Nakano Shiho, Ozasa Shiro, Yoshioka Kowashi, Fujii Isao, Mitsui Kouichi, Nomura Keiko, Kosuge Hirofumi, Endo Fumio, Matsukura Makoto, Kimura Shigemi
Department of Child Development Pediatrics, Kumamoto University Graduate School, Kumamoto, Japan.
Pediatr Int. 2011 Aug;53(4):524-9. doi: 10.1111/j.1442-200X.2011.03330.x.
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out-of-frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in-frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino-mediated exon skipping, which changes out-of-frame deletions to in-frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon-skipping event in cells of DMD patients before the clinical trial.
Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon-skipping event was analyzed on reverse transcription-polymerase chain reaction.
Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51-skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon-skipping patterns, which are total or partial exon 51-skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out-of-frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51-skipping event.
An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.
杜氏肌营养不良症（DMD）和贝克肌营养不良症（BMD）由DMD基因异常引起。大多数DMD患者存在框外缺失，这会破坏阅读框；而一些DMD病例由重复或无义突变引起。大多数BMD患者存在框内缺失，可保留阅读框。BMD的表型通常比DMD轻。反义吗啉代介导的外显子跳跃可将框外缺失转变为框内缺失，是一种有前景的DMD治疗方法。然而，在临床试验之前，有必要在DMD患者的细胞中确认外显子跳跃事件。
从4名DMD患者分离出的成纤维细胞通过感染Ad.CAGMyoD诱导分化为肌源性谱系。然后用两种吗啉代转染细胞。通过逆转录聚合酶链反应分析外显子跳跃事件。
位于DMD基因外显子51剪接增强子处的吗啉代B30在所有测试细胞缺失模式中均产生了所需的外显子51跳跃事件。位于外显子供体处的吗啉代I25诱导了两种不同的外显子跳跃模式，即外显子51完全或部分跳跃事件。根据序列分析，意外未跳跃区域是外显子51的95 bp片段和188 bp片段，表明使用I25时新产生了隐蔽剪接供体。不幸的是，这些隐蔽剪接供体产生了框外模式。基于这些体外结果，B30可能是一种有效的治疗方法。有趣的是，B30和I25的组合似乎产生了更有效的外显子51跳跃事件。
开发了一种体外系统，可轻松筛选反义序列的有效性并鉴定吗啉代治疗的良好候选物。
