GAA deficiency in Pompe disease is alleviated by exon inclusion in iPS cell-derived skeletal muscle cells

While there are many human skeletal muscle disorders, very few therapies have been developed. It has not been possible to generate large amounts of purified skeletal muscle cells from pluripotent stem cells, and to test therapies quantitatively. We therefore devised conditions for generating and expanding purified human myogenic progenitors from induced pluripotent stem (iPS) cells. The progenitors retained the capacity to differentiate into multinucleated myotubes and showed a normal karyotype throughout the expansion phase. We applied this method to Pompe disease, a metabolic myopathy caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). In a screen, we identified sequences that suppressed aberrant GAA exon 2 splicing caused by the frequent c.-32-13T>G (IVS1) GAA variant. Antisense oligonucleotides (AONs) that blocked these sequences promoted exon 2 inclusion in patient-derived myotubes. As this raised GAA enzymatic activity above the disease threshold, AON-mediated splicing correction may provide a treatment option for Pompe disease. RNA samples to be analyzed by microarrays were prepared using RNeasy columns with on-column DNA digestion (Qiagen). 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto HumanHT-12 v4 expression BeadChips (Illumina) following the manufacturer's instructions. After recommended washing, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data postprocessing and graphics was performed with in-house developed functions in Matlab. Hierarchical clustering of genes and samples was performed with one minus correlation metric and the unweighted average distance (UPGMA) (also known as group average) linkage method. 6 samples were analyzed: F134, Human fibroblast line F134, 1 replicate 237iPS, Human induced reprogrammed cells (iPSCs) from patient 1, 1 replicate 75iPS, Human induced reprogrammed cells (iPSCs) from patient 2, 1 replicate 1981iPS, Human induced reprogrammed cells (iPSCs) from patient 1, 1 replicate H1, Human Embryonic Stem Cell (ESC) H1, 1 replicate H9, Human Embryonic Stem Cell (ESC) H9, 1 replicate

尽管人类骨骼肌疾病谱广泛，目前可及的治疗方案却寥寥无几。此前学界尚未实现从多能干细胞中规模化制备纯化骨骼肌细胞，亦无法开展定量的治疗学评估。为此，本研究建立了从诱导多能干细胞（induced pluripotent stem cells，iPSCs）中制备并扩增纯化人类肌源性祖细胞的实验体系。该祖细胞可稳定分化为多核肌管，且在整个扩增过程中均保持正常核型。

本研究将该体系应用于庞贝病——一种因溶酶体酶酸性α-葡萄糖苷酶（acid alpha-glucosidase，GAA）缺乏引发的代谢性肌病。通过筛选实验，本研究鉴定出可抑制由常见c.-32-13T>G（IVS1）GAA突变引发的GAA基因2号外显子异常剪接的核酸序列。靶向阻断上述序列的反义寡核苷酸（antisense oligonucleotides，AONs）可促进患者来源肌管中GAA基因2号外显子的保留。经此处理后，GAA酶活性提升至疾病阈值以上，表明反义寡核苷酸介导的剪接校正有望成为庞贝病的潜在治疗方案。

用于芯片分析的RNA样品采用带有柱上DNA消化步骤的RNeasy柱（Qiagen）制备。每份样品取300 ng总RNA作为模板，采用Ambion的线性扩增体系进行扩增：首先合成带有T7启动子序列的双链cDNA，随后进行12小时的体外转录，掺入生物素标记的核苷酸。纯化并标记后的cRNA按照制造商说明书，在HumanHT-12 v4表达微珠芯片（Illumina）上进行18小时的杂交反应。按照推荐流程完成洗涤后，采用链霉亲和素-Cy3（GE Healthcare）对芯片进行染色，随后使用Illumina iScan扫描仪及其配套软件完成扫描。

所有样品均以生物学重复的形式进行杂交。采用BeadStudio 3.2（Illumina）将微珠荧光强度信号注释为基因表达信息。背景校正采用Affymetrix稳健多阵列分析（Affymetrix Robust Multi-array Analysis，RMA）模型进行。方差稳定化处理采用log2对数缩放，基因表达标准化则通过R-Bioconductor的lumi包实现。数据后处理及可视化绘图采用Matlab自研函数完成。基因与样品的层次聚类分析采用1-相关系数作为距离度量，以未加权平均距离（unweighted pair group method with arithmetic mean，UPGMA，亦称为组平均连接法）作为聚类连接方法。

本研究共分析6组样品：
1. F134：人类成纤维细胞系F134，1次生物学重复
2. 237iPS：患者1来源的诱导多能干细胞（iPSCs），1次生物学重复
3. 75iPS：患者2来源的诱导多能干细胞（iPSCs），1次生物学重复
4. 1981iPS：患者1来源的诱导多能干细胞（iPSCs），1次生物学重复
5. H1：人类胚胎干细胞（Embryonic Stem Cell，ESC）系H1，1次生物学重复
6. H9：人类胚胎干细胞（ESC）系H9，1次生物学重复