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

总结。 亨廷顿病(HD)是一种常染色体显性遗传性神经退行性疾病，其特征是 发病后20年内无休止地进展至死亡。先天性心脏病是由扩大的CAG引起的 在亨廷顿蛋白(Htt)基因的外显子1重复。疾病的发病在很大程度上是表达的结果 突变的转录本和蛋白质，它们具有神经毒性。抑制的表达 因此，突变的等位基因是一种很有前途的治疗方法，到目前为止一直在用抗体进行研究， 寡核苷酸和siRNA策略。就像任何击倒方法一样，特别是在中南欧， 使用这些方法的传递、可逆性和偏离目标效果的问题仍然是问题。 令人惊讶的是，人们对HD基因的调控知之甚少，尤其是对 调节HTT表达的机制。我们最近在链上发现了一种基因 在Hd位点反义HTT，我们称之为Huntingtin反义(HTT-AS)。我们有 提示HTT-AS的表达增加降低了HTT的表达，而HTT-AS的表达 表达可以用小分子来操纵。我们假设HTT本身，以及 调节其表达和与HTT相互作用的成分将提供新的治疗靶点 用于抑制HTT的表达，从而治疗HD。这一假设的推论是 更好地了解HD基因，在这种情况下，HTT-AS的作用将是至关重要的 解释任何HTT抑制策略。我们将通过一系列实验来验证这一假设 组织成三个具体目标，每个目标都建立在令人信服的初步数据基础上。在目标1中，我们将 确定是否存在额外的HTT-AS外显子、剪接变异体和启动子，确定 重复hTT-AS表达扩增，鉴定调节hTT-AS启动子的蛋白因子 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转录本抑制hTT的定量效应及染色质的作用 重塑对HTT表达的影响。在AIM 3中，我们将与NCATS和CHDI合作执行大型 扩大高通量筛选以寻找和表征通过以下方式降低hTT表达化合物 特异地增加HTT-AS的表达。选定的化合物将在细胞和小鼠中进行验证 模特们。

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.