The Center for Gene Therapy, Nationwide Children's Hospital, The Ohio State University, Columbus, OH, USA.
Perron Institute for Neurological and Translational Science, Center for Comparative Genomics, Murdoch University, Perth, Australia.
J Neuromuscul Dis. 2017;4(3):199-207. doi: 10.3233/JND-170233.
Exon skipping strategies in Duchenne muscular dystrophy (DMD) have largely been directed toward altering splicing of exons flanking out-of-frame deletions, with the goal of restoring an open mRNA reading frame that leads to production of an internally deleted but partially functional dystrophin protein.
We sought to apply exon skipping to duplication mutations, assuming that the inherently limited efficiency of antisense oligonucleotide-induced exon skipping would more frequently skip a single copy of a duplicated exon, rather than both and result in significant amounts of wild-type DMD mRNA.
We tested this hypothesis in fibroblast cell lines derived from patients with a variety of single or multiple exon duplications that have been modified to allow transdifferentiation into a myogenic lineage.
Using a variety of 2'O-methyl antisense oligonucleotides, significant skipping was induced for each duplication leading to a wild-type transcript as a major mRNA product.
This study provides another proof of concept for the feasibility of therapeutic skipping in patients carrying exon duplications in order to express wild-type, full-length mRNA, although careful evaluation of the skipping efficiency should be performed as some exons are easier to skip than others. Such a personalized strategy is expected to be highly beneficial for this subset of DMD patients, compared to inducing expression of an internally-deleted dystrophin.
Duchenne 肌营养不良症（DMD）中的外显子跳跃策略主要针对改变框架外缺失的侧翼外显子的剪接，目的是恢复开放的 mRNA 阅读框，从而产生内部缺失但部分功能的肌营养不良蛋白。
我们试图将外显子跳跃应用于重复突变，假设反义寡核苷酸诱导的外显子跳跃的固有效率有限，更有可能跳过重复外显子的单个拷贝，而不是两个拷贝，并导致大量野生型 DMD mRNA。
我们在源自具有各种单或多外显子重复的患者的成纤维细胞系中测试了这一假设，这些重复已被修改以允许向肌源性谱系转分化。
使用各种 2'O-甲基反义寡核苷酸，每个重复都能诱导显著的跳跃，从而产生野生型转录本作为主要的 mRNA 产物。
本研究为在携带外显子重复的患者中进行治疗性跳跃以表达野生型全长 mRNA 的可行性提供了另一个概念验证，尽管应仔细评估跳跃效率，因为有些外显子比其他外显子更容易跳跃。与诱导内部缺失的肌营养不良蛋白表达相比，这种个性化策略预计对这组 DMD 患者非常有益。
