Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.
Methods Mol Biol. 2023;2640:313-325. doi: 10.1007/978-1-0716-3036-5_22.
Antisense oligonucleotides (AOs) have demonstrated high potential as a therapy for treating genetic diseases like Duchene muscular dystrophy (DMD). As a synthetic nucleic acid, AOs can bind to a targeted messenger RNA (mRNA) and regulate splicing. AO-mediated exon skipping transforms out-of-frame mutations as seen in DMD into in-frame transcripts. This exon skipping approach results in the production of a shortened but still functional protein product as seen in the milder counterpart, Becker muscular dystrophy (BMD). Many potential AO drugs have advanced from laboratory experimentation to clinical trials with an increasing interest in this area. An accurate and efficient method for testing AO drug candidates in vitro, before implementation in clinical trials, is crucial to ensure proper assessment of efficacy. The type of cell model used to examine AO drugs in vitro establishes the foundation of the screening process and can significantly impact the results. Previous cell models used to screen for potential AO drug candidates, such as primary muscle cell lines, have limited proliferative and differentiation capacity, and express insufficient amounts of dystrophin. Recently developed immortalized DMD muscle cell lines effectively addressed this challenge allowing for the accurate measurement of exon-skipping efficacy and dystrophin protein production. This chapter presents a procedure used to assess DMD exons 45-55 skipping efficiency and dystrophin protein production in immortalized DMD patient-derived muscle cells. Exons 45-55 skipping in the DMD gene is potentially applicable to 47% of patients. In addition, naturally occurring exons 45-55 in-frame deletion mutation is associated with an asymptomatic or remarkably mild phenotype as compared to shorter in-frame deletions within this region. As such, exons 45-55 skipping is a promising therapeutic approach to treat a wider group of DMD patients. The method presented here allows for improved examination of potential AO drugs before implementation in clinical trials for DMD.
反义寡核苷酸 (AOs) 已被证明具有治疗杜氏肌营养不良症 (DMD) 等遗传疾病的巨大潜力。作为一种合成核酸，AOs 可以与靶向信使 RNA (mRNA) 结合并调节剪接。AO 介导的外显子跳跃将 DMD 中所见的无义突变转化为框架内转录本。这种外显子跳跃方法导致产生缩短但仍具有功能的蛋白质产物，如在较轻的贝克肌营养不良症 (BMD) 中所见。许多潜在的 AO 药物已经从实验室实验推进到临床试验，人们对这一领域的兴趣日益增加。在临床试验中实施之前，在体外对 AO 药物候选物进行准确和高效的测试是至关重要的，以确保对疗效进行适当评估。在体外检查 AO 药物的细胞模型的类型为筛选过程奠定了基础，并可能对结果产生重大影响。以前用于筛选潜在 AO 药物候选物的细胞模型，如原代肌肉细胞系，增殖和分化能力有限，并且表达的肌营养不良蛋白量不足。最近开发的永生化 DMD 肌肉细胞系有效地解决了这一挑战，允许准确测量外显子跳跃效率和肌营养不良蛋白的产生。本章介绍了一种用于评估永生化 DMD 患者来源肌肉细胞中外显子 45-55 跳跃效率和肌营养不良蛋白产生的程序。DMD 基因中外显子 45-55 的跳跃在 47%的患者中是潜在适用的。此外，与该区域内较短的框架内缺失突变相比，DMD 基因中外显子 45-55 的自然发生的框架内缺失突变与无症状或明显较轻的表型相关。因此，外显子 45-55 跳跃是一种有前途的治疗方法，可以治疗更广泛的 DMD 患者群体。这里提出的方法允许在 DMD 的临床试验中实施之前，对潜在的 AO 药物进行改进检查。
