Insight into the Role of Gut Microbiota in Duchenne Muscular Dystrophy: An Age-Related Study in Mdx Mice.

Dystrophin deficiency alters the sarcolemma structure, leading to muscle dystrophy, muscle disuse, and ultimately death. Beyond limb muscle deficits, patients with Duchenne muscular dystrophy have numerous transit disorders. Many studies have highlighted the strong relationship between gut microbiota and skeletal muscle. The aims of this study were: i) to characterize the gut microbiota composition over time up to 1 year in dystrophin-deficient mdx mice, and ii) to analyze the intestine structure and function and expression of genes linked to bacterial-derived metabolites in ileum, blood, and skeletal muscles to study interorgan interactions. Mdx mice displayed a significant reduction in the overall number of different operational taxonomic units and their abundance (α-diversity). Mdx genotype predicted 20% of ß-diversity divergence, with a large taxonomic modification of Actinobacteria, Proteobacteria, Tenericutes, and Deferribacteres phyla and the included genera. Interestingly, mdx intestinal motility and gene expressions of tight junction and Ffar2 receptor were down-regulated in the ileum. Concomitantly, circulating inflammatory markers related to gut microbiota (tumor necrosis factor, IL-6, monocyte chemoattractant protein-1) and muscle inflammation Tlr4/Myd88 pathway (Toll-like receptor 4, which recognizes pathogen-associated molecular patterns) were up-regulated. Finally, in mdx mice, adiponectin was reduced in blood and its receptor modulated in muscles. This study highlights a specific gut microbiota composition and highlights interorgan interactions in mdx physiopathology with gut microbiota as the potential central metabolic organ.

Sensitivity to behavioral stress impacts disease pathogenesis in dystrophin-deficient mice.

Mutation to the gene encoding dystrophin can cause Duchenne muscular dystrophy (DMD) and increase the sensitivity to stress in vertebrate species, including the mdx mouse model of DMD. Behavioral stressors can exacerbate some dystrophinopathy phenotypes of mdx skeletal muscle and cause hypotension-induced death. However, we have discovered that a subpopulation of mdx mice present with a wildtype-like response to mild (forced downhill treadmill exercise) and moderate (scruff restraint) behavioral stressors. These "stress-resistant" mdx mice are more physically active, capable of super-activating the hypothalamic-pituitary-adrenal and renin-angiotensin-aldosterone pathways following behavioral stress and they express greater levels of mineralocorticoid and glucocorticoid receptors in striated muscle relative to "stress-sensitive" mdx mice. Stress-resistant mdx mice also presented with a less severe striated muscle histopathology and greater exercise and skeletal muscle oxidative capacity at rest. Most interestingly, female mdx mice were more physically active following behavioral stressors compared to male mdx mice; a response abolished after ovariectomy and rescued with estradiol. We demonstrate that the response to behavioral stress greatly impacts disease severity in mdx mice suggesting the management of stress in patients with DMD be considered as a therapeutic approach to ameliorate disease progression.

A muscle fatigue-like contractile decline was recapitulated using skeletal myotubes from Duchenne muscular dystrophy patient-derived iPSCs.

Duchenne muscular dystrophy (DMD) is a muscle degenerating disease caused by dystrophin deficiency, for which therapeutic options are limited. To facilitate drug development, it is desirable to develop in vitro disease models that enable the evaluation of DMD declines in contractile performance. Here, we show MYOD1-induced differentiation of hiPSCs into functional skeletal myotubes in vitro with collagen gel and electrical field stimulation (EFS). Long-term EFS training (0.5 Hz, 20 V, 2 ms, continuous for 2 weeks) mimicking muscle overuse recapitulates declines in contractile performance in dystrophic myotubes. A screening of clinically relevant drugs using this model detects three compounds that ameliorate this decline. Furthermore, we validate the feasibility of adapting the model to a 96-well culture system using optogenetic technology for large-scale screening. Our results support a disease model using patient-derived iPSCs that allows for the recapitulation of the contractile pathogenesis of DMD and a screening strategy for drug development.

