Spinocerebellar ataxia type 3/Machado–Joseph disease (SCA3/MJD) is a progressive autosomal dominant neurodegenerative disease caused by abnormal CAG repeats in the exon 10 of ATXN3. The accumulation of the mutant ataxin-3 proteins carrying expanded polyglutamine (polyQ) leads to selective degeneration of neurons. Since the pathogenesis of SCA3 has not been fully elucidated, and no effective therapies have been identified, it is crucial to investigate the pathogenesis and seek new therapeutic strategies of SCA3. Induced pluripotent stem cells (iPSCs) can be used as the ideal cell model for the molecular pathogenesis of polyQ diseases. Abnormal CAG expansions mediated by CRISPR/Cas9 genome engineering technologies have shown promising potential for the treatment of polyQ diseases, including SCA3. In this study, SCA3-iPSCs can be corrected by the replacement of the abnormal CAG expansions (74 CAG) with normal repeats (17 CAG) using CRISPR/Cas9-mediated homologous recombination (HR) strategy. Besides, corrected SCA3-iPSCs retained pluripotent and normal karyotype, which can be differentiated into a neural stem cell (NSCs) and neuronal cells, and maintained electrophysiological characteristics. The expression of differentiation markers and electrophysiological characteristics were similar among the neuronal differentiation from normal control iPSCs (Ctrl-iPSCs), SCA3-iPSCs, and isogenic control SCA3-iPSCs. Furthermore, this study proved that the phenotypic abnormalities in SCA3 neurons, including aggregated IC2-polyQ protein, decreased mitochondrial membrane potential (MMP) and glutathione expressions, increased reactive oxygen species (ROS), intracellular Ca²⁺ concentrations, and lipid peroxidase malondialdehyde (MDA) levels, all were rescued in the corrected SCA3-NCs. For the first time, this study demonstrated the feasibility of CRISPR/Cas9-mediated HR strategy to precisely repair SCA3-iPSCs, and reverse the corresponding abnormal disease phenotypes. In addition, the importance of genetic control using CRISPR/Cas9-mediated iPSCs for disease modeling. Our work may contribute to providing a potential ideal model for molecular mechanism research and autologous stem cell therapy of SCA3 or other polyQ diseases, and offer a good gene therapy strategy for future treatment.

脊髓小脑性共济失调 3 型/马查多-约瑟夫病 (SCA3/MJD) 是一种进行性常染色体显性神经退行性疾病，由ATXN3外显子 10 中的异常 CAG 重复引起. 携带扩增的聚谷氨酰胺 (polyQ) 的突变体 ataxin-3 蛋白的积累导致神经元的选择性退化。由于SCA3的发病机制尚未完全阐明，也没有确定有效的治疗方法，因此研究SCA3的发病机制并寻求新的治疗策略至关重要。诱导多能干细胞 (iPSC) 可用作 polyQ 疾病分子发病机制的理想细胞模型。由 CRISPR/Cas9 基因组工程技术介导的异常 CAG 扩增已显示出治疗包括 SCA3 在内的 polyQ 疾病的潜力。在这项研究中，SCA3-iPSCs 可以通过使用 CRISPR/Cas9 介导的同源重组 (HR) 策略用正常重复 (17 CAG) 替换异常 CAG 扩增 (74 CAG) 来纠正。除了，校正后的 SCA3-iPSCs 保留了多能性和正常核型，可分化为神经干细胞 (NSCs) 和神经元细胞，并保持电生理特征。从正常对照 iPSC (Ctrl-iPSC)、SCA3-iPSC 和同基因对照 SCA3-iPSC 的神经元分化中，分化标志物的表达和电生理特征相似。此外，该研究证明 SCA3 神经元的表型异常，包括聚集的 IC2-polyQ 蛋白、线粒体膜电位 (MMP) 和谷胱甘肽表达降低、活性氧 (ROS) 增加、细胞内 Ca 从正常对照 iPSC (Ctrl-iPSC)、SCA3-iPSC 和同基因对照 SCA3-iPSC 的神经元分化中，分化标志物的表达和电生理特征相似。此外，该研究证明 SCA3 神经元的表型异常，包括聚集的 IC2-polyQ 蛋白、线粒体膜电位 (MMP) 和谷胱甘肽表达降低、活性氧 (ROS) 增加、细胞内 Ca 正常对照 iPSC (Ctrl-iPSC)、SCA3-iPSC 和同基因对照 SCA3-iPSC 的神经元分化之间的分化标志物和电生理特征的表达相似。此外，该研究证明 SCA3 神经元的表型异常，包括聚集的 IC2-polyQ 蛋白、线粒体膜电位 (MMP) 和谷胱甘肽表达降低、活性氧 (ROS) 增加、细胞内 Ca²⁺浓度和脂质过氧化物酶丙二醛 (MDA) 水平都在校正的 SCA3-NC 中得到拯救。本研究首次证明了CRISPR/Cas9介导的HR策略在精准修复SCA3-iPSCs，逆转相应异常疾病表型方面的可行性。此外，使用 CRISPR/Cas9 介导的 iPSC 进行疾病建模的遗传控制的重要性。我们的工作可能有助于为 SCA3 或其他 polyQ 疾病的分子机制研究和自体干细胞治疗提供潜在的理想模型，并为未来的治疗提供良好的基因治疗策略。