Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.
Proc Natl Acad Sci U S A. 2012 Aug 21;109(34):13763-8. doi: 10.1073/pnas.1204638109. Epub 2012 Aug 6.
Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.
杜氏肌营养不良症（DMD）是最常见的肌肉营养不良症，是由缺乏肌营养不良蛋白引起的。最有前途的治疗方法之一是通过外显子跳跃来消除框架破坏突变的反义介导。然而，这种方法面临两个主要障碍：每个靶外显子的应用有限，以及每个产生的截断肌营养不良蛋白的功能和稳定性不确定。在 DMD 基因突变热点跳过外显子 45-55 可以解决这两个问题。从理论上讲，它可以拯救超过 60%的缺失突变患者。此外，该特定区域的自发缺失与无症状或异常轻微的表型相关。然而，这种多个外显子 45-55 的跳跃已被证明在技术上具有挑战性。因此，我们设计了反义寡核苷酸（AO）混合物来最小化自或异双链体的形成。这些被测试为与穿透细胞部分（活体-吗啉代）的缀合物。我们已经测试了在 H2K-mdx52 肌管和缺乏外显子 52 的 mdx52 小鼠中跳过外显子 45-55 的可行性。令人鼓舞的是，我们使用 10 种 AO 的混合物，在体外、H2K-mdx52 肌管和肌肉内注射到 mdx52 小鼠中证明了所有 10 个外显子的跳跃。此外，在体内的 mdx52 小鼠中，10 种 AO 的全身注射在骨骼肌的亚肌膜中诱导广泛的肌营养不良蛋白表达，产生高达野生型肌营养不良蛋白水平的 15%，同时肌肉力量和组织病理学得到改善，没有任何可检测的毒性。这是在肌营养不良蛋白缺乏的动物模型中通过外显子 45-55 跳跃有效拯救的独特成功证明。