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart.

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

Loss of dystrophin expression in skeletal muscle is associated with senescence of macrophages and endothelial cells.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by cell cycle inhibitory proteins such as p16, p21, and p53. When cells enter senescence, they secrete a host of proinflammatory factors known as the senescence-associated secretory phenotype, which has deleterious effects on surrounding cells and tissues. Little is known of the role of senescence in Duchenne muscular dystrophy (DMD), the fatal X-linked neuromuscular disorder typified by chronic inflammation, extracellular matrix remodeling, and a progressive loss in muscle mass and function. Here, we demonstrate using C57-mdx (8-wk-old) and D2-mdx (4-wk-old and 8-wk-old) mice, two mouse models of DMD, that cells displaying canonical markers of senescence are found within the skeletal muscle. Eight-week-old D2-mdx mice, which display severe muscle pathology, had greater numbers of senescent cells associated with areas of inflammation, which were mostly Cdkn1a-positive macrophages, whereas in C57-mdx muscle, senescent populations were endothelial cells and macrophages localized to newly regenerated myofibers. Interestingly, this pattern was similar to cardiotoxin (CTX)-injured wild-type (WT) muscle, which experienced a transient senescent response. Dystrophic muscle demonstrated significant upregulations in senescence pathway genes [Cdkn1a (p21), Cdkn2a (p16INK4A), and Trp53 (p53)], which correlated with the quantity of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. These results highlight an underexplored role for cellular senescence in murine dystrophic muscle.

Complementary NAD+ replacement strategies fail to functionally protect dystrophin-deficient muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). METHODS: Using a metabolomics-based approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38. RESULTS: Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, nor improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery. CONCLUSIONS: In the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These molecular consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, we found no evidence that either small molecule strategy is sufficient to restore muscle contractile function or confer protection from eccentric injury, undermining the modulation of NAD metabolism as a therapeutic approach for DMD.

Using iRFP Genetic Labeling Technology to Track Tumorogenesis of Transplanted CRISPR/Cas9-Edited iPSC in Skeletal Muscle.

Tumorigenesis and attendant safety risks are significant concerns of induced pluripotent stem cell (iPSC)-based therapies. Thus, it is crucial to evaluate iPSC proliferation, differentiation, and tumor formation after transplantation. Several approaches have been employed for tracking the donor cells, including fluorescent protein and luciferase, but both have limitations. Here, we introduce a protocol using iRFP genetic labeling technology to track tumor formation of iPSCs in skeletal muscle after CRISPR/Cas9 gene editing.

Rescue of Defective Electroretinographic Responses in Dp71-Null Mice With AAV-Mediated Reexpression of Dp71.

Purpose: To study the potential effect of a gene therapy, designed to rescue the expression of dystrophin Dp71 in the retinas of Dp71-null mice, on retinal physiology. Methods: We recorded electroretinograms (ERGs) in Dp71-null and wild-type littermate mice. In dark-adapted eyes, responses to flashes of several strengths were measured. In addition, flash responses on a 25-candela/square meters background were measured. On- and Off-mediated responses to sawtooth stimuli and responses to photopic sine-wave modulation (3-30 Hz) were also recorded. After establishing the ERG phenotype, the ShH10-GFP adeno-associated virus (AAV), which has been previously shown to target specifically Müller glial cells (MGCs), was delivered intravitreously with or without (sham therapy) the Dp71 coding sequence under control of a CBA promoter. ERG recordings were repeated three months after treatment. Real-time quantitative PCR and Western blotting analyses were performed in order to quantify Dp71 expression in the retinas. Results: Dp71-null mice displayed reduced b-waves in dark- and light-adapted flash ERGs and smaller response amplitudes to photopic rapid-on sawtooth modulation and to sine-wave stimuli. Three months after intravitreal injections of the ShH10-GFP-2A-Dp71 AAV vector, ERG responses were completely recovered in treated eyes of Dp71-null mice. The functional rescue was associated with an overexpression of Dp71 in treated retinas. Conclusions: The present results show successful functional recovery accompanying the reexpression of Dp71. In addition, this experimental model sheds light on MGCs influencing ERG components, since previous reports showed that aquaporin 4 and Kir4.1 channels were mislocated in MGCs of Dp71-null mice, while their distribution could be normalized following intravitreal delivery of the same ShH10-GFP-2A-Dp71 vector.

