Center for Gene and Cell Therapy, Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Oncol Rep. 2011 Oct;26(4):769-75. doi: 10.3892/or.2011.1371. Epub 2011 Jul 1.
The two-step transcriptional amplification (TSTA) system was previously reported to enhance the tissue-specific gene expression driven by weak promoters, but the enhancement of the gene expression is limited to use in in vitro and in vivo experimental situations. To achieve robust tissue-specific gene expression using the TSTA system, we developed an advanced TSTA system which includes polyglutamines and rat glucocorticoid receptor sequences between the GAL4 and VP16 sequences in the region of the first step of transcription. We evaluated the advanced TSTA system as a method to enhance the human telomerase reverse transcriptase (hTERT) promoter-driving cancer-specific transcription in various cancer cell lines. As a result, the advanced TSTA enhanced cancer-specific luciferase gene expression in all of the examined cancer cell lines, when compared with both the one-step and conventional TSTA systems (an ~6- and ~17-fold enhancement, respectively). Notably, the enhancement of the hTERT driven expression by the conventional TSTA system was modest and even inferior to the one-step system in several cancer cell lines. We then constructed a luciferase gene encoding the adeno-associated virus vector in which the hTERT promoter-mediated expression was driven by the advanced TSTA or control systems. In an orthotopic liver tumor model, mice were treated with the vector via tail vein injection. An optical imaging device was used to visualize the in vivo luciferase expression in the orthotopic tumor. The advanced TSTA system significantly enhanced the luciferase expression compared with the one-step and conventional TSTA systems (18.0±1.0- and 15.9±0.85-fold gain, respectively). Therefore, the advanced TSTA system significantly improves hTERT-dependent cancer-specific gene expression both in vitro and in vivo when compared with the previous systems. Since the advanced TSTA method can also be applied to other site-specific gene expression systems using tissue-specific promoters, this approach is expected to become a valuable tool enabling in vivo site-specific targeting in the field of gene therapy and molecular imaging.
两步转录扩增（TSTA）系统以前被报道可以增强由弱启动子驱动的组织特异性基因表达，但基因表达的增强仅限于在体外和体内实验情况下使用。为了使用 TSTA 系统实现稳健的组织特异性基因表达，我们在转录的第一步区域的 GAL4 和 VP16 序列之间插入多聚谷氨酰胺和大鼠糖皮质激素受体序列，开发了一种先进的 TSTA 系统。我们评估了先进的 TSTA 系统作为一种增强人端粒酶逆转录酶（hTERT）启动子驱动的各种癌细胞系中癌症特异性转录的方法。结果，与一步法和传统 TSTA 系统相比，先进的 TSTA 增强了所有检测的癌细胞系中的癌症特异性荧光素酶基因表达（分别为6 倍和17 倍增强）。值得注意的是，在几种癌细胞系中，传统 TSTA 系统增强 hTERT 驱动的表达的效果适度，甚至不如一步法系统。然后，我们构建了一个荧光素酶基因编码的腺相关病毒载体，其中 hTERT 启动子介导的表达由先进的 TSTA 或对照系统驱动。在原位肝肿瘤模型中，通过尾静脉注射将载体递送给小鼠。使用光学成像设备可视化原位肿瘤中的体内荧光素酶表达。与一步法和传统 TSTA 系统相比，先进的 TSTA 系统显著增强了荧光素酶表达（分别为 18.0±1.0-和 15.9±0.85 倍增益）。因此，与以前的系统相比，先进的 TSTA 系统在体外和体内都显著提高了 hTERT 依赖性癌症特异性基因表达。由于先进的 TSTA 方法也可应用于使用组织特异性启动子的其他位点特异性基因表达系统，因此该方法有望成为基因治疗和分子成像领域中体内特异性靶向的有价值的工具。
