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

糖原贮积症II型 (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,导致溶酶体内糖原积累。该疾病已分为婴儿期和迟发性。大多数迟发性患者在GAA基因的内含子1中共享剪接突变c.-32-13T> G,从而阻止了剪接体对外显子2的有效识别。在这项研究中,我们绘制了GAA外显子2的剪接消音器,并开发了反义吗啉代寡核苷酸 (AMOs),以抑制这些区域并在c.-32-13T> G突变存在下挽救正常剪接。使用小基因方法和患者成纤维细胞,我们通过将特定的消音器与AMOs组合靶向,成功地增加了外显子2在mRNA和GAA酶产生中的包含。最重要的是,在患者肌管中使用这些AMOs会导致糖原积累减少。据我们所知,这是除酶替代疗法 (ERT) 和TFEB过表达外,导致患者组织中糖原积累减少的唯一治疗方法。因此,它可能代表了GSDII的一种非常新颖和有前途的治疗方法。

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