CRISPR/Cas9-Targeted Deletion of Polyglutamine in Spinocerebellar Ataxia Type 3-Derived Induced Pluripotent Stem Cells

AbstractSpinocerebellar 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.

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Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cells

PLoS ONE ◽

10.1371/journal.pone.0247434 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0247434

Author(s):

Richard J. Burman ◽

Lauren M. Watson ◽

Danielle C. Smith ◽

Joseph V. Raimondo ◽

Robea Ballo ◽

...

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

Induced Pluripotent Stem Cells ◽

Spinocerebellar Ataxia ◽

Pluripotent Stem Cells ◽

Spinocerebellar Ataxia Type ◽

Patient Specific ◽

Retinal Photoreceptors ◽

Spinocerebellar Ataxia Type 7 ◽

Induced Pluripotent

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the ATXN7 gene. Patients with this disease suffer from a degeneration of their cerebellar Purkinje neurons and retinal photoreceptors that result in a progressive ataxia and loss of vision. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated induced pluripotent stem cells (iPSCs) from a cohort of SCA7 patients in South Africa. First, we differentiated the SCA7 affected iPSCs into neurons which showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). We then performed electrophysiology on the SCA7 iPSC-derived neurons and found that these cells show features of functional aberrations. Lastly, we were able to differentiate the SCA7 iPSCs into retinal photoreceptors that also showed similar transcriptional aberrations to the SCA7 neurons. Our findings give technical insights on how iPSC-derived neurons and photoreceptors can be derived from SCA7 patients and demonstrate that these cells express molecular and electrophysiological differences that may be indicative of impaired neuronal health. We hope that these findings will contribute towards the ongoing efforts to establish the cell-derived models of neurodegenerative diseases that are needed to develop patient-specific treatments.

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Generation of Human-Induced Pluripotent Stem Cells to Model Spinocerebellar Ataxia Type 2 In vitro

Journal of Molecular Neuroscience ◽

10.1007/s12031-012-9930-2 ◽

2012 ◽

Vol 51 (2) ◽

pp. 237-248 ◽

Cited By ~ 30

Author(s):

Guangbin Xia ◽

Katherine Santostefano ◽

Takashi Hamazaki ◽

Jilin Liu ◽

S. H. Subramony ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Spinocerebellar Ataxia ◽

Pluripotent Stem Cells ◽

Spinocerebellar Ataxia Type ◽

Spinocerebellar Ataxia Type 2 ◽

Induced Pluripotent

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CRISPR/Cas9 Mediated Gene Modification Ameliorates Abnormal Phenotypes in SCA3/MJD Disease Patient Derived Induced Pluripotent Stem Cells

10.21203/rs.3.rs-88222/v1 ◽

2020 ◽

Author(s):

Lang He ◽

Shang Wang ◽

Huifang Zhao ◽

Shuai Li ◽

Xiaobo Han ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Patient ◽

Therapeutic Strategies ◽

Cag Repeats ◽

Spinocerebellar Ataxia Type ◽

Differentiation Markers ◽

Spinocerebellar Ataxia Type 3 ◽

Induced Pluripotent

Abstract Background：Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a progressive autosomal dominant neurodegenerative disease caused by abnormal CAG repeats in exon 10 of ATXN3. The accumulation of the mutant ataxin3 proteins carrying polyglutamine (polyQ) lead to selective degeneration of neurons. Therapeutic strategies were used to inhibit mutant ATXN3 expression, including antisense oligonucleotides, RNA interference and more recently CRISPR/Cas9 genome-editing based approaches. Since the pathogenesis of SCA3 has not been fully elucidated, and no effective therapies can be used, it is crucial to investigate the pathogenesis and seek new therapeutic strategies of SCA3/MJD. Methods: Here we used the paired sgRNA/Cas9 nickases and Cre-loxP mediated homologous recombination (HR) strategy to precisely modify the abnormal CAG expansions in the ATXN3 of SCA3/MJD patient derived induced pluripotent stem cells (SCA3/MJD-iPSCs). Meanwhile, we investigated the disease related phenotypes in differentiated neurons, including electrophysiological characteristics, IC2-positive aggregations, mitochondrial membrane potentials (MMPs), glutathione (GSH) expressions, intracellular reactive oxygen species (ROS), Ca2+ concentrations and malondialdehyde (MDA) levels. Results: SCA3/MJD-iPSCs can be corrected by the replacement of the abnormal CAG expansions with normal repeats using HR. Besides, corrected SCA3/MJD-iPSCs retained pluripotent and normal karyotype, which could be differentiated into neuron cells (NCs) and maintained electrophysiological characteristics. The expression of differentiation markers and electrophysiological characteristics were similar among the control individuals, SCA3/MJD patients and isogenic control SCA3/MJD groups. Furthermore, this study proved that the phenotypic abnormalities in SCA3/MJD-iPSCs derived NCs, including aggregated polyQ toxic protein, decreased MMPs and GSH expressions, increased ROS, Ca2+ concentrations and MDA levels, all were rescued in the corrected SCA3/MJD-NCs. Conclusion: The present study firstly suggested that the genetically corrected SCA3/MJD-iPSCs and associated phenotypic abnormalities, which will provide an ideal models for molecular mechanism research and autologous stem cell therapy.

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Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cells

10.1101/288480 ◽

2018 ◽

Author(s):

RJ Burman ◽

LM Watson ◽

DC Smith ◽

JV Raimondo ◽

R Ballo ◽

...

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

Induced Pluripotent Stem Cells ◽

Spinocerebellar Ataxia ◽

Pluripotent Stem Cells ◽

Spinocerebellar Ataxia Type ◽

Patient Specific ◽

Retinal Photoreceptors ◽

Spinocerebellar Ataxia Type 7 ◽

Induced Pluripotent

AbstractSpinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the ATXN7 gene. Patients with this disease suffer from a degeneration of their cerebellar Purkinje neurons and retinal photoreceptors that result in a progressive ataxia and loss of vision. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated induced pluripotent stem cells (iPSCs) from a cohort of SCA7 patients in South Africa. First, we differentiated the SCA7 affected iPSCs into neurons which showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). We then performed electrophysiology on the SCA7 iPSC-derived neurons and found that these cells show features of functional aberrations. Lastly, we were able to differentiate the SCA7 iPSCs into retinal photoreceptors that also showed similar transcriptional aberrations to the SCA7 neurons. Our findings demonstrate that iPSC-derived neurons and photoreceptors from SCA7 patients express molecular and electrophysiological differences that are indicative of impaired neuronal health. We hope that these findings will contribute towards the ongoing efforts to establish the cell-derived models of neurodegenerative diseases that are needed to develop patient-specific treatments.

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