GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells.
GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells.
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DOI：
10.1016/j.omtn.2017.03.002
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发表时间：
2017-06-16
期刊：
影响因子：
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通讯作者：
Pijnappel WWMP
中科院分区：
文献类型：
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作者：
van der Wal E;Bergsma AJ;van Gestel TJM;In 't Groen SLM;Zaehres H;Araúzo-Bravo MJ;Schöler HR;van der Ploeg AT;Pijnappel WWMP
庞贝氏症是一种由酸性α-葡萄糖苷酶（GAA）缺乏引起的代谢性肌病，导致骨骼肌细胞进行性萎缩。C. 32- 13 T>G（IVS 1）GAA变体在前体mRNA剪接期间促进外显子2跳跃，并且是儿童/成人疾病形式的最常见变体。我们以前确定的反义寡核苷酸（AON），促进GAA外显子2纳入患者来源的成纤维细胞。目前尚不清楚这些AON如何影响骨骼肌细胞中的GAA剪接。为了测试这一点，我们扩增了诱导多能干细胞（iPSC）衍生的肌源性祖细胞，并将其分化为多核肌管。AON在肌管中恢复剪接的程度与成纤维细胞相似，这表明它们通过调节共享剪接调节因子的作用来起作用。AON靶向一个隐蔽的剪接受体位点的推定的多聚嘧啶道，该位点是GAA内含子1中的假外显子的一部分。用AON阻断假外显子的隐蔽剪接供体同样促进了GAA外显子2的包含。同时阻断隐蔽受体和隐蔽供体位点恢复了大部分典型剪接，并减轻了GAA酶缺乏。这些结果突出了隐蔽剪接在人类疾病中的相关性及其作为使用AON进行剪接调节的治疗靶点的潜力。
Pompe disease is a metabolic myopathy caused by deficiency of the acid α-glucosidase (GAA) enzyme and results in progressive wasting of skeletal muscle cells. The c.-32-13T>G (IVS1) GAA variant promotes exon 2 skipping during pre-mRNA splicing and is the most common variant for the childhood/adult disease form. We previously identified antisense oligonucleotides (AONs) that promoted GAA exon 2 inclusion in patient-derived fibroblasts. It was unknown how these AONs would affect GAA splicing in skeletal muscle cells. To test this, we expanded induced pluripotent stem cell (iPSC)-derived myogenic progenitors and differentiated these to multinucleated myotubes. AONs restored splicing in myotubes to a similar extent as in fibroblasts, suggesting that they act by modulating the action of shared splicing regulators. AONs targeted the putative polypyrimidine tract of a cryptic splice acceptor site that was part of a pseudo exon in GAA intron 1. Blocking of the cryptic splice donor of the pseudo exon with AONs likewise promoted GAA exon 2 inclusion. The simultaneous blocking of the cryptic acceptor and cryptic donor sites restored the majority of canonical splicing and alleviated GAA enzyme deficiency. These results highlight the relevance of cryptic splicing in human disease and its potential as therapeutic target for splicing modulation using AONs.