Preclinical models, biomarkers, and therapy for myotonic dystrophy type 1

强直性肌营养不良1型（DM 1），由3'非翻译区CTG扩增（CTGexp）引起 DMPK基因，已被用作RNA介导的疾病机制的模型，与其他疾病相关。 微卫星扩增疾病，包括脆性X震颤/共济失调综合征（FXTAS）和C9 orf 72 肌萎缩侧索硬化症和额颞叶痴呆症（C9-ALS/FTD）。在DM 1中， CTGexp突变导致CUGexp RNA改变前mRNA加工的发育调节 以及由RNA结合蛋白的MBNL和CELF家族介导的mRNA定位事件。然而，在这方面， 另外的细胞途径，如miRNA加工和重复相关的非AUG翻译，也 与DM 1发病机制有关。最重要的是，没有有效的治疗方法来治疗这种情况。 神经肌肉疾病为了解决这些缺陷，该项目旨在产生更多的信息 DM 1小鼠实验模型，以阐明每种建议的相对贡献， 病理机制，并将RNA剪接缺陷作为治疗反应的反应性生物标志物， 目标是开发有效的治疗方法以降低CUGexp RNA的毒性负担。Aim 1构建 在我们最近开发的Dmpk CTGexp敲入小鼠中，使用滚环结合 在一些实施方案中，CRISPR/Cas9介导的基因组修饰包括扩增以在体外产生大重复序列和CRISPR/Cas9介导的基因组修饰。使用 CTG重复长度增加的等位基因系列，代表DM 1的晚发型至先天性谱 致病范围，我们将确定CTG对骨骼和心肌的长度依赖性影响 结构/功能，RNA加工/定位/周转和RAN翻译。转录组分析将是 在目标2中进一步追求，这是基于我们先前的观察，即患者功能障碍 对应于RNA剪接缺陷和MBNL功能丧失，以确定剪接缺陷是否符合 对CUGexp水平、MBNL活性和治疗干预有反应的有效生物标志物。在Aim中 3、我们将扩大治疗范围，评估多种策略，包括反义寡核苷酸， （阿索）介导的CUGexp敲低和抑制突变体Dmpk转录的小分子方法 CTGexp基因。该项目的总体目标是为DM领域提供更稳健的小鼠模型 同时还评估剪接缺陷作为疾病状态的生物标志物， 分子策略

Myotonic dystrophy type 1 (DM1), which is caused by CTG expansions (CTGexp) in the 3' untranslated region of the DMPK gene, has been used as a model for RNA-mediated disease mechanisms associated with other microsatellite expansion diseases, including fragile X tremor/ataxia syndrome (FXTAS) and C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). In DM1, transcription of the CTGexp mutation results in CUGexp RNAs that alter the developmental regulation of pre-mRNA processing and mRNA localization events mediated by the MBNL and CELF families of RNA binding proteins. However, additional cellular pathways, such as miRNA processing and repeat-associated non-AUG translation, have also been implicated in DM1 pathogenesis. Most importantly, no effective therapies exist to treat this neuromuscular disease. To address these deficiencies, this project is designed to generate more informative mouse experimental models for DM1 to elucidate the relative contribution of each of the proposed pathomechanisms and qualify RNA splicing defects as responsive biomarkers of therapeutic response with the goal of developing effective therapeutic approaches to decrease the toxic burden of CUGexp RNAs. Aim 1 builds upon our recent development of Dmpk CTGexp knockin mice generated using a combination of rolling circle amplification to generate large repeats in vitro and CRISPR/Cas9-mediated genome modification. Using an allelic series of increasing CTG repeat lengths that represent the late-onset to congenital spectrum of the DM1 pathogenic range, we will determine CTG length-dependent effects on skleletal and heart muscle structure/function, RNA processing/localization/turnover and RAN translation. Transcriptome analysis will be pursued further in Aim 2, which is based upon our prior observations that patient functional impairment corresponds to RNA splicing defects and MBNL loss of function, to determine if splicing defects qualify as effective biomarkers that are responsive to CUGexp levels, MBNL activity and therapeutic intervention. In Aim 3, we will broaden this therapeutic scope and evaluate multiple strategies, including antisense oligonucleotide (ASO)-mediated CUGexp knockdown and small molecule approaches to inhibit transcription of mutant Dmpk CTGexp genes. The overall objective of this project is to provide the DM field with more robust mouse models of DM1 while also evaluating splicing defects as biomarkers of disease status and developing single small molecule strategies