Shi Chang-Xin, Graham Frank L, Hitt Mary M
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
J Gene Med. 2006 Apr;8(4):442-51. doi: 10.1002/jgm.867.
Helper-dependent (HD) adenovirus (Ad) vectors, deleted of all viral coding sequences, have a higher cloning capacity, improved performance of tissue-specific promoters, and reduced toxicity in animals relative to first-generation Ad vectors, making these vectors promising tools for gene transfer in vitro and in vivo. However, the large size of HDAd precursor plasmids renders them relatively difficult to manipulate due to the paucity of unique restriction enzyme sites suitable for transgene insertion and to the size constraints imposed by the viral packaging machinery.
We have constructed a series of HDAd precursor plasmids that allows cassette insertion at a unique site in the vector backbone. We have tested whether these vector backbones will support the tissue-specificity of inserted expression cassettes in a study of the activity of the potentially breast-cancer-specific mammaglobin promoter and enhancer.
We report here the generation of a series of HDAd precursor plasmids, both with and without an additional reporter expression cassette, that were designed to accommodate a wide range in size of inserted DNA. The system was validated for transcriptional targeting studies by demonstrating the tissue-specificity and activity of the mammaglobin promoter rescued using this precursor system. In addition, we have extended our previous studies on the mammaglobin promoter by demonstrating that two copies of the mammaglobin enhancer fused to the minimal promoter surpassed the activity of the single enhancer/promoter by at least 10-fold in breast cancer cells while maintaining only minimal expression in normal cells both in vitro and in a mouse tumor model.
This versatile plasmid system simplifies the construction of HDAd vectors and was valuable in demonstrating the targeting potential of the mammaglobin promoter for breast cancer gene therapy.
辅助依赖型（HD）腺病毒（Ad）载体删除了所有病毒编码序列，与第一代Ad载体相比，具有更高的克隆能力、组织特异性启动子性能更佳以及在动物体内毒性更低的特点，这使得这些载体成为体内外基因转移的有前景的工具。然而，HDAd前体质粒体积较大，由于适合转基因插入的独特限制性酶切位点较少以及病毒包装机制所施加的大小限制，使得它们相对难以操作。
我们构建了一系列HDAd前体质粒，允许在载体骨架的一个独特位点插入盒式结构。在一项关于潜在乳腺癌特异性乳腺珠蛋白启动子和增强子活性的研究中，我们测试了这些载体骨架是否支持插入的表达盒式结构的组织特异性。
我们在此报告了一系列HDAd前体质粒的产生，有或没有额外的报告基因表达盒式结构，其设计目的是容纳大小范围广泛的插入DNA。通过证明使用该前体系统拯救的乳腺珠蛋白启动子的组织特异性和活性，验证了该系统用于转录靶向研究。此外，我们扩展了之前对乳腺珠蛋白启动子的研究，证明与最小启动子融合的两个乳腺珠蛋白增强子拷贝在乳腺癌细胞中的活性比单个增强子/启动子至少高10倍，同时在体外和小鼠肿瘤模型中的正常细胞中仅维持最低表达。
这种通用的质粒系统简化了HDAd载体的构建，并且在证明乳腺珠蛋白启动子对乳腺癌基因治疗的靶向潜力方面具有重要价值。
