Project description:Duchenne 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 associated cell types. Here, we describe the creation of a new mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with the nuclei of myofibers, as well as other mononucleated cell types that contribute to DMD disease pathogenesis, we performed single nucleus transcriptomics of skeletal muscle of mice with 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 new insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.
Project description:Duchenne 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 associated cell types. Here, we describe the creation of a new mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with the nuclei of myofibers, as well as other mononucleated cell types that contribute to DMD disease pathogenesis, we performed single nucleus transcriptomics of skeletal muscle of mice with 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 new insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.
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.
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
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.
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.
Project description:To investigate the role of the circadian clock gene Bmal1 in skeletal muscle, we compared the circadian transcriptomes of fast tibialis anterior (TA) and slow soleus (SOL) skeletal muscles from muscle-specific Bmal1 KO (mKO) and their control Cre- littermates (Ctrl). Keyword: Circadian Transcriptome, time course 72 samples were analyzed, comprised of 4 experimental groups (Ctrl SOL, mKO SOL, Ctrl TA, mKO TA), with 3 biological replicates for each time point sampled every 4 hours for 24 hours. SOL and TA muscles were collected from the same animals, as indicated by Source Animal ID data column
Project description:The extraocular muscles (EOMs) are a unique group of muscles that are anatomically and physiologically distinct from other skeletal muscles. Previously, we and others have shown that EOMs have a unique transcriptome and proteome. Here, we investigated the expression pattern of microRNAs (miRNAs) in EOM, as they may play a role in generating the unique EOM allotype. We screened LC Sciences miRNA microarrays covering the sequences of miRBase 10.0 to define the microRNAome of normal mouse EOM and tibialis anterior (TA) limb muscle. 74 miRNAs were found to be differentially regulated (p-value < 0.05) and 31 miRNAs (14 up-regulated and 17 down-regulated) were found to be differentially regulated at a signal strength > 500 including the muscle-specific miR-206, miR-1, miR-133a, miR-133b and miR-499. qPCR analysis was used to validate the differential expression. Bioinformatic tools were used to identify potential miRNA-mRNA-protein interactions and integrate data with previous transcriptome and proteomic profiling data. Luciferase assays using co-transfection of precursor miRNAs (pre-miRNAs) along with reporter constructs containing the 3â-untranslated region (3âUTR) of their predicted target genes were used to validate targeting by identified miRNAs. The definition of the EOM microRNAome complements existing transcriptome and proteome data about the molecular make-up of EOM and provides further insight into regulation of muscle genes. These data will also help to further explain the unique EOM muscle allotype and its differential sensitivity to diseases such as Duchenne's muscular dystrophy (DMD) and may assist in development of therapeutic strategies. Total RNA from four EOM and four TA tissue samples dissected from four adult male C57/Bl10 mice were used (TA served as control) to screen four LC Sciences microRNA Microarray chips. The chips contained microRNA sequences based on miRBase content 10.0 totalling 568 different miRNAs. Samples were labelled with Cy3 and Cy5 using dye-swap. Relative differences of miRNA expression was expressed as fold-changes EOM/TA, which were calculated after normalization across all four arrays.
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.