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Correction of the Exon 2, Exon 2-8 and Exons 8-9 duplications in DMD patient myogenic cells by a Single CRISPR/Cas9 system

ABSTRACT: Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guideRNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2 to 9, and exons 8 to 9 in the DMD gene. Immunostaining on CRISPR corrected derived myotubes demonstrated the rescue of dystrophin protein. RNA sequencing of the corrected differentiated myogenic derivatives indicated rescue of dystrophin related molecular pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we demonstrated a single guide CRISPR strategy that can be utilized to delete multi-exons duplication in patient DMD genes without significant off target effect, leading to restoration of transcriptome in the corrected patient derived cells. This study further expands the application of single guide CRISPR strategy as a potential therapeutic approach for patients with larger DNA region duplication in DMD patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE248257 | GEO | 2024/01/01

REPOSITORIES: GEO

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Correction of Exon 2, Exon 2-9 and Exons 8-9 duplications in DMD patient myogenic cells by a Single CRISPR/Cas9 system

Project description:Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2 to 9, and exons 8 to 9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the corrected myotubes indicated rescue of dystrophin related molecular pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. This resulted in the restoration of dystrophin expression and reversal of transcriptome pathological signatures in the corrected patient-derived cells. Our study contributes valuable insights into the safety and efficacy about the application of the single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.

2024-10-08 | GSE272233 | GEO

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

Project description:Exonic duplications account for 10-15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a novel CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one gRNA directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications independently from the duplication extension.

2017-06-23 | GSE83658 | GEO

A novel CRISPR-Cas9 strategy to target DYSTROPHIN mutations downstream of exon 44 in patient-specific DMD iPSCs

Project description:Here, we performed CRISPR-cas9 mediated genetic correction in DMD patient derived iPSC and then differentiated them into in vitro myotubes. Pairing bulk RNA sequencing between Wild-type (WT), DMD and the corrected counterparts we were able to differentiate the molecular changes that occurs during the absence of dystrophin.

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In vivo genome editing restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers [WES]

Project description:Duchenne muscular dystrophy (DMD) is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. In this study we developed a novel strategy for reframing DMD mutations by CRISPR-mediated large-scale excision of exons 46–54. We compared this approach to other DMD rescue strategies using DMD patient-derived primary muscle-derived stem cells (MDSCs) and found that it showed the highest efficiency in terms of restoring of dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cpf1)-mediated genome editing could correct DMD mutation with higher specificity than CRISPR-associated protein 9 (Cas9). Furthermore, A patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Dystrophin expression levels were increased by 10%–30% in human DMD muscle fibers. The restored dystrophin in vivo was functional, as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. This study provides a sensitive indicator for in vivo efficacy of gene editing and lays the foundation for a clinical trial of DMD treatment with gene editing technology.

2021-02-28 | GSE167586 | GEO

In vivo genome editing restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers [RNA-seq]

2021-02-28 | GSE167585 | GEO

In vivo genome editing restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers [Nanopore sequencing]

2021-02-28 | GSE167584 | GEO

In vivo genome editing restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers [Amplicon-seq]

2021-02-28 | GSE167572 | GEO

In vivo genome editing restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers [GUIDE-seq]

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Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52

Project description:Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the dystrophin (DMD) gene, leading to the complete absence of DMD and progressive degeneration of skeletal and heart muscles. Expression of an internally shortened dystrophin in DMD subjects (DMDΔ52) can be achieved by skipping DMD exon 51 to reframe the transcript. To predict the best possible outcome of this therapeutic strategy, we generated transgenic pigs lacking DMD exon 51 and 52, additionally representing a new model for Becker muscular dystrophy (BMD). To inspect the proteome alterations caused by the different dystrophin mutations in an unbiased and comprehensive manner, we performed a label-free liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) of myocardial and skeletal muscle samples from wild-type (WT), DMDΔ52 and DMDΔ51-52 pigs.

2023-07-11 | PXD039533 | Pride

Targeting Duchenne Muscular Dystrophy by Skipping DMD Exon 45 with Base Editors

Project description:Duchenne muscular dystrophy is an X-linked monogenic disease caused by mutations in the dystrophin gene (DMD) and characterized by progressive muscle weakness leading to loss of ambulation and significantly decreased life expectancy. Since the current standard of care for Duchenne muscular dystrophy is to merely treat symptoms, there is a dire need for novel treatment modalities that can correct the underlying genetic mutations. While several gene replacement therapies are being explored in clinical trials, one emerging approach that can directly correct mutations in genomic DNA is base editing. We have recently developed CRISPR-SKIP, a base editing strategy to induce permanent exon skipping by introducing C>T or A>G mutations at splice acceptors in genomic DNA, which can be utilized therapeutically to recover dystrophin expression when a genomic deletion leads to an out-of-frame DMD transcript. We now demonstrate that CRISPR-SKIP can be adapted to correct some forms of Duchenne muscular dystrophy by disrupting the splice acceptor in human DMD exon 45 with high efficiency, which enables open reading frame recovery and restoration of dystrophin expression. We also demonstrate that AAV-delivered split-intein base editors edit the splice acceptor of DMD exon 45 in cultured human cells and in vivo, highlighting the therapeutic potential of this strategy.

2023-07-20 | GSE235673 | GEO

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