Durocher Yves, Pham Phuong Lan, St-Laurent Gilles, Jacob Danielle, Cass Brian, Chahal Parminder, Lau Cara J, Nalbantoglu Joséphine, Kamen Amine
Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
J Virol Methods. 2007 Sep;144(1-2):32-40. doi: 10.1016/j.jviromet.2007.03.014. Epub 2007 Apr 30.
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.
重组腺相关病毒（rAAV）近年来已成为一种有前景的基因治疗载体，可用于治疗多种人类疾病。扩大rAAV载体使用范围的主要障碍仍然是现有生产系统提供足够数量的rAAV用于临床前和临床试验的能力有限。扩大常用贴壁细胞系的不切实际性是大规模生产的一个限制因素。本文描述了在无血清培养基中使用悬浮生长的人胚肾（HEK293）细胞成功生产2型rAAV的过程。所开发的工艺基于采用聚乙烯亚胺（PEI）作为DNA转运体的三重转染，实现了AAV的无血清生产，在3.5-L生物反应器中产生的病毒载体滴度高达4.5×10¹¹感染性病毒颗粒（IVP）。获得了约200:1的最大VG:IVP比率，表明病毒载体在HEK293细胞中有效包装。研究了三种质粒比例变化的影响以及转染时细胞密度的影响。条件培养基不会限制或抑制rAAV的生产；因此，消除转染前后的培养基更换步骤大大简化了rAAV生产的放大过程。在生物反应器悬浮培养中获得的细胞特异性病毒滴度与对照贴壁细胞培养中获得的滴度相似或更高，这进一步支持了该工艺的可扩展性。从工艺简单性、可扩展性和低运营成本等多个方面来看，这种转染方法似乎是大规模生产rAAV最有前景的技术。
