 3.5-L bioreactor. A maximum ratio of VG:IVP in the order of 200:1 was obtained, indicating the efficient encapsidation of viral vectors in HEK293 cells. The effect of varying the ratio of three plasmids and the influence of cell density at transfection were studied. The conditioned medium did not limit or inhibit the rAAV production; therefore, the elimination of the medium exchange step before or after transfection greatly simplified the scale-up of rAAV production. The cell-specific viral titers obtained in bioreactor suspension cultures were similar or higher than those obtained with control adherent cell cultures which further supported the scalability of the process. From multiple aspects including process simplicity, scalability, and low operating costs, this transfection method appears to be the most promising technology for large-scale production of rAAV.
authors.affiliation: Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
authors.forename: Yves
authors.initials: Y
authors.lastname: Durocher
authors.forename: Phuong Lan
authors.initials: PL
authors.lastname: Pham
authors.forename: Gilles
authors.initials: G
authors.lastname: St-Laurent
authors.forename: Danielle
authors.initials: D
authors.lastname: Jacob
authors.forename: Brian
authors.initials: B
authors.lastname: Cass
authors.forename: Parminder
authors.initials: P
authors.lastname: Chahal
authors.forename: Cara J
authors.initials: CJ
authors.lastname: Lau
authors.forename: Joséphine
authors.initials: J
authors.lastname: Nalbantoglu
authors.forename: Amine
authors.initials: A
authors.lastname: Kamen
mesh_terms: D019149:Bioreactors; D018929:Cell Culture Techniques; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics / Q000254:growth & development*; D005822:Genetic Vectors; D006801:Humans; D014162:Transfection; D014776:Virus Cultivation / Q000379:methods*