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van der Lei MB, Kooy RF. From Discovery to Innovative Translational Approaches in 80 Years of Fragile X Syndrome Research. Biomedicines 2025; 13:805. [PMID: 40299377 PMCID: PMC12024745 DOI: 10.3390/biomedicines13040805] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2025] [Revised: 03/21/2025] [Accepted: 03/25/2025] [Indexed: 04/30/2025] Open
Abstract
Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and a major genetic contributor to autism spectrum disorder. It is caused by a CGG trinucleotide repeat expansion in the FMR1 gene, resulting in gene silencing and the loss of FMRP, an RNA-binding protein essential for synaptic plasticity. This review covers over 80 years of FXS research, highlighting key milestones, clinical features, genetic and molecular mechanisms, the FXS mouse model, disrupted molecular pathways, and current therapeutic strategies. Additionally, we discuss recent advances including AI-driven combination therapies, CRISPR-based gene editing, and antisense oligonucleotides (ASOs) therapies. Despite these scientific breakthroughs, translating preclinical findings into effective clinical treatments remains challenging. Clinical trials have faced several difficulties, including patient heterogeneity, inconsistent outcome measures, and variable therapeutic responses. Standardized preclinical testing protocols and refined clinical trial designs are required to overcome these challenges. The development of FXS-specific biomarkers could also improve the precision of treatment assessments. Ultimately, future therapies will need to combine pharmacological and behavioral interventions tailored to individual needs. While significant challenges remain, ongoing research continues to offer hope for transformative breakthroughs that could significantly improve the quality of life for individuals with FXS and their families.
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Affiliation(s)
| | - R. Frank Kooy
- Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, 2650 Edegem, Belgium;
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2
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Azeez SS, Hamad RS, Hamad BK, Shekha MS, Bergsten P. Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine. Front Genome Ed 2024; 6:1509924. [PMID: 39726634 PMCID: PMC11669675 DOI: 10.3389/fgeed.2024.1509924] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Accepted: 11/28/2024] [Indexed: 12/28/2024] Open
Abstract
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.
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Affiliation(s)
- Sarkar Sardar Azeez
- Department of Medical Laboratory Technology, Soran Technical College, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq
| | - Rahin Shareef Hamad
- Nursing Department, Soran Technical College, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq
| | - Bahra Kakamin Hamad
- Department of Medical Laboratory Technology, Erbil Health and Medical Technical College, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq
| | - Mudhir Sabir Shekha
- Department of Biology, College of Science, Salahaddin University, Erbil, Kurdistan Region, Iraq
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Peter Bergsten
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
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3
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Sledzinski P, Nowaczyk M, Smielowska MI, Olejniczak M. CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair. BMC Biol 2024; 22:282. [PMID: 39627841 PMCID: PMC11616332 DOI: 10.1186/s12915-024-02079-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2024] [Accepted: 11/22/2024] [Indexed: 12/06/2024] Open
Abstract
BACKGROUND The expansion of CAG/CTG repeats in functionally unrelated genes is a causative factor in many inherited neurodegenerative disorders, including Huntington's disease (HD), spinocerebellar ataxias (SCAs), and myotonic dystrophy type 1 (DM1). Despite many years of research, the mechanism responsible for repeat instability is unknown, and recent findings indicate the key role of DNA repair in this process. The repair of DSBs induced by genome editing tools results in the shortening of long CAG/CTG repeats in yeast models. Understanding this mechanism is the first step in developing a therapeutic strategy based on the controlled shortening of repeats. The aim of this study was to characterize Cas9-induced DSB repair products at the endogenous HTT locus in human cells and to identify factors affecting the formation of specific types of sequences. RESULTS The location of the cleavage site and the surrounding sequence influence the outcome of DNA repair. DSBs within CAG repeats result in shortening of the repeats in frame in ~ 90% of products. The mechanism of this contraction involves MRE11-CTIP and RAD51 activity and DNA end resection. We demonstrated that a DSB located upstream of CAG repeats induces polymerase theta-mediated end joining, resulting in deletion of the entire CAG tract. Furthermore, using proteomic analysis, we identified novel factors that may be involved in CAG sequence repair. CONCLUSIONS Our study provides new insights into the complex mechanisms of CRISPR/Cas9-induced shortening of CAG repeats in human cells.
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Affiliation(s)
- Pawel Sledzinski
- Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704, Poznan, Poland
| | - Mateusz Nowaczyk
- Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704, Poznan, Poland
| | - Marianna Iga Smielowska
- Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704, Poznan, Poland
| | - Marta Olejniczak
- Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704, Poznan, Poland.
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4
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Zhang Y, Liu X, Li Z, Li H, Miao Z, Wan B, Xu X. Advances on the Mechanisms and Therapeutic Strategies in Non-coding CGG Repeat Expansion Diseases. Mol Neurobiol 2024; 61:10722-10735. [PMID: 38780719 DOI: 10.1007/s12035-024-04239-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2023] [Accepted: 05/02/2024] [Indexed: 05/25/2024]
Abstract
Non-coding CGG repeat expansions within the 5' untranslated region are implicated in a range of neurological disorders, including fragile X-associated tremor/ataxia syndrome, oculopharyngeal myopathy with leukodystrophy, and oculopharyngodistal myopathy. This review outlined the general characteristics of diseases associated with non-coding CGG repeat expansions, detailing their clinical manifestations and neuroimaging patterns, which often overlap and indicate shared pathophysiological traits. We summarized the underlying molecular mechanisms of these disorders, providing new insights into the roles that DNA, RNA, and toxic proteins play. Understanding these mechanisms is crucial for the development of targeted therapeutic strategies. These strategies include a range of approaches, such as antisense oligonucleotides, RNA interference, genomic DNA editing, small molecule interventions, and other treatments aimed at correcting the dysregulated processes inherent in these disorders. A deeper understanding of the shared mechanisms among non-coding CGG repeat expansion disorders may hold the potential to catalyze the development of innovative therapies, ultimately offering relief to individuals grappling with these debilitating neurological conditions.
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Affiliation(s)
- Yutong Zhang
- Departments of Neurology, The First Affiliated Hospital of Soochow University, Suzhou City, China
| | - Xuan Liu
- Departments of Neurology, The First Affiliated Hospital of Soochow University, Suzhou City, China
| | - Zeheng Li
- Departments of Neurology, The First Affiliated Hospital of Soochow University, Suzhou City, China
| | - Hao Li
- Departments of Neurology, The First Affiliated Hospital of Soochow University, Suzhou City, China
- Department of Neurology, The Fourth Affiliated Hospital of Soochow University, Suzhou, 215124, China
| | - Zhigang Miao
- The Institute of Neuroscience, Soochow University, Suzhou City, China
| | - Bo Wan
- The Institute of Neuroscience, Soochow University, Suzhou City, China
| | - Xingshun Xu
- Departments of Neurology, The First Affiliated Hospital of Soochow University, Suzhou City, China.
- The Institute of Neuroscience, Soochow University, Suzhou City, China.
- Department of Neurology, The First Affiliated Hospital of Soochow University, Suzhou, 215000, China.
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5
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Broniarek I, Niewiadomska D, Sobczak K. Contribution of DNA/RNA Structures Formed by Expanded CGG/CCG Repeats Within the FMR1 Locus in the Pathogenesis of Fragile X-Associated Disorders. WILEY INTERDISCIPLINARY REVIEWS. RNA 2024; 15:e1874. [PMID: 39523485 DOI: 10.1002/wrna.1874] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/14/2024] [Revised: 09/05/2024] [Accepted: 09/09/2024] [Indexed: 11/16/2024]
Abstract
Repeat expansion disorders (REDs) encompass over 50 inherited neurological disorders and are characterized by the expansion of short tandem nucleotide repeats beyond a specific repeat length. Particularly intriguing among these are multiple fragile X-associated disorders (FXds), which arise from an expansion of CGG repeats in the 5' untranslated region of the FMR1 gene. Despite arising from repeat expansions in the same gene, the clinical manifestations of FXds vary widely, encompassing developmental delays, parkinsonism, dementia, and an increased risk of infertility. FXds also exhibit molecular mechanisms observed in other REDs, that is, gene- and protein-loss-of-function and RNA- and protein-gain-of-function. The heterogeneity of phenotypes and pathomechanisms in FXds results from the different lengths of the CGG tract. As the number of repeats increases, the structures formed by RNA and DNA fragments containing CGG repeats change significantly, contributing to the diversity of FXd phenotypes and mechanisms. In this review, we discuss the role of RNA and DNA structures formed by expanded CGG repeats in driving FXd pathogenesis and how the genetic instability of CGG repeats is mediated by the complex interplay between transcription, DNA replication, and repair. We also discuss therapeutic strategies, including small molecules, antisense oligonucleotides, and CRISPR-Cas systems, that target toxic RNA and DNA involved in the development of FXds.
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Affiliation(s)
- Izabela Broniarek
- Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
| | - Daria Niewiadomska
- Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
| | - Krzysztof Sobczak
- Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
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Wu Y, Ma B, Liu C, Li D, Sui G. Pathological Involvement of Protein Phase Separation and Aggregation in Neurodegenerative Diseases. Int J Mol Sci 2024; 25:10187. [PMID: 39337671 PMCID: PMC11432175 DOI: 10.3390/ijms251810187] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2024] [Revised: 09/19/2024] [Accepted: 09/20/2024] [Indexed: 09/30/2024] Open
Abstract
Neurodegenerative diseases are the leading cause of human disability and immensely reduce patients' life span and quality. The diseases are characterized by the functional loss of neuronal cells and share several common pathogenic mechanisms involving the malfunction, structural distortion, or aggregation of multiple key regulatory proteins. Cellular phase separation is the formation of biomolecular condensates that regulate numerous biological processes, including neuronal development and synaptic signaling transduction. Aberrant phase separation may cause protein aggregation that is a general phenomenon in the neuronal cells of patients suffering neurodegenerative diseases. In this review, we summarize the pathological causes of common neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, among others. We discuss the regulation of key amyloidogenic proteins with an emphasis of their aberrant phase separation and aggregation. We also introduce the approaches as potential therapeutic strategies to ameliorate neurodegenerative diseases through intervening protein aggregation. Overall, this review consolidates the research findings of phase separation and aggregation caused by misfolded proteins in a context of neurodegenerative diseases.
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Affiliation(s)
- Yinuo Wu
- Aulin College, Northeast Forestry University, Harbin 150040, China;
| | - Biao Ma
- College of Life Science, Northeast Forestry University, Harbin 150040, China; (B.M.); (C.L.)
| | - Chang Liu
- College of Life Science, Northeast Forestry University, Harbin 150040, China; (B.M.); (C.L.)
| | - Dangdang Li
- College of Life Science, Northeast Forestry University, Harbin 150040, China; (B.M.); (C.L.)
| | - Guangchao Sui
- College of Life Science, Northeast Forestry University, Harbin 150040, China; (B.M.); (C.L.)
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Sirois CL, Guo Y, Li M, Wolkoff NE, Korabelnikov T, Sandoval S, Lee J, Shen M, Contractor A, Sousa AMM, Bhattacharyya A, Zhao X. CGG repeats in the human FMR1 gene regulate mRNA localization and cellular stress in developing neurons. Cell Rep 2024; 43:114330. [PMID: 38865241 PMCID: PMC11240841 DOI: 10.1016/j.celrep.2024.114330] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2023] [Revised: 04/18/2024] [Accepted: 05/22/2024] [Indexed: 06/14/2024] Open
Abstract
The human genome has many short tandem repeats, yet the normal functions of these repeats are unclear. The 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene contains polymorphic CGG repeats, the length of which has differing effects on FMR1 expression and human health, including the neurodevelopmental disorder fragile X syndrome. We deleted the CGG repeats in the FMR1 gene (0CGG) in human stem cells and examined the effects on differentiated neurons. 0CGG neurons have altered subcellular localization of FMR1 mRNA and protein, and differential expression of cellular stress proteins compared with neurons with normal repeats (31CGG). In addition, 0CGG neurons have altered responses to glucocorticoid receptor (GR) activation, including FMR1 mRNA localization, GR chaperone HSP90α expression, GR localization, and cellular stress protein levels. Therefore, the CGG repeats in the FMR1 gene are important for the homeostatic responses of neurons to stress signals.
