RNA-seq data for edited and unedited GAA repeats in FRDA patient iPSC-derived neurons

Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by a homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that a single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in the prevention of the neurologic and cardiac complications of FRDA in YG8R mice, and the rescue was mediated by FXN transfer from tissue engrafted HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation of this approach, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients' CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions in the cells. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. To this end, we generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. FRDA neurons generated from these iPSCs displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. These results were validated by molecular and functional studies, and gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe structural damage to the ER in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage and function in FRDA. In addition, these results validate the efficacy profile of our FXN gene editing approach, in a disease relevant model improving mitochondrial and cellular, and supporting our approach as an effective strategy for therapeutic intervention for Friedreich's ataxia. Overall design: To investigate the normalization effect of the excision of the GAA repeats in FRDA patient neurons, we generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. We then performed gene expression profiling analysis using data obtained from RNA-seq of 5 cell lines for both edited and unedited GAA repeats in FRDA patient iPSC-derived neurons at 2 weeks of age. Comparative gene expression profiling analysis of RNA-seq data for edited and unedited GAA repeats in FRDA patient iPSC-derived neurons was performed.

弗里德赖希共济失调（Friedreich ataxia, FRDA）是一类多系统受累的常染色体隐性遗传病，致病根源为frataxin（FXN）基因第1内含子发生纯合GAA扩增突变。FXN是参与铁硫簇生物合成的关键线粒体蛋白，其表达缺失会损伤线粒体电子传递链功能，并破坏细胞器内的铁稳态。目前，针对FRDA尚无获批的有效治疗手段。
我们此前的研究证实，单次输注野生型造血干细胞与祖细胞（hematopoietic stem and progenitor cells, HSPCs）可预防YG8R小鼠出现FRDA相关的神经与心脏并发症，该保护效应由组织定植的HSPC来源小胶质细胞/巨噬细胞，向病变神经元/肌细胞传递FXN所介导。为推动该疗法向临床转化，我们开发了一套自体干细胞移植策略：利用CRISPR/Cas9技术敲除FRDA患者CD34阳性造血干细胞与祖细胞中的GAA重复序列，该策略可提升细胞内FXN的表达水平，并改善线粒体功能。
本研究旨在于疾病相关模型中，验证我们的基因编辑策略的有效性与安全性。为此，我们构建了一组FRDA患者来源的诱导多能干细胞（induced pluripotent stem cells, iPSCs）及其同基因编辑株，均采用我们的CRISPR/Cas9策略完成基因编辑。由上述iPSCs诱导生成的FRDA神经元呈现该病特征性的凋亡与线粒体表型：与健康对照组相比，其神经突内微管染色不均一、半胱天冬酶-3（caspase-3）表达上调、线粒体超氧化物水平升高、线粒体碎片化及线粒体嵴部分降解。经基因编辑的神经元则完全消除了上述病理缺陷。
对FRDA神经元与基因编辑神经元的RNA测序（RNA-seq）分析显示，同基因编辑株中与内质网（endoplasmic reticulum, ER）应激相关的基因簇表达得到显著改善。上述结果经分子与功能实验验证：与FRDA神经元相比，基因编辑神经元的内质网-钙离子释放功能得到改善、内质网应激应答基因XBP-1恢复至正常水平，且内质网与线粒体间的接触位点显著增多——该位点对两种细胞器的功能稳态至关重要。对上述接触位点的超微结构分析显示，FRDA神经元的内质网存在严重结构损伤，而基因编辑神经元中未观察到此病理改变。
综上，本研究揭示了FRDA发病机制中内质网结构与功能严重受损这一新的病理特征。此外，本研究于疾病相关模型中证实了我们的FXN基因编辑策略的有效性，该策略可改善线粒体与细胞功能，支持其成为治疗弗里德赖希共济失调的有效临床干预手段。
研究设计：为探究敲除GAA重复序列对FRDA患者神经元的归一化调控效应，我们构建了一组FRDA患者来源的诱导多能干细胞及其同基因编辑株，均采用CRISPR/Cas9策略完成基因编辑。随后，我们针对体外培养2周的FRDA患者iPSC诱导神经元，对5株分别携带编辑后与未编辑GAA重复序列的细胞系的RNA-seq数据开展基因表达谱分析。本研究完成了FRDA患者iPSC诱导神经元中，编辑与未编辑GAA重复序列的RNA-seq数据对比基因表达谱分析。