Small Molecule-Mediated Cleavage of RNA In Cells. Application to Incurable Human Genetic Disease

小分子介导的细胞内 RNA 切割。

• 批准号: 318757958
• 负责人: Dr. Andrei Ursu
• 金额: --
• 依托单位: Department of Chemistry
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/318757958?language=en
• 关键词: Small; Molecule; Mediated; Cleavage; RNA

Microsatellite disorders are devastating diseases caused by specific nucleotide repeats present within the genome of affected patients. Whereas these repeat expansions are not pathogenic within a certain length, they can become pathogenic when exceeding a precise threshold. When transcribed, they initiate various toxic mechanisms which are the basis of more than 20 neuromuscular disorders including myotonic dystrophy type 1 (DM1) (caused by CUG repeats) and chromosome 9p21-linked amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) (triggered by GGGGCC repeats), for which at present no effective treatment exists. The current research proposal presents two therapeutic strategies meant to improve the symptoms of DM1 and c9ALS/FTD by employing chemical nucleases (without turnover!) that specifically degrade toxic RNA repeats. A chemical nuclease consists of three modules: (1) RNA binding module, a multivalent construct composed of monomeric units previously shown to interact with the repeats; (2) linker unit; (3) RNA cleavage module, responsible for the degradation of toxic RNA expansions. We will employ (-)-Pyrimidoblamic acid, the cleavage unit of a structurally complex antibiotic Bleomycin, which was previously shown to efficiently cleave ribonucleic acids. The first strategy will focus on DM1 and consists of assembling the chemical nuclease modules in vitro using standard amide coupling reactions. The second approach addresses c9ALS/FTD and attempts to generate the chemical nuclease in cellulo from monomer units functionalized with alkyne and azide moieties. The assembly process occurs within the diseased cells by means of in situ click chemistry catalyzed by the structure of the toxic RNA which acts as a template bringing the monomeric units in close proximity. These approaches offer a generalized platform to approach other microsatellite diseases that were so far not efficiently addressed. Moreover, the cleaving approach via chemical nucleases could be applied to a host of other RNAs, outside of repeats, such as cancer-causing RNAs. Altogether, the results of this research will: (1) provide a small molecule-based therapeutic alternative to antisense technology; (2) define basic rules and principles for effective targeting of toxic RNA in cellulo and potentially in animal model; (3) reveal structural features of the small molecules or small molecule assemblies required to promote efficient and selective RNA cleavage in cellulo; (4) help transform RNA into a druggable target.

微卫星疾病是由受影响患者基因组内存在的特定核苷酸重复序列引起的破坏性疾病。尽管这些重复扩增在一定长度内不是致病性的，但当超过精确的阈值时，它们可以变成致病性的。当转录时，它们启动各种毒性机制，这些机制是20多种神经肌肉疾病的基础，包括强直性肌营养不良1型（DM 1）（由CUG重复序列引起）和染色体9 p21连锁的肌萎缩性侧索硬化症和额颞叶痴呆（c9 ALS/FTD）（由GGGGCC重复序列触发），目前还没有有效的治疗方法。目前的研究提案提出了两种治疗策略，旨在通过采用化学核酸酶（无周转！）能特异性降解有毒的RNA重复序列化学核酸酶由三个模块组成：（1）RNA结合模块，由先前显示与重复序列相互作用的单体单元组成的多价构建体;（2）接头单元;（3）RNA切割模块，负责降解毒性RNA扩增。我们将使用（-）-嘧啶核酸，一种结构复杂的抗生素博莱霉素的切割单位，它以前被证明可以有效地切割核糖核酸。第一种策略将集中在DM 1上，包括使用标准酰胺偶联反应在体外组装化学核酸酶模块。第二种方法解决了c9 ALS/FTD，并试图在纤维素中从用炔和叠氮化物部分官能化的单体单元产生化学核酸酶。组装过程在患病细胞内通过由毒性RNA的结构催化的原位点击化学发生，毒性RNA的结构充当模板，使单体单元紧密接近。这些方法提供了一个通用的平台，以接近其他微卫星疾病，迄今尚未有效地解决。此外，通过化学核酸酶的切割方法可以应用于重复序列之外的许多其他RNA，例如致癌RNA。总之，本研究的结果将：（1）为反义技术提供一种基于小分子的治疗方法;（2）确定细胞内和潜在动物模型中有效靶向毒性RNA的基本规则和原则;（3）揭示促进细胞内有效和选择性RNA切割所需的小分子或小分子组装体的结构特征;（4）帮助将RNA转化为可药用靶标。