Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.
Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.
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DOI：
10.1242/dmm.027367
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发表时间：
2017-04-01
影响因子：
4.3
通讯作者：
Furling D
中科院分区：
文献类型：
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作者：
Arandel L;Polay Espinoza M;Matloka M;Bazinet A;De Dea Diniz D;Naouar N;Rau F;Jollet A;Edom-Vovard F;Mamchaoui K;Tarnopolsky M;Puymirat J;Battail C;Boland A;Deleuze JF;Mouly V;Klein AF;Furling D
强直性肌营养不良1型（DM 1）和2型（DM 2）是由微卫星扩增引起的常染色体显性遗传神经肌肉疾病，属于RNA显性遗传疾病家族。DM突变在其自然环境中表达的细胞模型的可用性对于促进鉴定新治疗化合物的努力是至关重要的。在这里，我们产生了永生化的DM 1和DM 2人类肌肉细胞系，其显示了扩展重复序列的核RNA聚集体，这是强直性肌营养不良的标志。与对照和DM 2细胞相比，DM 1和DM 2永生化成肌细胞的选定克隆表现为亲本原代成肌细胞，具有永生化DM 1成肌细胞的融合能力降低。在分化的DM 1肌细胞系中观察到选择性剪接缺陷，但在DM 2系中没有观察到。剪接改变并没有导致分化延迟，因为在永生化的DM 1转分化成纤维细胞中发现了类似的变化，其中肌原性分化已被MYOD 1的过表达所迫。作为概念验证，我们表明反义方法减轻了疾病相关的缺陷，并且RNA-seq分析证实了永生化DM 1肌细胞中绝大多数错误剪接事件受到反义治疗的影响，其中一半在治疗的DM 1细胞中得到了显著拯救。显示出特征性疾病相关分子特征如核RNA聚集体和剪接缺陷的永生化DM 1肌细胞系可用作筛选治疗性化合物的稳健读数。因此，永生化的DM 1和DM 2肌细胞系代表了研究分子病理生理学机制和评价化合物对与强直性肌营养不良突变相关的RNA毒性的体外作用的新模型和工具。总结：强直性肌营养不良的肌肉细胞模型显示特征性疾病相关的分子特征，可用于研究分子病理生理机制和评估治疗方法。
Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. Summary: Myotonic dystrophy muscle cell models displaying characteristic disease-associated molecular features can be used to investigate molecular pathophysiological mechanisms and evaluate therapeutic approaches.