An enhanced U6 promoter for synthesis of short hairpin RNA
An enhanced U6 promoter for synthesis of short hairpin RNA
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DOI：
10.1093/nar/gng098
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发表时间：
2003-09-01
影响因子：
14.9
通讯作者：
Xu, ZS
中科院分区：
文献类型：
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作者：
Xia, XG;Zhou, HX;Xu, ZS
由 RNA 聚合酶 III (Pol III) 启动子转录的短发夹 RNA (shRNA) 可以在培养物和体内触发序列选择性基因沉默，因此可用于治疗由显性、功能获得型基因突变引起的疾病。这些疾病发生在携带一种突变型和一种野生型基因等位基因的人身上。虽然突变体有毒，但野生型却发挥着重要的功能。因此，理想的治疗必须选择性地沉默突变体但维持野生型表达。为了实现这一目标，我们设计了一种 shRNA，可以选择性沉默导致肌萎缩侧索硬化症的突变铜、锌超氧化物歧化酶 (SOD1(G93A)) 等位基因。然而，这种 shRNA 的功效相对较小。由于等位基因特异性 shRNA 必须针对突变位点，因此我们无法扫描 SOD1 mRNA 的其他区域来找到最佳的沉默子。为了克服这个问题，我们试图通过增强 Pol III 启动子来增加该 shRNA 的剂量。在这里，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的合成和RNA干扰（RNAi）的功效。因此，当 shRNA 序列的选择有限而无法选择高效的 RNAi 靶区域时，这种增强的 U6 启动子非常有用。
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.