Ulasov Ilya V, Rivera Angel A, Sonabend Adam M, Rivera Lisa B, Wang Ming, Zhu Zeng B, Lesniak Maciej S
Division of Neurosurgery, The University of Chicago, Chicago, Illinois 60637, USA.
Cancer Biol Ther. 2007 May;6(5):679-85. doi: 10.4161/cbt.6.5.3957. Epub 2007 Feb 3.
Transcriptional targeting is a key strategy to enhance therapeutic efficacy of gene therapy applications. In the context of oncolytic virotherapy, transcriptional promoter elements are used from genes that are over expressed in a variety of malignant cancers. In the present study, we examined the feasibility of transcriptional targeting to glioma cells by comparing the activity of survivin, midkine, and CXCR4 tumor-specific promoters.
To evaluate the expression level of several glioma related genes, we performed quantitative RT-PCR analyses on samples obtained from cell lines and patients. To determine specific level of gene expression mediated by selective promoter elements, we measured luciferase expression in glioma samples transduced with replication deficient adenoviral vectors. Finally, we incorporated the optimal promoters into a conditionally replicative adenoviral vector, CRAd-5/3, and examined the cytopathic effect in vitro.
The survivin promoter demonstrated the highest level of mRNA expression in primary tumor samples and cell lines. Transcriptional targeting was confirmed by infection of glioma cells with an adenovirus expression vector containing a surviving-driven luciferase reporter gene. Of the tested promoters, minimal level of survivin activity was detected in normal human liver and brain. A novel vector, CRAd-survivin5/3, with E1a under the control of the survivin promoter, exhibited enhanced cytopathic effect in vitro.
Our data demonstrate that the survivin promoter element is very active in glioma samples and has low activity in normal human brain and liver. A novel oncolytic virus, CRAd-survivin-5/3, was effective against a panel of glioma cell lines in vitro. Our results suggest that employing the survivin promoter element in the context of CRAd-5/3 may present a new opportunity for the development of glioma specific oncolytic vectors.
转录靶向是提高基因治疗应用疗效的关键策略。在溶瘤病毒疗法中，转录启动子元件取自多种恶性肿瘤中过表达的基因。在本研究中，我们通过比较生存素、中期因子和CXCR4肿瘤特异性启动子的活性，研究了转录靶向胶质瘤细胞的可行性。
为评估几种胶质瘤相关基因的表达水平，我们对从细胞系和患者获取的样本进行了定量逆转录聚合酶链反应分析。为确定选择性启动子元件介导的基因表达的特定水平，我们测量了用复制缺陷型腺病毒载体转导的胶质瘤样本中的荧光素酶表达。最后，我们将最佳启动子整合到条件性复制腺病毒载体CRAd-5/3中，并在体外检测其细胞病变效应。
生存素启动子在原发性肿瘤样本和细胞系中显示出最高水平的mRNA表达。通过用含有由生存素驱动的荧光素酶报告基因的腺病毒表达载体感染胶质瘤细胞，证实了转录靶向。在测试的启动子中，在正常人类肝脏和大脑中检测到的生存素活性水平最低。一种新型载体CRAd-生存素5/3，其E1a受生存素启动子控制，在体外表现出增强的细胞病变效应。
我们的数据表明，生存素启动子元件在胶质瘤样本中非常活跃，而在正常人类大脑和肝脏中活性较低。一种新型溶瘤病毒CRAd-生存素-5/3在体外对一组胶质瘤细胞系有效。我们的结果表明，在CRAd-5/3的背景下使用生存素启动子元件可能为开发胶质瘤特异性溶瘤载体提供新的机会。
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