Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington’s disease (HD). CAG repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.

聚谷氨酰胺（polyQ）疾病是由CAG在特定基因的开放阅读框（ORF）中重复扩增引起的不可治愈的神经系统疾病。HTT中的这种突变该基因负责亨廷顿舞蹈病（HD）。CAG重复靶向人工miRNA（art-miRNA）被证明是针对polyQ疾病的有吸引力的治疗方法，因为它们引起等位基因选择性降低突变蛋白的水平。在这里，我们使用polyQ疾病模型，旨在证明基于miRNA的基因表达调控如何依赖于靶序列特征。我们表明，沉默的效率和art-miRNA的选择性受转录本内的CAG重复道的定位和特定序列环境的影响。此外，我们旨在揭示导致突变体polyQ蛋白下调的事件，并发现翻译抑制和HTT的激活非常迅速转录腺苷酸化。正如在用CRISPR-Cas9技术生成的AGO2敲除细胞中所确定的那样，在此过程中AGO2的切片机活性是必需的。我们还显示了人类高清神经祖细胞（NP）中亨廷顿蛋白的高度等位基因选择性下调。综上所述，art-miRNA活性可以作为内源性miRNA协同作用和靶向ORF区域的模型。