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

微卫星疾病是由受影响患者基因组中存在的特定核苷酸重复引起的毁灭性疾病。虽然这些重复扩增在一定长度内不具有致病性，但当超过一个精确的阈值时，它们就会变得具有致病性。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1型肌强直性营养不良（DM1）（由CUG重复引起）和染色体9p21相关的肌萎缩性侧索硬化症和额颞叶痴呆（c9ALS/FTD）（由GGGGCC重复触发），目前尚无有效的治疗方法。目前的研究方案提出了两种治疗策略，旨在通过使用化学核酸酶（无转换！）特异性降解有毒RNA重复序列来改善DM1和c9ALS/FTD的症状。化学核酸酶由三个模块组成：(1)RNA结合模块，这是一种多价结构，由先前显示与重复序列相互作用的单体单元组成；(2)连接单元；(3) RNA裂解模块，负责降解毒性RNA扩增物。我们将使用(-)-嘧啶藻酸，这是一种结构复杂的抗生素博莱霉素的裂解单元，它以前被证明可以有效地裂解核糖核酸。第一种策略将集中在DM1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c9ALS/FTD，并试图从具有炔和叠氮基团的单体单元中产生纤维素中的化学核酸酶。装配过程发生在病变细胞内，通过原位点击化学的方式，由有毒RNA的结构催化，有毒RNA作为模板，使单体单元接近。这些方法为处理迄今尚未得到有效处理的其他微卫星疾病提供了一个通用平台。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，这项研究的结果将：(1)提供一种基于小分子的治疗替代反义技术；(2)明确在细胞和潜在动物模型中有效靶向有毒RNA的基本规则和原则；(3)揭示小分子或小分子组合的结构特征，以促进细胞中高效和选择性的RNA切割；(4)帮助将RNA转化为可药物靶标。

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.