Argyros O, Wong S P, Niceta M, Waddington S N, Howe S J, Coutelle C, Miller A D, Harbottle R P
Department of Chemistry, Imperial College Genetic Therapies Centre, Imperial College London, London, UK.
Gene Ther. 2008 Dec;15(24):1593-605. doi: 10.1038/gt.2008.113. Epub 2008 Jul 17.
An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo.
理想的基因治疗载体应能实现持续的转基因表达，且不受安全性和可重复性的限制。在此，我们报告了一种非病毒附加体质粒DNA（pDNA）载体的研发情况，该载体似乎满足了这些标准。这种pDNA载体将一个支架/基质附着区域（S/MAR）与一个人肝脏特异性启动子（α1-抗胰蛋白酶（AAT））相结合，使得在水动力注射后，可在小鼠肝脏中实现长期表达。通过原位生物发光成像监测长达6个月的纵向荧光素酶表达谱，证明了长期表达。所有表达荧光素酶的相关对照pDNA构建体在给药后1周以上均无法维持显著的转基因表达。我们确定这种表达的关闭是由于启动子甲基化。相比之下，S/MAR元件似乎抑制了AAT启动子的甲基化，从而防止转基因沉默。尽管该载体似乎始终作为附加体维持存在，但我们没有证据表明它能作为一个复制实体得以确立。我们得出结论，哺乳动物组织特异性启动子与S/MAR元件的组合足以驱动基因在体内的长期附加体pDNA表达。
