Combination of baculovirus-mediated gene delivery and packed-bed reactor for scalable production of adeno-associated virus

• Published in: Human gene therapy Vol. 18; no. 11; p. 1161
• Main Authors: Huang, Kuo-Shiang, Lo, Wen-Hsin, Chung, Yao-Chi, Lai, Yiu-Kay, Chen, Chi-Yuan, Chou, Szu-Ting, Hu, Yu-Chen
• Format: Journal Article
• Language: English
• Published: United States 01.11.2007
• Subjects: Animals; Baculoviridae - genetics; Bioreactors - economics; Cell Line; Dependovirus - genetics; Dependovirus - growth & development; Gene Transfer Techniques; Humans; Insecta - cytology; Recombinant Proteins - genetics; Virus Cultivation - methods; Virus Replication
• ISSN: 1043-0342; 1557-7422
• DOI: 10.1089/hum.2007.107

The production of recombinant adeno-associated virus (rAAV) commonly requires plasmid cotransfection, which hinders its mass production. Herein we describe the development of a novel process for rAAV production by combining the advantages of baculovirus-mediated gene delivery and BelloCell bioreactor (a novel packed-bed reactor for animal cell culture; CESCO Bioengineering, Hsinchu, Taiwan). We constructed three baculoviral vectors: Bac-LacZ carries the lacZ gene flanked by AAV inverted terminal repeats, Bac-RC harbors AAV rep and cap genes, and Bac-Helper carries helper genes derived from adenovirus. Cotransduction of HEK-293 cells with these three baculoviruses resulted in successful production of rAAV, and the protein and rAAV yield did not decrease with Bac-RC passage for up to four passages. By adjusting the dose ratio of Bac-LacZ to Bac-RC, adding sodium butyrate, and transferring the production process to the BelloCell-500-AP (500 ml), which allowed for high-density culture and effective baculovirus-mediated transduction of HEK-293 cells, the maximal specific rAAV yield reached approximately 3.8 x 10(4) vector genome (VG) or 247 infectious viral particles (IVP) per cell, which corresponded to approximately 1 x 10(14) VG or 8.5 x 10(11) IVP per reactor run. The yield was comparable or superior to those obtained with other production systems. Baculoviral transduction is simple and cost-effective and the BelloCell-500-AP offers high-density culture of HEK-293 cells. Altogether, the combination of baculoviral transduction and BelloCell reactor culture provides a novel and economically viable approach for rAAV production.