Martin John, Frederick Amy, Luo Yuxia, Jackson Robert, Joubert Michelle, Sol Bruce, Poulin Francis, Pastor Eric, Armentano Donna, Wadsworth Samuel, Vincent Karen
Genzyme, a Sanofi company , Framingham, MA 01701-9322, USA.
Hum Gene Ther Methods. 2013 Aug;24(4):253-69. doi: 10.1089/hgtb.2013.046. Epub 2013 Aug 9.
Adeno-associated virus (AAV) producer cell lines represent an effective method for large-scale production of AAV vectors. We set out to evaluate and characterize the use of an abbreviated protocol to generate "masterwells" (MWs; a nonclonal cell population) as a platform for research and preclinical vector production. In this system, a single plasmid containing three components, the vector sequence, the AAV rep, and cap genes, and a selectable marker gene is stably transfected into HeLaS3 cells. Producer cell lines generating an AAV2 vector expressing a secreted form of human placental alkaline phosphatase (SEAP) have been created. Several MWs showed vector yields in the 5×10(4) to 2×10(5) DNase-resistant particles/cell range, and the productivity was stable over >60 population doublings. Integrated plasmid copy number in three high-producing MWs ranged from approximately 12 to 50; copies were arranged in a head-to-tail configuration. Upon infection with adenovirus, rep/cap copy number was amplified approximately 100-fold and high yield appeared to be dependent on the extent of amplification. Rep/cap gene expression and vector packaging both reached a peak at 48 hr postinfection. AAV2-SEAP vector was produced in 1-liter shaker culture and purified for assessment of vector quality and potency. The data showed that the majority of the capsids from the MWs contained vector DNA (≥70%) and that purified vector was free of replication-competent AAV. In vitro and in vivo analyses demonstrated that potency of the producer cell-derived vector was comparable to vector generated via the standard transfection method.
腺相关病毒（AAV）生产细胞系是大规模生产AAV载体的一种有效方法。我们着手评估和表征使用简化方案来生成“母孔”（MWs；非克隆细胞群体）作为研究和临床前载体生产平台的情况。在这个系统中，将包含三个组件（载体序列、AAV rep和cap基因以及一个选择标记基因）的单个质粒稳定转染到HeLaS3细胞中。已经创建了产生表达人胎盘碱性磷酸酶分泌形式（SEAP）的AAV2载体的生产细胞系。几个母孔显示载体产量在5×10⁴至2×10⁵抗DNA酶颗粒/细胞范围内，并且生产力在超过60次群体倍增中保持稳定。三个高产母孔中的整合质粒拷贝数范围约为12至50；拷贝以头对头的方式排列。用腺病毒感染后，rep/cap拷贝数扩增约100倍，高产似乎取决于扩增程度。rep/cap基因表达和载体包装在感染后48小时均达到峰值。在1升摇瓶培养中生产AAV2-SEAP载体并进行纯化，以评估载体质量和效力。数据表明，来自母孔的大多数衣壳含有载体DNA（≥70%），并且纯化的载体不含具有复制能力的AAV。体外和体内分析表明，生产细胞衍生载体的效力与通过标准转染方法产生的载体相当。
