van Deutekom J C, Bremmer-Bout M, Janson A A, Ginjaar I B, Baas F, den Dunnen J T, van Ommen G J
Department of Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
Hum Mol Genet. 2001 Jul 15;10(15):1547-54. doi: 10.1093/hmg/10.15.1547.
Due to frame-shifting mutations in the DMD gene that cause dystrophin deficiency, Duchenne muscular dystrophy (DMD) patients suffer from lethal muscle degeneration. In contrast, mutations in the allelic Becker muscular dystrophy (BMD) do not disrupt the translational reading frame, resulting in a less severe phenotype. In this study, we explored a genetic therapy aimed at restoring the reading frame in muscle cells from DMD patients through targeted modulation of dystrophin pre-mRNA splicing. Considering that exon 45 is the single most frequently deleted exon in DMD, whereas exon (45+46) deletions cause only a mild form of BMD, we set up an antisense-based system to induce exon 46 skipping from the transcript in cultured myotubes of both mouse and human origin. In myotube cultures from two unrelated DMD patients carrying an exon 45 deletion, the induced skipping of exon 46 in only approximately 15% of the mRNA led to normal amounts of properly localized dystrophin in at least 75% of myotubes. Our results provide first evidence of highly effective restoration of dystrophin expression from the endogenous gene in DMD patient-derived muscle cells. This strategy may be applicable to not only >65% of DMD mutations, but also many other genetic diseases.
由于DMD基因中的移码突变导致肌营养不良蛋白缺乏，杜兴氏肌营养不良症（DMD）患者会遭受致命的肌肉退化。相比之下，等位基因贝克尔肌营养不良症（BMD）中的突变不会破坏翻译阅读框，从而导致较轻的表型。在本研究中，我们探索了一种基因疗法，旨在通过对肌营养不良蛋白前体mRNA剪接的靶向调控来恢复DMD患者肌肉细胞中的阅读框。鉴于外显子45是DMD中最常缺失的单个外显子，而外显子（45 + 46）缺失仅导致轻度形式的BMD，我们建立了一个基于反义的系统，以诱导来自小鼠和人类来源的培养肌管中转录本中外显子46的跳跃。在来自两名携带外显子45缺失的无关DMD患者的肌管培养物中，仅约15%的mRNA中诱导的外显子46跳跃导致至少75%的肌管中出现正常量的正确定位的肌营养不良蛋白。我们的结果首次证明了在DMD患者来源的肌肉细胞中从内源基因高效恢复肌营养不良蛋白表达。这种策略不仅可能适用于>65%的DMD突变，也可能适用于许多其他遗传疾病。
