Elusive sources of variability of dystrophin rescue by exon skipping.
Elusive sources of variability of dystrophin rescue by exon skipping.
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DOI：
10.1186/s13395-015-0070-6
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发表时间：
2015
期刊：
影响因子：
4.9
通讯作者：
Nagaraju K
中科院分区：
文献类型：
--
作者：
Vila MC;Klimek MB;Novak JS;Rayavarapu S;Uaesoontrachoon K;Boehler JF;Fiorillo AA;Hogarth MW;Zhang A;Shaughnessy C;Gordish-Dressman H;Burki U;Straub V;Lu QL;Partridge TA;Brown KJ;Hathout Y;van den Anker J;Hoffman EP;Nagaraju K
将反义寡核苷酸全身递送至杜氏肌营养不良症（DMD）患者以诱导肌营养不良蛋白在肌肉中的从头表达（外显子跳跃）是一种有前途的治疗方法。用磷酰二胺吗啉代寡聚物（PMO）治疗导致在动物模型和缺乏肌营养不良蛋白的DMD男孩中的新生肌营养不良蛋白质更短;然而，已经观察到肌营养不良蛋白的恢复是高度可变的。了解导致临床前模型中肌养蛋白表达的高度可变诱导的因素可能会导致在临床前研究和人类临床试验中更有效的外显子跳跃手段。在本研究中，我们调查了可能导致在mdx小鼠模型中使用吗啉代药物的外显子跳跃可变成功的可能因素。我们测试了特定的肌肉群或纤维类型是否比其他肌肉群或纤维类型表现出更好的成功率，并将肌肉中残留的PMO浓度与单次高剂量吗啉代注射（800 mg/kg）后1个月的从头肌营养不良蛋白的量相关联。我们使用三种不同的抗肌萎缩蛋白定量方法比较了六个肌肉群的结果：免疫染色，免疫印迹和质谱分析。三头肌显示出最大程度的拯救（通过免疫染色平均38 ± 28%）。所有三种抗肌萎缩蛋白检测方法对于所有肌肉通常是一致的。我们发现，肌营养不良蛋白救援发生在一个零星的斑块模式，具有较高的地理变异性，在肌肉部分。我们没有发现肌肉组织中残留的吗啉药物与肌营养不良蛋白表达程度之间的相关性。虽然我们发现了一些肌肉群增强和成功拯救的证据，但我们的数据也表明，其他尚未确定的因素可能是外显子跳跃成功的变异性的基础。我们的研究强调了在临床试验中定量肌营养不良蛋白的挑战，其中单个小肌肉活检取自DMD患者。本文的在线版本（doi：10.1186/s13395 - 015 - 0070 - 6）包含补充材料，可供授权用户使用。
Systemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials. In the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays. The triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression. While we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient. The online version of this article (doi:10.1186/s13395-015-0070-6) contains supplementary material, which is available to authorized users.