Golden Retriever Muscular Dystrophy Dogs Research Articles

Bradykinin restores left ventricular function, sarcomeric protein phosphorylation, and e/nNOS levels in dogs with Duchenne muscular dystrophy cardiomyopathy

Cardiomyopathy is a lethal result of Duchenne muscular dystrophy (DMD), but its characteristics remain elusive. The golden retriever muscular dystrophy (GRMD) dogs produce DMD pathology and mirror DMD patient's symptoms, including cardiomyopathy. We previously showed that bradykinin slows the development of pacing-induced heart failure. Therefore, the goals of this research were to characterize dystrophin-deficiency cardiomyopathy and to examine cardiac effects of bradykinin in GRMD dogs. At baseline, adult GRMD dogs had reduced fractional shortening (28 ± 2 vs. 38 ± 2% in control dogs, P < 0.001) and left ventricular (LV) subendocardial dysfunction leading to impaired endo-epicardial gradient of radial systolic velocity (1.3 ± 0.1 vs. 3.8 ± 0.2 cm/s in control dogs, P < 0.001) measured by echocardiography. These changes were normalized by bradykinin infusion (1 µg/min, 4 weeks). In isolated permeabilized LV subendocardial cells of GRMD dogs, tension-calcium relationships were shifted downward and force-generating capacity and transmural gradient of myofilament length-dependent activation were impaired compared with control dogs. Concomitantly, phosphorylation of sarcomeric regulatory proteins and levels of endothelial and neuronal nitric oxide synthase (e/nNOS) in LV myocardium were significantly altered in GRMD dogs. All these abnormalities were normalized in bradykinin-treated GRMD dogs. Cardiomyopathy in GRMD dogs is characterized by profound LV subendocardial dysfunction, abnormal sarcomeric protein phosphorylation, and impaired e/nNOS, which can be normalized by bradykinin treatment. These data provide new insights into the pathophysiological mechanisms accounting for DMD cardiomyopathy and open new therapeutic perspectives.

MRI roadmap-guided transendocardial delivery of exon-skipping recombinant adeno-associated virus restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open-reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were performed with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with MRI (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30±10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a three-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.

Functional decline, remodeling, and compensation in the respiratory system of a canine model of Duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD) experience a progressive loss of pulmonary performance and eventual insufficiency. The clinical course is well known, but the inability of the DMD patient to raise his metabolic rate has prevented respiratory reserve from being measured directly. The golden retriever muscular dystrophy (GRMD) dog model of DMD exhibits many of the hallmarks of the human disease, including respiratory muscle dysfunction. We used the respiratory stimulant doxapram in anesthetized dogs to simulate the effects of increased metabolic demand on respiratory drive while measuring multiple respiratory parameters. We found indicators of reduced reserve – reduced maximum tidal volume and ventilatory rate – and altered chest wall kinematics. GRMD diaphragms were severely hypertrophic and foreshortened. The abdominal contribution to inspired volume, an indicator of diaphragm involvement, was severely reduced in GRMD dogs. We propose that this remodeling of the diaphragm is adaptive, and allows the GRMD dog to employ accessory respiratory muscles to store elastic energy during expiration, aiding inspiratory pressure and flow and delaying the onset of respiratory insufficiency.

Chronic Administration of a Leupeptin-Derived Calpain Inhibitor Fails to Ameliorate Severe Muscle Pathology in a Canine Model of Duchenne Muscular Dystrophy

