Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT) that results in toxic gain of function and cell death. Despite its monogenic cause, the pathogenesis of HD is highly complex, and increasing evidence indicates that, in addition to the full-length (FL) mutant HTT protein, the expanded exon 1 HTT (HTTexon1) protein that is translated from the HTT1a transcript generated by aberrant splicing is prone to aggregate and might contribute to HD pathology. This finding suggests that reducing the expression of HTT1a might achieve a greater therapeutic benefit than targeting only FL mutant HTT. Conversely, strategies that exclusively target FL HTT might not completely prevent the pathogenesis of HD. We have developed an engineered microRNA targeting the HTT exon 1 sequence (miHTT), delivered via adeno-associated virus serotype 5 (AAV5). The target sequence of miHTT is present in both FL HTT and HTT1a transcripts. Preclinical studies with AAV5-miHTT have demonstrated efficacy in several rodent and large animal models by reducing FL HTT mRNA and protein and rescuing HD-like phenotypes and have been the rationale for phase I/II clinical studies now ongoing in the USA and Europe. In the present study, we evaluated the ability of AAV5-miHTT to reduce the levels of aberrantly spliced HTT1a mRNA and the HTTexon1 protein in the brain of two mouse models of HD (heterozygous zQ175 knock-in mice and humanized Hu128/21 mice). Polyadenylated HTT1a mRNA and HTTexon1 protein were detected in the striatum and cortex of heterozygous zQ175 knock-in mice, but not in wild-type littermate control mice. Intrastriatal administration of AAV5-miHTT resulted in dose-dependent expression of mature miHTT microRNA in cortical brain regions, accompanied by significant lowering of both FL HTT and HTT1a mRNA expression at 2 months postinjection. Mutant HTT and HTTexon1 protein levels were also significantly reduced in the striatum and cortex of heterozygous zQ175 knock-in mice at 2 months after AAV5-miHTT treatment and in humanized Hu128/21 mice 7 months post-treatment. The effects were confirmed in primary Hu128/21 neuronal cultures. These results demonstrate that AAV5-miHTT gene therapy is an effective approach to lower both FL HTT and the pathogenic HTTexon1 levels, which could potentially have an additive therapeutic benefit in comparison to other HTT-targeting modalities.

中文翻译：

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靶向外显子 1 的 miRNA 可降低亨廷顿舞蹈症模型中的致病性外显子 1 HTT 蛋白


亨廷顿舞蹈症 （HD） 是一种致命的神经退行性疾病，由亨廷顿基因 （HTT） 外显子 1 的三核苷酸重复扩增引起，导致功能中毒增加和细胞死亡。尽管HD是单基因的，但其发病机制非常复杂，越来越多的证据表明，除了全长（FL）突变HTT蛋白外，从异常剪接产生的HTT1a转录本翻译而来的扩增外显子1 HTT（HTTexon1）蛋白容易聚集，并可能导致HD病理学。这一发现表明，降低 HTT1a 的表达可能比仅靶向 FL 突变型 HTT 获得更大的治疗益处。相反，专门针对 FL HTT 的策略可能无法完全阻止 HD 的发病机制。我们开发了一种靶向 HTT 外显子 1 序列 （miHTT） 的工程化 microRNA，通过腺相关病毒血清型 5 （AAV5） 递送。miHTT 的靶序列存在于 FL HTT 和 HTT1a 转录本中。AAV5-miHTT 的临床前研究通过减少 FL HTT mRNA 和蛋白质以及挽救 HD 样表型，在几种啮齿动物和大型动物模型中证明了疗效，并且已成为目前正在美国和欧洲进行的 I/II 期临床研究的基本原理。在本研究中，我们评估了 AAV5-miHTT 降低两种 HD 小鼠模型 （杂合子 zQ175 敲入小鼠和人源化 Hu128/21 小鼠） 大脑中异常剪接的 HTT1a mRNA 和 HTTexon1 蛋白水平的能力。在杂合子 zQ175 敲入小鼠的纹状体和皮层中检测到多聚腺苷酸化 HTT1a mRNA 和 HTTexon1 蛋白，但在野生型同窝对照小鼠中未检测到。 AAV5-miHTT 的纹状体内给药导致成熟 miHTT microRNA 在皮质脑区的剂量依赖性表达，同时在注射后 2 个月时 FL HTT 和 HTT1a mRNA 表达显着降低。在 AAV5-miHTT 处理后 2 个月和治疗后 7 个月，杂合 zQ175 敲入小鼠的纹状体和皮层中突变的 HTT 和 HTTexon1 蛋白水平也显著降低。在原代 Hu128/21 神经元培养物中证实了这种影响。这些结果表明，AAV5-miHTT 基因治疗是降低 FL HTT 和致病性 HTTexon1 水平的有效方法，与其他 HTT 靶向方式相比，这可能具有附加的治疗益处。