Mitrpant Chalermchai, Fletcher Sue, Iversen Patrick L, Wilton Steve D
Centre for Neuromuscular and Neurological Disorders, University of Western Australia, QE II Medical Centre, Nedlands, Australia.
J Gene Med. 2009 Jan;11(1):46-56. doi: 10.1002/jgm.1265.
Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein-truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg-Dmd(mdx-4Cv)/J (4(CV)) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame.
A series of 2'-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels.
Compared to the original mdx mouse studies, induction of exon skipping from the 4(CV) dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near-full length dystrophin protein in cultured 4(CV) myogenic cells and in vivo, after a single intramuscular injection.
By-passing the protein-truncating mutation in the 4(CV) mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo.
杜兴氏肌营养不良症（DMD）是一种严重的神经肌肉疾病，由肌营养不良蛋白基因中的蛋白质截短突变引起。功能性肌营养不良蛋白的缺失使肌纤维更容易受到损伤和坏死。我们报告了反义寡聚物（AO）诱导B6Ros.Cg-Dmd(mdx-4Cv)/J（4(CV)）小鼠外显子跳跃，该小鼠是一种因肌营养不良蛋白外显子53中的无义突变而产生的肌营养不良模型。必须切除外显子52和53才能去除突变并维持阅读框。
设计了一系列基于硫代磷酸酯骨架的2'-O-甲基修饰寡聚物（2OMeAO），并评估其对每个外显子的去除效果，然后将最有效的化合物组合起来，在成肌细胞培养物和体内诱导双外显子跳跃。在RNA和蛋白质水平上，在体外和体内评估了2OMeAO和磷酰胺吗啉代寡聚物（PMO）的外显子跳跃效率。
与最初的mdx小鼠研究相比，从4(CV)肌营养不良蛋白mRNA诱导外显子跳跃要困难得多。单次肌肉注射后，PMO混合物可以在培养的4(CV)成肌细胞和体内恢复近全长肌营养不良蛋白的合成。
在4(CV)肌营养不良小鼠模型中，仅用靶向两个外显子的单个寡聚物无法绕过蛋白质截短突变，只有在应用AO混合物去除外显子52和53后才能实现。与之前的研究一样，在体内持续稳定地诱导蛋白质方面，PMO的稳定性和效率优于2OMeAO。