Core Transcription Factors Promote Induction of PAX3-Positive Skeletal Muscle Stem Cells.

The use of adult skeletal muscle stem cells (MuSCs) for cell therapy has been attempted for decades, but still encounters considerable difficulties. MuSCs derived from human induced pluripotent stem cells (hiPSCs) are promising candidates for stem cell therapy to treat Duchenne muscular dystrophy (DMD). Here we report that four transcription factors, HEYL, KLF4, MYOD, and PAX3, selected by comprehensive screening of different MuSC populations, enhance the derivation of PAX3-positive myogenic progenitors from fibroblasts and hiPSCs, using medium that promotes the formation of presomitic mesoderm. These induced PAX3-positive cells contribute efficiently to the repair of DMD-damaged myofibers and also reconstitute the MuSC population. These studies demonstrate how a combination of core transcription factors can fine-tune the derivation of MuSCs capable of contributing to the repair of adult skeletal muscle.

Dystrophin deficiency promotes leukocyte recruitment in mdx mice.

BACKGROUND: A growing body of evidence defines inflammation as a hallmark feature of disease pathogenesis of Duchenne muscular dystrophy. To tailor potential immune modulatory interventions, a better understanding of immune dysregulation in Duchenne muscular dystrophy is needed. We now asked whether dystrophin deficiency affects the cascade of leukocyte recruitment. METHODS: We performed intravital microscopy on the cremaster muscle of wild-type and dystrophin-deficient mdx mice. Recruitment was triggered by preparation alone (traumatic inflammation) or in combination with scrotal TNFα injections. Neutrophilic infiltration of the cremaster muscle was assessed on tissue sections. Integrin expression on circulating neutrophils and serum levels of pro-inflammatory cytokines were measured by flow cytometry. RESULTS: Mdx mice show increased rolling and adhesion at baseline (traumatic inflammation) and a more profound response upon TNFα injection compared with wild-type animals. In both models, neutrophilic infiltration of the cremaster muscle is increased. Upregulation of the integrins LFA-1 and Mac-1 on circulating leukocytes and pro-inflammatory cytokines IL-6 and CCL2 in the serum points toward systemically altered immune regulation in mdx mice. CONCLUSION: We are the first to show exaggerated activation of the leukocyte recruitment cascade in a dystrophin-deficient organism in vivo.

A modified diet does not ameliorate muscle pathology in a mouse model for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by a lack of dystrophin protein. Next to direct effects on the muscles, this has also metabolic consequences. The influence of nutrition on disease progression becomes more and more recognized. Protein intake by DMD patients may be insufficient to meet the increased demand of the constantly regenerating muscle fibers. This led to the hypothesis that improving protein uptake by the muscles could have therapeutic effects. The present study examined the effects of a modified diet, which composition might stimulate muscle growth, on disease pathology in the D2-mdx mouse model. D2-mdx males were fed with either a control diet or modified diet, containing high amounts of branched-chain amino acids, vitamin D3 and ursolic acid, for six weeks. Our study indicates that the modified diet could not ameliorate the muscle pathology. No effects on bodyweight or weight of individual muscles were observed. Neither did the diet affect severity of fibrosis or calcification of the muscles.

