CRISPR-mediated correction of skeletal muscle Ca2+handling in a novel DMD patient-derived pluripotent stem cell model (original) (raw)
Related papers
Cell Stem Cell, 2016
Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed for evaluation of dystrophin in disease relevant cell types. Cardiomyocytes and skeletal muscle *
Nature communications, 2017
Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx(4cv) mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that A...
CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy
Genes, 2020
Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created...
. CRISPR-Cas9-mediated genomic and transcript deletion of exon 23 through intramuscular AAV-CRISPR administration. (A) The Cas9 nuclease is targeted to introns 22 and 23 by two gRNAs. Simultaneous generation of double-stranded breaks (DSBs) by Cas9 leads to excision of the region surrounding the mutated exon 23. The distal ends are repaired through nonhomologous end joining (NHEJ). The reading frame of the dystrophin gene is recovered and protein expression is restored. (B) PCR across the genomic deletion region shows the smaller-deletion PCR product in treated muscles. Sequencing of the deletion band shows perfect ligation of Cas9 target sites (+, AAV-injected muscles; -, contralateral muscles). (C) ddPCR of genomic DNA shows 2% genome editing efficiency (n = 6 muscles, mean + SEM). (D) RT-PCR across exons 22 and 24 of dystrophin cDNA shows a smaller band that does not include exon 23 in treated muscles. Sanger sequencing confirmed exon 23 deletion. (E) ddPCR of intact dystrophin transcripts and D23 transcripts shows that 59% of transcripts do not have exon 23 (n = 6 muscles, mean + SEM). Asterisk, significantly different from the sham group (P < 0.05).
Altered Gene Expression Pathways in Duchenne Muscular Dystrophy
2012
Ca 2+ is a highly versatile second messenger that can regulate several cellular functions. Skeletal muscles use Ca 2+ for contraction process and as regulatory signaling molecule. Subsequently, muscle plasticity is closely related with calcium signals .
In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy
Science (New York, N.Y.), 2015
Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the CRISPR/Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR/Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR/Cas9-based genome editing as a potential therapy to treat DMD.
Nucleic acids research, 2018
CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components wi...
Common therapeutic advances for Duchenne muscular dystrophy (DMD)
International Journal of Neuroscience, 2020
Background and purpose: Duchenne muscular dystrophy (DMD), a lethal X-linked recessive muscle dystrophy, is resulted in by different mutations including mostly frame-shifting gross deletions and duplications and rarely point mutations in DMD gene. Increasing weakness, progressive loss of skeletal muscle mass, and later-onset cardiomyopathy are serious clinical symptoms which ultimately lead to cardiac and respiratory failure, and premature death in DMD patients by age of 30. DMD is a prevalent genetic disorder and considers as an interesting target for gene therapy approaches. Massive gene size and existence of enormous number of muscle tissues are terrific hindrance against DMD treatments, nevertheless enormous efforts have been executed in the fields of gene replacement therapy, gene editing strategies, cellbased treatments, and small drug medications. Hot spot exons skipping and suppression of premature stop codons are the most interesting treatments for restoring functional DMD product, dystrophin protein. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPRassociated protein (Cas) systems are the most interesting genome editing platforms that are able to restore open reading frame of DMD gene. CRISPR-Cas9 and CRISPR-Cpf1 are two main genome editing sub-types that successfully used in mdx mice. Conclusions: This review aims to present recent progresses and future prospects over three main DMD therapeutic subgroups including gene therapy, cell therapy, and pharmacological therapy.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2002
Calcium is the most ubiquitous second messenger. Its concentration inside the cell is tightly regulated by a series of mechanisms, among which some have been extensively studied in nonmuscle cells. This is the case of the "store-operated entry of Ca(2+)", the uptake of Ca(2+) by mitochondria and the inositol 1,4,5-trisphosphate (IP(3)) cascade. These processes were recently found to be also present in skeletal muscle and are reviewed here. The "store-operated entry of Ca(2+)" allows the refilling of the stores after muscle fiber depolarization and is activated even after a partial depletion of the sarcoplasmic reticulum (SR). The uptake of Ca(2+) by mitochondria accelerates muscle relaxation and allows the adaptation of ATP supply to the increased energy demand. IP(3) receptors are found in the nuclear envelope and are involved in Ca(2+) waves propagating from one nucleus to another. This pathway is possibly involved in gene expression regulation. Finally, cytosolic Ca(2+) buffers like parvalbumins modify [Ca(2+)](i) transients and, therefore, muscle mechanics.The importance of these regulation mechanisms is also evaluated in Duchenne muscular dystrophy (DMD), a disease in which impairment of [Ca(2+)](i) homeostasis has been postulated but remains, however, controversial. This genetic disease is indeed characterized by the absence of a cytoskeletal protein called dystrophin, a situation leading to a disorganization of the cytoskeleton and to an abnormal influx of Ca(2+). How this increased entry of Ca(2+) affects the local concentration of Ca(2+) in subcellular compartmen...
Scientific reports, 2015
Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca(2+)) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca(2+) overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These resul...