Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA326658 | ENA |
REPOSITORIES: ENA
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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: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
Project description:Duchenne Muscular Dystrophy (DMD) is a lethal muscle disease caused by mutations in the dystrophin gene. CRISPR/Cas9 genome-editing has been used to correct DMD mutations in animal models at young ages. However, the longevity and durability of CRISPR/Cas9 editing remained to be determined. To address these issues, we subjected DEx44 DMD mice to systemic delivery of AAV9 expressing CRISPR/Cas9 gene editing components to reframe exon 45 of the dystrophin gene, allowing robust dystrophin expression and maintenance of muscle structure and function. We showed that genome correction by CRISPR/Cas9 confers lifelong expression of dystrophin in mice and that corrected skeletal muscle is highly durable and resistant to myofiber necrosis and fibrosis, even in response to chronic injury. In contrast, when muscle fibers were ablated by barium chloride injection, satellite cells were unable to restore dystrophin expression due to restriction of gene editing components. Analysis of on-target and off-target gene editing in aged mice confirmed the stability of gene correction and the lack of significant off-target editing at 18 months of age. These findings demonstrate the long-term durability of CRISPR/Cas9 genome editing as a therapy for maintaining the integrity and function of DMD muscle, even under conditions of stress.
2021-07-27 | GSE180771 | GEO
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 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.
2024-01-01 | GSE248257 | GEO
Project description:CRISPR-Cas9 has tremendous potential as a therapeutic tool for treating human diseases. However, prolonged expression of the nuclease and gRNA from viral vectors in an in vivo context may cause off-target activity and immunogenicity. While extracellular vesicles have been recently demonstrated to be a promising option to transiently deliver the CRISPR-system, sufficient packaging of both Cas9 protein and gRNA is critical to achieve efficient genome editing in hard-to-transfect cells and tissues, such as skeletal muscle. Here, we developed a novel ribonucleoprotein delivery system utilizing two distinct homing mechanisms. The first is by chemical induced dimerization to recruit Cas9 protein into extracellular nanovesicles. The second utilizes a viral RNA packaging signal and two self-cleaving riboswitches to tether and release sgRNA into nanovesicles. We term our fully engineered delivery system NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo) and demonstrate efficient genome editing in various hard-to-transfect cell types, including human iPS cells and myoblasts. Furthermore, NanoMEDIC production is scalable for industrial production as a xeno-free suspension culture system. As a disease model, therapeutic exon skipping in the dystrophin gene locus was targeted and resulted in over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy patient iPS cells. Finally, we generated novel luciferase-based reporter mice to demonstrate that NanoMEDIC could induce exon skipping and sustain skipping activity for over 160 days, even though NanoMEDIC itself was rapidly degraded within 3 days, indicating its utility for transient in vivo genome editing therapy of DMD and beyond.
2020-01-30 | PXD014527 | JPOST Repository
Project description:Exon skipping is an effective strategy for the treatment of Duchenne Muscular Dystrophy (DMD). Natural exon skipping identified in several DMD cases can help with identifying novel therapeutic tools. Here, we demonstrate that CRISPR/Cas9 inactivation of the splicing factor Celf2a in a DMD-D44 background induces skipping of exon-45 and dystrophin rescue. Celf2a ablation could be compatible with life and curative since a DMD case with a milder symptomatology exists where exon-45 skipping occurs because of a Celf2a deficiency. We show that in this individual, Celf2a absence is due to inherited epigenetic silencing and that its repression is controlled by the lncRNA DUXAP8.
2020-07-01 | GSE139571 | GEO
Project description:Based on the hypothesis that, enhancing the local concentration of donor oligos could increase the correction rates, we generated and tested novel CRISPR-Cas9 systems, in which the DNA repair template is covalently conjugated to Cas9 (RNPD system). To validate our results from the HEK293T reporter cells, we here tested our approach at different endogenous genomic loci and in different cell types. We first targeted the human beta globin (HBB) locus in the K562 cell line, and analyzed correction- and editing frequencies using next generation sequencing (NGS). Next we targeted the Rosa26 and proprotein convertase subtilisin/kexin type 9 (Pcsk9) locus in mouse embryonic stem cells (mESCs). Here, RNPD system was always compared to Cas9 SNAP-tag fusion proteins with uncoupled donor oligos. To also directly compare the engineered RNPD system to the classical CRISPR-Cas9 system, we performed experiments where we used wild-type Cas9 with the uncoupled donor oligos as a control. We therefore targeted the fluorescent reporter locus as well as the endogenous loci HBB, empty spiracles homeobox 1 (EMX1), and C-X-C chemokine receptor type 4 (CXCR4) in HEK293T cells. Finally, we performed the analysis of three computationally predicted off-target sites of the reporter locus.
2018-06-04 | E-MTAB-6808 | biostudies-arrayexpress
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:Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected mouse DMD-induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD-iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Remarkably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation, and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.
2024-04-25 | GSE254371 | GEO