Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system
Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system
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DOI：
10.1016/j.ymthe.2005.08.018
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发表时间：
2005-12-01
影响因子：
12.4
通讯作者：
Zolotukhin, S
中科院分区：
文献类型：
--
作者：
Kohlbrenner, E;Aslanidi, G;Zolotukhin, S
rAAV载体的可规模化生产仍然是这种原型基因治疗载体临床应用的主要障碍。最近开发的基于杆状病毒的生产方案（M. Urabe等人，2002年，J. Gene Ther. 1935年至1943年）发现有限的应用，由于系统的设计。在这里，我们报告了一个详细的分析的稳定性的原始杆状病毒系统组件BacRep，BacVP，和转基因盒含有BacGFP。分析的所有杆状病毒辅助病毒都倾向于发生rAAV依赖性功能缺失，导致rAAV滴度显著降低。为了减轻不稳定性并将杆状病毒平台扩展到其他rAAV血清型，我们修改了原始系统的Rep和Cap编码组件。修饰包括细小病毒磷脂酶A2结构域交换，允许在体内产生感染性rAAV8载体。或者，使用新型杆状病毒载体产生在具有AAV8衣壳蛋白的嵌合载体颗粒中掺入AAV2 VP1衣壳蛋白的感染性rAAV8（或rAAV5）载体。在该载体中，AAV2 VP1表达水平由“核糖开关”控制，“核糖开关”是一种由丰霉素以“开启”模式控制的自切割核酶。重新设计的杆状病毒系统通过使该生产平台更适用于其他现有血清型来提高我们的rAAV生产能力。
Scalable production of rAAV vectors remains a major obstacle to the clinical application of this prototypical gene therapy vector. A recently developed baculovirus-based production protocol (M. Urabe et al., 2002, Hum. Gene Ther. 13, 1935-1943) found limited applications due to the system's design. Here we report a detailed analysis of the stability of the original baculovirus system components BacRep, BacVP, and transgene cassette-containing BacGFP. All of the baculovirus helpers analyzed were prone to passage-dependent loss-of-function deletions resulting in considerable decreases in rAAV titers. To alleviate the instability and to extend the baculovirus platform to other rAAV serotypes, we have modified both Rep- and Cap-encoding components of the original system. The modifications include a parvoviral phospholipase A2 domain swap allowing production of infectious rAAV8 vectors in vivo. Alternatively, an infectious rAAV8 (or rAAV5) vector incorporating the AAV2 VP1 capsid protein in a mosaic vector particle with AAV8 capsid proteins was produced using a novel baculovirus vector. In this vector, the level of AAV2 VP1 expression is controlled with a "riboswitch," a self-cleaving ribozyme controlled by toyocamycin in the "ON" mode. The redesigned baculovirus system improves our capacity for rAAV manufacturing by making this production platform more applicable to other existing serotypes.