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Affiliation(s)
- Carissa L Sirois
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Yu Guo
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Meng Li
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Natalie E Wolkoff
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Tomer Korabelnikov
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Soraya Sandoval
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA; Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Jiyoun Lee
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA; Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Minjie Shen
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Amaya Contractor
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Andre M M Sousa
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Anita Bhattacharyya
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Xinyu Zhao
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA.
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8
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Handal T, Juster S, Abu Diab M, Yanovsky-Dagan S, Zahdeh F, Aviel U, Sarel-Gallily R, Michael S, Bnaya E, Sebban S, Buganim Y, Drier Y, Mouly V, Kubicek S, van den Broek WJAA, Wansink DG, Epsztejn-Litman S, Eiges R. Differentiation shifts from a reversible to an irreversible heterochromatin state at the DM1 locus. Nat Commun 2024; 15:3270. [PMID: 38627364 PMCID: PMC11021500 DOI: 10.1038/s41467-024-47217-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2021] [Accepted: 03/25/2024] [Indexed: 04/19/2024] Open
Abstract
Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Here we show by the analysis of myotonic dystrophy type 1 (DM1)-related locus that in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (CTG2000 expansion), whereas in patient myoblasts (CTG2600 expansion), repeat deletion fails to do so. This distinction between undifferentiated and differentiated cells arises during cell differentiation, and can be reversed by reprogramming of gene-edited myoblasts. We demonstrate that abnormal methylation in DM1 is distinctively maintained in the undifferentiated state by the activity of the de novo DNMTs (DNMT3b in tandem with DNMT3a). Overall, the findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, thus underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus.
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Affiliation(s)
- Tayma Handal
- Stem Cell Research Laboratory, Medical Genetics Institute, The Eisenberg R&D Authority, Shaare Zedek Medical Center, Jerusalem, 91031, Israel
- The Hebrew University School of Medicine, Jerusalem, 91120, Israel
| | - Sarah Juster
- Stem Cell Research Laboratory, Medical Genetics Institute, The Eisenberg R&D Authority, Shaare Zedek Medical Center, Jerusalem, 91031, Israel
- The Hebrew University School of Medicine, Jerusalem, 91120, Israel
| | - Manar Abu Diab
- Stem Cell Research Laboratory, Medical Genetics Institute, The Eisenberg R&D Authority, Shaare Zedek Medical Center, Jerusalem, 91031, Israel
- The Hebrew University School of Medicine, Jerusalem, 91120, Israel
| | - Shira Yanovsky-Dagan
- Stem Cell Research Laboratory, Medical Genetics Institute, The Eisenberg R&D Authority, Shaare Zedek Medical Center, Jerusalem, 91031, Israel
- The Hebrew University School of Medicine, Jerusalem, 91120, Israel
| | - Fouad Zahdeh
- Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, 91031, Israel
| | - Uria Aviel
- Stem Cell Research Laboratory, Medical Genetics Institute, The Eisenberg R&D Authority, Shaare Zedek Medical Center, Jerusalem, 91031, Israel
- The Hebrew University School of Medicine, Jerusalem, 91120, Israel
| | - Roni Sarel-Gallily
- The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, The Life Sciences Institute, The Hebrew University, Jerusalem, 91904, Israel
| | - Shir Michael
- Stem Cell Research Laboratory, Medical Genetics Institute, The Eisenberg R&D Authority, Shaare Zedek Medical Center, Jerusalem, 91031, Israel
- The Hebrew University School of Medicine, Jerusalem, 91120, Israel
| | - Ester Bnaya
- Stem Cell Research Laboratory, Medical Genetics Institute, The Eisenberg R&D Authority, Shaare Zedek Medical Center, Jerusalem, 91031, Israel
- The Hebrew University School of Medicine, Jerusalem, 91120, Israel
| | - Shulamit Sebban
- Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel
| | - Yosef Buganim
- Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel
| | - Yotam Drier
- The Lautenberg Center for Immunology and Cancer Research, IMRIC, Faculty of Medicine, The Hebrew University, Jerusalem, Israel
| | - Vincent Mouly
- Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, F-75013, Paris, France
| | - Stefan Kubicek
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, AKH BT 25.3, 1090, Vienna, Austria
| | - Walther J A A van den Broek
- Department of Medical BioSciences, Research Institute for Medical Innovation, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Derick G Wansink
- Department of Medical BioSciences, Research Institute for Medical Innovation, Radboud University Medical Center, Nijmegen, The Netherlands.
| | - Silvina Epsztejn-Litman
- Stem Cell Research Laboratory, Medical Genetics Institute, The Eisenberg R&D Authority, Shaare Zedek Medical Center, Jerusalem, 91031, Israel
| | - Rachel Eiges
- Stem Cell Research Laboratory, Medical Genetics Institute, The Eisenberg R&D Authority, Shaare Zedek Medical Center, Jerusalem, 91031, Israel.
- The Hebrew University School of Medicine, Jerusalem, 91120, Israel.
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9
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Fang M, Deibler SK, Krishnamurthy PM, Wang F, Rodriguez P, Banday S, Virbasius CM, Sena-Esteves M, Watts JK, Green MR. EZH2 inhibition reactivates epigenetically silenced FMR1 and normalizes molecular and electrophysiological abnormalities in fragile X syndrome neurons. Front Neurosci 2024; 18:1348478. [PMID: 38449737 PMCID: PMC10915284 DOI: 10.3389/fnins.2024.1348478] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2023] [Accepted: 02/09/2024] [Indexed: 03/08/2024] Open
Abstract
Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here, using a candidate-based shRNA screen, we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors, or FMR1- SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells, neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2, for which an FDA-approved small molecule inhibitor, EPZ6438 (also known as tazemetostat), is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately, EZH2 inhibitors do not efficiently cross the blood-brain barrier, limiting their therapeutic use for FXS. Recently, antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons, and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively, our results establish EZH2 inhibition in general, and EZH2 ASOs in particular, as a therapeutic approach for FXS.
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Affiliation(s)
- Minggang Fang
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Sara K. Deibler
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | | | - Feng Wang
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Paola Rodriguez
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Shahid Banday
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Ching-Man Virbasius
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Miguel Sena-Esteves
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Jonathan K. Watts
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Michael R. Green
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, United States
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10
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Zeng T, Chen Y, Huang H, Li S, Huang J, Xie H, Lin S, Chen S, Chen G, Yang D. Neuronal Intranuclear Inclusion Disease with NOTCH2NLC GGC Repeat Expansion: A Systematic Review and Challenges of Phenotypic Characterization. Aging Dis 2024; 16:AD.2024.0131-1. [PMID: 38377026 PMCID: PMC11745434 DOI: 10.14336/ad.2024.0131-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Accepted: 01/31/2024] [Indexed: 02/22/2024] Open
Abstract
Neuronal intranuclear inclusion disease (NIID) is a highly clinically heterogeneous neurodegenerative disorder primarily attributed to abnormal GGC repeat expansions in the NOTCH2NLC gene. This study aims to comprehensively explore its phenotypic characteristics and genotype-phenotype correlation. A literature search was conducted in PubMed, Embase, and the Cochrane Library from September 1, 2019, to December 31, 2022, encompassing reported NIID cases confirmed by pathogenic NOTCH2NLC mutations. Linear regressions and trend analyses were performed. Analyzing 635 cases from 85 included studies revealed that familial cases exhibited significantly larger GGC repeat expansions than sporadic cases (p < 0.001), and this frequency significantly increased with expanding GGC repeats (p trend < 0.001). Age at onset (AAO) showed a negative correlation with GGC repeat expansions (p < 0.001). The predominant initial symptoms included tremor (31.70%), cognitive impairment (14.12%), and muscle weakness (10.66%). The decreased or absent tendon reflex (DTR/ATR) emerged as a notable clinical indicator of NIID due to its high prevalence. U-fiber was observed in 79.11% of patients, particularly prominent in paroxysmal disease-dominant (87.50%) and dementia-dominant cases (81.08%). Peripheral neuropathy-dominant cases exhibited larger GGC repeat expansions (median = 123.00) and an earlier AAO (median = 33.00) than other phenotypes. Moreover, a significant genetic anticipation of 3.5 years was observed (p = 0.039). This study provides a comprehensive and up-to-date compilation of genotypic and phenotypic information on NIID since the identification of the causative gene NOTCH2NLC. We contribute a novel diagnostic framework for NIID to support clinical practice.
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Affiliation(s)
- Tian Zeng
- The First School of Medicine, School of Information and Engineering, Wenzhou Medical University, Wenzhou, China.
| | - Yiqun Chen
- The First School of Medicine, School of Information and Engineering, Wenzhou Medical University, Wenzhou, China.
| | - Honghao Huang
- Department of Cardiology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
| | - Shengqi Li
- The First School of Medicine, School of Information and Engineering, Wenzhou Medical University, Wenzhou, China.
| | - Jiaqi Huang
- The First School of Medicine, School of Information and Engineering, Wenzhou Medical University, Wenzhou, China.
| | - Haobo Xie
- The First School of Medicine, School of Information and Engineering, Wenzhou Medical University, Wenzhou, China.
| | - Shenyi Lin
- The First School of Medicine, School of Information and Engineering, Wenzhou Medical University, Wenzhou, China.
| | - Siyao Chen
- The First School of Medicine, School of Information and Engineering, Wenzhou Medical University, Wenzhou, China.
| | - Guangyong Chen
- Department of Neurology, The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
| | - Dehao Yang
- Department of Neurology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
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11
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Malachowski T, Chandradoss KR, Boya R, Zhou L, Cook AL, Su C, Pham K, Haws SA, Kim JH, Ryu HS, Ge C, Luppino JM, Nguyen SC, Titus KR, Gong W, Wallace O, Joyce EF, Wu H, Rojas LA, Phillips-Cremins JE. Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome. Cell 2023; 186:5840-5858.e36. [PMID: 38134876 PMCID: PMC10794044 DOI: 10.1016/j.cell.2023.11.019] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2022] [Revised: 07/31/2023] [Accepted: 11/16/2023] [Indexed: 12/24/2023]
Abstract
Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X chromosome in FXS patient-derived iPSCs, iPSC-derived neural progenitors, EBV-transformed lymphoblasts, and brain tissue with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication-stress-induced double-strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the mutation-length CGG to premutation length reverses H3K9me3 on the X chromosome and multiple autosomes, refolds TADs, and restores gene expression. H3K9me3 domains can also arise in normal-length iPSCs created with perturbations linked to genome instability, suggesting their relevance beyond FXS. Our results reveal Mb-scale heterochromatinization and trans interactions among loci susceptible to instability.
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Affiliation(s)
- Thomas Malachowski
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Keerthivasan Raanin Chandradoss
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Ravi Boya
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Linda Zhou
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Ashley L Cook
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Chuanbin Su
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Kenneth Pham
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Spencer A Haws
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Ji Hun Kim
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
| | - Han-Seul Ryu
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Chunmin Ge
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
| | - Jennifer M Luppino
- Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Son C Nguyen
- Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Katelyn R Titus
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Wanfeng Gong
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Owen Wallace
- Fulcrum Therapeutics Incorporated, Cambridge, MA, USA
| | - Eric F Joyce
- Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA
| | - Hao Wu
- Fulcrum Therapeutics Incorporated, Cambridge, MA, USA
| | | | - Jennifer E Phillips-Cremins
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA.
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12
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Lotfi M, Morshedi Rad D, Mashhadi SS, Ashouri A, Mojarrad M, Mozaffari-Jovin S, Farrokhi S, Hashemi M, Lotfi M, Ebrahimi Warkiani M, Abbaszadegan MR. Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells. Stem Cell Rev Rep 2023; 19:2576-2596. [PMID: 37723364 PMCID: PMC10661828 DOI: 10.1007/s12015-023-10585-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/30/2023] [Indexed: 09/20/2023]
Abstract
Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.
