Abstract：
Fukuyama congenital muscular dystrophy (FCMD) is an autosomal recessive disorder caused by fukutin (FKTN) gene mutations. FCMD is the second most common form of childhood muscular dystrophy in Japan, and the most patients possess a homozygous retrotransposal SINE-VNTR-Alu insertion in the 3\'-untranslated region of FKTN. A deep-intronic variant (DIV) was previously identified as the second most prevalent loss-of-function mutation in Japanese patients with FCMD. The DIV creates a new splicing donor site in intron 5 that causes aberrant splicing and the formation of a 64-base pair pseudoexon in the mature mRNA, resulting in a truncated nonfunctional protein. Patients with FCMD carrying the DIV present a more severe symptoms, and currently, there is no radical therapy available for this disorder. In the present study, we describe in vitro evaluation of antisense oligonucleotide mediated skipping of pseudoexon inclusion and restoration of functional FKTN protein. A total of 16 19-26-mer antisense oligonucleotide sequences were designed with a 2\'-O-methyl backbone and were screened in patient-derived fibroblasts, lymphoblast cells and minigene splice assays. One antisense oligonucleotide targeting the exonic splice enhancer region significantly induced pseudoexon skipping and increased the expression of normal mRNA. It also rescued FKTN protein production in lymphoblast cells and restored functional O-mannosyl glycosylation of alpha-dystroglycan in patient-derived myotubes. Based on our results, antisense oligonucleotide-based splicing correction should be investigated further as a potential treatment for patients with FCMD carrying the DIV.
One Sentence Summary Antisense oligonucleotide treatment restored normal FKTN protein production and functional O-mannosyl glycosylation of alpha-dystroglycan via pseudoexon skipping in patient-derived cells carrying the compound heterozygous deep-intronic variant of Fukuyama muscular dystrophy.
One Sentence Summary Antisense oligonucleotide treatment restored normal FKTN protein production and functional O-mannosyl glycosylation of alpha-dystroglycan via pseudoexon skipping in patient-derived cells carrying the compound heterozygous deep-intronic variant of Fukuyama muscular dystrophy.
摘要：
福山先天性肌营养不良症（FCMD）是由福库丁（FKTN）基因突变引起的常染色体隐性遗传疾病。FCMD是日本儿童肌肉萎缩症的第二常见形式，大多数患者在FKTN的3'-非翻译区具有纯合逆转录SINE-VNTR-Alu插入。深度内含子变体（DIV）先前被确定为日本FCMD患者中第二普遍的功能丧失突变。DIV在内含子5中创建了一个新的剪接供体位点，导致成熟mRNA中的异常剪接和64个碱基对假外显子的形成，导致截短的无功能蛋白质。携带DIV的FCMD患者表现出更严重的症状，目前,目前尚无针对这种疾病的根治疗法。在本研究中,我们描述了反义寡核苷酸介导的假外显子包涵体的跳跃和功能性FKTN蛋白的恢复的体外评估。总共设计了16个19-26聚体反义寡核苷酸序列，具有2'-O-甲基骨架，并在患者来源的成纤维细胞中进行了筛选，淋巴母细胞,和小基因剪接分析。一种靶向外显子剪接增强子区的反义寡核苷酸显着诱导假外显子跳跃并增加正常mRNA的表达。它还挽救了淋巴母细胞中FKTN蛋白的产生，并恢复了患者来源的肌管中α-肌聚糖的功能性O-甘露糖糖基化。根据我们的结果,应进一步研究基于ASO的剪接校正作为携带DIV的FCMD患者的潜在治疗方法。
