PDF(Pubmed)
Abstract：
Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by 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 single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice, and rescue was mediated by FXN transfer from tissue engrafted, HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation, 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. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons 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. 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 ER structural damage 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 in FRDA, validate the efficacy profile of our FXN gene editing approach in a disease relevant model, and support our approach as an effective strategy for therapeutic intervention for Friedreich\'s ataxia.
摘要：
弗里德赖希共济失调(FRDA)是一种多系统的共济失调，共济失调蛋白（FXN）基因第一个内含子中的纯合GAA扩展突变引起的常染色体隐性遗传疾病。FXN是对铁-硫簇生物合成至关重要的线粒体蛋白，缺乏会损害线粒体电子传递链功能和细胞器内的铁稳态。目前,FRDA没有有效的治疗方法。我们以前已经证明，单次输注野生型造血干细胞和祖细胞（HSPCs）可预防YG8R小鼠的FRDA的神经和心脏并发症，拯救是由FXN从移植的组织转移介导的，HSPC衍生的小胶质细胞/巨噬细胞对患病的神经元/肌细胞。对于未来的临床翻译，我们开发了一种使用CRISPR/Cas9的自体干细胞移植方法,用于切除FRDA患者CD34+HSPCs中的GAA重复序列;该策略导致FXN表达增加和线粒体功能改善.当前研究的目的是验证我们的基因编辑方法在疾病相关模型中的效率和安全性。我们生成了一组FRDA患者来源的iPSC和等基因系，这些系使用我们的CRISPR/Cas9方法进行了基因编辑。iPSC来源的FRDA神经元表现出特征性的凋亡和线粒体表型的疾病，如神经突的非均质微管染色，caspase-3表达增加，线粒体超氧化物水平，线粒体片段化，与健康对照相比，cr的部分降解。这些缺陷在基因编辑的神经元中被完全预防。FRDA和基因编辑的神经元的RNASeq分析表明，等基因系中与内质网（ER）应激相关的基因簇有显着改善。基因编辑的神经元显示出改善的ER-钙释放，ER应激反应基因的正常化，XBP-1，并显着增加ER-线粒体接触，这对两个细胞器的功能稳态至关重要，与FRDA神经元相比。这些接触部位的超微结构分析显示FRDA神经元中严重的ER结构损伤，在基因编辑的神经元中没有发现。一起来看,这些结果代表了疾病发病机制的新发现，显示FRDA中明显的ER结构损伤，在疾病相关模型中验证我们的FXN基因编辑方法的功效概况，并支持我们的方法作为弗里德赖希共济失调治疗干预的有效策略。