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Affiliation(s)
- Malihe Lotfi
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Dorsa Morshedi Rad
- School of Biomedical Engineering, University of Technology Sydney, Sydney, Australia
| | - Samaneh Sharif Mashhadi
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Atefeh Ashouri
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Majid Mojarrad
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Sina Mozaffari-Jovin
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Shima Farrokhi
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Maryam Hashemi
- Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Marzieh Lotfi
- Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran
| | - Majid Ebrahimi Warkiani
- School of Biomedical Engineering, University of Technology Sydney, Sydney, Australia.
- Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney, Sydney, Australia.
| | - Mohammad Reza Abbaszadegan
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
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13
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Réthelyi JM, Vincze K, Schall D, Glennon J, Berkel S. The role of insulin/IGF1 signalling in neurodevelopmental and neuropsychiatric disorders - Evidence from human neuronal cell models. Neurosci Biobehav Rev 2023; 153:105330. [PMID: 37516219 DOI: 10.1016/j.neubiorev.2023.105330] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2022] [Revised: 07/15/2023] [Accepted: 07/26/2023] [Indexed: 07/31/2023]
Abstract
Insulin and insulin-like growth factor 1 (IGF1) signalling play a central role in the development and maintenance of neurons in the brain, and human neurodevelopmental as well as neuropsychiatric disorders have been linked to impaired insulin and IGF1 signalling. This review focuses on the impairments of the insulin and IGF1 signalling cascade in the context of neurodevelopmental and neuropsychiatric disorders, based on evidence from human neuronal cell models. Clear evidence was obtained for impaired insulin and IGF1 receptor downstream signalling in neurodevelopmental disorders, while the evidence for its role in neuropsychiatric disorders was less substantial. Human neuronal model systems can greatly add to our knowledge about insulin/IGF1 signalling in the brain, its role in restoring dendritic maturity, and complement results from clinical studies and animal models. Moreover, they represent a useful model for the development of new therapeutic strategies. Further research is needed to systematically investigate the exact role of the insulin/IGF1 signalling cascades in neurodevelopmental and neuropsychiatric disorders, and to elucidate the respective therapeutic implications.
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Affiliation(s)
- János M Réthelyi
- Department of Psychiatry and Psychotherapy, Semmelweis University, Budapest, Hungary
| | - Katalin Vincze
- Department of Psychiatry and Psychotherapy, Semmelweis University, Budapest, Hungary; Doctoral School of Mental Health Sciences, Semmelweis University, Budapest, Hungary
| | - Dorothea Schall
- Institute of Human Genetics, Heidelberg University, Heidelberg, Germany
| | - Jeffrey Glennon
- Conway Institute of Biomedical and Biomolecular Research, School of Medicine, University College Dublin, Dublin, Ireland
| | - Simone Berkel
- Institute of Human Genetics, Heidelberg University, Heidelberg, Germany; Interdisciplinary Centre of Neurosciences (IZN), Heidelberg University, Germany.
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14
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Shah S, Sharp KJ, Raju Ponny S, Lee J, Watts JK, Berry-Kravis E, Richter JD. Antisense oligonucleotide rescue of CGG expansion-dependent FMR1 mis-splicing in fragile X syndrome restores FMRP. Proc Natl Acad Sci U S A 2023; 120:e2302534120. [PMID: 37364131 PMCID: PMC10319035 DOI: 10.1073/pnas.2302534120] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2023] [Accepted: 05/26/2023] [Indexed: 06/28/2023] Open
Abstract
Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here, we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissues of individuals with fragile X syndrome (FXS). Surprisingly, the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene is transcribed in >70% of the FXS tissues. In all FMR1-expressing FXS tissues, FMR1 RNA itself is mis-spliced in a CGG expansion-dependent manner to generate the little-known FMR1-217 RNA isoform, which is comprised of FMR1 exon 1 and a pseudo-exon in intron 1. FMR1-217 is also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissues. We show that in cells aberrantly expressing mis-spliced FMR1, antisense oligonucleotide (ASO) treatment reduces FMR1-217, rescues full-length FMR1 RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, FMR1 gene reactivation in transcriptionally silent FXS cells using 5-aza-2'-deoxycytidine (5-AzadC), which prevents DNA methylation, increases FMR1-217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length FMR1 expression and restores FMRP. These findings indicate that misregulated RNA-processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing FMR1-217, ASO treatment may offer a therapeutic approach to mitigate the disorder.
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Affiliation(s)
- Sneha Shah
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA01605
| | - Kevin J. Sharp
- Department of Pediatrics, Rush University Medical Center, Chicago, IL60612
| | - Sithara Raju Ponny
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA01605
| | - Jonathan Lee
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA01605
| | - Jonathan K. Watts
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA01605
- Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA01605
- Li Weibo Rare Disease Institute, University of Massachusetts Chan Medical School, Worcester, MA01605
| | - Elizabeth Berry-Kravis
- Department of Pediatrics, Rush University Medical Center, Chicago, IL60612
- Department of Neurological Sciences, Rush University Medical Center, Chicago, IL60612
- Department of Anatomy and Cell Biology, Rush University Medical Center, Chicago, IL60612
| | - Joel D. Richter
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA01605
- Li Weibo Rare Disease Institute, University of Massachusetts Chan Medical School, Worcester, MA01605
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15
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Sandhu A, Kumar A, Rawat K, Gautam V, Sharma A, Saha L. Modernising autism spectrum disorder model engineering and treatment via CRISPR-Cas9: A gene reprogramming approach. World J Clin Cases 2023; 11:3114-3127. [PMID: 37274051 PMCID: PMC10237133 DOI: 10.12998/wjcc.v11.i14.3114] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/28/2022] [Revised: 02/13/2023] [Accepted: 04/06/2023] [Indexed: 05/16/2023] Open
Abstract
A neurological abnormality called autism spectrum disorder (ASD) affects how a person perceives and interacts with others, leading to social interaction and communication issues. Limited and recurring behavioural patterns are another feature of the illness. Multiple mutations throughout development are the source of the neurodevelopmental disorder autism. However, a well-established model and perfect treatment for this spectrum disease has not been discovered. The rising era of the clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) system can streamline the complexity underlying the pathogenesis of ASD. The CRISPR-Cas9 system is a powerful genetic engineering tool used to edit the genome at the targeted site in a precise manner. The major hurdle in studying ASD is the lack of appropriate animal models presenting the complex symptoms of ASD. Therefore, CRISPR-Cas9 is being used worldwide to mimic the ASD-like pathology in various systems like in vitro cell lines, in vitro 3D organoid models and in vivo animal models. Apart from being used in establishing ASD models, CRISPR-Cas9 can also be used to treat the complexities of ASD. The aim of this review was to summarize and critically analyse the CRISPR-Cas9-mediated discoveries in the field of ASD.
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Affiliation(s)
- Arushi Sandhu
- Department of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh 0172, Chandigarh, India
| | - Anil Kumar
- Department of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh 0172, Chandigarh, India
| | - Kajal Rawat
- Department of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh 0172, Chandigarh, India
| | - Vipasha Gautam
- Department of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh 0172, Chandigarh, India
| | - Antika Sharma
- Department of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh 0172, Chandigarh, India
| | - Lekha Saha
- Department of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh 0172, Chandigarh, India
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16
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Datta A, Sarmah D, Kaur H, Chaudhary A, Vadak N, Borah A, Shah S, Wang X, Bhattacharya P. Advancement in CRISPR/Cas9 Technology to Better Understand and Treat Neurological Disorders. Cell Mol Neurobiol 2023; 43:1019-1035. [PMID: 35751791 PMCID: PMC11414438 DOI: 10.1007/s10571-022-01242-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2022] [Accepted: 06/09/2022] [Indexed: 11/26/2022]
Abstract
Neurological disorders have complicated pathophysiology that may involve several genetic mutations. Conventional treatment has limitations as they only treat apparent symptoms. Although, personalized medicine is emerging as a promising neuro-intervention, lack of precision is the major pitfall. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is evolving as a technological platform that may overcome the therapeutic limitations towards precision medicine. In the future, targeting genes in neurological disorders may be the mainstay of modern therapy. The present review on CRISPR/Cas9 and its application in various neurological disorders may provide a platform for its future clinical relevance towards developing precise and personalized medicine.
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Affiliation(s)
- Aishika Datta
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, 382355, India
| | - Deepaneeta Sarmah
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, 382355, India
| | - Harpreet Kaur
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, 382355, India
| | - Antra Chaudhary
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, 382355, India
| | - Namrata Vadak
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, 382355, India
| | - Anupom Borah
- Cellular and Molecular Neurobiology Laboratory, Department of Life Science and Bioinformatics, Assam University, Silchar, Assam, 788011, India
| | - Sudhir Shah
- Department of Neurology, SVPIMSR and NHL Municipal Medical College & Sterling Hospital, Ahmedabad, Gujarat, 380006, India
| | - Xin Wang
- Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA
| | - Pallab Bhattacharya
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gandhinagar, Gujarat, 382355, India.
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17
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Zhang R, Xu H, Lu J, Chen Y, Zhang Y, Xiao L. Accelerated Apoptosis and Down-Regulated FMRP in Human Neuroblastoma Cells with CRISPR/ Cas9 Genome Editing. IRANIAN JOURNAL OF PUBLIC HEALTH 2023; 52:703-712. [PMID: 37551173 PMCID: PMC10404333 DOI: 10.18502/ijph.v52i4.12438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Accepted: 12/19/2022] [Indexed: 08/09/2023]
Abstract
Background Fragile X syndrome (FXS) is a genetic disease with intellectual disabilities. FXS is often caused by the CGG-repeat expansion mutation in the FMR1 gene with suppressed FMR1 transcription and decreased protein levels in the brain of the patients. The RNA-guided CRISPR/Cas9 system is a promising targeted genomic editing tool in gene therapy of FXS. In order to evaluate its feasibility, the present study used CRISPR/Cas9 system to target the FMR1 5'-UTR sites in cultured human neuroblastoma cells. Methods PCR and DNA clone were used to construct plasmids. CRISPR function was tested by Western blot and flow cytometry. Data were analyzed by a two-tailed unpaired Student's t-test using GraphPad software. This research was conducted from 2020 to 2022 in the Second Affiliated Hospital of Soochow University, Suzhou, China. Results Cell cycle analysis showed significant differences in G1, S and G2/M phases between the two groups (P<0.05). In the knockout cells, apoptosis was accelerated (P<0.05) with a significantly down-regulated (P<0.05) expression of FMRP as compared with the control group. Conclusion This study provides further understanding about the FMRP function and molecular mechanism of FMR1 gene in nerve cells, and suggests the feasibility of gene therapy in FXS by CRISPR/Cas9 gene editing system.
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Affiliation(s)
- Rong Zhang
- Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Soochow University, Suzhou, 215004, China
- State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, 215123, China
| | - Huifen Xu
- Department of Pharmacy, Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, 310057, China
| | - Jin Lu
- Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Soochow University, Suzhou, 215004, China
| | - Ying Chen
- Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Soochow University, Suzhou, 215004, China
| | - Yahui Zhang
- Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Soochow University, Suzhou, 215004, China
| | - Li Xiao
- Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Soochow University, Suzhou, 215004, China
- State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, 215123, China
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18
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Simpson BP, Yrigollen CM, Izda A, Davidson BL. Targeted long-read sequencing captures CRISPR editing and AAV integration outcomes in brain. Mol Ther 2023; 31:760-773. [PMID: 36617193 PMCID: PMC10014281 DOI: 10.1016/j.ymthe.2023.01.004] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2022] [Revised: 12/07/2022] [Accepted: 01/04/2023] [Indexed: 01/09/2023] Open
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing is an emerging therapeutic modality that shows promise in Huntington's disease and spinocerebellar ataxia (SCA) mouse models. However, advancing CRISPR-based therapies requires methods to fully define in vivo editing outcomes. Here, we use polymerase-free, targeted long-read nanopore sequencing and evaluate single- and dual-gRNA AAV-CRISPR editing of human ATXN2 in transgenic mouse models of SCA type 2 (SCA2). Unbiased high sequencing coverage showed 10%-25% editing. Along with intended edits there was AAV integration, 1%-2% of which contained the entire AAV genome and were largely unmethylated. More than 150 kb deletions at target loci and rearrangements of the transgenic allele (1%) were also found. In contrast, PCR-based nanopore sequencing showed bias for partial AAV fragments and inverted terminal repeats (ITRs) and failed to detect full-length AAV. Cumulatively this work defines the spectrum of outcomes of CRISPR editing in mouse brain after AAV gene transfer using an unbiased long-read sequencing approach.
