Inherited epigenetic repression of the Celf2a splicing factor mediates the amelioration of a Duchenne phenotype
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE139571 | GEO | 2020/07/01
REPOSITORIES: GEO
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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
Project description:Purpose: Duchenne muscular dystrophy (DMD) is typically caused by mutations that disrupt the DMD reading frame, but nonsense mutations in the 5’ part of the gene lead to the utilization of an internal ribosomal entry site (IRES) in exon 5, resulting in expression of a highly functional N-truncated dystrophin. We have developed an AAV9 vector expressing U7 small nuclear RNAs targeting DMD exon 2 and tested it in a mouse model containing a duplication of exon 2, in which skipping of both exon 2 copies results in IRES-induced expression, and skipping of only one exon 2 leads to wild-type dystrophin expression. Methods: One-time intravascular injection of an AAV9 vector expressing U7 small nuclear RNAs targeting Dmd exon 2 at either P0-P1 or at 2 months of age results in efficient exon skipping and dystrophin expression, and significant protection from functional and pathologic deficits. Results: In mice treated neonatally, dystrophin immunofluorescence reached 49-85% of normal signal intensity and 76-99% dystrophin-positive fibers, with near-complete correction of dystrophic pathology, and these beneficial effects showed no significant signs of declining for at least 6 months. RNA sequencing (RNA-Seq) and Ribosome Profiling (RPF-Seq) indicated that treatment restored a ‘non-dystrophic’ gene expression profile by reversing the direction and magnitude of differentially expressed genes previously identified in dystrophic skeletal muscle from DMD patients. The dystrophin transcript showed clearly increased RPF reads in the treated sample, indicating that the treatment specifically increased dystrophin translation. Conclusions: The results demonstrate the robustness, durability, and safety of exon 2 skipping following delivery of scAAV9.U7snRNA.ACCA, supporting its clinical use.
2022-01-29 | GSE195480 | GEO
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
Project description:Absence of the dystrophin gene in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory medicine, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the efficacy of correction of DMD using the CRISPR/Cas9 system in mitigating the cardiomyopathy phenotype in DMD. To define the effects of CRISPR/Cas9 genome editing on structural, functional and transcriptional dysregulation in DMD-associated cardiomyopathy. We generated induced pluripotent stem cells (iPSCs) from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. Here, we targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD (cDMD) iPSC lines, wherein the DMD open reading frame was restored via reframing (RF) or exon skipping (ES). While DMD cardiomyocytes (CMs) demonstrated morphologic, structural and functional deficits compared to control CMs, CMs from both cDMD lines were similar to control CMs. Bulk RNA-sequencing of DMD CMs showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in cDMD CMs. We then corrected DMD CMs by adenoviral delivery of Cas9/gRNA and showed that postnatal correction of DMD CMs reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts showed reduced transcriptional dysregulation in CMs and fibroblasts in corrected mice compared with DMD mice, consistent with reduced histopathologic changes.We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional and transcriptional dysregulation consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in iPSC-CMs and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.
2021-08-02 | GSE169551 | GEO
Project description:Rationale – Absence of the dystrophin gene in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory medicine, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the efficacy of correction of DMD using the CRISPR/Cas9 system in mitigating the cardiomyopathy phenotype in DMD. Objective – To define the effects of CRISPR/Cas9 genome editing on structural, functional and transcriptional dysregulation in DMD-associated cardiomyopathy. Methods and Results – We generated induced pluripotent stem cells (iPSCs) from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. Here, we targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD (cDMD) iPSC lines, wherein the DMD open reading frame was restored via reframing (RF) or exon skipping (ES). While DMD cardiomyocytes (CMs) demonstrated morphologic, structural and functional deficits compared to control CMs, CMs from both cDMD lines were similar to control CMs. Bulk RNA-sequencing of DMD CMs showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in cDMD CMs. We then corrected DMD CMs by adenoviral delivery of Cas9/gRNA and showed that postnatal correction of DMD CMs reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts showed reduced transcriptional dysregulation in CMs and fibroblasts in corrected mice compared with DMD mice, consistent with reduced histopathologic changes. Conclusions – We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional and transcriptional dysregulation consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in iPSC-CMs and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.
2021-08-06 | GSE169190 | GEO
Project description:Targeting Duchenne Muscular Dystrophy by Skipping DMD Exon 45 with Base Editors
| PRJNA986884 | ENA
Project description:The Dystrophin gene (DMD) is the largest gene in the human genome, mapping on Xp21, spanning 2.2Mb and accounting for approximately 1% of the entire human genome. Mutations in this gene cause Duchenne and Becker muscular dystrophy, X-linked dilated cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16 hours, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA pol II along the entire 2.2 Mb of the DMD locus with just one site of pausing around intron 52. More importantly, epigenetic marks highlighted the existence of four novel cis-DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies.
2018-03-01 | GSE66571 | GEO
Project description:Dystrophin proteomic regulation in Muscular Dystrophies (MD) remains unclear. We report that a long noncoding RNA (lncRNA) H19 associates with dystrophin. To investigate the biological roles of this interaction in vivo, we performed mass spectrometry analysis of dystrophin and its associated proteins in H19-proficient and -deficient C2C12 myotubes. Mass spectrometry data indicated that in H19-proficient myotubes, dystrophin associates with components of dystrophin-associated protein complex (DPC); however, in H19-deficient myotubes, dystrophin associated with UBA1, UB2G1, TRIM63 ubiquitin E3 ligase and ubiquitin. In H19-deficient myotubes, dystrophin was post-translationally modified with K48-linked poly-ubiquitination at Lys3577 (referred to as Ub-DMD). This mass spectrometry study demonstrated that lncRNA H19, associates with dystrophin and inhibits E3 ligase-dependent Ub-DMD formation and its subsequent proteasomal degradation. Based on this study, H19 RNA oligonucleotides conjugated with a muscle homing ligand Agrin (referred to as AGR-H19) and Nifenazone, a TRIM63-specific small molecule inhibitor, reverses the dystrophin degradation in iPSC-derived skeletal muscle cells from Becker Muscular Dystrophy patients. Furthermore,treatment of mdx mice with exon-skipping reagent, in combination with either AGR-H19 or Nifenazone, dramatically stablized dystrophin, preserved skeletal/cardiac muscle histology, and improved strength/heart function. In summary, this mass spectrometry study paves the way to meaningful targeted therapeutics for BMD and certain DMD patients.
2020-08-19 | PXD020566 | Pride
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
Project description:Purpose: DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated in 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Methods: Reverse transcription PCR (RT-PCR) or high-throughput RNA sequencing (RNA-Seq) methods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. Results: In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3’-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. Conclusions: We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.
2022-05-06 | GSE175861 | GEO