Efficient and Precise CRISPR/Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent Stem Cells

Le, Thi Thanh Huong; Tran, Ngoc Tung; Dao, Thi Mai Lan; Nguyen, Dinh Dung; Duong, Huy; Ha, Thi Lien; Kühn, Ralf; Nguyen, Thanh Phuc; Rajewsky, Klaus; Chu, Van Trung

doi:10.3389/fgene.2019.00625

Cited by 25 publications

(16 citation statements)

References 35 publications

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“…Delivery was achieved via plasmid DNA (pDNA) transfection, wherein one plasmid carries the Cas9 enzyme and a second plasmid carries the sgRNA and HDR template. As pDNA transfection never reaches every cell of an in vitro population and edit efficiency of CRISPR-HDR is low, iPSCs were sorted based on fluorescence, leading to editing efficiencies of 20% for R270X [13] and 80% for T158M [14]. CRISPR-HDR was also used to correct the nonsense mutation R841X in SHANK2 [20], which leads to monogenic ASD.…”

Section: Permanent Gene Therapies That Alter the Genomementioning

confidence: 99%

Gene Therapies for Monogenic Autism Spectrum Disorders

Weuring

Geerligs

Koeleman

2021

Genes

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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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Section: Permanent Gene Therapies That Alter the Genomementioning

confidence: 99%

Gene Therapies for Monogenic Autism Spectrum Disorders

Weuring

Geerligs

Koeleman

2021

Genes

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“…Rett syndrome CRISPR-Cas9 HR in human iPS cells to correct disease variants. [525,526] Fragile X syndrome CRISPR-dCas9Tet1 demethylation of the FMR1 gene Lentiviral delivery.…”

Section: Mouse Activation Of Psd95 Leads To Memory Rescue In Aged Andmentioning

confidence: 99%

Chromatin Alterations in Neurological Disorders and Strategies of (Epi)Genome Rescue

et al. 2021

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Neurological disorders (NDs) comprise a heterogeneous group of conditions that affect the function of the nervous system. Often incurable, NDs have profound and detrimental consequences on the affected individuals’ lives. NDs have complex etiologies but commonly feature altered gene expression and dysfunctions of the essential chromatin-modifying factors. Hence, compounds that target DNA and histone modification pathways, the so-called epidrugs, constitute promising tools to treat NDs. Yet, targeting the entire epigenome might reveal insufficient to modify a chosen gene expression or even unnecessary and detrimental to the patients’ health. New technologies hold a promise to expand the clinical toolkit in the fight against NDs. (Epi)genome engineering using designer nucleases, including CRISPR-Cas9 and TALENs, can potentially help restore the correct gene expression patterns by targeting a defined gene or pathway, both genetically and epigenetically, with minimal off-target activity. Here, we review the implication of epigenetic machinery in NDs. We outline syndromes caused by mutations in chromatin-modifying enzymes and discuss the functional consequences of mutations in regulatory DNA in NDs. We review the approaches that allow modifying the (epi)genome, including tools based on TALENs and CRISPR-Cas9 technologies, and we highlight how these new strategies could potentially change clinical practices in the treatment of NDs.

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“…Potential methods to achieve this goal include, but are not limited to: Gene correction, virus-mediated MECP2 delivery, and MeCP2 expression from the inactivated X-chromosome [ 3 ]. More recently, clustered regularly interspaced short palindromic repeats (CRISPR) technology was used to attempt to correct the disease relevant regions in the MECP2 sequence [ 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

Expression, Purification, Characterization and Cellular Uptake of MeCP2 Variants

et al. 2022

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The transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2) is an intrinsically disordered protein, mutations in which, are implicated in the onset of Rett Syndrome, a severe and debilitating neurodevelopmental disorder. Delivery of this protein fused to the cell-penetrating peptide TAT could allow for the intracellular replenishment of functional MeCP2 and hence potentially serve as a prospective Rett Syndrome therapy. This work outlines the expression, purification and characterization of various TAT-MeCP2 constructs as well as their full-length and shortened eGFP fusion variants. The latter two constructs were used for intracellular uptake studies with subsequent analysis via western blotting and live-cell imaging. All purified MeCP2 samples exhibited high degree of stability and very little aggregation propensity. Full length and minimal TAT-MeCP2-eGFP were found to efficiently transduce into human dermal and murine fibroblasts and localize to cell nuclei. These findings clearly support the utility of MeCP2-based protein replacement therapy as a potential Rett Syndrome treatment option.

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Efficient and Precise CRISPR/Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent Stem Cells

Cited by 25 publications

References 35 publications

Gene Therapies for Monogenic Autism Spectrum Disorders

Gene Therapies for Monogenic Autism Spectrum Disorders

Chromatin Alterations in Neurological Disorders and Strategies of (Epi)Genome Rescue

Expression, Purification, Characterization and Cellular Uptake of MeCP2 Variants

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