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Affiliation(s)
- Bryan P Simpson
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA; Cell and Molecular Biology Graduate Group, Biomedical Graduate Studies, University of Pennsylvania, Philadelphia, PA, USA
| | - Carolyn M Yrigollen
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Aleksandar Izda
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Beverly L Davidson
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.
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19
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Hong D, Iakoucheva LM. Therapeutic strategies for autism: targeting three levels of the central dogma of molecular biology. Transl Psychiatry 2023; 13:58. [PMID: 36792602 PMCID: PMC9931756 DOI: 10.1038/s41398-023-02356-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2022] [Revised: 02/01/2023] [Accepted: 02/03/2023] [Indexed: 02/17/2023] Open
Abstract
The past decade has yielded much success in the identification of risk genes for Autism Spectrum Disorder (ASD), with many studies implicating loss-of-function (LoF) mutations within these genes. Despite this, no significant clinical advances have been made so far in the development of therapeutics for ASD. Given the role of LoF mutations in ASD etiology, many of the therapeutics in development are designed to rescue the haploinsufficient effect of genes at the transcriptional, translational, and protein levels. This review will discuss the various therapeutic techniques being developed from each level of the central dogma with examples including: CRISPR activation (CRISPRa) and gene replacement at the DNA level, antisense oligonucleotides (ASOs) at the mRNA level, and small-molecule drugs at the protein level, followed by a review of current delivery methods for these therapeutics. Since central nervous system (CNS) penetrance is of utmost importance for ASD therapeutics, it is especially necessary to evaluate delivery methods that have higher efficiency in crossing the blood-brain barrier (BBB).
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Affiliation(s)
- Derek Hong
- Department of Psychiatry, University of California San Diego, La Jolla, CA, USA
- Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - Lilia M Iakoucheva
- Department of Psychiatry, University of California San Diego, La Jolla, CA, USA.
- Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA.
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20
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Dhuriya YK, Naik AA. CRISPR: a tool with potential for genomic reprogramming in neurological disorders. Mol Biol Rep 2023; 50:1845-1856. [PMID: 36507966 DOI: 10.1007/s11033-022-08136-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2022] [Accepted: 11/17/2022] [Indexed: 12/14/2022]
Abstract
The intricate neural circuitry of the brain necessitates precise and synchronized transcriptional programs. Any disturbance during embryonic or adult development, whether caused by genetic or environmental factors, may result in refractory and recurrent neurological disorders. Inadequate knowledge of the pathogenic mechanisms underlying neurological disorders is the primary obstacle to the development of effective treatments, necessitating the development of alternative therapeutic approaches to identify rational molecular targets. Recently, with the evolution of CRISPR-Cas9 technology, an engineered RNA system provides precise and highly effective correction or silencing of disease-causing mutations by modulating expression and thereby avoiding the limitations of the RNA interference strategy. This article discusses the CRISPR-Cas9 technology, its mechanisms, and the limitations of the new technology. We provide a glimpse of how the far-reaching implications of CRISPR can open new avenues for the development of tools to combat neurological disorders, as well as a review of recent attempts by neuroscientists to launch therapeutic correction.
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Affiliation(s)
| | - Aijaz A Naik
- National Institute of Mental Health (NIMH), Bethesda, USA.
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21
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Krzisch MA, Wu H, Yuan B, Whitfield TW, Liu XS, Fu D, Garrett-Engele CM, Khalil AS, Lungjangwa T, Shih J, Chang AN, Warren S, Cacace A, Andrykovich KR, Rietjens RGJ, Wallace O, Sur M, Jain B, Jaenisch R. Fragile X Syndrome Patient-Derived Neurons Developing in the Mouse Brain Show FMR1-Dependent Phenotypes. Biol Psychiatry 2023; 93:71-81. [PMID: 36372569 DOI: 10.1016/j.biopsych.2022.08.020] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Revised: 08/11/2022] [Accepted: 08/11/2022] [Indexed: 01/10/2023]
Abstract
BACKGROUND Fragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. METHODS To mimic human neuron development in vivo, we coinjected neural precursor cells derived from FXS patient-derived induced pluripotent stem cells and neural precursor cells derived from corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. RESULTS The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Immunofluorescence and single and bulk RNA sequencing analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, we found increased percentages of Arc- and Egr-1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons. CONCLUSIONS This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3-dimensional context.
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Affiliation(s)
- Marine A Krzisch
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.
| | - Hao Wu
- Full Circles Therapeutics, Inc., Cambridge, Massachusetts
| | - Bingbing Yuan
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - Troy W Whitfield
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - X Shawn Liu
- Department of Physiology and Cellular Biophysics, Columbia University Medical Center, New York, New York
| | - Dongdong Fu
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | | | - Andrew S Khalil
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - Tenzin Lungjangwa
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - Jennifer Shih
- Picower Institute for Learning and Memory, Cambridge, Massachusetts
| | | | - Stephen Warren
- Departments of Human Genetics, Biochemistry, and Pediatrics, Emory University School of Medicine, Atlanta, Georgia
| | | | | | | | | | - Mriganka Sur
- Picower Institute for Learning and Memory, Cambridge, Massachusetts
| | - Bhav Jain
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
| | - Rudolf Jaenisch
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts.
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22
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Operto FF, Pastorino GMG, Viggiano A, Dell’Isola GB, Dini G, Verrotti A, Coppola G. Epilepsy and Cognitive Impairment in Childhood and Adolescence: A Mini-Review. Curr Neuropharmacol 2023; 21:1646-1665. [PMID: 35794776 PMCID: PMC10514538 DOI: 10.2174/1570159x20666220706102708] [Citation(s) in RCA: 21] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2022] [Revised: 02/28/2022] [Accepted: 04/26/2022] [Indexed: 11/22/2022] Open
Abstract
Managing epilepsy in people with an intellectual disability remains a therapeutic challenge and must take into account additional issues such as diagnostic difficulties and frequent drug resistance. Advances in genomic technologies improved our understanding of epilepsy and raised the possibility to develop patients-tailored treatments acting on the key molecular mechanisms involved in the development of the disease. In addition to conventional antiseizure medications (ASMs), ketogenic diet, hormone therapy and epilepsy surgery play an important role, especially in cases of drugresistance. This review aims to provide a comprehensive overview of the mainfactors influencing cognition in children and adolescents with epilepsy and the main therapeutic options available for the epilepsies associated with intellectual disability.
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Affiliation(s)
- Francesca Felicia Operto
- Child and Adolescent Neuropsychiatry Unit, Department of Medicine, Surgery and Dentistry, University of Salerno, Baronissi, SA, Italy
| | - Grazia Maria Giovanna Pastorino
- Child and Adolescent Neuropsychiatry Unit, Department of Medicine, Surgery and Dentistry, University of Salerno, Baronissi, SA, Italy
| | - Andrea Viggiano
- Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Baronissi, SA, Italy
| | | | - Gianluca Dini
- Department of Pediatrics, University of Perugia, Giorgio Menghini Square, 06129 Perugia, Italy
| | - Alberto Verrotti
- Department of Pediatrics, University of Perugia, Giorgio Menghini Square, 06129 Perugia, Italy
| | - Giangennaro Coppola
- Child and Adolescent Neuropsychiatry Unit, Department of Medicine, Surgery and Dentistry, University of Salerno, Baronissi, SA, Italy
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23
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Shahcheraghi SH, Ayatollahi J, Lotfi M, Aljabali AAA, Al-Zoubi MS, Panda PK, Mishra V, Satija S, Charbe NB, Serrano-Aroca Á, Bahar B, Takayama K, Goyal R, Bhatia A, Almutary AG, Alnuqaydan AM, Mishra Y, Negi P, Courtney A, McCarron PA, Bakshi HA, Tambuwala MM. Gene Therapy for Neuropsychiatric Disorders: Potential Targets and Tools. CNS & NEUROLOGICAL DISORDERS DRUG TARGETS 2023; 22:51-65. [PMID: 35249508 DOI: 10.2174/1871527321666220304153719] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Revised: 01/16/2022] [Accepted: 01/16/2022] [Indexed: 01/01/2023]
Abstract
Neuropsychiatric disorders that affect the central nervous system cause considerable pressures on the health care system and have a substantial economic burden on modern societies. The present treatments based on available drugs are mostly ineffective and often costly. The molecular process of neuropsychiatric disorders is closely connected to modifying the genetic structures inherited or caused by damage, toxic chemicals, and some current diseases. Gene therapy is presently an experimental concept for neurological disorders. Clinical applications endeavor to alleviate the symptoms, reduce disease progression, and repair defective genes. Implementing gene therapy in inherited and acquired neurological illnesses entails the integration of several scientific disciplines, including virology, neurology, neurosurgery, molecular genetics, and immunology. Genetic manipulation has the power to minimize or cure illness by inducing genetic alterations at endogenous loci. Gene therapy that involves treating the disease by deleting, silencing, or editing defective genes and delivering genetic material to produce therapeutic molecules has excellent potential as a novel approach for treating neuropsychiatric disorders. With the recent advances in gene selection and vector design quality in targeted treatments, gene therapy could be an effective approach. This review article will investigate and report the newest and the most critical molecules and factors in neuropsychiatric disorder gene therapy. Different genome editing techniques available will be evaluated, and the review will highlight preclinical research of genome editing for neuropsychiatric disorders while also evaluating current limitations and potential strategies to overcome genome editing advancements.
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Affiliation(s)
- Seyed H Shahcheraghi
- Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
- Infectious Diseases Research Center, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
| | - Jamshid Ayatollahi
- Infectious Diseases Research Center, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
| | - Marzieh Lotfi
- Abortion Research Center, Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
| | - Alaa A A Aljabali
- Department of Pharmaceutics & Pharmaceutical Technology, Yarmouk University, Irbid, Jordan
| | - Mazhar S Al-Zoubi
- Yarmouk University, Faculty of Medicine, Department of Basic Medical Sciences, Irbid, Jordan
| | - Pritam K Panda
- Condensed Matter Theory Group, Materials Theory Division, Department of Physics and Astronomy, Uppsala University, Box 516, 75120 Uppsala, Sweden
| | - Vijay Mishra
- School of Pharmaceutical Sciences, Lovely Professional University, Phagwara 144411, Punjab, India
| | - Saurabh Satija
- School of Pharmaceutical Sciences, Lovely Professional University, Phagwara 144411, Punjab, India
| | - Nitin B Charbe
- Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX 78363, USA
| | - Ángel Serrano-Aroca
- Biomaterials and Bioengineering Lab, Translational Research Centre San Alberto Magno, Catholic University of Valencia San Vicente Mártir, C/Guillem de Castro 94, 46001 Valencia, Spain
| | - Bojlul Bahar
- Nutrition Sciences and Applied Food Safety Studies, Research Centre for Global Development, School of Sport & Health Sciences, University of Central Lancashire, Preston, PR1 2HE, UK
| | - Kazuo Takayama
- Center for IPS Cell Research and Application, Kyoto University, Kyoto, 606-8397, Japan
| | - Rohit Goyal
- Neuropharmacology Laboratory, School of Pharmaceutical Sciences, Shoolini University, Post Box No. 9, Solan, Himachal Pradesh 173212, India
| | - Amit Bhatia
- Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical University, Punjab 151001, India
| | - Abdulmajeed G Almutary
- Department of Medical Biotechnology, College of Applied Medical Sciences, Qassim University, Saudi Arabia
| | - Abdullah M Alnuqaydan
- Department of Medical Biotechnology, College of Applied Medical Sciences, Qassim University, Saudi Arabia
| | - Yachana Mishra
- Shri Shakti Degree College, Sankhahari, Ghatampur 209206, India
| | - Poonam Negi
- Shoolini University of Biotechnology and Management Sciences, Solan 173 212, India
| | - Aaron Courtney
- School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, County Londonderry, BT52 1SA, Northern Ireland, United Kingdom
| | - Paul A McCarron
- School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, County Londonderry, BT52 1SA, Northern Ireland, United Kingdom
| | - Hamid A Bakshi
- School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, County Londonderry, BT52 1SA, Northern Ireland, United Kingdom
| | - Murtaza M Tambuwala
- School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, County Londonderry, BT52 1SA, Northern Ireland, United Kingdom
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24
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Megagiannis P, Suresh R, Rouleau GA, Zhou Y. Reversibility and therapeutic development for neurodevelopmental disorders, insights from genetic animal models. Adv Drug Deliv Rev 2022; 191:114562. [PMID: 36183904 DOI: 10.1016/j.addr.2022.114562] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2022] [Revised: 08/30/2022] [Accepted: 09/24/2022] [Indexed: 01/24/2023]
Abstract
Neurodevelopmental Disorders (NDDs) encompass a broad spectrum of conditions resulting from atypical brain development. Over the past decades, we have had the fortune to witness enormous progress in diagnosis, etiology discovery, modeling, and mechanistic understanding of NDDs from both fundamental and clinical research. Here, we review recent neurobiological advances from experimental models of NDDs. We introduce several examples and highlight breakthroughs in reversal studies of phenotypes using genetically engineered models of NDDs. The in-depth understanding of brain pathophysiology underlying NDDs and evaluations of reversibility in animal models paves the foundation for discovering novel treatment options. We discuss how the expanding property of cutting-edge technologies, such as gene editing and AAV-mediated gene delivery, are leveraged in animal models for the therapeutic development of NDDs. We envision opportunities and challenges toward faithful modeling and fruitful clinical translation.
