A novel CRISPR-Cas9 strategy to target DYSTROPHIN mutations downstream of exon 44 in patient-specific DMD iPSCs
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE262976 | GEO | 2024/05/30
REPOSITORIES: GEO
Similar Datasets
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
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: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:Duchenne muscular dystrophy (DMD) is caused by mutations in the X-linked dystrophin (DMD) gene. The absence of dystrophin protein leads to progressive muscle weakness and wasting, disability and death. To establish a tailored large animal model of DMD, we deleted DMD exon 52 in male pig cells by gene targeting and generated offspring by nuclear transfer. DMD pigs exhibit absence of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive dystrophic changes of skeletal muscles, impaired mobility, muscle weakness, and a maximum life span of 3 months due to respiratory impairment. To address the accelerated development of muscular dystrophy in DMD pigs as compared to human patients, we performed a genome-wide transcriptome study of M. biceps femoris samples from 2-day-old and 3-month-old DMD and age-matched wild-type pigs. The transcriptome changes in 3-month-old DMD pigs were in good accordance with the findings of gene expression profiles in human DMD, reflecting the processes of degeneration, regeneration, inflammation, fibrosis, and impaired metabolic activity. The transcriptome profile of 2-day-old DMD pigs pointed towards increased protein and DNA catabolism, reduced extracellular matrix formation and cell proliferation and showed similarities with transcriptome changes induced by exercise injury in muscle. Our transcriptome studies provide new insights into congenital changes associated with dystrophin deficiency and secondary complications arising during postnatal development. Thus the DMD pig is a useful model to determine the hierarchy of physiological derangements in dystrophin-deficient muscle. 13 samples, two conditions, two age-groups, 3-4 biological replicates
2013-08-06 | E-GEOD-44096 | biostudies-arrayexpress
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 caused by mutations in the X-linked dystrophin (DMD) gene. The absence of dystrophin protein leads to progressive muscle weakness and wasting, disability and death. To establish a tailored large animal model of DMD, we deleted DMD exon 52 in male pig cells by gene targeting and generated offspring by nuclear transfer. DMD pigs exhibit absence of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive dystrophic changes of skeletal muscles, impaired mobility, muscle weakness, and a maximum life span of 3 months due to respiratory impairment. To address the accelerated development of muscular dystrophy in DMD pigs as compared to human patients, we performed a genome-wide transcriptome study of M. biceps femoris samples from 2-day-old and 3-month-old DMD and age-matched wild-type pigs. The transcriptome changes in 3-month-old DMD pigs were in good accordance with the findings of gene expression profiles in human DMD, reflecting the processes of degeneration, regeneration, inflammation, fibrosis, and impaired metabolic activity. The transcriptome profile of 2-day-old DMD pigs pointed towards increased protein and DNA catabolism, reduced extracellular matrix formation and cell proliferation and showed similarities with transcriptome changes induced by exercise injury in muscle. Our transcriptome studies provide new insights into congenital changes associated with dystrophin deficiency and secondary complications arising during postnatal development. Thus the DMD pig is a useful model to determine the hierarchy of physiological derangements in dystrophin-deficient muscle.
2013-08-06 | GSE44096 | GEO
Project description:The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (ncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of DMD symptomatic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD-specific lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by selective targeting and down-modulation of dystrophin promoter transcriptional activity.
2011-06-05 | GSE27068 | GEO
Project description:The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (ncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of DMD symptomatic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD-specific lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by selective targeting and down-modulation of dystrophin promoter transcriptional activity. We tiled the entire DMD gene, in both sense and antisense directions, using the web-based Agilent eArray database, Version 4.5 (Agilent Technologies), with 60-mer oligos every 66 bp of repeat-masked genome sequence. We defined probe sets for both orientations, encompassing the DMD exons, promoters, introns, predicted MiRNA (identified by PromiRII) and conserved non-coding sequences (CNSs) identified within dystrophin introns using the VISTA programme (http://genome.lbl.gov/vista/index.shtml). Two specific sets of probes were designed to cover, in both directions, the cDNA sequences of a group of control genes identified in the Gene Expression Omnibus (GEO) database http://www.ncbi.nlm.nih.gov/geo/) and expressed equally in both normal and dystrophic muscles. Each probe set was opportunely distributed and replicated several times in order to obtain two 4x44k microarrays, referred to as DMD GEx Sense and DMD GEx Antisense, respectively, able to detect transcripts in the same and opposite directions as that of DMD gene transcription. Three commercial poly A+ RNAs from normal human brain, heart and skeletal muscle tissues were utilised (Ambion). Skin poly A+ RNA was isolated from total Skin RNA (Stratagene) using the Qiagen Oligotex kit. All RNA samples were checked for purity using a ND-1000 spectrophotometer (NanoDrop Technologies), and for integrity by electrophoresis on a 2100 BioAnalyzer (Agilent Technologies). Sample labelling and hybridisation were performed according to the protocols provided by Agilent (One-Color Microarray-Based Gene Expression Analysis version 5.0.1). The array was analysed using the Agilent scanner and Feature Extraction software (v9.1).
2011-06-05 | E-GEOD-27068 | biostudies-arrayexpress
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: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