National Research Council of Canada. NRC Biotechnology Research Institute
Recombinant adeno-associated viruses (rAAV) represent a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broaden their usage is the availability of a production process able to provide sufficient quantities for preclinical and human trials. We present here a successful process for rAAV-2 production in low-serum and serum-free medium performed in a 3L stirred-tank bioreactor. The process is based on the triple transfection of suspension-growing HEK293 cells employing polyethylenimine (PEI) as the DNA carrier. Production of AAV-GFP in a 3L serum-free medium bioreactor yielded titers of 5.1 x 1011 infectious viral particles (IVP), corresponding to 2 x 1012 viral genome (VG) or 6.8 x 1012 viral particles (VP). The cell-specific and total viral titers obtained in suspension culture were about three-fold higher to those obtained with adherent cells. The process is very simple and robust as it does not require a medium exchange, either before or after transfection, making it particularly attractive for the generation of large and homogeneous stocks of rAAV vectors.