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Affiliation(s)
- Platon Megagiannis
- Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital; Faculty of Medicine and Health Sciences, McGill University, Montreal, Quebec H3A 2B4, Canada
| | - Rahul Suresh
- Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital; Faculty of Medicine and Health Sciences, McGill University, Montreal, Quebec H3A 2B4, Canada
| | - Guy A Rouleau
- Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital; Faculty of Medicine and Health Sciences, McGill University, Montreal, Quebec H3A 2B4, Canada
| | - Yang Zhou
- Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital; Faculty of Medicine and Health Sciences, McGill University, Montreal, Quebec H3A 2B4, Canada.
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25
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Human Brain Models of Intellectual Disability: Experimental Advances and Novelties. Int J Mol Sci 2022; 23:ijms23126476. [PMID: 35742919 PMCID: PMC9224308 DOI: 10.3390/ijms23126476] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Revised: 05/20/2022] [Accepted: 06/07/2022] [Indexed: 11/16/2022] Open
Abstract
Intellectual disability (ID) is characterized by deficits in conceptual, social and practical domains. ID can be caused by both genetic defects and environmental factors and is extremely heterogeneous, which complicates the diagnosis as well as the deciphering of the underlying pathways. Multiple scientific breakthroughs during the past decades have enabled the development of novel ID models. The advent of induced pluripotent stem cells (iPSCs) enables the study of patient-derived human neurons in 2D or in 3D organoids during development. Gene-editing tools, such as CRISPR/Cas9, provide isogenic controls and opportunities to design personalized gene therapies. In practice this has contributed significantly to the understanding of ID and opened doors to identify novel therapeutic targets. Despite these advances, a number of areas of improvement remain for which novel technologies might entail a solution in the near future. The purpose of this review is to provide an overview of the existing literature on scientific breakthroughs that have been advancing the way ID can be studied in the human brain. The here described human brain models for ID have the potential to accelerate the identification of underlying pathophysiological mechanisms and the development of therapies.
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26
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Lee A, Xu J, Wen Z, Jin P. Across Dimensions: Developing 2D and 3D Human iPSC-Based Models of Fragile X Syndrome. Cells 2022; 11:1725. [PMID: 35681419 PMCID: PMC9179297 DOI: 10.3390/cells11111725] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Revised: 05/11/2022] [Accepted: 05/13/2022] [Indexed: 02/01/2023] Open
Abstract
Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism spectrum disorder. FXS is caused by a cytosine-guanine-guanine (CGG) trinucleotide repeat expansion in the untranslated region of the FMR1 gene leading to the functional loss of the gene's protein product FMRP. Various animal models of FXS have provided substantial knowledge about the disorder. However, critical limitations exist in replicating the pathophysiological mechanisms. Human induced pluripotent stem cells (hiPSCs) provide a unique means of studying the features and processes of both normal and abnormal human neurodevelopment in large sample quantities in a controlled setting. Human iPSC-based models of FXS have offered a better understanding of FXS pathophysiology specific to humans. This review summarizes studies that have used hiPSC-based two-dimensional cellular models of FXS to reproduce the pathology, examine altered gene expression and translation, determine the functions and targets of FMRP, characterize the neurodevelopmental phenotypes and electrophysiological features, and, finally, to reactivate FMR1. We also provide an overview of the most recent studies using three-dimensional human brain organoids of FXS and end with a discussion of current limitations and future directions for FXS research using hiPSCs.
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Affiliation(s)
- Azalea Lee
- Neuroscience Graduate Program, Emory University, Atlanta, GA 30322, USA;
- MD/PhD Program, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Jie Xu
- Genetics and Molecular Biology Graduate Program, Emory University, Atlanta, GA 30322, USA;
| | - Zhexing Wen
- Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA 30322, USA
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Peng Jin
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA
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27
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Nambiar TS, Baudrier L, Billon P, Ciccia A. CRISPR-based genome editing through the lens of DNA repair. Mol Cell 2022; 82:348-388. [PMID: 35063100 PMCID: PMC8887926 DOI: 10.1016/j.molcel.2021.12.026] [Citation(s) in RCA: 89] [Impact Index Per Article: 29.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2021] [Revised: 12/18/2021] [Accepted: 12/20/2021] [Indexed: 01/22/2023]
Abstract
Genome editing technologies operate by inducing site-specific DNA perturbations that are resolved by cellular DNA repair pathways. Products of genome editors include DNA breaks generated by CRISPR-associated nucleases, base modifications induced by base editors, DNA flaps created by prime editors, and integration intermediates formed by site-specific recombinases and transposases associated with CRISPR systems. Here, we discuss the cellular processes that repair CRISPR-generated DNA lesions and describe strategies to obtain desirable genomic changes through modulation of DNA repair pathways. Advances in our understanding of the DNA repair circuitry, in conjunction with the rapid development of innovative genome editing technologies, promise to greatly enhance our ability to improve food production, combat environmental pollution, develop cell-based therapies, and cure genetic and infectious diseases.
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Affiliation(s)
- Tarun S Nambiar
- Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY 10032, USA
| | - Lou Baudrier
- Department of Biochemistry and Molecular Biology, Robson DNA Science Centre, Arnie Charbonneau Cancer Institute, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada
| | - Pierre Billon
- Department of Biochemistry and Molecular Biology, Robson DNA Science Centre, Arnie Charbonneau Cancer Institute, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada.
| | - Alberto Ciccia
- Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY 10032, USA.
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28
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Romagnoli A, Di Marino D. The Use of Peptides in the Treatment of Fragile X Syndrome: Challenges and Opportunities. Front Psychiatry 2021; 12:754485. [PMID: 34803767 PMCID: PMC8599826 DOI: 10.3389/fpsyt.2021.754485] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/06/2021] [Accepted: 10/11/2021] [Indexed: 01/17/2023] Open
Abstract
Fragile X Syndrome (FXS) is the most frequent cause of inherited intellectual disabilities and autism spectrum disorders, characterized by cognitive deficits and autistic behaviors. The silencing of the Fmr1 gene and consequent lack of FMRP protein, is the major contribution to FXS pathophysiology. FMRP is an RNA binding protein involved in the maturation and plasticity of synapses and its absence culminates in a range of morphological, synaptic and behavioral phenotypes. Currently, there are no approved medications for the treatment of FXS, with the approaches under study being fairly specific and unsatisfying in human trials. Here we propose peptides/peptidomimetics as candidates in the pharmacotherapy of FXS; in the last years this class of molecules has catalyzed the attention of pharmaceutical research, being highly selective and well-tolerated. Thanks to their ability to target protein-protein interactions (PPIs), they are already being tested for a wide range of diseases, including cancer, diabetes, inflammation, Alzheimer's disease, but this approach has never been applied to FXS. As FXS is at the forefront of efforts to develop new drugs and approaches, we discuss opportunities, challenges and potential issues of peptides/peptidomimetics in FXS drug design and development.
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Affiliation(s)
| | - Daniele Di Marino
- Department of Life and Environmental Sciences, New York-Marche Structural Biology Center (NY-MaSBiC), Polytechnic University of Marche, Ancona, Italy
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29
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Weuring W, Geerligs J, Koeleman BPC. Gene Therapies for Monogenic Autism Spectrum Disorders. Genes (Basel) 2021; 12:genes12111667. [PMID: 34828273 PMCID: PMC8617899 DOI: 10.3390/genes12111667] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Revised: 10/20/2021] [Accepted: 10/21/2021] [Indexed: 12/26/2022] Open
Abstract
Novel genome editing and transient gene therapies have been developed the past ten years, resulting in the first in-human clinical trials for monogenic disorders. Syndromic autism spectrum disorders can be caused by mutations in a single gene. Given the monogenic aspect and severity of syndromic ASD, it is an ideal candidate for gene therapies. Here, we selected 11 monogenic ASD syndromes, validated by animal models, and reviewed current gene therapies for each syndrome. Given the wide variety and novelty of some forms of gene therapy, the best possible option must be decided based on the gene and mutation.
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30
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Kang Y, Zhou Y, Li Y, Han Y, Xu J, Niu W, Li Z, Liu S, Feng H, Huang W, Duan R, Xu T, Raj N, Zhang F, Dou J, Xu C, Wu H, Bassell GJ, Warren ST, Allen EG, Jin P, Wen Z. A human forebrain organoid model of fragile X syndrome exhibits altered neurogenesis and highlights new treatment strategies. Nat Neurosci 2021; 24:1377-1391. [PMID: 34413513 PMCID: PMC8484073 DOI: 10.1038/s41593-021-00913-6] [Citation(s) in RCA: 110] [Impact Index Per Article: 27.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2020] [Accepted: 07/15/2021] [Indexed: 02/07/2023]
Abstract
Fragile X syndrome (FXS) is caused by the loss of fragile X mental retardation protein (FMRP), an RNA-binding protein that can regulate the translation of specific mRNAs. In this study, we developed an FXS human forebrain organoid model and observed that the loss of FMRP led to dysregulated neurogenesis, neuronal maturation and neuronal excitability. Bulk and single-cell gene expression analyses of FXS forebrain organoids revealed that the loss of FMRP altered gene expression in a cell-type-specific manner. The developmental deficits in FXS forebrain organoids could be rescued by inhibiting the phosphoinositide 3-kinase pathway but not the metabotropic glutamate pathway disrupted in the FXS mouse model. We identified a large number of human-specific mRNAs bound by FMRP. One of these human-specific FMRP targets, CHD2, contributed to the altered gene expression in FXS organoids. Collectively, our study revealed molecular, cellular and electrophysiological abnormalities associated with the loss of FMRP during human brain development.
