Glycogen storage disease type II (GSDII) is a lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme, leading to the accumulation of glycogen within the lysosomes. The disease has been classified in infantile and late-onset forms. Most late-onset patients share a splicing mutation c.-32-13T > G in intron 1 of the GAA gene that prevents efficient recognition of exon 2 by the spliceosome. In this study, we have mapped the splicing silencers of GAA exon 2 and developed antisense morpholino oligonucleotides (AMOs) to inhibit those regions and rescue normal splicing in the presence of the c.-32-13T > G mutation. Using a minigene approach and patient fibroblasts, we successfully increased inclusion of exon 2 in the mRNA and GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, the use of these AMOs in patient myotubes results in a decreased accumulation of glycogen. To our knowledge, this is the only therapeutic approach resulting in a decrease of glycogen accumulation in patient tissues beside enzyme replacement therapy (ERT) and TFEB overexpression. As a result, it may represent a highly novel and promising therapeutic line for GSDII.

译文

:II型糖原贮积病(GSDII)是一种溶酶体疾病,由酸性α-葡萄糖苷酶(GAA)酶的活性不足引起,导致糖原在溶酶体内积累。该疾病已被分类为婴儿期和晚期发作形式。大多数晚期发病的患者在GAA基因内含子1中共有一个剪接突变c.-32-13T> G,该剪接突变阻止剪接体有效识别外显子2。在这项研究中,我们已经绘制了GAA外显子2的剪接沉默基因,并开发了反义吗啉代寡核苷酸(AMO)以抑制这些区域并在存在c.-32-13T> G突变的情况下拯救正常剪接。通过使用小基因方法和患者成纤维细胞,我们通过结合AMO靶向特定的沉默子,成功增加了外显子2在mRNA和GAA酶生产中的含量。最重要的是,在患者的肌管中使用这些AMO会导致糖原积累减少。据我们所知,这是唯一一种减少酶替代疗法(ERT)和TFEB过表达的导致患者组织中糖原积累减少的治疗方法。结果,它可能代表了GSDII的一种非常新颖和有希望的治疗方法。

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