Targeting angiogenesis in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) represents one of the most devastating types of muscular dystrophies which affect boys already at early childhood. Despite the fact that the primary cause of the disease, namely the lack of functional dystrophin is known already for more than 30 years, DMD still remains an incurable disease. Thus, an enormous effort has been made during recent years to reveal novel mechanisms that could provide therapeutic targets for DMD, especially because glucocorticoids treatment acts mostly symptomatic and exerts many side effects, whereas the effectiveness of genetic approaches aiming at the restoration of functional dystrophin is under the constant debate. Taking into account that dystrophin expression is not restricted to muscle cells, but is present also in, e.g., endothelial cells, alterations in angiogenesis process have been proposed to have a significant impact on DMD progression. Indeed, already before the discovery of dystrophin, several abnormalities in blood vessels structure and function have been revealed, suggesting that targeting angiogenesis could be beneficial in DMD. In this review, we will summarize current knowledge about the angiogenesis status both in animal models of DMD as well as in DMD patients, focusing on different organs as well as age- and sex-dependent effects. Moreover, we will critically discuss some approaches such as modulation of vascular endothelial growth factor or nitric oxide related pathways, to enhance angiogenesis and attenuate the dystrophic phenotype. Additionally, we will suggest the potential role of other mediators, such as heme oxygenase-1 or statins in those processes.

EGFR-Aurka Signaling Rescues Polarity and Regeneration Defects in Dystrophin-Deficient Muscle Stem Cells by Increasing Asymmetric Divisions.

Loss of dystrophin expression in Duchenne muscular dystrophy (DMD) causes progressive degeneration of skeletal muscle, which is exacerbated by reduced self-renewing asymmetric divisions of muscle satellite cells. This, in turn, affects the production of myogenic precursors and impairs regeneration and suggests that increasing such divisions may be beneficial. Here, through a small-molecule screen, we identified epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation.

Various effects of AAV9-mediated ßARKct gene therapy on the heart in dystrophin-deficient (mdx) mice and δ-sarcoglycan-deficient (Sgcd-/-) mice.

So far effective strategies to treat cardiomyopathy in patients with muscular dystrophies are still not clearly defined. Previously, treatment with ß-blockers showed beneficial effects on the development of cardiomyopathy in dystrophin-deficient (mdx) mice, but not in δ-sarcoglycan-deficient (Sgcd-/-) mice. We therefore aimed to study a more specific approach to target maladaptive ß-adrenergic signalling in these mice. It has been shown that lowering cardiac G-protein-coupled-receptor-kinase-2 (GRK2) activity with ßARKct expression, a peptide inhibitor of protein-coupled-receptor-kinase-2 (GRK2), results in improvement of heart failure in several different animal models. We therefore investigated whether adeno-associated virus type 9 (AAV9)-mediated gene delivery of ßARKct, could ameliorate cardiac pathology in mdx and Sgcd-/- mice. We found that long-term treatment with AAV9- ßARKct-cDNA with a cardiac-specific promoter significantly improves left ventricular systolic function and reduces myocardial hypertrophy in mdx mice, whereas only mild beneficial effects on cardiac function is observed in Sgcd-/- mice. Interestingly, in contrast to mdx mice neither GRK2 nor nuclear-factor-kappaB (NFκB) were upregulated in Sgcd-/- mice. Taken together, effectiveness of AAV-mediated ßARKct therapy may vary between different genetic mutations and presumably depend on the state of adrenergic dysregulation mediated through the upregulation of GRK2.

Cmah-dystrophin deficient mdx mice display an accelerated cardiac phenotype that is improved following peptide-PMO exon skipping treatment.

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids-the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah-/-;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah-/-;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah-/-;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah-/-;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah-/-;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics.

Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle.

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or ßcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.

Xanthine oxidase is hyper-active in Duchenne muscular dystrophy.