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Affiliation(s)
- Yunhee Kang
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA;,Department of Biostatistics and Bioinformatics, Emory University School of Public Health, Atlanta, GA 30322, USA
| | - Ying Zhou
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA;,Department of Biostatistics and Bioinformatics, Emory University School of Public Health, Atlanta, GA 30322, USA
| | - Yujing Li
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA;,Department of Biostatistics and Bioinformatics, Emory University School of Public Health, Atlanta, GA 30322, USA
| | - Yanfei Han
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Jie Xu
- The Graduate Program in Genetics and Molecular Biology, Emory University, GA 30322, USA
| | - Weibo Niu
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Ziyi Li
- Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030
| | - Shiying Liu
- Department of Population and Quantitative Health Sciences, Case Western Reserve University, OH 44106, USA
| | - Hao Feng
- Department of Population and Quantitative Health Sciences, Case Western Reserve University, OH 44106, USA
| | - Wen Huang
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, 410078, China
| | - Ranhui Duan
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, 410078, China
| | - Tianmin Xu
- Department of Gynecology and Obstetrics, The Second Hospital of Jilin University, Changchun, Jilin, China
| | - Nisha Raj
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Feiran Zhang
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Juan Dou
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Chongchong Xu
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Hao Wu
- Department of Biostatistics and Bioinformatics, Emory University School of Public Health, Atlanta, GA 30322, USA
| | - Gary J Bassell
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Stephen T Warren
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Emily G Allen
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Peng Jin
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA;,To whom correspondence should be addressed: (P.J.) and (Z.W.)
| | - Zhexing Wen
- Department of Psychiatry and Behavioral Scieces, Emory University School of Medicine, Atlanta, GA 30322, USA;,Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA;,To whom correspondence should be addressed: (P.J.) and (Z.W.)
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31
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Xu K, Li Y, Allen EG, Jin P. Therapeutic Development for CGG Repeat Expansion-Associated Neurodegeneration. Front Cell Neurosci 2021; 15:655568. [PMID: 34054431 PMCID: PMC8149615 DOI: 10.3389/fncel.2021.655568] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2021] [Accepted: 04/12/2021] [Indexed: 12/16/2022] Open
Abstract
Non-coding repeat expansions, such as CGG, GGC, CUG, CCUG, and GGGGCC, have been shown to be involved in many human diseases, particularly neurological disorders. Of the diverse pathogenic mechanisms proposed in these neurodegenerative diseases, dysregulated RNA metabolism has emerged as an important contributor. Expanded repeat RNAs that form particular structures aggregate to form RNA foci, sequestering various RNA binding proteins and consequently altering RNA splicing, transport, and other downstream biological processes. One of these repeat expansion-associated diseases, fragile X-associated tremor/ataxia syndrome (FXTAS), is caused by a CGG repeat expansion in the 5'UTR region of the fragile X mental retardation 1 (FMR1) gene. Moreover, recent studies have revealed abnormal GGC repeat expansion within the 5'UTR region of the NOTCH2NLC gene in both essential tremor (ET) and neuronal intranuclear inclusion disease (NIID). These CGG repeat expansion-associated diseases share genetic, pathological, and clinical features. Identification of the similarities at the molecular level could lead to a better understanding of the disease mechanisms as well as developing novel therapeutic strategies. Here, we highlight our current understanding of the molecular pathogenesis of CGG repeat expansion-associated diseases and discuss potential therapeutic interventions for these neurological disorders.
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Affiliation(s)
- Keqin Xu
- Department of Human Genetics, School of Medicine, Emory University, Atlanta, GA, United States.,Department of Neurology, Xiangya Hospital, Central South University, Changsha, China
| | - Yujing Li
- Department of Human Genetics, School of Medicine, Emory University, Atlanta, GA, United States
| | - Emily G Allen
- Department of Human Genetics, School of Medicine, Emory University, Atlanta, GA, United States
| | - Peng Jin
- Department of Human Genetics, School of Medicine, Emory University, Atlanta, GA, United States
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Ricci R, Colasante G. CRISPR/dCas9 as a Therapeutic Approach for Neurodevelopmental Disorders: Innovations and Limitations Compared to Traditional Strategies. Dev Neurosci 2021; 43:253-261. [PMID: 33940579 DOI: 10.1159/000515845] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2021] [Accepted: 03/10/2021] [Indexed: 11/19/2022] Open
Abstract
Brain development is a complex process that requires a series of precise and coordinated events to take place. When alterations in some of those events occur, neurodevelopmental disorders (NDDs) may appear, with their characteristic symptoms, including cognitive, social motor deficits, and epilepsy. While pharmacologic treatments have been the only therapeutic options for many years, more recently the research is turning to the direct removal of the underlying genetic cause of each specific NDD. This is possible thanks to the increased knowledge of genetic basis of those diseases and the enormous advances in genome-editing tools. Together with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategies, there is a great development also of nuclease defective Cas9 (dCas9) tools that, with an extreme flexibility, allow the recruitment of specific protein functions to the desired genomic sites. In this work, we review dCas9-based tools and discuss all the published applications in the setting of therapeutic approaches for NDDs at the preclinical level. In particular, dCas9-based therapeutic strategies for Dravet syndrome, transcallosal dysconnectivity caused by mutations in C11orf46 gene, and Fragile X syndrome are presented and discussed. A direct comparison with other possible therapeutic strategies, such as classic gene replacement or CRISPR/Cas9-based strategies, is provided. We also highlight not only those aspects that constitute a clear advantage compared to previous strategies but also the main technical hurdles related to their applications that need to be overcome.
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Affiliation(s)
- Raffaele Ricci
- Stem Cell and Neurogenesis Unit, Division of Neuroscience, Ospedale San Raffaele, Milan, Italy.,Translational and Molecular Medicine PhD Program, DIMET, University of Milan-Bicocca, Milan, Italy
| | - Gaia Colasante
- Stem Cell and Neurogenesis Unit, Division of Neuroscience, Ospedale San Raffaele, Milan, Italy
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Handal T, Eiges R. Correction of Heritable Epigenetic Defects Using Editing Tools. Int J Mol Sci 2021; 22:ijms22083966. [PMID: 33921346 PMCID: PMC8070094 DOI: 10.3390/ijms22083966] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Revised: 04/02/2021] [Accepted: 04/07/2021] [Indexed: 11/21/2022] Open
Abstract
Epimutations refer to mistakes in the setting or maintenance of epigenetic marks in the chromatin. They lead to mis-expression of genes and are often secondary to germline transmitted mutations. As such, they are the cause for a considerable number of genetically inherited conditions in humans. The correction of these types of epigenetic defects constitutes a good paradigm to probe the fundamental mechanisms underlying the development of these diseases, and the molecular basis for the establishment, maintenance and regulation of epigenetic modifications in general. Here, we review the data to date, which is limited to repetitive elements, that relates to the applications of key editing tools for addressing the epigenetic aspects of various epigenetically regulated diseases. For each approach we summarize the efforts conducted to date, highlight their contribution to a better understanding of the molecular basis of epigenetic mechanisms, describe the limitations of each approach and suggest perspectives for further exploration in this field.
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Affiliation(s)
- Tayma Handal
- Stem Cell Research Laboratory, Medical Genetics Institute Shaare Zedek Medical Center, Jerusalem 91031, Israel;
- School of Medicine, The Hebrew University, Campus Ein Kerem, Jerusalem 91120, Israel
| | - Rachel Eiges
- Stem Cell Research Laboratory, Medical Genetics Institute Shaare Zedek Medical Center, Jerusalem 91031, Israel;
- School of Medicine, The Hebrew University, Campus Ein Kerem, Jerusalem 91120, Israel
- Correspondence:
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Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis. Cell Rep 2021; 35:108991. [PMID: 33852833 PMCID: PMC8133829 DOI: 10.1016/j.celrep.2021.108991] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Revised: 03/08/2021] [Accepted: 03/23/2021] [Indexed: 02/06/2023] Open
Abstract
Transcriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to the loss of the RNA-binding protein FMRP. In addition to regulating mRNA translation and protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether loss of FMRP-mediated translational control is related to impaired cell fate specification in the developing human brain remains unknown. Here, we use human patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells and organoids to model neurogenesis in FXS. We developed a high-throughput, in vitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development. Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells. Raj et al. developed a multiparametric assay to measure cellular and molecular phenotypes during neurogenesis in fragile X syndrome iPSC-derived neural cells. Relative to controls, FXS patient cultures have more proliferative cells with increased protein synthesis. Defects in cell fate acquisition can be normalized by inhibiting overactive PI3K signaling.
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Local Protein Translation and RNA Processing of Synaptic Proteins in Autism Spectrum Disorder. Int J Mol Sci 2021; 22:ijms22062811. [PMID: 33802132 PMCID: PMC8001067 DOI: 10.3390/ijms22062811] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Revised: 03/05/2021] [Accepted: 03/06/2021] [Indexed: 12/12/2022] Open
Abstract
Autism spectrum disorder (ASD) is a heritable neurodevelopmental condition associated with impairments in social interaction, communication and repetitive behaviors. While the underlying disease mechanisms remain to be fully elucidated, dysfunction of neuronal plasticity and local translation control have emerged as key points of interest. Translation of mRNAs for critical synaptic proteins are negatively regulated by Fragile X mental retardation protein (FMRP), which is lost in the most common single-gene disorder associated with ASD. Numerous studies have shown that mRNA transport, RNA metabolism, and translation of synaptic proteins are important for neuronal health, synaptic plasticity, and learning and memory. Accordingly, dysfunction of these mechanisms may contribute to the abnormal brain function observed in individuals with autism spectrum disorder (ASD). In this review, we summarize recent studies about local translation and mRNA processing of synaptic proteins and discuss how perturbations of these processes may be related to the pathophysiology of ASD.
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36
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Mani I. CRISPR-Cas9 for treating hereditary diseases. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 181:165-183. [PMID: 34127193 DOI: 10.1016/bs.pmbts.2021.01.017] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
This chapter analyzes to use of the genome editing tool to the treatment of various genetic diseases. The genome editing method could be used to change the DNA in cells or organisms to understand their physiological response. Therefore, a key objective is to present general information about the use of the genome editing tool in a pertinent way. An emerging genome editing technology like a clustered regularly short palindromic repeats (CRISPR) is an extensively expended in biological sciences. CRISPR and CRISPR-associated protein 9 (CRISPR-Cas9) technique is being utilized to edit any DNA mutations associated with hereditary diseases to study in cells (in vitro) and animals (in vivo). Interestingly, CRISPR-Cas9 could be used to the investigation of treatments of various human hereditary diseases such as hemophila, β-thalassemia, cystic fibrosis, Alzheimer's, Huntington's, Parkinson's, tyrosinemia, Duchnene muscular dystrophy, Tay-Sachs, and fragile X syndrome disorders. Furthermore, CRISPR-Cas9 could also be used in other diseases to the improvement of human health. Finally, this chapter discuss current progress to treatment for hereditary diseases using CRISPR-Cas9 technology and highlights associated challenges and future prospects.
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Affiliation(s)
- Indra Mani
- Department of Microbiology, Gargi College, University of Delhi, New Delhi, India.
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Lubroth P, Colasante G, Lignani G. In vivo Genome Editing Therapeutic Approaches for Neurological Disorders: Where Are We in the Translational Pipeline? Front Neurosci 2021; 15:632522. [PMID: 33679313 PMCID: PMC7930815 DOI: 10.3389/fnins.2021.632522] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Accepted: 01/29/2021] [Indexed: 01/10/2023] Open
Abstract
In vivo genome editing tools, such as those based on CRISPR, have been increasingly utilized in both basic and translational neuroscience research. There are currently nine in vivo non-CNS genome editing therapies in clinical trials, and the pre-clinical pipeline of major biotechnology companies demonstrate that this number will continue to grow. Several biotechnology companies commercializing in vivo genome editing and modification technologies are developing therapies for CNS disorders with accompanying large partnering deals. In this review, the authors discuss the current genome editing and modification therapy pipeline and those in development to treat CNS disorders. The authors also discuss the technical and commercial limitations to translation of these same therapies and potential avenues to overcome these hurdles.
