Duchenne muscular dystrophy (DMD) is a lethal, X-chromosome linked muscle-wasting disease affecting about 1 in 3500-6000 boys worldwide. Myofibre necrosis and subsequent loss of muscle mass are due ...to several molecular sequelae, such as inflammation and oxidative stress. We have recently shown increased neutrophils, highly reactive oxidant hypochlorous acid (HOCl) generation by myeloperoxidase (MPO), and associated oxidative stress in muscle from the GRMD dog and mdx mouse models for DMD. These findings have led us to hypothesise that generation of HOCl by myeloperoxidase released from neutrophils has a significant role in dystropathology. Since access to muscle from DMD patients is limited, the aim of this study was to develop methods to study this pathway in urine. Using immunoblotting to measure markers of protein oxidation, we show increased labelling of proteins with antibodies to dinitrophenylhydrazine (DNP, oxidative damage) and DiBrY (halogenation by reactive oxidants from myeloperoxidase) in GRMD and mdx urine. A strong positive correlation was observed between DiBrY labelling in dog urine and muscle. A strong positive correlation was also observed when comparing DNP and DiBrY labelling (in muscle and urine) to markers of dystropathology (plasma creatine kinase) and neutrophil presence (muscle MPO). Our results indicate the presence of neutrophil mediated oxidative stress in both models, and suggest that urine is a suitable bio-fluid for the measurement of such biomarkers. These methods could be employed in future studies into the role of neutrophil mediated oxidative stress in DMD and other inflammatory pathologies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The success of many gene therapy applications hinges on efficient whole body transduction. In the case of muscular dystrophies, a therapeutic vector has to reach every muscle in the body. Recent ...studies suggest that vectors based on adeno-associated virus (AAV) are capable of body-wide transduction in rodents. However, translating this finding to large animals remains a challenge. Here we explored systemic gene delivery with AAV serotype-9 (AAV-9) in neonatal dogs. Previous attempts to directly deliver AAV to adult canine muscle have yielded minimal transduction due to a strong cellular immune response. However, in neonatal dogs we observed robust skeletal muscle transduction throughout the body after a single intravenous injection. Importantly, systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and it lasted for at least 6 months (the duration of study). We also observed several unique features not predicted by murine studies. In particular, cardiac muscle was barely transduced in dogs. Many muscular dystrophy patients can be identified by neonatal screening. The technology described here may lead to an effective early intervention in these patients.
Duchenne muscular dystrophy (DMD) is a devastating disease that dramatically decreases the lifespan and
abilities of affected young people. The primary molecular cause of the disease is the absence ...of functional dystrophin protein,
which is critical to proper muscle function. Those with DMD vary in disease presentation and dystrophin mutation;
the same causal mutation may be associated with drastically different levels of disease severity. Also contributing to this
variation are the influences of additional modifying genes and/or changes in functional elements governing such modifiers.
This genetic heterogeneity complicates the efficacy of treatment methods and to date medical interventions are limited
to treating symptoms. Animal models of DMD have been instrumental in teasing out the intricacies of DMD disease and
hold great promise for advancing knowledge of its variable presentation and treatment. This review addresses the utility of
comparative genomics in elucidating the complex background behind phenotypic variation in a canine model of DMD,
Golden Retriever muscular dystrophy (GRMD). This knowledge can be exploited in the development of improved, more
personalized treatments for DMD patients, such as therapies that can be tailor-matched to the disease course and genomic
background of individual patients.
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the
gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino ...oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the
reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig
gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.
Duchenne (DMD) and golden retriever (GRMD) muscular dystrophy are caused by genetic mutations in the dystrophin gene and afflict striated muscles. We investigated systemic gene delivery in 4-day-old ...GRMD dogs given a single intravenous injection of an AAV9 vector (1.5 × 1014 vector genomes/kg) carrying a human codon-optimized human mini-dystrophin gene under control of the cytomegalovirus (CMV) promoter. One of the three treated dogs was euthanized 9 days later due to pre-existing conditions. Scattered mini-dystrophin-positive myofibers were seen by immunofluorescent (IF) staining in numerous muscles. At the end of the 16-week study, the other two dogs showed generalized muscle expression of mini-dystrophin in ∼15% to nearly 100% of myofibers. Western blot and vector DNA quantitative PCR results agreed with the IF data. Delayed growth and pelvic limb muscle atrophy and contractures were seen several weeks after vector delivery. T-2 weighted magnetic resonance imaging (MRI) at 8 weeks showed increased signal intensity compatible with inflammation in several pelvic limb muscles. This marked early inflammatory response raised concerns regarding methodology. Use of the ubiquitous CMV promoter, extra-high vector dose, and marked expression of a human protein in canine muscles may have contributed to the pathologic changes seen in the pelvic limbs.
