Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells
Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells
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DOI：
10.1016/j.jviromet.2007.03.014
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发表时间：
2007-09-01
影响因子：
3.1
通讯作者：
Kamen, Amine
中科院分区：
文献类型：
--
作者：
Durocher, Yves;Pham, Phuong Lan;Kamen, Amine
重组腺相关病毒（rAAV）是近年来出现的一种有前途的基因治疗载体，可用于治疗多种人类疾病。扩大rAAV载体使用的主要障碍仍然是可用生产系统提供足够的rAAV量用于临床前和临床试验的能力有限。扩增常用的贴壁细胞系的不切实际性代表了对大规模生产的限制。本文描述了使用悬浮生长的人胚肾（HEK 293）细胞在无血清培养基中成功生产rAAV 2型。开发的工艺基于采用聚乙烯亚胺（PEI）作为DNA转运蛋白的三重转染，允许无血清生产AAV，在3.5 L生物反应器中产生高达4.5 x 10（11）个感染性病毒颗粒（IVP）的病毒载体滴度。获得了约200：1的VG：IVP的最大比率，表明病毒载体在HEK 293细胞中的有效包埋。研究了改变三种质粒的比例的效果和转染时细胞密度的影响。条件培养基不限制或抑制rAAV生产;因此，在转染之前或之后消除培养基交换步骤大大简化了rAAV生产的规模化。生物反应器悬浮培养物中获得的细胞特异性病毒滴度与对照贴壁细胞培养物中获得的病毒滴度相似或更高，这进一步支持了该工艺的可扩展性。从工艺简单、可扩展性和低操作成本等多个方面来看，这种转染方法似乎是大规模生产rAAV的最有前途的技术。皇冠版权所有（c）2007年出版的爱思唯尔B. V.保留所有权利。
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 4.5 x 10(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. Crown Copyright (c) 2007 Published by Elsevier B.V. All rights reserved.