Design of precision small molecules targeting RNA repeating transcripts to manipulate and study disease biology

项目摘要：一个极具挑战性的问题是开发针对缺陷的通用方法 或者选择性地导致疾病的RNAs故障。目前针对RNA的治疗策略是基于 关于寡核苷酸识别特定序列的研究。然而，许多人类疾病是由高度 传统碱基配对不容易靶向的结构化RNA，特别是 导致或促成无法治愈的神经肌肉疾病和基因定义的痴呆症。因此，等位基因- 这些微卫星疾病的特定ASOS模式已经通过靶向多态被开发出来 在重复序列之外。这种方法的后果是只有患有 多态从治疗中受益，并且必须为每种疾病开发ASO，即使是引起的 通过相同的重复序列。如果这些疾病中的毒素，即扩大的重复序列，可以成为靶点 选择性地与结构特定的小分子结合，那么单一的模式可以是治疗或化学药物 为多种疾病和所有患者进行探查。 在过去的14年里，我们已经证明了rna结构可以被小分子选择性地作为靶标。 在原位和体内，比寡核苷酸更有选择性。事实上，我们已经设计了针对许多人的化合物 RNA重复扩增选择性地识别靶子的结构并拯救与疾病相关的 原位和活体病理生物学。此外，这些化学探针阐明了新的疾病机制， 包括一条先前未知的RNA介导的转录沉默途径，该途径在脆性X 综合症。这些研究，以及我们在疾病现场合成药物和 设计具有新活性的小分子，包括反义或类似CRISPR的作用模式，为 为我们提出的研究计划提供了基础。 在此，我们提出了一种全面的策略来研究RNA重复序列扩张的分子识别 通过在原位和体内的小分子，使新的化学生物学框架得以建立靶向 利用小分子RNA和开发临床前候选药物。我们的研究涵盖了许多类型的 重复扩展，在序列和基因环境中都不同(内含子、非翻译区或开放阅读 帧)。我们设计了创新的策略：(I)利用RNA重复序列的结构来诱骗疾病- 使RNA合成自己的药物；(Ii)使小分子与自然的RNA衰变和QC途径相连接； 和(Iii)招募内源性核酸酶到具有小分子的重复序列中。我们不仅会提供证据- 概念小分子修复原位和活体疾病相关缺陷，但有新发现 关于如何用小分子给核糖核酸下药。因此，我们提议的工作将得到一个 R35奖项，因为这一奖项赋予的灵活性对于确保可持续的长期供资是真正必要的 以及开发一种新的代码所需的投资，该代码描述了分子与RNA在健康和疾病中的相互作用。

PROJECT SUMMARY: An extraordinarily challenging problem is to develop general methods to target defective or malfunctioning RNAs that cause disease selectively. Current therapeutic strategies to target RNAs are based on specific sequence recognition by oligonucleotides. However, many human disorders are caused by highly structured RNAs not readily targetable by conventional base pairing, in particular RNA repeat expansions that cause or contribute to >30 incurable neuromuscular diseases and genetically defined dementia. Thus, allele- specific ASOs modalities for these microsatellite disorders have been developed by targeting polymorphisms outside of the repeating sequence. The consequences of this approach are that only patients with the polymorphisms benefit from treatment and that an ASO has to be developed for each disease, even if caused by the same repeating sequence. If the toxin in these diseases, the expanded repeat, could be targeted selectively with a structure-specific small molecule, then a single modality could be a therapeutic or chemical probe for multiple diseases and for all patients. Over the past 14 years, we have shown that RNA structures can be targeted selectively with small molecules in situ and in vivo, more selectively than oligonucleotides. Indeed, we have designed compounds against many RNA repeat expansions that selectively recognize the target’s structure and rescue disease-associated pathobiology in situ and in vivo. Further, these chemical probes have elucidated new mechanisms of disease, including a previously unknown RNA-mediated transcriptional silencing pathway that operates in fragile X syndrome. These studies, along with our innovative strategies to synthesize drugs at the site of disease and to engineer small molecules with novel activities, including antisense- or CRISPR-like modes of action, lay the foundation for our proposed research program. Herein, we propose a comprehensive strategy to study the molecular recognition of RNA repeat expansions by small molecules in situ and in vivo, enabling the establishment of new chemical biology frameworks to target RNA using small molecules and the development of preclinical candidates. Our studies span many types of repeat expansions, differing in both sequence and gene contexts (intron, untranslated region, or open reading frame). We have devised innovative strategies to: (i) exploit the structures of RNA repeats to coax the disease- causing RNA to synthesize its own drug; (ii) interface small molecules with natural RNA decay and QC pathways; and (iii) recruit endogenous nuclease to the repeats with small molecules. We will not only deliver proof-of- concept small molecules that rescue disease-associated defects in situ and in vivo, but make new discoveries about how to drug RNA using small molecules. Our proposed work would therefore be well supported by an R35 award, as the flexibility conferred by this award is truly necessary to ensure sustainable, long-term funding and the investment required to develop a new code for how molecules interface with RNA in health and disease.