There is a great demand for accurate non‐invasive measures to better define the natural history of disease progression or treatment outcome in Duchenne muscular dystrophy (DMD) and to facilitate the ...inclusion of a large range of participants in DMD clinical trials. This review aims to investigate which MRI sequences and analysis methods have been used and to identify future needs. Medline, Embase, Scopus, Web of Science, Inspec, and Compendex databases were searched up to 2 November 2019, using keywords “magnetic resonance imaging” and “Duchenne muscular dystrophy.” The review showed the trend of using T1w and T2w MRI images for semi‐qualitative inspection of structural alterations of DMD muscle using a diversity of grading scales, with increasing use of T2map, Dixon, and MR spectroscopy (MRS). High‐field (>3T) MRI dominated the studies with animal models. The quantitative MRI techniques have allowed a more precise estimation of local or generalized disease severity. Longitudinal studies assessing the effect of an intervention have also become more prominent, in both clinical and animal model subjects. Quality assessment of the included longitudinal studies was performed using the Newcastle‐Ottawa Quality Assessment Scale adapted to comprise bias in selection, comparability, exposure, and outcome. Additional large clinical trials are needed to consolidate research using MRI as a biomarker in DMD and to validate findings against established gold standards. This future work should use a multiparametric and quantitative MRI acquisition protocol, assess the repeatability of measurements, and correlate findings to histologic parameters.
•Dogs with GRMD have elevated tidal expiratory flow at 5–7 months of age.•Expiratory flow increases between 2–3 and 5–7 months of age in dogs with GRMD.•Paradoxical abdominal motion was not noted in ...young dogs with GRMD at rest.•Pulmonary function testing is difficult in unsedated dogs at 2–3 months of age.
Respiratory disease is a leading cause of morbidity in people with Duchenne muscular dystrophy and also occurs in the golden retriever muscular dystrophy (GRMD) model. We have previously shown that adult GRMD dogs have elevated expiratory flow as measured non-invasively during tidal breathing. This abnormality likely results from increased chest and diaphragmatic recoil associated with fibrosis and remodeling. Treatments must reverse pathologic effects on the diaphragm and other respiratory muscles to maximally reduce disease morbidity and mortality. Here, we extended our work in adults to younger GRMD dogs to define parameters that would be helpful in preclinical trials. Tidal breathing spirometry and respiratory inductance plethysmography were performed in GRMD dogs at approximately 3 and 6 months of age, corresponding to approximately 5–10 years in DMD, when clinical trials are often conducted. Expiratory flows were markedly elevated in GRMD versus normal dogs at 6 months. Values increased in GRMD dogs between 3 and 6 months, providing a 3-month window to assess treatment efficacy. These changes in breathing mechanics have not been previously identified at such an early age. Expiratory flow measured during tidal breathing of unsedated young GRMD dogs could be a valuable marker of respiratory mechanics during preclinical trials.
Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin (DMD) gene, is associated with fatal muscle degeneration and atrophy. Patients with DMD have progressive reductions in ...skeletal muscle strength and resistance to eccentric muscle stretch. Using the DE50-MD dog model of DMD, we assessed tibiotarsal joint (TTJ) flexor and extensor force dynamics, and the resistance of dystrophic muscle to eccentric stretch. Male DE50-MD and wild-type (WT) dogs were analysed every 3 months until 18 months of age. There was an age-associated decline in eccentric contraction resistance in DE50-MD TTJ flexors that discriminated, with high statistical power, WT from DE50-MD individuals. For isometric contraction, at the majority of timepoints, DE50-MD dogs had lower maximum absolute and relative TTJ flexor force, reduced TTJ muscle contraction times and prolonged relaxation compared to those in WT dogs. Cranial tibial muscles, the primary TTJ flexor, of 18-month-old DE50-MD dogs had significant numbers of regenerating fibres as expected, but also fewer type I fibres and more hybrid fibres than those in WT dogs. We conclude that these parameters, in particular, the eccentric contraction decrement, could be used as objective outcome measures for pre-clinical assessment in DE50-MD dogs.
Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration caused by lack of the cytoskeletal protein dystrophin. Dystrophin deficiency causes muscle membrane instability, ...skeletal muscle wasting, cardiomyopathy, and heart failure. Advances in palliative respiratory care have increased the incidence of heart disease in DMD patients, for which there is no cure or effective therapy. Here we have shown that chronic infusion of membrane-sealing poloxamer to severely affected dystrophic dogs reduced myocardial fibrosis, blocked increased serum cardiac troponin I (cTnI) and brain type natriuretic peptide (BNP), and fully prevented left-ventricular remodeling. Mechanistically, we observed a markedly greater primary defect of reduced cell compliance in dystrophic canine myocytes than in the mildly affected mdx mouse myocytes, and this was associated with a lack of utrophin upregulation in the dystrophic canine cardiac myocytes. Interestingly, after chronic poloxamer treatment, the poor compliance of isolated canine myocytes remained evident, but this could be restored to normal upon direct application of poloxamer. Collectively, these findings indicate that dystrophin and utrophin are critical to membrane stability-dependent cardiac myocyte mechanical compliance and that poloxamer confers a highly effective membrane-stabilizing chemical surrogate in dystrophin/utrophin deficiency. We propose that membrane sealant therapy is a potential treatment modality for DMD heart disease and possibly other disorders with membrane defect etiologies.
The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin
, a rod-like protein
that protects striated myocytes from contraction-induced injury
. Dystrophin-related protein (or ...utrophin) retains most of the structural and protein binding elements of dystrophin
. Importantly, normal thymic expression in DMD patients
should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.
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