Precise small molecule cleavage of a r(CUG) repeat expansion in a myotonic dystrophy mouse model

Myotonic dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by an expanded CTG repeat that is transcribed into r(CUG)exp. The RNA repeat expansion sequesters regulatory proteins such as Muscleblind-like protein 1 (MBNL1), which causes pre-mRNA splicing defects. The disease-causing r(CUG)exp has been targeted by antisense oligonucleotides, CRISPR-based approaches, and RNA-targeting small molecules. Herein, we describe a designer small molecule, Cugamycin, that targets the structure of r(CUG)exp and cleaves it in cells and in vivo. Cugamycin selectively cleaves r(CUG)exp while leaving short repeats of r(CUG) untouched. In contrast, oligonucleotides that recognize r(CUG) sequence rather than structure cleave both long and short r(CUG)-containing transcripts. In the HSALR mouse model of DM1, Cugamycin selectively ablates r(CUG)exp in disease-affected muscle. Transcriptomic, histological, and phenotypic studies demonstrate that Cugamycin broadly and specifically relieves DM1-associated defects without detectable off-targets. Thus, small molecules that bind and cleave RNA may have utility as lead chemical probes and medicines. Overall design: RNA-Seq was perfomed on tibialis anterior muscle from HSALR and WT mice treated with PBS or small molecule drug.

1型强直性肌营养不良（Myotonic dystrophy type 1, DM1）是一种由扩增CTG重复序列转录生成r(CUG)exp引发的无法治愈的神经肌肉疾病。该RNA重复扩增片段会结合并隔离肌肉盲样蛋白1（Muscleblind-like protein 1, MBNL1）等调控蛋白，进而引发前体mRNA剪接缺陷。目前，研究人员已通过反义寡核苷酸、基于CRISPR的技术以及靶向RNA的小分子化合物，针对致病的r(CUG)exp展开靶向干预。本文报道了一种经设计合成的小分子化合物Cugamycin，它可靶向识别r(CUG)exp的二级结构，并在细胞及活体环境中对其进行切割。Cugamycin能够选择性切割长链r(CUG)exp，而对短链r(CUG)重复序列无作用。与之相反，仅识别r(CUG)序列而非其结构的寡核苷酸，会同时切割含长、短r(CUG)的转录本。在DM1的HSALR小鼠模型中，Cugamycin可选择性清除病变受累肌肉中的r(CUG)exp。转录组学、组织学及表型研究证实，Cugamycin可广泛且特异性地缓解DM1相关的病理缺陷，且未检测到脱靶效应。由此可见，能够结合并切割RNA的小分子化合物有望成为优质化学探针与候选药物。
整体实验设计：对经磷酸盐缓冲液（PBS）或小分子药物处理的HSALR模型小鼠与野生型（Wild Type, WT）小鼠的胫骨前肌进行RNA测序（RNA-Seq）实验。