Koldej R, Cmielewski P, Stocker A, Parsons D W, Anson D S
Department of Genetic Medicine, Children, Youth and Women's Health Service, 72 King William Road, North Adelaide, South Australia, 5006.
J Gene Med. 2005 Nov;7(11):1390-9. doi: 10.1002/jgm.803.
We have previously described a five-plasmid HIV-1 vector system that utilises a codon-optimised gagpol gene. While this system was shown to be safer than systems using proviral type helpers, the titre of virus produced was relatively low. Therefore, a process of optimising all aspects of virus production was initiated.
A systematic approach was taken to the optimisation of virus production by transient expression using a five-plasmid packaging system. Codon-manipulation was used to reduce homology between helper and vector constructs. Ultrafiltration and ultracentrifugation were used for large-scale virus production.
We describe codon-optimised reading frames for Tat and Rev and the optimisation of virus production. The optimisation process resulted in an increase in virus titre of 7- to 8-fold. Several other approaches to increasing viral titre described by others proved ineffective in our system after it had been optimised. In addition, we show that by varying the ratio of the GagPol helper construct to vector, the infectivity of the virus could be controlled. The use of a novel codon-optimised HIV-1 GagPol expression construct with reduced homology to vector sequences significantly reduced transfer of gagpol sequences to transduced cells. Virus could be collected in serum-free medium without a significant loss of titre, which facilitated subsequent processing. Processing using a combination of ultrafiltration and ultracentrifugation allowed efficient and rapid processing of litre volumes of virus supernatant.
By taking a systematic approach to optimising all aspects of our five-plasmid lentiviral vector system we improved titre, safety, large-scale production, and demonstrated that infectivity could be specifically controlled.
我们之前描述了一种利用密码子优化的gagpol基因的五质粒HIV-1载体系统。虽然该系统被证明比使用前病毒型辅助质粒的系统更安全，但产生的病毒滴度相对较低。因此，我们启动了一个优化病毒生产各个方面的过程。
采用系统方法，通过使用五质粒包装系统进行瞬时表达来优化病毒生产。使用密码子操作来降低辅助质粒和载体构建体之间的同源性。超滤和超速离心用于大规模病毒生产。
我们描述了Tat和Rev的密码子优化阅读框以及病毒生产的优化。优化过程使病毒滴度提高了7至8倍。在我们的系统优化后，其他人描述的几种提高病毒滴度的其他方法被证明无效。此外，我们表明，通过改变GagPol辅助质粒与载体的比例，可以控制病毒的感染性。使用与载体序列同源性降低的新型密码子优化HIV-1 GagPol表达构建体可显著减少gagpol序列向转导细胞的转移。病毒可以在无血清培养基中收集，而滴度没有显著损失，这便于后续处理。结合超滤和超速离心进行处理，可以高效快速地处理数升体积的病毒上清液。
通过系统地优化我们的五质粒慢病毒载体系统的各个方面，我们提高了滴度、安全性、大规模生产能力，并证明可以特异性地控制感染性。
