1,721,025 research outputs found
Correction of muscular dystrophies by CRISPR gene editing
Full text linkMuscular dystrophies are debilitating disorders that result in progressive weakness and degeneration of skeletal muscle. Although the genetic mutations and clinical abnormalities of a variety of neuromuscular diseases are well known, no curative therapies have been developed to date. The advent of genome editing technology provides new opportunities to correct the underlying mutations responsible for many monogenic neuromuscular diseases. For example, Duchenne muscular dystrophy, which is caused by mutations in the dystrophin gene, has been successfully corrected in mice, dogs, and human cells through CRISPR/Cas9 editing. In this Review, we focus on the potential for, and challenges of, correcting muscular dystrophies by editing disease-causing mutations at the genomic level. Ideally, because muscle tissues are extremely long-lived, CRISPR technology could offer a one-time treatment for muscular dystrophies by correcting the culprit genomic mutations and enabling normal expression of the repaired gene
CRISPR-Editing Therapy for Duchenne Muscular Dystrophy
No full textDuchenne muscular dystrophy (DMD) is a debilitating genetic disorder that results in progressive muscle degeneration and premature death. DMD is caused by mutations in the gene encoding dystrophin protein, a membrane-associated protein required for maintenance of muscle structure and function. Although the genetic mutations causing the disease are well known, no curative therapies have been developed to date. The advent of genome-editing technologies provides new opportunities to correct the underlying mutations responsible for DMD. These mutations have been successfully corrected in human cells, mice, and large animal models through different strategies based on CRISPR-Cas9 gene editing. Ideally, CRISPR-editing could offer a one-time treatment for DMD by correcting the genetic mutations and enabling normal expression of the repaired gene. However, numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene-editing components to all the muscles of the body, and the suppression of possible immune responses to the CRISPR-editing therapy. This review provides an overview of the recent advances toward CRISPR-editing therapy for DMD and discusses the opportunities and the remaining challenges in the path to clinical translation
Tissues & organs: Biochemistry of development: Striated muscle
No full textStriated muscles are the skeletal and cardiac muscles that have distinct bands when viewed by a microscope. Although skeletal and cardiac muscles appear similar, they originate from different progenitor cells and use different evolutionarily conserved networks of transcription factors and non-coding RNAs to regulate the programs controlling cell differentiation and morphogenesis during development. Elucidating the genetic networks that govern striated muscle development not only yields insights into general principles of organogenesis, but also facilitates therapies for skeletal and cardiac muscle diseases
Myogenin and class II histone deacetylases control skeletal muscle atrophy by inducing E3 ubiquitin ligases.
No full textMice lacking class II HDACs are resistant to skeletal muscle atrophy following denervation. Making Muscle in the Embryo and Adult.
No full textDegenerative and regenerative pathways underlying Duchenne muscular dystrophy revealed by single-nucleus RNA sequencing
Full text linkDuchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei
Single-swap editing for the correction of common Duchenne muscular dystrophy mutations
Full text linkDuchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease of progressive muscle weakness and wasting caused by the absence of dystrophin protein. Current gene therapy approaches using antisense oligonucleotides require lifelong dosing and have limited efficacy in restoring dystrophin production. A gene editing approach could permanently correct the genome and restore dystrophin protein expression. Here, we describe single-swap editing, in which an adenine base editor edits a single base pair at a splice donor site or splice acceptor site to enable exon skipping or reframing. In human induced pluripotent stem cell-derived cardiomyocytes, we demonstrate that single-swap editing can enable beneficial exon skipping or reframing for the three most therapeutically relevant exons—DMD exons 45, 51, and 53—which could be beneficial for 30% of all DMD patients. Furthermore, an adeno-associated virus delivery method for base editing components can efficiently restore dystrophin production locally and systemically in skeletal and cardiac muscles of a DMD mouse model containing a deletion of Dmd exon 44. Our studies demonstrate single-swap editing as a potential gene editing therapy for common DMD mutations
Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing
Full text linkDuchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD
Ablation of CaMKIId oxidation by CRISPR-Cas9 base editing as a therapy for cardiac disease
Full text linkCRISPR-Cas9 gene editing is emerging as a prospective therapy for genomic mutations. However, current editing approaches are directed primarily toward relatively small cohorts of patients with specific mutations. Here, we describe a cardioprotective strategy potentially applicable to a broad range of patients with heart disease. We used base editing to ablate the oxidative activation sites of CaMKIId, a primary driver of cardiac disease. We show in cardiomyocytes derived from human induced pluripotent stem cells that editing the CaMKIId gene to eliminate oxidation-sensitive methionine residues confers protection from ischemia/reperfusion (IR) injury. Moreover, CaMKIId editing in mice at the time of IR enables the heart to recover function from otherwise severe damage. CaMKIId gene editing may thus represent a permanent and advanced strategy for heart disease therapy
Elimination of CaMKIIδ Autophosphorylation by CRISPR-Cas9 Base Editing Improves Survival and Cardiac Function in Heart Failure in Mice
No full textBACKGROUND: Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. METHODS: To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. RESULTS: Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+transients and an increased frequency of arrhythmias after chronic β-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. CONCLUSIONS: Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease
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