Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neuroscience Programme, UCL Great Ormond Street Institute of Child Health, London, United Kingdom.
Department of Neurodegenerative Diseases, UCL Institute of Neurology, London, United Kingdom.
PLoS One. 2018 Mar 26;13(3):e0194540. doi: 10.1371/journal.pone.0194540. eCollection 2018.
Clinical trials using strategies aimed at inducing dystrophin expression in Duchenne muscular dystrophy (DMD) are underway or at advanced planning stage, including splice switching antisense oligonucleotides (AON), drugs to induce read-through of nonsense mutations and viral mediated gene therapy. In all these strategies, different dystrophin proteins, often internally deleted, are produced, similar to those found in patients with the milder DMD allelic variant, Becker muscular dystrophy (BMD). The primary biological endpoint of these trials is to induce functional dystrophin expression. A reliable and reproducible method for quantification of dystrophin protein expression at the sarcolemma is crucial to monitor the biochemical outcome of such treatments. We developed a new high throughput semi quantitative fluorescent immunofluorescence method for quantifying dystrophin expression in transverse sections of skeletal muscle. This technique is completely operator independent as it based on an automated scanning system and an image processing script developed with Definiens software. We applied this new acquisition-analysis method to quantify dystrophin and sarcolemma-related proteins using paediatric control muscles from cases without a neuromuscular disorder as well as DMD and BMD samples. The image analysis script was instructed to recognize myofibres immunostained for spectrin or laminin while dystrophin was quantified in each identified myofibre (from 2,000 to over 20,000 fibres, depending on the size of the biopsy). We were able to simultaneously extrapolate relevant parameters such as mean sarcolemmal dystrophin, mean spectrin and laminin intensity, fibre area and diameter. In this way we assessed dystrophin production in each muscle fibre in samples of DMD, BMD and controls. This new method allows the unbiased quantification of dystrophin in every myofibre within a transverse muscle section and will be of help for translational research projects as a biological outcome in clinical trials in DMD and BMD.
正在进行或处于高级规划阶段的临床试验使用旨在诱导杜氏肌营养不良症（DMD）中肌营养不良蛋白表达的策略，包括剪接转换反义寡核苷酸（AON）、诱导无义突变通读的药物和病毒介导的基因治疗。在所有这些策略中，都会产生不同的肌营养不良蛋白，通常是内部缺失的，类似于在较轻的 DMD 等位变体 Becker 肌营养不良症（BMD）患者中发现的那些。这些试验的主要生物学终点是诱导功能性肌营养不良蛋白表达。一种可靠且可重复的方法来定量肌细胞膜上的肌营养不良蛋白表达对于监测此类治疗的生化结果至关重要。我们开发了一种新的高通量半定量荧光免疫荧光方法来定量骨骼肌横切面上的肌营养不良蛋白表达。该技术完全不依赖于操作人员，因为它基于自动扫描系统和使用 Definiens 软件开发的图像处理脚本。我们应用这种新的获取-分析方法来定量儿科对照组肌肉中的肌营养不良蛋白和与肌膜相关的蛋白，这些肌肉来自没有神经肌肉疾病的病例以及 DMD 和 BMD 样本。图像分析脚本被指示识别免疫染色 spectrin 或 laminin 的肌纤维，同时在每个识别的肌纤维中定量肌营养不良蛋白（取决于活检的大小，从 2000 到 20000 多个纤维不等）。我们能够同时推断出相关参数，如平均肌膜肌营养不良蛋白、平均 spectrin 和 laminin 强度、纤维面积和直径。通过这种方式，我们评估了 DMD、BMD 和对照组样本中每个肌纤维的肌营养不良蛋白产生情况。这种新方法允许在横切肌肉切片中的每根肌纤维中进行无偏置的肌营养不良蛋白定量，并将有助于转化研究项目，作为 DMD 和 BMD 临床试验中的生物学结果。