Calpains likely play a role in the pathogenesis of Duchenne muscular dystrophy (DMD). Accordingly, calpain inhibition may provide therapeutic benefit to DMD patients. In the present study, we sought to measure benefit from administration of a novel calpain inhibitor, C101, in a canine muscular dystrophy model. Specifically, we tested the hypothesis that treatment with C101 mitigates progressive weakness and severe muscle pathology observed in young dogs with golden retriever muscular dystrophy (GRMD). Young (6-week-old) GRMD dogs were treated daily with either C101 (17 mg/kg twice daily oral dose, n = 9) or placebo (vehicle only, n = 7) for 8 weeks. A battery of functional tests, including tibiotarsal joint angle, muscle/fat composition, and pelvic limb muscle strength were performed at baseline and every 2 weeks during the 8-week study. Results indicate that C101-treated GRMD dogs maintained strength in their cranial pelvic limb muscles (tibiotarsal flexors) while placebo-treated dogs progressively lost strength. However, concomitant improvement was not observed in posterior pelvic limb muscles (tibiotarsal extensors). C101 treatment did not mitigate force drop following repeated eccentric contractions and no improvement was seen in the development of joint contractures, lean muscle mass, or muscle histopathology. Taken together, these data do not support the hypothesis that treatment with C101 mitigates progressive weakness or ameliorates severe muscle pathology observed in young dogs with GRMD.

Vascular endothelial dysfunction in Duchenne muscular dystrophy is restored by bradykinin through upregulation of eNOS and nNOS

Little is known about the vascular function and expression of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) in Duchenne muscular dystrophy (DMD). Bradykinin is involved in the regulation of eNOS expression induced by angiotensin-converting enzyme inhibitors. We characterized the vascular function and eNOS and nNOS expression in a canine model of DMD and evaluated the effects of chronic bradykinin treatment. Vascular function was examined in conscious golden retriever muscular dystrophy (GRMD) dogs with left ventricular dysfunction (measured by echocardiography) and in isolated coronary arteries. eNOS and nNOS proteins in carotid arteries were measured by western blot and cyclic guanosine monophosphate (cGMP) content was analyzed by radioimmunoassay. Compared with controls, GRMD dogs had an impaired vasodilator response to acetylcholine. In isolated coronary artery, acetylcholine-elicited relaxation was nearly absent in placebo-treated GRMD dogs. This was explained by reduced nNOS and eNOS proteins and cGMP content in arterial tissues. Chronic bradykinin infusion (1 μg/min, 4 weeks) restored in vivo and in vitro vascular response to acetylcholine to the level of control dogs. This effect was NO-mediated through upregulation of eNOS and nNOS expression. In conclusion, this study is the first to demonstrate that DMD is associated with NO-mediated vascular endothelial dysfunction linked to an altered expression of eNOS and nNOS, which can be overcome by bradykinin.

Systemic Delivery of Allogenic Muscle Stem Cells Induces Long-Term Muscle Repair and Clinical Efficacy in Duchenne Muscular Dystrophy Dogs

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.

P1.24 Microarray analysis of two exceptional Golden Retriever Muscular Dystrophy (GRMD) dogs with no dystrophin and a mild course

P1.24 Microarray analysis of two exceptional Golden Retriever Muscular Dystrophy (GRMD) dogs with no dystrophin and a mild course

Longitudinal ambulatory measurements of gait abnormality in dystrophin-deficient dogs

BackgroundThis study aimed to measure the gait abnormalities in GRMD (Golden retriever muscular dystrophy) dogs during growth and disease progression using an ambulatory gait analyzer (3D-accelerometers) as a possible tool to assess the effects of a therapeutic intervention.MethodsSix healthy and twelve GRMD dogs were evaluated twice monthly, from the age of two to nine months. The evolution of each gait variable previously shown to be modified in control and dystrophin-deficient adults was assessed using two-ways variance analysis (age, clinical status) with repeated measurements. A principal component analysis (PCA) was applied to perfect multivariate data interpretation.ResultsSpeed, stride length, total power and force significantly already decreased (p < 0.01) at the age of 2 months. The other gait variables (stride frequency, relative power distributions along the three axes) became modified at later stages. Using the PCA analysis, a global gait index taking into account the main gait variables was calculated, and was also consistent to detect the early changes in the GRMD gait patterns, as well as the progressive degradation of gait quality.ConclusionThe gait variables measured by the accelerometers were sensitive to early detect and follow the gait disorders and mirrored the heterogeneity of clinical presentations, giving sense to monitor gait in GRMD dogs during progression of the disease and pre-clinical therapeutic trials.

