Allele-Selective Inhibitors for Expanded Trinucleotide Repeat Genes

简介(申请人提供)：背景。扩大的三核苷酸重复导致亨廷顿病(HD)和其他退行性疾病。这些毁灭性的疾病没有治愈的方法，迫切需要治疗。三核苷酸重复疾病是由一个基因的突变引起的，而阻断突变基因表达的药物提供了一个有希望的治疗选择。反义寡核苷酸(ASO)或双链RNA可以在动物模型中抑制三核苷酸重复基因的表达，但对突变和野生型等位基因的抑制在患者中可能是不能容忍的。仅对突变蛋白进行选择性抑制 这将是理想的，但需要创新的方法来基因沉默，因为需要区分两个相似的等位基因。在之前的资助期间，我们使用单链反义寡核苷酸、单链干扰RNA(SsiRNAs)和双链RNA来实现对突变HTT的选择性抑制。目标。我们建议通过优化这些新发现的化合物并更好地了解它们的作用机制来最大限度地发挥临床开发的潜力。我们将把我们的策略扩展到其他三核苷酸重复疾病，以确定更多的治疗前导分子。三核苷酸重复基因的mRNAs在许多方面不同，抑制剂开发的挑战将导致对重复序列如何影响识别机制的深入了解。在最基本的水平上，我们使用的核酸的化学性质和设计特征是新颖的，他们的研究将为ASO、ssRNAs和双链RNAs的基因沉默提供重要的一般性见解。目的1.优化双链RNAs、ASO和单链RNAs的效力和选择性。我们将设计和测试新一代化合物，并测试它们对HTT表达的抑制作用。我们开发的结构活性关系将使我们能够为动物研究选择最好的化合物，并提供对等位基因选择性的机械性见解。目的2.探讨等位基因选择性抑制的机制。了解等位基因选择性的机制将有助于设计高效的HTT表达抑制剂。我们将使用生化和基于细胞的方法来表征在我们的抑制分子识别HTT mRNA过程中发生的蛋白质和核酸的相互作用。目的3.抑制剂在含有扩展重复序列的其他疾病基因中的应用。其他疾病基因包含扩展的三联体重复序列，包括ataxin-3、强直性肌营养不良蛋白激酶和ataxin-7。开发化合物以实现对这些基因的等位基因选择性抑制将扩大该方法的治疗潜力，并为机制提供新的视角。

DESCRIPTION (provided by applicant): Background. Expanded trinucleotide repeats cause Huntington's Disease (HD) and other degenerative disorders. There are no cures for these devastating illnesses and treatments are urgently needed. Trinucleotide repeat disorders are due to mutations in just one gene, and agents that block expression of the mutant gene offer a promising option for treatment. Antisense oligonucleotides (ASOs) or duplex RNAs can inhibit expression of trinucleotide repeat genes in animal models, but inhibition of both the mutant and wild-type alleles may not be tolerated in patients. Selective inhibition of only the mutant protein would be ideal, but requires innovative approaches to gene silencing because of the need to discriminate between the two similar alleles. During the previous funding period we used single-stranded antisense oligonucleotides, single-stranded interfering RNAs (ssiRNAs), and duplex RNAs to achieve selective inhibition of mutant HTT. Objective. We propose to maximize the potential for clinical development by optimizing these newly discovered compounds and better understanding their mechanisms of action. We will extend our strategy to other trinucleotide-repeat diseases to identify additional therapeutic lead molecules. The mRNAs of trinucleotide repeat genes differ in many ways, and the challenge of inhibitor development will lead to insights into how repetitive sequence affects the mechanism of recognition. At the most fundamental level, the chemical properties and design features of the nucleic acids we use are novel and their study will offer important general insights into gene silencing by ASOs, ssRNAs, and duplex RNAs. Aim 1. Optimize potency and selectivity of duplex RNAs, ASOs, and ssRNAs. We will design and test new generations of compounds and assay their inhibition of HTT expression. The structure activity relationships we develop will allow us to select the best compounds for animal studies and offer mechanistic insights into allele-selectivity. Aim 2. Investigate the mechanism of allele-selective inhibition. Understanding the mechanism of allele-selectivity will facilitate the design of highly effective inhibitors of HTT expression. We will us biochemical and cell-based approaches to characterize the protein- and nucleic acid- interactions that occur during recognition of HTT mRNA by our inhibitory molecules. Aim 3. Application of inhibitors to other disease genes containing expanded repeats. Other disease genes contain expanded triplet repeats including ataxin-3, myotonic dystrophy protein kinase, and ataxin-7. Developing compounds to achieve allele-selective inhibition of these genes would widen the therapeutic potential of the approach and offer new perspective on mechanisms.