Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers
Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers
复制标题
DOI：
10.1038/sj.gt.3301177
复制
发表时间：
2000-05-01
期刊：
影响因子：
5.1
通讯作者：
Bicknell, R
中科院分区：
文献类型：
--
作者：
Modlich, U;Pugh, CW;Bicknell, R
目前基因治疗的挑战之一是构建靶向特定组织的载体。靶向内皮表达在几种病理的治疗中特别令人感兴趣。我们以前已经表明，E-选择素和KDR启动子的确定区域赋予逆转录病毒递送后的内皮细胞特异性表达。然而，表达水平较低。在试图增加表达，但要保持组织特异性，我们已经检查了低氧和尼古丁诱导的增强子元件与KDR和E-选择素启动子的组合。这两种增强子应该在肿瘤环境中是活性的，促进表达并在肿瘤内皮中提供额外的基因表达特异性。将鼠磷酸甘油酸激酶-1（PGK-1）启动子的缺氧反应元件（HRE）用作缺氧增强子，并将鼠血管细胞粘附分子-1（VCAM-1）启动子的NF κ B B的串联结合位点用作精氨酸诱导的增强子。HRE赋予低氧诱导KDR和E-选择素启动子。KDR启动子保留了内皮特异性表达，而E-选择素启动子则没有。NF κ B结合位点赋予KDR启动子对TNF-α的反应性，但诱导水平低于HRE。结合两种增强子元件的逆转录病毒通过缺氧和TNF-α诱导转移，并在刺激后达到最高表达水平。这些结果证实了异源增强子元件可以对单个内皮细胞特异性启动子起作用。这些发现使得使用诱导型增强子成为增加组织特异性基因表达的有希望的策略。
One of the current challenges in gene therapy is to construct a vector that will target specific tissues. Targeting expression to endothelium is of particular interest in the treatment of several pathologies. We have shown previously that defined regions of the E-selectin and KDR promoters confer endothelial cell specific expression following retroviral delivery. However, the levels of expression were low. In an attempt to increase expression but to preserve the tissue specificity we have examined hypoxic and cytokine-inducible enhancer elements in combination with the KDR and E-selectin promoters. Both enhancers should be active in the tumour environment, boosting expression and giving additional specificity of gene expression in the tumour endothelium. The hypoxia response element (HRE) of the murine phosphoglycerate kinase-1 (PGK-1) promoter was used as a hypoxic enhancer and the tandem-binding site for NF kappa B from the murine Vascular cell adhesion molecule-1 (VCAM-1) promoter as a cytokine-inducible enhancer. The HRE conferred hypoxia inducibility to the KDR and E-selectin promoters. Endothelial specificity of expression was retained with the KDR but not the E-selectin promoter The NF kappa B-binding site conferred responsiveness to TNF-alpha to the KDR promoter, however the level of induction was less than that achieved with the HRE. Retrovirus combining both enhancer elements transferred inducibility by hypoxia and TNF-alpha, and reached the highest expression levels upon stimulation. These results confirm that heterologous enhancer elements may operate on a single endothelial cell specific promoter These findings make the use of inducible enhancers a promising strategy for increasing tissue specific gene expression.