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

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.

Advances in Modeling Polyglutamine Diseases Using Genome Editing Tools

“…It is vital to exclude interference caused by different genetics and epigenetics [129] as subtle differences in DNA may influence somatic instability or disease onset [130]. Isogenic models of polyQ disease were generated by targeted HR [131] and genome-editing tools, mainly CRISPR-Cas9 technology [130,[132][133][134][135][136][137][138][139].…”

“…Genome-wide sequencing and proteomics assays revealed transcriptional differences between both CAG lengths and cell types [63]. Increased MDA levels [133] Aa-amino acids, AAV-adeno-associated virus; BDNF-brain-derived neurotrophic factor; CE-capillary electrophoresis; ER-endoplasmic reticulum; FC-flow cytometry; FISH-fluorescence in situ hybridization; H&E-hematoxylin and eosin; IHC-immunohistochemistry; MDA-malondialdehyde; NSCs-neural stem cells; RANrepeat-associated non-AUG translation; RT-qPCR-reverse transcription-quantitative polymerase chain reaction; SCNT-somatic cell nuclear transfer; T7E1-T7 endonuclease I; TGF-β1-transforming growth factor β1; WB-western blot, WT-wild type.…”

“…CRISPR-Cas9 technology is used to introduce or repair a mutant allele by inducing homologous recombination (HR), as well as to excise a mutant DNA sequence. In either case, single-molecule (one-hit method) [62,130,[134][135][136][137]146] or two-molecule (two-hit method) [130,132,133,[137][138][139]147,148,151] gRNA may be used. The use of modified Streptococcus pyogenes Cas9 (SpCas9) nucleases further enhances the modeling possibilities of polyQ diseases.…”

“…An et al were first to correct iPSCs derived from HD patient fibroblasts by using HR, creating the basis and guideline for further research in this field [131]. iPSC lines were subsequently corrected using CRISPR-Cas9 technology to generate isogenic HD [130,139], SCA2 [134][135][136] and SCA3 models [132,133].…”

“…Two SCA3 iPSC models with corrected CAG repeats in exon 10 of the ATXN3 gene have been created so far [132,133]. Ouyang et al (2018) excised the CAG repeat tract by using CRISPR-Cas9 technology with paired gRNAs to promote the production of a truncated ATXN3 protein without the toxic polyQ domain [132].…”