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

强直性肌营养不良1型(DM1)，由3‘非翻译区CTG扩张(CTGexp)引起 DMPK基因，已被用作RNA介导的与其他疾病相关的疾病机制的模型 微卫星扩张性疾病，包括脆性X震颤/共济失调综合征(FXTAS)和C9orf72 肌萎缩侧索硬化症和额颞叶痴呆(C9-ALS/FTD)。在DM1中，转录的 CTGexp突变导致CUGexp RNA改变前mRNA加工的发育调节 以及由MBNL和CELF家族的RNA结合蛋白介导的mRNA定位事件。然而， 其他细胞途径，如miRNA加工和重复相关的非AUG翻译，也 与DM1的发病机制有牵连。最重要的是，目前还没有有效的治疗方法。 神经肌肉疾病。为了解决这些不足，这个项目旨在产生更多的信息 为阐明小鼠DM1实验模型的相对贡献，提出了 RNA剪接缺陷作为治疗反应的生物标志物的病理机制和资格 开发有效的治疗方法以减少CUGexp RNA的毒性负担的目标。目标1构建 根据我们最近开发的DMPK CTGexp敲门小鼠，使用滚动圆组合产生 体外扩增产生大重复序列和CRISPR/Cas9介导的基因组修饰。使用 一系列CTG重复长度增加的等位基因，代表了DM1的晚发型到先天性谱 致病范围，我们将确定CTG对骨骼和心肌的长度依赖效应 结构/功能、RNA加工/本地化/周转和RAN翻译。转录组分析将是 在目标2中进一步探讨，这是基于我们之前观察到的患者功能障碍 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活性和治疗干预作出反应的有效生物标志物。在AIM 3、我们将扩大这种治疗范围，并评估包括反义寡核苷酸在内的多种策略 (ASO)介导的CUGexp基因敲除和小分子途径抑制突变的DMPK转录 CTGexp基因。该项目的总体目标是为DM领域提供更健壮的老鼠模型 同时也评估剪接缺陷作为疾病状态的生物标记物，并发展成单个小的 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