Generation of superoxide by xanthine oxidase can be stimulated under ischemic and aberrant calcium homeostasis. Because patients and mice with Duchenne muscular dystrophy (DMD) suffer from ischemia and excessive calcium influx, we tested the hypothesis that xanthine oxidase activity is elevated and contributes to disease pathology. Xanthine oxidase activity was measured by urinary isoxanthopterin in DMD patients at rest and in response to exercise. Urinary isoxanthopterin/creatinine was elevated compared to age-matched controls and Becker muscular dystrophy (BMD) patients. Concentrations were also increased after a six minute walk test in ambulatory patients. We also measured urinary isoxanthopterin in wildtype mice and a number of dystrophic mouse models; the DMD mouse model (mdx), mdx mice overexpressing a variety of transgenic miniaturized and chimeric skeletal muscle-specific dystrophins and utrophin and the ß-sarcoglycan deficient (Scgb-/-) mouse which represents type 2E human limb-girdle muscular dystrophy. Mdx and Scgb-/-mice had greater urinary isoxanthopterin/creatinine than wildtype mice while mdx mice expressing dystrophin or utrophin linking the extracellular matrix to the actin cytoskeleton were not different than wildtype. We also measured higher levels of urinary ortho-tyrosine in humans and mice deficient for dystrophin to confirm elevated oxidative stress. Surprisingly, mdx had lower xanthine oxidase protein levels and higher mRNA in gastrocnemius muscle compared to wildtype mice, however, the enzymatic activity of skeletal muscle xanthine oxidase was elevated above wildtype and a transgenic rescued mdx mouse (DysΔMTB-mdx). Downhill treadmill running also caused significant increases in mdx urinary isoxanthopterin that was prevented with the xanthine oxidase inhibitor allopurinol. Similarly, in vitro eccentric contraction-induced force drop of mdx muscle was attenuated by the allopurinol metabolite, oxypurinol. Together, our data suggests hyper-activity of xanthine oxidase in DMD, identifies xanthine oxidase activity as a contributing factor in eccentric contraction-induced force drop of dystrophin-deficient skeletal muscle and highlights the potential of isoxanthopterin as a noninvasive biomarker in DMD.

Necroptosis mediates myofibre death in dystrophin-deficient mice.

Duchenne muscular dystrophy (DMD) is a severe degenerative disorder caused by mutations in the dystrophin gene. Dystrophin-deficient muscles are characterised by progressive myofibre necrosis in which inflammation plays a deleterious role. However, the molecular mechanisms underlying inflammation-induced necrosis in muscle cells are unknown. Here we show that necroptosis is a mechanism underlying myofibre death in dystrophin-deficient muscle. RIPK1, RIPK3 and MLKL are upregulated in dystrophic mouse myofibres. In human DMD samples, there is strong immunoreactivity to RIPK3 and phospho-MLKL in myofibres. In vitro, TNFα can elicit necroptosis in C2C12 myoblasts, and RIPK3 overexpression sensitises myoblasts to undergo TNF-induced death. Furthermore, genetic ablation of Ripk3 in mdx mice reduces myofibre degeneration, inflammatory infiltrate, and muscle fibrosis, and eventually improves muscle function. These findings provide the first evidence of necroptotic cell death in a disease affecting skeletal muscle and identify RIPK3 as a key player in the degenerative process in dystrophin-deficient muscles.

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases.

Variable rescue of microtubule and physiological phenotypes in mdx muscle expressing different miniaturized dystrophins.

Delivery of miniaturized dystrophin genes via adeno-associated viral vectors is one leading approach in development to treat Duchenne muscular dystrophy. Here we directly compared the functionality of five mini- and micro-dystrophins via skeletal muscle-specific transgenic expression in dystrophin-deficient mdx mice. We evaluated their ability to rescue defects in the microtubule network, passive stiffness and contractility of skeletal muscle. Transgenic mdx mice expressing the short dystrophin isoform Dp116 served as a negative control. All mini- and micro-dystrophins restored elevated detyrosinated α-tubulin and microtubule density of mdx muscle to values not different from C57BL/10, however, only mini-dystrophins restored the transverse component of the microtubule lattice back to C57BL/10. Passive stiffness values in mdx muscles expressing mini- or micro-dystrophins were not different from C57BL/10. While all mini- and micro-dystrophins conferred significant protection from eccentric contraction-induced force loss in vivo and ex vivo compared to mdx, removal of repeats two and three resulted in less protection from force drop caused by eccentric contraction ex vivo. Our data reveal subtle yet significant differences in the relative functionalities for different therapeutic constructs of miniaturized dystrophin in terms of protection from ex vivo eccentric contraction-induced force loss and restoration of an organized microtubule lattice.