Klein Reinhard, Ruttkowski Bärbel, Knapp Elzbieta, Salmons Brian, Günzburg Walter H, Hohenadl Christine
AUSTRIANOVA Biotechnology GmbH, Veterinärplatz 1, A-1210 Vienna, Austria.
Gene. 2006 May 10;372:153-61. doi: 10.1016/j.gene.2005.12.018. Epub 2006 Feb 20.
The success of gene therapy approaches relies on sufficiently high levels of expression of the therapeutic gene. However, if tissue specific or tumour specific gene expression is desired, a lower level of transgene expression usually has to be accepted due to the weakness of the majority of available tissue or tumour specific promoters. This obstacle can in part be overcome by the insertion of viral cis-acting elements that enhance gene expression in various expression vector contexts regardless of the respective promoter. We designed a series of murine leukaemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors that contain the woodchuck hepatitis post-transcriptional regulatory element (WPRE) and evaluated its use by measuring enhanced green fluorescent protein (EGFP) levels and viral titres. In viral vector packaging cells, when the EGFP encoding gene was transcribed from the MLV promoter, incorporation of the WPRE resulted in a marked improvement of the vectors in terms of EGFP expression and virus titres. However, in infected cells after promoter conversion had taken place, the effect of the WPRE became promoter and cell line dependent. When the EGFP gene was transcribed from the heterologous mouse mammary tumour virus (MMTV) promoter the same beneficial role of the WPRE on transgene expression was observed in all eight cell lines tested. In contrast, when EGFP gene expression was driven by the murine whey acidic protein (WAP) promoter, the positive effect of the WPRE could only be observed in two cell lines whereas expression was actually reduced in the six other cell lines tested. This decrease of EGFP expression was not only demonstrated at the protein level but also manifested on the RNA level.
基因治疗方法的成功依赖于治疗性基因足够高的表达水平。然而，如果需要组织特异性或肿瘤特异性基因表达，由于大多数可用的组织或肿瘤特异性启动子的活性较弱，通常不得不接受较低水平的转基因表达。通过插入病毒顺式作用元件可以部分克服这一障碍，这些元件可在各种表达载体环境中增强基因表达，而与各自的启动子无关。我们设计了一系列基于鼠白血病病毒（MLV）的逆转录病毒启动子转换（ProCon）载体，这些载体包含旱獭肝炎病毒转录后调控元件（WPRE），并通过测量增强型绿色荧光蛋白（EGFP）水平和病毒滴度来评估其用途。在病毒载体包装细胞中，当EGFP编码基因从MLV启动子转录时，WPRE的掺入导致载体在EGFP表达和病毒滴度方面有显著改善。然而，在发生启动子转换后的感染细胞中，WPRE的作用变得依赖于启动子和细胞系。当EGFP基因从异源小鼠乳腺肿瘤病毒（MMTV）启动子转录时，在所有测试的8种细胞系中都观察到WPRE对转基因表达具有相同的有益作用。相反，当EGFP基因表达由鼠乳清酸性蛋白（WAP）启动子驱动时，WPRE的积极作用仅在两种细胞系中观察到，而在其他六种测试细胞系中表达实际上降低了。EGFP表达的这种降低不仅在蛋白质水平上得到证实，在RNA水平上也有体现。