Quantitative Proteomic Analysis of Dystrophic Dog Muscle

Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene, leading to progressive and unrelenting muscle loss. Although the genetic basis of DMD is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. Increasing evidence suggests that secondary mechanisms, as the alteration of key signaling pathways, may play an important role. In order to identify reliable biomarkers and potential therapeutic targets, and taking advantage of the clinically relevant Golden Retriever Muscular Dystrophy (GRMD) dog model, a proteomic study was performed. Isotope-coded affinity tag (ICAT) profiling was used to compile quantitative changes in protein expression profiles of the vastus lateralis muscles of 4-month old GRMD vs healthy dogs. Interestingly, the set of under-expressed proteins detected appeared primarily composed of metabolic proteins, many of which have been shown to be regulated by the transcriptional peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α). Subsequently, we were able to showed that PGC1-α expression is dramatically reduced in GRMD compared to healthy muscle. Collectively, these results provide novel insights into the molecular pathology of the clinically relevant animal model of DMD, and indicate that defective energy metabolism is a central hallmark of the disease in the canine model.

Ringo: Discordance between the molecular and clinical manifestation in a Golden Retriever Muscular Dystrophy dog

Of the various genetic homologues to Duchenne Muscular Dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog, which presents a variable but usually severe and progressive muscle weakness, has the closest relevance to DMD in both clinical severity and histopathological change. Among 77 GRMD dogs born in our colony in Brazil, we have identified a very mildly affected dog, Ringo, born July 2003. Among his descendants, at least one male, Suflair, is also showing a mild course. In an attempt to better characterize these two dogs, we studied the pattern of muscle proteins expression in Ringo and Suflair, as compared to severely affected and normal control dogs. Dystrophin was absent in both and utrophin was overexpressed in a pattern similar to the observed in severely affected dogs. Understanding the mechanism that is protecting Ringo and Suflair from the deleterious effect of the dystrophin gene mutation is of utmost interest. In addition it points out that the clinical impact of therapeutic trials should be interpreted with caution.

Kinematics of gait in Golden Retriever Muscular Dystrophy

The goal of this study was to quantify the two-dimensional kinematics of pathologic gait during over-ground walking at a self-selected speed at the stifle (knee) and hock (ankle) joints in six Golden Retriever Muscular Dystrophy (GRMD) dogs and six carrier littermates (controls). We found that GRMD dogs walked significantly slower than controls ( p < 0.01). At the stifle joint, both groups displayed similar ROM (range of motion), but compared to controls, GRMD dogs walked with the stifle joint relatively more extended. At the hock joint, GRMD dogs displayed less ROM (range of motion) and walked with the joint relatively less flexed compared to controls. We controlled for gait speed in all analyses, so the differences we observed in joint kinematics between groups cannot be attributed solely to the slower walking speed of the GRMD dogs. This is the first kinematic study of gait in the GRMD dog, an important step in using this model in pre-clinical trials.

Gait analysis using accelerometry in dystrophin-deficient dogs

Dogs affected with Golden Retriever Muscular Dystrophy (GRMD) exhibit striking clinical similarities with patients suffering from Duchenne muscular dystrophy (DMD), particularly gait impairments. The purpose of this study was to describe the use and reliability of accelerometry in gait assessment of dogs with muscular dystrophy. Eight healthy and 11 GRMD adult dogs underwent three gait assessment sessions, using accelerometry. Three-axial recordings of accelerations were performed, and gait variables calculated. Total power, force and regularity of accelerations, stride length and speed, normalized by height at withers, stride frequency, and cranio-caudal power were significantly decreased, whereas medio-lateral power was significantly increased in GRMD dogs. Moreover, these variables were repeatable within and between sessions. Accelerometry provides reliable variables which highlight specific gait patterns of GRMD dogs, describing objectively and quantitatively their slow, short-stepped, and swaying gait. As it is easy to set-up, quick to perform and inexpensive, accelerometry represents a useful tool, to assess locomotion during pre-clinical trials.