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Affiliation(s)
| | - Gaia Colasante
- Stem Cell and Neurogenesis Unit, Division of Neuroscience, Ospedale San Raffaele, Milan, Italy
| | - Gabriele Lignani
- Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology, University College London, London, United Kingdom
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Karapurkar JK, Antao AM, Kim KS, Ramakrishna S. CRISPR-Cas9 based genome editing for defective gene correction in humans and other mammals. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 181:185-229. [PMID: 34127194 DOI: 10.1016/bs.pmbts.2021.01.018] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR/Cas9), derived from bacterial and archean immune systems, has received much attention from the scientific community as a powerful, targeted gene editing tool. The CRISPR/Cas9 system enables a simple, relatively effortless and highly specific gene targeting strategy through temporary or permanent genome regulation or editing. This endonuclease has enabled gene correction by taking advantage of the endogenous homology directed repair (HDR) pathway to successfully target and correct disease-causing gene mutations. Numerous studies using CRISPR support the promise of efficient and simple genome manipulation, and the technique has been validated in in vivo and in vitro experiments, indicating its potential for efficient gene correction at any genomic loci. In this chapter, we detailed various strategies related to gene editing using the CRISPR/Cas9 system. We also outlined strategies to improve the efficiency of gene correction via the HDR pathway and to improve viral and non-viral mediated gene delivery methods, with an emphasis on their therapeutic potential for correcting genetic disorder in humans and other mammals.
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Affiliation(s)
| | - Ainsley Mike Antao
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea
| | - Kye-Seong Kim
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea.
| | - Suresh Ramakrishna
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea.
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Nobile V, Pucci C, Chiurazzi P, Neri G, Tabolacci E. DNA Methylation, Mechanisms of FMR1 Inactivation and Therapeutic Perspectives for Fragile X Syndrome. Biomolecules 2021; 11:biom11020296. [PMID: 33669384 PMCID: PMC7920310 DOI: 10.3390/biom11020296] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 02/04/2021] [Accepted: 02/06/2021] [Indexed: 12/13/2022] Open
Abstract
Among the inherited causes of intellectual disability and autism, Fragile X syndrome (FXS) is the most frequent form, for which there is currently no cure. In most FXS patients, the FMR1 gene is epigenetically inactivated following the expansion over 200 triplets of a CGG repeat (FM: full mutation). FMR1 encodes the Fragile X Mental Retardation Protein (FMRP), which binds several mRNAs, mainly in the brain. When the FM becomes methylated at 10-12 weeks of gestation, the FMR1 gene is transcriptionally silent. The molecular mechanisms involved in the epigenetic silencing are not fully elucidated. Among FXS families, there is a rare occurrence of males carrying a FM, which remains active because it is not methylated, thus ensuring enough FMRPs to allow for an intellectual development within normal range. Which mechanisms are responsible for sparing these individuals from being affected by FXS? In order to answer this critical question, which may have possible implications for FXS therapy, several potential epigenetic mechanisms have been described. Here, we focus on current knowledge about the role of DNA methylation and other epigenetic modifications in FMR1 gene silencing.
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Affiliation(s)
- Veronica Nobile
- Sezione di Medicina Genomica, Dipartimento Scienze della Vita e Sanità Pubblica, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (V.N.); (C.P.); (P.C.); (G.N.)
| | - Cecilia Pucci
- Sezione di Medicina Genomica, Dipartimento Scienze della Vita e Sanità Pubblica, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (V.N.); (C.P.); (P.C.); (G.N.)
| | - Pietro Chiurazzi
- Sezione di Medicina Genomica, Dipartimento Scienze della Vita e Sanità Pubblica, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (V.N.); (C.P.); (P.C.); (G.N.)
- Fondazione Policlinico Universitario A. Gemelli IRCCS, UOC Genetica Medica, 00168 Rome, Italy
| | - Giovanni Neri
- Sezione di Medicina Genomica, Dipartimento Scienze della Vita e Sanità Pubblica, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (V.N.); (C.P.); (P.C.); (G.N.)
- Greenwood Genetic Center, JC Self Research Institute, Greenwood, SC 29646, USA
| | - Elisabetta Tabolacci
- Sezione di Medicina Genomica, Dipartimento Scienze della Vita e Sanità Pubblica, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (V.N.); (C.P.); (P.C.); (G.N.)
- Correspondence: ; Tel.: +39-06-30154606
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40
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Loss of the fragile X syndrome protein FMRP results in misregulation of nonsense-mediated mRNA decay. Nat Cell Biol 2021; 23:40-48. [PMID: 33420492 PMCID: PMC8273690 DOI: 10.1038/s41556-020-00618-1] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Accepted: 11/29/2020] [Indexed: 01/28/2023]
Abstract
Loss of the fragile X protein FMRP is a leading cause of intellectual disability and autism1,2, but the underlying mechanism remains poorly understood. We report that FMRP deficiency results in hyperactivated nonsense-mediated mRNA decay (NMD)3,4 in human SH-SY5Y neuroblastoma cells and fragile X syndrome (FXS) fibroblast-derived induced pluripotent stem cells (iPSCs). We examined the underlying mechanism and found that the key NMD factor UPF1 binds directly to FMRP, promoting FMRP binding to NMD targets. Our data indicate that FMRP acts as an NMD repressor. In the absence of FMRP, NMD targets are relieved from FMRP-mediated translational repression so that their half-lives are decreased and, for those NMD targets encoding NMD factors, increased translation produces abnormally high factor levels despite their hyperactivated NMD. Transcriptome-wide alterations caused by NMD hyperactivation have a role in the FXS phenotype. Consistent with this, small-molecule-mediated inhibition of hyperactivated NMD, which typifies iPSCs derived from patients with FXS, restores a number of neurodifferentiation markers, including those not deriving from NMD targets. Our mechanistic studies reveal that many molecular abnormalities in FMRP-deficient cells are attributable-either directly or indirectly-to misregulated NMD.
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41
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Sabitha KR, Shetty AK, Upadhya D. Patient-derived iPSC modeling of rare neurodevelopmental disorders: Molecular pathophysiology and prospective therapies. Neurosci Biobehav Rev 2020; 121:201-219. [PMID: 33370574 DOI: 10.1016/j.neubiorev.2020.12.025] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2020] [Revised: 12/18/2020] [Accepted: 12/19/2020] [Indexed: 12/12/2022]
Abstract
The pathological alterations that manifest during the early embryonic development due to inherited and acquired factors trigger various neurodevelopmental disorders (NDDs). Besides major NDDs, there are several rare NDDs, exhibiting specific characteristics and varying levels of severity triggered due to genetic and epigenetic anomalies. The rarity of subjects, paucity of neural tissues for detailed analysis, and the unavailability of disease-specific animal models have hampered detailed comprehension of rare NDDs, imposing heightened challenge to the medical and scientific community until a decade ago. The generation of functional neurons and glia through directed differentiation protocols for patient-derived iPSCs, CRISPR/Cas9 technology, and 3D brain organoid models have provided an excellent opportunity and vibrant resource for decoding the etiology of brain development for rare NDDs caused due to monogenic as well as polygenic disorders. The present review identifies cellular and molecular phenotypes demonstrated from patient-derived iPSCs and possible therapeutic opportunities identified for these disorders. New insights to reinforce the existing knowledge of the pathophysiology of these disorders and prospective therapeutic applications are discussed.
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Affiliation(s)
- K R Sabitha
- Centre for Molecular Neurosciences, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India
| | - Ashok K Shetty
- Institute for Regenerative Medicine, Department of Molecular and Cellular Medicine, Texas A&M University College of Medicine, College Station, TX, USA.
| | - Dinesh Upadhya
- Centre for Molecular Neurosciences, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.
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42
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Nakayama Y, Adachi K, Shioda N, Maeta S, Nanba E, Kugoh H. Establishment of FXS-A9 panel with a single human X chromosome from fragile X syndrome-associated individual. Exp Cell Res 2020; 398:112419. [PMID: 33296661 DOI: 10.1016/j.yexcr.2020.112419] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2020] [Revised: 11/20/2020] [Accepted: 11/26/2020] [Indexed: 11/29/2022]
Abstract
Fragile X syndrome (FXS) is the most common inheritable form of intellectual disability. FMR1, the gene responsible for FXS, is located on human chromosome Xq27.3 and contains a stretch of CGG trinucleotide repeats in its 5' untranslated region. FXS is caused by CGG repeats that expand beyond 200, resulting in FMR1 silencing via promoter hypermethylation. The molecular mechanism underlying CGG repeat expansion, a fundamental cause of FXS, remains poorly understood, partly due to a lack of experimental systems. Accumulated evidence indicates that the large chromosomal region flanking a CGG repeat is critical for repeat dynamics. In the present study, we isolated and introduced whole human X chromosomes from healthy, FXS premutation carriers, or FXS patients who carried disease condition-associated CGG repeat lengths, into mouse A9 cells via microcell-mediated chromosome transfer. The CGG repeat length-associated methylation status and human FMR1 expression in these monochromosomal hybrid cells mimicked those in humans. Thus, this set of A9 cells containing CGG repeats from three different origins (FXS-A9 panel) may provide a valuable resource for investigating a series of genetic and epigenetic CGG repeat dynamics during FXS pathogenesis.
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Affiliation(s)
- Yuji Nakayama
- Division of Radioisotope Science, Research Initiative Center, Organization for Research Initiative and Promotion, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan
| | - Kaori Adachi
- Division of Genomic Science, Research Initiative Center, Organization for Research Initiative and Promotion, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan
| | - Nofirifumi Shioda
- Department of Genomic Neurology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, 860-0811, Japan
| | - Shoya Maeta
- Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan
| | - Eiji Nanba
- Office for Research Strategy, Organization for Research Initiative and Promotion, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan
| | - Hiroyuki Kugoh
- Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan; Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan.
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43
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Winkelsas AM, Fischbeck KH. Nucleic acid therapeutics in neurodevelopmental disease. Curr Opin Genet Dev 2020; 65:112-116. [PMID: 32623324 PMCID: PMC11441424 DOI: 10.1016/j.gde.2020.05.022] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2020] [Accepted: 05/22/2020] [Indexed: 12/16/2022]
Abstract
Nucleic acid therapeutics allow sequence-based targeting of mutation-harboring genes. They can be used to increase the expression and function of disease genes or to decrease the expression of toxic gene products. Antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and gene-replacement therapies have received FDA approval, and in vivo gene editing applications are currently under development. Special consideration should be given to target engagement in neurons and amelioration of neurological phenotypes. Here we discuss the uses and limitations of different nucleic acid therapeutics, highlighting examples in the clinical and pre-clinical application of these modalities for the treatment of neurodevelopmental diseases.
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Affiliation(s)
- Audrey M Winkelsas
- Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, United States; Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Kenneth H Fischbeck
- Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, United States.
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Turner TJ, Zourray C, Schorge S, Lignani G. Recent advances in gene therapy for neurodevelopmental disorders with epilepsy. J Neurochem 2020; 157:229-262. [PMID: 32880951 PMCID: PMC8436749 DOI: 10.1111/jnc.15168] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Revised: 08/18/2020] [Accepted: 08/20/2020] [Indexed: 12/14/2022]
Abstract
Neurodevelopmental disorders can be caused by mutations in neuronal genes fundamental to brain development. These disorders have severe symptoms ranging from intellectually disability, social and cognitive impairments, and a subset are strongly linked with epilepsy. In this review, we focus on those neurodevelopmental disorders that are frequently characterized by the presence of epilepsy (NDD + E). We loosely group the genes linked to NDD + E with different neuronal functions: transcriptional regulation, intrinsic excitability and synaptic transmission. All these genes have in common a pivotal role in defining the brain architecture and function during early development, and when their function is altered, symptoms can present in the first stages of human life. The relationship with epilepsy is complex. In some NDD + E, epilepsy is a comorbidity and in others seizures appear to be the main cause of the pathology, suggesting that either structural changes (NDD) or neuronal communication (E) can lead to these disorders. Furthermore, grouping the genes that cause NDD + E, we review the uses and limitations of current models of the different disorders, and how different gene therapy strategies are being developed to treat them. We highlight where gene replacement may not be a treatment option, and where innovative therapeutic tools, such as CRISPR‐based gene editing, and new avenues of delivery are required. In general this group of genetically defined disorders, supported increasing knowledge of the mechanisms leading to neurological dysfunction serve as an excellent collection for illustrating the translational potential of gene therapy, including newly emerging tools.
