Molecular biological study to establish the treatment for Duchenne muscular dystrophy

杜氏肌营养不良症（DMD）是一种快速进展的疾病，通常在20岁左右导致死亡，而贝克尔肌营养不良症（BMD）是一种临床上不太严重的疾病，通常只有轻微的衰弱作用。DMD和BMD是由肌营养不良蛋白基因突变引起的等位基因疾病，该基因由79个外显子组成。在三分之二的DMD/BMD病例中发现了缺失突变，DMD或BMD患者的临床进展可以通过缺失是否维持或破坏mRNA的翻译阅读框来预测。基因治疗作为一种可行的治疗DMD的方法正引起越来越多的关注。而用阅读框置换来纠正读框移位对翻译的影响是其中一种方法。在这项研究中，我们首次报道了肌营养不良蛋白基因转录本的外显子跳跃可以通过与外显子识别序列（ERS）互补的反义寡脱氧核苷酸（ODN）在活细胞中发生。对抗肌营养不良蛋白外显子19富含嘌呤区域具有反义ODN的淋巴母细胞样细胞进行孵育，导致抗肌营养不良蛋白转录本的外显子跳过。第19外显子在孵育6小时后开始出现跳变，孵育24小时后出现完全跳变。其他78个抗肌营养不良蛋白外显子均未被跳过，第19外显子的跳过既不能由正ODN引起，也不能由另一个ERS对应的反义ODN引起。这些结果表明，即使在活细胞中，针对ERS的反义ODN也能诱导外显子跳变。这为建立DMD基因疗法铺平了道路。

Duchenne muscular dystrophy (DMD) is a rapid progressive disease which usually results in death at around the age of 20, while Becker muscular dystrophy (BMD) is a clinically less severe form of the disease which often has only slight debilitating effects. DMD and BMD are allelic diseases caused by mutations of the dystrophin gene, which consists of 79 exons. Deletion mutations have been identified in two thirds of DMD/BMD cases, and the clinical progression of DMD or BMD patients can be predicted from whether or not the deletion maintains or disrupts the translational reading frame of the mRNA.Gene therapy is attracting increasing attention as a plausible treatment for DMD.And reading frame displacement to correct the effects of frame-shift deletions on translation is one of approaches.In this study we first report that exon skipping from the dystrophin gene transcript could in living cells by an antisense oligodeoxynucleotide (ODN) complementary to an exon recognition sequence (ERS). Incubation of lymphoblastoid cells with an antisense ODN against the purine-rich region of dystrophin exon 19 resulted in skipping of the exon from the dystrophin transcript. Skipping of exon 19 started to appear after 6 hours of incubation, and complete skipping was observed after 24 hours of incubation. None of the other 78 dystrophin exons were skipped, and exon 19 skipping could not be induced by the sense ODN or by an antisense ODN corresponding to another ERS.These results showed that antisense ODN against ERS induces exon skipping even in living cells. This paved the road to establish gene therapy for DMD.