An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells
An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells
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DOI：
10.1073/pnas.0810614106
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发表时间：
2009-03-31
影响因子：
11.1
通讯作者：
Zolotukhin, Sergei
中科院分区：
文献类型：
--
作者：
Aslanidi, George;Lamb, Kenneth;Zolotukhin, Sergei
生产用于人体试验的临床级基因治疗载体仍然是推进许多其他不治之症治疗的主要障碍。我们描述了一个基于稳定转化的昆虫细胞系的系统，这些细胞系含有载体生产所需的辅助基因。整合基因保持沉默，直到细胞被单一杆状病毒表达载体（BEV）感染。表达的诱导是由整合的驻留基因扩增（每个细胞多达1200个拷贝）和BEV编码的即时早期反式调节因子1 （IE-1）介导的表达增强的结合而产生的。整合盒包含来自野生型加利福尼亚多核多角体病毒的IE-1结合靶序列，这是一个同源区域2 （hr2）。一个前馈循环由其中一种诱导蛋白Rep78启动，促进了整合基因的扩增。对该系统进行了重组腺相关病毒（rAAV）2和rAAV1包装所需的7种蛋白的协调表达测试。与先前描述的杆状病毒/rAAV生产系统相比，所描述的排列提供了高水平的Rep和Cap蛋白，从而将rAAV产量提高了10倍。
Production of clinical-grade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases. We describe a system based on a stably transformed insect cell lines harboring helper genes required for vector production. Integrated genes remain silent until the cell is infected with a single baculovirus expression vector (BEV). The induction of expression results from a combination of the amplification of integrated resident genes (up to 1,200 copies per cell) and the enhancement of the expression mediated by the immediate-early trans-regulator 1 (IE-1) encoded by BEV. The integration cassette incorporates an IE-1 binding target sequence from wild-type Autographa califomica multiple nuclear polyhedrosis virus, a homologous region 2 (hr2). A feed-forward loop is initiated by one of the induced proteins, Rep78, boosting the amplification of the integrated genes. The system was tested for the coordinated expression of 7 proteins required to package recombinant adeno-associated virus (rAAV)2 and rAAV1. The described arrangement provided high levels of Rep and Cap proteins, thus improving rAAV yield by 10-fold as compared with the previously described baculovirus/rAAV production system.