PTEN Contributes to Profound PI3K/Akt Signaling Pathway Deregulation in Dystrophin-Deficient Dog Muscle

Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3beta (GSK3beta), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38delta and p38gamma kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3beta, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3beta and PTEN activities. PTEN/GSK3beta-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3beta and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency.

Hydrodynamic Limb Vein Injection of Adeno-Associated Virus Serotype 8 Vector Carrying Canine Myostatin Propeptide Gene into Normal Dogs Enhances Muscle Growth

Inhibition or blockade of myostatin, a negative growth factor of skeletal muscle, enhances muscle growth and therefore is considered a promising strategy for the treatment of muscle-wasting diseases such as the muscular dystrophies. Previously, we showed that myostatin blockade in both normal and dystrophin-deficient mdx mice by systemic delivery of the myostatin propeptide (MPRO) gene by an adeno-associated virus serotype 8 (AAV8) vector could enhance muscle growth and ameliorate dystrophic lesions. Here, we further investigate whether the muscle growth effect of myostatin blockade can be achieved in dogs by gene transfer. First, we cloned the canine MPRO gene, packaged it in the AAV8 vector, and showed robust muscle-enhancing effects after systemic delivery into neonatal mice. This vector was then further tested in two 3-month-old normal dogs (weighing 9.7 and 6.3 kg). The vector was delivered to one limb by hydrodynamic vein injection, and the contralateral limb served as a control. The delivery procedure was safe, without discernible adverse effects. AAV vector DNA and MPRO gene expression were detected by quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining of muscle biopsies. Overexpression of MPRO resulted in enhanced muscle growth without a cytotoxic T lymphocytic immune response, as evidenced by larger myofibers in multiple muscles, increased muscle volume determined by magnetic resonance imaging, and the lack of CD4+ and CD8+ T cell infiltration in the vector-injected limbs. Our preliminary study thus supports further investigation of this therapeutic strategy in the dystrophin-deficient golden retriever muscular dystrophy dog model.

Skeletal Muscle Ultrasound: Correlation Between Fibrous Tissue and Echo Intensity

In this study, we examined the correlation between muscle ultrasound and muscle structure. Echo intensity (EI) of 14 muscles of two golden retriever muscular dystrophy dogs was correlated to the percentage interstitial fibrous tissue and fat in muscle biopsy. A significant correlation between interstitial fibrous tissue and EI was found ( r = 0.87; p < 0.001). The separate influence of interstitial fat on muscle EI could not be established as only little fat was present. We conclude that fibrous tissue causes increased muscle EI. The high correlation between interstitial fibrous tissue and EI makes ultrasound a reliable method to determine severity of structural muscle changes. (E-mail: s.pillen@cukz.umcn.nl)

230 COMPARISON OF SEMEN SAMPLES FROM NORMAL AND MUSCULAR DYSTROPHY AFFECTED ANIMAL MODELS BY THIOBARBITURIC ACID REACTIVE SUBSTANCES TEST AND FLUORESCENCE-ACTIVATED CELL SORTING ANALYSIS

