Oh Taekeun, Bajwa Ali, Jia Guangfu, Park Frank
Department of Medicine, Kidney Disease Center, Medical College of Wisconsin, Milwaukee, WI, USA.
Retrovirology. 2007 Jun 5;4:38. doi: 10.1186/1742-4690-4-38.
Lentiviral vectors have been designed with complex RNA export sequences in both the integrating and packaging plasmids in order to co-ordinate efficient vector production. Recent studies have attempted to replace the existing complex rev/RRE system with a more simplistic RNA export system from simple retroviruses to make these vectors in a rev-independent manner.
Towards this end, lentiviral transfer plasmids were modified with various cis-acting DNA elements that co-ordinate RNA export during viral production to determine their ability to affect the efficiency of vector titer and transduction in different immortalized cell lines in vitro. It was found that multiple copies of the constitutive transport element (CTE) originating from different simian retroviruses, including simian retrovirus type 1 (SRV-1) and type-2 (SRV-2) and Mason-Pfizer (MPV) could be used to eliminate the requirement for the rev responsive element (RRE) in the transfer and packaging plasmids with titers >106 T.U./mL (n = 4-8 preparations). The addition of multiple copies of the murine intracisternal type A particle, the woodchuck post-regulatory element (WPRE), or single and dual copies of the simian CTE had minimal effect on viral titer. Immortalized cell lines from different species were found to be readily transduced by VSV-G pseudotyped lentiviral vectors containing the multiple copies of the CTE similar to the findings in HeLa cells, although the simian-derived CTE were found to have a lower infectivity into murine cell lines compared to the other species.
These studies demonstrated that the rev-responsive element (RRE) could be replaced with other constitutive transport elements to produce equivalent titers using lentivectors containing the RRE sequence in vitro, but that concatemerization of the CTE or the close proximity of RNA export sequences was needed to enhance vector production.
慢病毒载体在整合质粒和包装质粒中设计了复杂的RNA输出序列，以协调高效的载体生产。最近的研究试图用来自简单逆转录病毒的更简单的RNA输出系统取代现有的复杂的rev/RRE系统，从而以不依赖rev的方式生产这些载体。
为此，用各种顺式作用DNA元件修饰慢病毒转移质粒，这些元件在病毒生产过程中协调RNA输出，以确定它们影响体外不同永生化细胞系中载体滴度和转导效率的能力。研究发现，源自不同猴逆转录病毒（包括1型猴逆转录病毒（SRV-1）、2型猴逆转录病毒（SRV-2）和梅森-辉瑞猴病毒（MPV））的组成型转运元件（CTE）的多个拷贝，可用于消除转移质粒和包装质粒中对rev反应元件（RRE）的需求，滴度>106转导单位/毫升（n = 4 - 8个制备物）。添加多个小鼠A型颗粒、土拨鼠调节后元件（WPRE）的拷贝，或猴CTE的单拷贝和双拷贝对病毒滴度的影响最小。发现不同物种的永生化细胞系很容易被含有CTE多个拷贝的VSV-G假型慢病毒载体转导，这与在HeLa细胞中的发现相似，尽管与其他物种相比，源自猴的CTE对小鼠细胞系的感染性较低。
这些研究表明，在体外使用含有RRE序列的慢病毒载体时，rev反应元件（RRE）可以被其他组成型转运元件取代以产生相当的滴度，但需要CTE的串联或RNA输出序列的紧密相邻来提高载体产量。
