Helmholtz Center for Infection Research (HZI), 38124 Braunschweig, Germany.
Hum Gene Ther. 2010 Aug;21(8):979-91. doi: 10.1089/hum.2009.089.
The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line.
由于生产依赖于不可控的载体整合事件，因此标准逆转录病毒包装细胞系中生产的逆转录病毒载体的安全性和滴度的提高受到阻碍。染色体周围环境的影响使得难以从各自整合位点的染色体周围环境中剖析特定载体的性能。利用一种依赖于使用具有预定义整合位点的包装细胞系的技术，我们系统地评估了几种逆转录病毒载体的性能。在具有单个定义的染色体整合位点的两个先前建立的模块化包装细胞系（Flp293A 和 293 FLEX）中，通过 Flp 介导的位点特异性重组将逆转录病毒载体整合。通过正向或反向取向引入具有不同长末端重复启动子的载体。结果表明，启动子、病毒载体取向和整合位点是滴度的主要决定因素。此外，我们利用病毒生产系统来评估通读活性。通读被认为是由于载体转录的终止效率低下而引起的，并且是逆转录病毒载体的固有性质。我们评估了来自两个整合位点的侧翼逆转录病毒载体序列的转导频率。这里提出的方法为系统设计和评估针对给定生产细胞系优化的逆转录病毒载体的效率和安全性提供了一个平台。
