Xia Xu Gang, Zhou Hongxia, Ding Hongliu, Affar El Bashir, Shi Yang, Xu Zuoshang
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.
Nucleic Acids Res. 2003 Sep 1;31(17):e100. doi: 10.1093/nar/gng098.
Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III (Pol III) promoters can trigger sequence-selective gene silencing in culture and in vivo and, therefore, may be developed to treat diseases caused by dominant, gain-of-function type of gene mutations. These diseases develop in people bearing one mutant and one wild-type gene allele. While the mutant is toxic, the wild-type performs important functions. Thus, the ideal therapy must selectively silence the mutant but maintain the wild-type expression. To achieve this goal, we designed an shRNA that selectively silenced a mutant Cu,Zn superoxide dismutase (SOD1(G93A)) allele that causes amyotrophic lateral sclerosis. However, the efficacy of this shRNA was relatively modest. Since the allele-specific shRNA has to target the mutation site, we could not scan other regions of SOD1 mRNA to find the best silencer. To overcome this problem, we sought to increase the dose of this shRNA by enhancing the Pol III promoter. Here we demonstrate that the enhancer from the cytomegalovirus immediate-early promoter can enhance the U6 promoter activity, the synthesis of shRNA and the efficacy of RNA interference (RNAi). Thus, this enhanced U6 promoter is useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region.
由RNA聚合酶III（Pol III）启动子转录的短发夹RNA（shRNA）可在培养物和体内引发序列选择性基因沉默，因此，可开发用于治疗由显性、功能获得型基因突变引起的疾病。这些疾病在携带一个突变基因和一个野生型基因等位基因的人群中发生。虽然突变体具有毒性，但野生型发挥着重要功能。因此，理想的治疗方法必须选择性地沉默突变体，但保持野生型的表达。为了实现这一目标，我们设计了一种shRNA，它能选择性地沉默导致肌萎缩侧索硬化症的突变型铜锌超氧化物歧化酶（SOD1(G93A)）等位基因。然而，这种shRNA的效果相对一般。由于等位基因特异性shRNA必须靶向突变位点，我们无法扫描SOD1 mRNA的其他区域以找到最佳沉默子。为了克服这个问题，我们试图通过增强Pol III启动子来增加这种shRNA的剂量。在此我们证明，来自巨细胞病毒立即早期启动子的增强子可以增强U6启动子活性、shRNA的合成以及RNA干扰（RNAi）的效果。因此，当shRNA序列选择有限而无法选择高效RNAi靶区域时，这种增强的U6启动子是有用的。
