CRISPR/Cas9 mediated gene correction ameliorates abnormal phenotypes in spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cells

Lang He,Zhiyuan Li,Hongyu Yuan,Shuai Li,Hongxia Peng ,Jean De Dieu Habimana,Qing Deng ,Guangdong Zou,Yue Xie,Yun Peng,Linliu Peng,Zhao Chen,Shang Wang,Huifang Zhao,Na Wan,Yiqing Gong,Linlin Wan,Chunrong Wang,Xiaobo Han,Beisha Tang,Lijing Lei,Hong Jiang

doi:10.1038/s41398-021-01605-2

Abstract

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.

Highlights

Spinocerebellar ataxia type 3/Machado–Joseph disease (SCA3/ MJD) is the most common subtype of spinocerebellar ataxias (SCAs), accounting for about 60–70% of SCAs in the Chinese population [1,2,3]
We further demonstrated that a series of abnormal phenotypes in SCA3-NCs, including IC2-polyQ aggregations, decreased the levels of membrane potential (MMP) and GSH, H2O2induced oxidative stress activation, and significantly increased reactive oxygen species (ROS), Ca2+, and MDA levels, above phenomenons all are rescued in isogenic control SCA3 neurons
In summary, this study proved for the first time that SCA3-Induced pluripotent stem cells (iPSCs) could be accurately repaired by using the paired sgRNAs/Cas9 nickase (Cas9n) and Cre-loxP-mediated homologous recombination (HR) strategy, and genetically repaired SCA3-iPSCs did not have the expression of the mutant ataxin-3 protein

Summary

Introduction

Spinocerebellar ataxia type 3/Machado–Joseph disease (SCA3/ MJD) is the most common subtype of spinocerebellar ataxias (SCAs), accounting for about 60–70% of SCAs in the Chinese population [1,2,3]. The pathogenesis of SCA3 is caused by the abnormal CAG repeats in the encoding region of ATXN3. CAG expansions result in abnormal polyglutamine (polyQ) tract in the encoded ataxin-3 protein, forming neuronal intranuclear inclusions (NIIs) selectively accumulated in the specific regions of the nervous system (cerebral cortex, cerebellum, brain stem and spinal cord, etc.) [7]. This disease is one of the representative diseases of polyQ diseases. Previous studies on neuropathology have been conducted in animal models of polyQ diseases, but they cannot fully simulate all aspects of human neuronal degeneration. It is very important to select a disease model that effectively simulates neuronopathy. humaninduced pluripotent stem cells (hiPSCs) carry the entire genetic background of patients, which have the potential to proliferate

Results

Discussion

Conclusion