Duchenne’s muscular dystrophy (DMD) is the most common human muscular affliction with high frequency in male individuals. The condition is characterized by progressive muscle degeneration, weakness, and loss of motion capacity. These individuals exhibit longer lifespans resulting from improvements in technical patient care and now raise new questions ranging from ethical to physiological issues never observed before, such as the possibility of reproduction. Thus, this study used Golden Retriever muscular dystrophy (GRMD)-affected dogs, a natural experimental model of DMD, to evaluate semen quality and oxidative stress by thiobarbituric acid reactive substances (TBARS) test. Thirty-seven ejaculates from 4 non-affected Golden Retriever and 5 GRMD-affected dogs (from 1.5 to 4.5 years old) were collected monthly and evaluated as 5 different replicates. Semen samples were processed, and initial analysis comprised of sperm concentration, percent of straightforward motility and morphology. Samples were then diluted to a final concentration of 106 spermatozoa mL–1 of prior incubation with each fluorescent probe. Acrosome integrity assessment was conducted with PSAFITC (100 g mL–1), and mitochondrial activity was assessed by JC1 (50 g mL–1). Fluorescence-activated cell sorting was performed in 103 spermatozoa, from a pre-selected gate that contained only these cells. Seminal plasma was submitted to TBARS quantification by spectrophotometer under normal and oxidative conditions (stress). Parametric data were compared by Student’s t-test. There were no significant differences in ejaculate volume, sperm concentration, acrosomal integrity, mithocondrial activity, and ejaculate TBARS tests between GRMD and normal dogs. Data are summarized in the table. GRMD does not affect the production and quality of semen of dogs. This animal model suggests that ejaculates from DMD men can be obtained by the proper stimulation and that conventional in vitro fertilization techniques combined with preimplantation genetic diagnosis for single gene disorders may be able to satisfy the desire of paternity of DMD patients with the birth of non-affected children. Table 1.Sperm analysis and TBARS tests of ejaculates from GRMD and normal dogs FAPESP for the financial support (06/50272-3); Surgery Department and Reproduction Department of FMVZ-USP; Canil GRMD Brazil.

G.P.6.09 Skeletal muscle characterization in golden retriever muscular dystrophy dogs by a nuclear magnetic resonance imaging longitudinal study

G.P.6.09 Skeletal muscle characterization in golden retriever muscular dystrophy dogs by a nuclear magnetic resonance imaging longitudinal study

Ringo, a Golden Retriever Muscular Dystrophy (GRMD) dog with absent dystrophin but normal strength

The closest model to human Duchenne Muscular Dystrophy (DMD) is the Golden Retriever Muscular Dystrophy (GRMD) dog, which carries a point mutation in the splice acceptor site in intron 6 of the orthologe X-linked dystrophin gene, leading to the absence of protein in the muscles. These dogs present clinical signs within the first weeks of life involving the limbs as well as masticatory muscles. Diaphragmatic and intercostal muscles impairment leads to progressive respiratory failure. Death occurs from bronchopneumonia and cardiac arrest, usually before 2 years of age.

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

BackgroundThe golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression.MethodsHuman Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes.Results and DiscussionWe analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age.ConclusionOur data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.

A canine minidystrophin is functional and therapeutic in mdx mice

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder lacking a curative treatment. We wish to use the dystrophin-deficient golden retriever muscular dystrophy (GRMD) dog, a canine model of DMD, to investigate adeno-associated virus (AAV) vector-mediated minidystrophin gene therapy. The dog model is useful in evaluating vector dose requirement and immunological consequences owing to its large size and outbred nature. In this study, we have cloned and constructed a canine minidystrophin gene vector. Owing to limited availability of the GRMD dogs, here we first examined the functions and therapeutic effects of the canine minidystrophin in the mdx mouse model. We observed efficient minigene expression without cellular immune responses in mdx mice after AAV1-cMinidys vector intramuscular injection. We also observed restoration of the missing dystrophin-associated protein complex (DPC) onto the sarcolemma, including sarcoglycans and dystrobrevin, and a partial restoration of alpha-syntrophin and neural nitric oxide synthase (nNOS). In addition, minidystrophin treatment ameliorated dystrophic pathology, such as fibrosis and myofiber central nucleation (CN). CN remained minimal (<2%) after AAV injection in the neonatal mdx mice and was reduced from more than 75% to about 25% after AAV injection in adult mdx mice. Finally, in vivo cell membrane leakage test with Evans blue dye showed that the canine minidystrophin could effectively protect the myofiber plasma membrane integrity. Our results, thus, demonstrated the functionality and therapeutic potential of the canine minidystrophin and paved its way for further testing in the GRMD dog model.