Choi Byung Hyune, Ha Yoon, Ahn Cheol-Hee, Huang Xian, Kim Jin-Mo, Park So Ra, Park Hyeonseon, Park Hyung Chun, Kim Sung Wan, Lee Minhyung
Inha Research Institute for Medical Sciences, Inha University College of Medicine, Incheon, South Korea.
Neurosci Lett. 2007 Jan 29;412(2):118-22. doi: 10.1016/j.neulet.2006.11.015. Epub 2006 Dec 18.
Many neurologic disorders are accompanied by ischemic injury during the pathologic process. To develop a controllable and injury-specific gene therapy system for the neurologic disorders, we constructed a hypoxia inducible plasmid with the erythropoietin (Epo) 3' untranslated region (UTR), which can enhance the stability of target mRNAs in response to hypoxia. The Epo 3' UTR was inserted at the 3' flanking region of luciferase gene in pSV-Luc, resulting in the construction of pSV-Luc-EpoUTR. In pEpo-SV-Luc-EpoUTR, the Epo enhancer was inserted into the upstream of the SV40 promoter to increase the hypoxia inducibility. The plasmids were evaluated in N2a mouse neuroblastoma cells under hypoxic conditions and in a rat spinal cord injury (SCI) model. The results showed that the Epo 3' UTR alone showed a three-fold increase in luciferase activity in hypoxic N2a cells as well as in the rat SCI model when compared to the sham control. In contrast, the Epo 3' UTR showed no effect on the luciferase activity in the presence of the Epo enhancer, probably because the Epo enhancer was more sensitive to hypoxia and showed a dominant effect. However, the Epo enhancer itself showed high level of luciferase activity even in normoxia (about five to eight-folds increase), while the Epo 3' UTR did not show enhanced background activity. Immunohistochemical staining showed expression of luciferase from pSV-Luc-EpoUTR both in neurons and astrocytes around the injured spinal cord of rat. These results suggest that the Epo 3' UTR could provide a specific and safe system for the hypoxia-inducible gene therapy of the neurologic disorders including SCI.
许多神经系统疾病在病理过程中伴有缺血性损伤。为了开发一种用于神经系统疾病的可控且损伤特异性的基因治疗系统，我们构建了一种带有促红细胞生成素（Epo）3'非翻译区（UTR）的缺氧诱导质粒，该质粒可在缺氧时增强靶mRNA的稳定性。将Epo 3'UTR插入pSV-Luc中荧光素酶基因的3'侧翼区域，从而构建了pSV-Luc-EpoUTR。在pEpo-SV-Luc-EpoUTR中，将Epo增强子插入SV40启动子的上游以增加缺氧诱导性。在缺氧条件下的N2a小鼠神经母细胞瘤细胞和大鼠脊髓损伤（SCI）模型中对这些质粒进行了评估。结果表明，与假手术对照组相比，单独的Epo 3'UTR在缺氧的N2a细胞以及大鼠SCI模型中荧光素酶活性增加了三倍。相反，在存在Epo增强子的情况下，Epo 3'UTR对荧光素酶活性没有影响，这可能是因为Epo增强子对缺氧更敏感并表现出主导作用。然而，即使在常氧条件下，Epo增强子本身也显示出高水平的荧光素酶活性（增加约五至八倍），而Epo 3'UTR并未显示出增强的背景活性。免疫组织化学染色显示pSV-Luc-EpoUTR的荧光素酶在大鼠脊髓损伤周围的神经元和星形胶质细胞中均有表达。这些结果表明，Epo 3'UTR可为包括SCI在内的神经系统疾病的缺氧诱导基因治疗提供一个特异性且安全的系统。