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Affiliation(s)
- Thomas J Turner
- Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology, London, UK
| | - Clara Zourray
- Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology, London, UK.,Department of Pharmacology, UCL School of Pharmacy, London, UK
| | | | - Gabriele Lignani
- Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology, London, UK
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45
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High rate of HDR in gene editing of p.(Thr158Met) MECP2 mutational hotspot. Eur J Hum Genet 2020; 28:1231-1242. [PMID: 32332872 PMCID: PMC7609331 DOI: 10.1038/s41431-020-0624-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Revised: 03/16/2020] [Accepted: 03/24/2020] [Indexed: 02/06/2023] Open
Abstract
Rett syndrome is a progressive neurodevelopmental disorder which affects almost exclusively girls, caused by variants in MECP2 gene. Effective therapies for this devastating disorder are not yet available and the need for tight regulation of MECP2 expression for brain to properly function makes gene replacement therapy risky. For this reason, gene editing with CRISPR/Cas9 technology appears as a preferable option for the development of new therapies. To study the disease, we developed and characterized a human neuronal model obtained by genetic reprogramming of patient-derived primary fibroblasts into induced Pluripotent Stem Cells. This cellular model represents an important source for our studies, aiming to correct MECP2 variants in neurons which represent the primarily affected cell type. We engineered a gene editing toolkit composed by a two-plasmid system to correct a hotspot missense variant in MECP2, c.473 C > T (p.(Thr158Met)). The first construct expresses the variant-specific sgRNA and the Donor DNA along with a fluorescent reporter system. The second construct brings Cas9 and targets for auto-cleaving, to avoid long-term Cas9 expression. NGS analysis on sorted cells from four independent patients demonstrated an exceptionally high editing efficiency, with up to 80% of HDR and less than 1% of indels in all patients, outlining the relevant potentiality of the approach for Rett syndrome therapy.
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46
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Ramezankhani R, Minaei N, Haddadi M, Torabi S, Hesaraki M, Mirzaei H, Vosough M, Verfaillie CM. Gene editing technology for improving life quality: A dream coming true? Clin Genet 2020; 99:67-83. [PMID: 32506418 DOI: 10.1111/cge.13794] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2020] [Revised: 06/02/2020] [Accepted: 06/03/2020] [Indexed: 12/13/2022]
Abstract
The fact that monogenic diseases are related to mutations in one specific gene, make gene correction one of the promising strategies in the future to treat genetic diseases or alleviate their symptoms. From this perspective, and along with recent advances in technology, genome editing tools have gained momentum and developed fast. In fact, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) are regarded as novel technologies which are able to correct a number of genetic aberrations in vitro and in vivo. The number of ongoing clinical trials employing these tools has been increased showing the encouraging outcomes of these tools. However, there are still some major challenges with respect to the safety profile and directed delivery of them. In this paper, we provided updated information regarding the history, nature, methods of delivery, and application of the above-mentioned gene editing tools along with the meganucleases (an older similar tool) based on published in vitro and in vivo studies and introduced clinical trials which employed these technologies.
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Affiliation(s)
- Roya Ramezankhani
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.,Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.,Department of Development and Regeneration, KU Leuven Stem Cell Institute, Leuven, Belgium
| | - Neda Minaei
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.,Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
| | - Mahnaz Haddadi
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
| | - Shukoofeh Torabi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.,Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
| | - Mahdi Hesaraki
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
| | - Hamed Mirzaei
- Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran
| | - Massoud Vosough
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.,Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
| | - Catherine M Verfaillie
- Department of Development and Regeneration, Stem Cell Institute, KU Leuven, Leuven, Belgium
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Agote-Aran A, Schmucker S, Jerabkova K, Jmel Boyer I, Berto A, Pacini L, Ronchi P, Kleiss C, Guerard L, Schwab Y, Moine H, Mandel JL, Jacquemont S, Bagni C, Sumara I. Spatial control of nucleoporin condensation by fragile X-related proteins. EMBO J 2020; 39:e104467. [PMID: 32706158 PMCID: PMC7560220 DOI: 10.15252/embj.2020104467] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 06/16/2020] [Accepted: 06/22/2020] [Indexed: 01/14/2023] Open
Abstract
Nucleoporins (Nups) build highly organized nuclear pore complexes (NPCs) at the nuclear envelope (NE). Several Nups assemble into a sieve‐like hydrogel within the central channel of the NPCs. In the cytoplasm, the soluble Nups exist, but how their assembly is restricted to the NE is currently unknown. Here, we show that fragile X‐related protein 1 (FXR1) can interact with several Nups and facilitate their localization to the NE during interphase through a microtubule‐dependent mechanism. Downregulation of FXR1 or closely related orthologs FXR2 and fragile X mental retardation protein (FMRP) leads to the accumulation of cytoplasmic Nup condensates. Likewise, models of fragile X syndrome (FXS), characterized by a loss of FMRP, accumulate Nup granules. The Nup granule‐containing cells show defects in protein export, nuclear morphology and cell cycle progression. Our results reveal an unexpected role for the FXR protein family in the spatial regulation of nucleoporin condensation.
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Affiliation(s)
- Arantxa Agote-Aran
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.,Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France.,Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Stephane Schmucker
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.,Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France.,Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Katerina Jerabkova
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.,Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France.,Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Inès Jmel Boyer
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.,Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France.,Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Alessandro Berto
- Institut Jacques Monod, CNRS UMR7592-Université Paris Diderot, Sorbonne Paris Cité, Paris, France.,Ecole Doctorale SDSV, Université Paris Sud, Orsay, France
| | - Laura Pacini
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
| | - Paolo Ronchi
- European Molecular Biology Laboratory, Electron Microscopy Core Facility, Heidelberg, Germany
| | - Charlotte Kleiss
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.,Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France.,Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Laurent Guerard
- Imaging Core Facility, Biozentrum, University of Basel, Basel, Switzerland
| | - Yannick Schwab
- European Molecular Biology Laboratory, Electron Microscopy Core Facility, Heidelberg, Germany.,European Molecular Biology Laboratory, European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Heidelberg, Germany
| | - Hervé Moine
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.,Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France.,Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Jean-Louis Mandel
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.,Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France.,Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
| | - Sebastien Jacquemont
- Service de Génétique Médicale, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.,CHU Sainte-Justine Research Centre, University of Montreal, Montreal, QC, Canada
| | - Claudia Bagni
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.,Department of Fundamental Neuroscience, University of Lausanne, Lausanne, Switzerland
| | - Izabela Sumara
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.,Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France.,Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France.,Université de Strasbourg, Strasbourg, France
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48
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Mystery of Expansion: DNA Metabolism and Unstable Repeats. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2020; 1241:101-124. [PMID: 32383118 DOI: 10.1007/978-3-030-41283-8_7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/11/2023]
Abstract
The mammalian genome mostly contains repeated sequences. Some of these repeats are in the regulatory elements of genes, and their instability, particularly the propensity to change the repeat unit number, is responsible for 36 well-known neurodegenerative human disorders. The mechanism of repeat expansion has been an unsolved question for more than 20 years. There are a few hypotheses describing models of mutation development. Every hypothesis is based on assumptions about unusual secondary structures that violate DNA metabolism processes in the cell. Some models are based on replication errors, and other models are based on mismatch repair or base excision repair errors. Additionally, it has been shown that epigenetic regulation of gene expression can influence the probability and frequency of expansion. In this review, we consider the molecular bases of repeat expansion disorders and discuss possible mechanisms of repeat expansion during cell metabolism.
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49
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Poeta L, Drongitis D, Verrillo L, Miano MG. DNA Hypermethylation and Unstable Repeat Diseases: A Paradigm of Transcriptional Silencing to Decipher the Basis of Pathogenic Mechanisms. Genes (Basel) 2020; 11:E684. [PMID: 32580525 PMCID: PMC7348995 DOI: 10.3390/genes11060684] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Revised: 06/18/2020] [Accepted: 06/19/2020] [Indexed: 12/12/2022] Open
Abstract
Unstable repeat disorders comprise a variable group of incurable human neurological and neuromuscular diseases caused by an increase in the copy number of tandem repeats located in various regions of their resident genes. It has become clear that dense DNA methylation in hyperexpanded non-coding repeats induces transcriptional silencing and, subsequently, insufficient protein synthesis. However, the ramifications of this paradigm reveal a far more profound role in disease pathogenesis. This review will summarize the significant progress made in a subset of non-coding repeat diseases demonstrating the role of dense landscapes of 5-methylcytosine (5mC) as a common disease modifier. However, the emerging findings suggest context-dependent models of 5mC-mediated silencing with distinct effects of excessive DNA methylation. An in-depth understanding of the molecular mechanisms underlying this peculiar group of human diseases constitutes a prerequisite that could help to discover novel pathogenic repeat loci, as well as to determine potential therapeutic targets. In this regard, we report on a brief description of advanced strategies in DNA methylation profiling for the identification of unstable Guanine-Cytosine (GC)-rich regions and on promising examples of molecular targeted therapies for Fragile X disease (FXS) and Friedrich ataxia (FRDA) that could pave the way for the application of this technique in other hypermethylated expansion disorders.
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Affiliation(s)
- Loredana Poeta
- Institute of Genetics and Biophysics “Adriano Buzzati-Traverso”, CNR, 80131 Naples, Italy; (L.P.); (D.D.); (L.V.)
| | - Denise Drongitis
- Institute of Genetics and Biophysics “Adriano Buzzati-Traverso”, CNR, 80131 Naples, Italy; (L.P.); (D.D.); (L.V.)
| | - Lucia Verrillo
- Institute of Genetics and Biophysics “Adriano Buzzati-Traverso”, CNR, 80131 Naples, Italy; (L.P.); (D.D.); (L.V.)
- Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, 81100 Caserta, Italy
| | - Maria Giuseppina Miano
- Institute of Genetics and Biophysics “Adriano Buzzati-Traverso”, CNR, 80131 Naples, Italy; (L.P.); (D.D.); (L.V.)
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50
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Croci S, Carriero ML, Capitani K, Daga S, Donati F, Papa FT, Frullanti E, Lopergolo D, Lamacchia V, Tita R, Giliberti A, Benetti E, Niccheri F, Furini S, Lo Rizzo C, Conticello SG, Renieri A, Meloni I. AAV-mediated FOXG1 gene editing in human Rett primary cells. Eur J Hum Genet 2020; 28:1446-1458. [PMID: 32541681 PMCID: PMC7608362 DOI: 10.1038/s41431-020-0652-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2019] [Revised: 04/16/2020] [Accepted: 04/24/2020] [Indexed: 12/21/2022] Open
Abstract
Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry+/EGFP+ transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20–35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.
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Affiliation(s)
| | | | - Katia Capitani
- Medical Genetics, University of Siena, Siena, Italy.,Molecular Mechanisms of Oncogenesis, ISPRO Core Research Laboratory (CRL), Firenze, Italy
| | - Sergio Daga
- Medical Genetics, University of Siena, Siena, Italy
| | - Francesco Donati
- Medical Genetics, University of Siena, Siena, Italy.,Molecular Mechanisms of Oncogenesis, ISPRO Core Research Laboratory (CRL), Firenze, Italy
| | | | | | - Diego Lopergolo
- Medical Genetics, University of Siena, Siena, Italy.,Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy
| | - Vittoria Lamacchia
- Medical Genetics, University of Siena, Siena, Italy.,Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy
| | - Rossella Tita
- Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy
| | | | - Elisa Benetti
- Medical Genetics, University of Siena, Siena, Italy.,Department of Medical Biotechnologies, University of Siena, Siena, Italy
| | - Francesca Niccheri
- Molecular Mechanisms of Oncogenesis, ISPRO Core Research Laboratory (CRL), Firenze, Italy
| | - Simone Furini
- Department of Medical Biotechnologies, University of Siena, Siena, Italy
| | - Caterina Lo Rizzo
- Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy
| | | | - Alessandra Renieri
- Medical Genetics, University of Siena, Siena, Italy. .,Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy.
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