Highly efficient in vivo delivery of PMO into regenerating myotubes and rescue in laminin-α2 chain-null congenital muscular dystrophy mice
Highly efficient in vivo delivery of PMO into regenerating myotubes and rescue in laminin-α2 chain-null congenital muscular dystrophy mice
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DOI：
10.1093/hmg/ddt341
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发表时间：
2013-12-15
影响因子：
3.5
通讯作者：
Takeda, Shin'ichi
中科院分区：
文献类型：
--
作者：
Aoki, Yoshitsugu;Nagata, Tetsuya;Takeda, Shin'ichi
磷酰二胺吗啉寡聚体（PMO）介导的外显子跳跃是治疗包括杜氏肌营养不良症在内的几种神经肌肉疾病的最有前途的方法之一。这种方法的主要弱点来自于将电荷中性PMO递送到肌纤维中的低效率和零星性质，其机制尚不清楚。在这项研究中，为了验证我们的假设，肌纤维采取PMO更有效地在肌管形成过程中，我们诱导同步肌肉再生注射心脏毒素到胫骨前肌的Dmd外显子52缺陷mdx 52和野生型小鼠。有趣的是，通过原位杂交，我们检测到PMO主要在胚胎肌球蛋白重链阳性再生纤维。此外，我们表明，PMO或2 '-O-甲基硫代磷酸酯在诱导分化后24-72小时转染时被有效地摄取到C2 C12肌管中，但被不好地摄取到未分化的C2 C12成肌细胞中，表明在C2 C12肌管形成的早期阶段PMO的有效摄取。接下来，我们测试了PMO对层粘连蛋白-α 2链缺失dy（3 K）/dy（3 K）小鼠的治疗潜力：具有主动肌肉再生的缺裂蛋白先天性肌营养不良症（MDC 1A）模型。我们证实了在dy（3 K）/dy（3 K）小鼠中跳跃突变的外显子4后，层粘连蛋白-α 2链的恢复和寿命的略微延长。这些发现支持了PMO进入纤维依赖于肌发生的发育阶段而不是无肌营养不良蛋白的肌肉膜的观点，并为开发PMO介导的治疗各种肌肉疾病（如MDC 1A）提供了一个平台，这些肌肉疾病涉及主动肌肉再生。
Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches to the treatment of several neuromuscular disorders including Duchenne muscular dystrophy. The main weakness of this approach arises from the low efficiency and sporadic nature of the delivery of charge-neutral PMO into muscle fibers, the mechanism of which is unknown. In this study, to test our hypothesis that muscle fibers take up PMO more efficiently during myotube formation, we induced synchronous muscle regeneration by injection of cardiotoxin into the tibialis anterior muscle of Dmd exon 52-deficient mdx52 and wild-type mice. Interestingly, by in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. In addition, we showed that PMO or 2'-O-methyl phosphorothioate is taken up efficiently into C2C12 myotubes when transfected 24-72 h after the induction of differentiation but is poorly taken up into undifferentiated C2C12 myoblasts suggesting efficient uptake of PMO in the early stages of C2C12 myotube formation. Next, we tested the therapeutic potential of PMO for laminin-alpha 2 chain-null dy(3K)/dy(3K) mice: a model of merosin-deficient congenital muscular dystrophy (MDC1A) with active muscle regeneration. We confirmed the recovery of laminin-alpha 2 chain and slightly prolonged life span following skipping of the mutated exon 4 in dy(3K)/dy(3K) mice. These findings support the idea that PMO entry into fibers is dependent on a developmental stage in myogenesis rather than on dystrophinless muscle membranes and provide a platform for developing PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration.