Endogenous regulation of huntingtin expression as a therapeutic target for Huntington's disease

摘要 亨廷顿病（HD）是一种常染色体显性遗传的神经退行性疾病，其特征在于 在发病后~20年内持续进展至死亡。HD是由扩张的CAG引起的 在亨廷顿蛋白（HTT）基因的外显子1中重复。疾病的发病机制在很大程度上是由于表达 突变的转录本和蛋白质，它们具有神经毒性。抑制表达 因此，突变等位基因是一种有希望的治疗方法，迄今为止用抗体， 寡核苷酸和siRNA策略。与任何击倒方法一样，特别是在CNS中， 使用这些方法的递送、可逆性和脱靶效应的问题仍然存在。 令人惊讶的是，关于HD基因座的调控，特别是对 调节HTT表达的机制。我们最近发现了一种基因 在HD基因座处与HTT反义，我们称之为亨廷顿蛋白反义（HTT-AS）。我们有 表明增加HTT-AS的表达会降低HTT的表达，并且HTT-AS 可以使用小分子操纵表达。我们假设HTT-AS本身和 调节其表达和与HTT相互作用的组分，将提供新的治疗靶点 用于抑制HTT表达并因此治疗HD。这个假设的一个推论是， 更好地了解HD基因座，在这种情况下，HTT-AS的作用，将是至关重要的， 任何HTT抑制策略的解释。我们将用一系列实验来检验这一假设 这三个目标都建立在令人信服的初步数据基础上。在目标1中，我们 确定是否存在额外的HTT-AS外显子、剪接变体和启动子，确定 重复扩增HTT-AS表达，并鉴定调节HTT-AS启动子的蛋白质因子 活动在目标2中，我们将确定HTT-AS抑制HTT的机制，包括 不同HTT-AS转录物对HTT抑制的定量作用，以及染色质的作用 重塑HTT表达。在目标3中，我们将与NCATS和CHDI合作， 大规模高通量筛选，以发现和表征通过以下途径降低HTT表达的化合物： 特别是增加HTT-AS的表达。选定的化合物将在细胞和小鼠中进行验证 模型

Summary. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by relentless progression to death ~20 years after disease onset. HD is caused by an expanded CAG repeat in exon 1 of the huntingtin (HTT) gene. Disease pathogenesis is largely a result of expression of the mutant transcript and protein, which have neurotoxic properties. Suppressing the expression of the mutant allele is therefore a promising therapeutic approach, thus far pursued with antibody, oligonucleotide and siRNA strategies. As with any knockdown approach, especially in the CNS, problems of delivery, reversibility, and off-target effects using these methods remain problematic. Surprisingly, relatively little is known about the regulation of the HD locus, and in particular on the mechanisms that regulate HTT expression. We have recently discovered a gene on the strand antisense to HTT at the HD locus, which we have termed huntingtin antisense (HTT-AS). We have demonstrated that increasing expression of HTT-AS decreases expression of HTT, and that HTT-AS expression can be manipulated using small molecules. We hypothesize that HTT-AS itself, and the components that regulate its expression and interaction with HTT, will provide novel therapeutic targets for suppression of HTT expression and hence treatment of HD. A corollary to this hypothesis is that a better understanding of the HD locus, in this case the role of HTT-AS, will be critical in the interpretation of any HTT suppression strategy. We will test this hypothesis with a series of experiments organized into three specific aims, each built on compelling preliminary data. In Aim 1, we will determine if additional HTT-AS exons, splice variants, and promoters exist, determine the effect of repeat expansion on HTT-AS expression, and identify protein factors that regulate HTT-AS promoter activity. In Aim 2, we will determine the mechanisms by which HTT-AS suppresses HTT, including the quantitative effect of different HTT-AS transcripts on the suppression of HTT, and the role of chromatin remodeling on HTT expression. In Aim 3, we will collaborate with NCATS and CHDI to perform a large scale high throughput screen to find and characterize compounds that decrease expression of HTT by specifically increasing expression of HTT-AS. Selected compounds will be validated in cell and mouse models.