Dystrophic Dogs Research Articles

Adaptive Immune Response Impairs the Efficacy of Autologous Transplantation of Engineered Stem Cells in Dystrophic Dogs.

Duchenne muscular dystrophy is the most common genetic muscular dystrophy. It is caused by mutations in the dystrophin gene, leading to absence of muscular dystrophin and to progressive degeneration of skeletal muscle. We have demonstrated that the exon skipping method safely and efficiently brings to the expression of a functional dystrophin in dystrophic CD133+ cells injected scid/mdx mice. Golden Retriever muscular dystrophic (GRMD) dogs represent the best preclinical model of Duchenne muscular dystrophy, mimicking the human pathology in genotypic and phenotypic aspects. Here, we assess the capacity of intra-arterial delivered autologous engineered canine CD133+ cells of restoring dystrophin expression in Golden Retriever muscular dystrophy. This is the first demonstration of five-year follow up study, showing initial clinical amelioration followed by stabilization in mild and severe affected Golden Retriever muscular dystrophy dogs. The occurrence of T-cell response in three Golden Retriever muscular dystrophy dogs, consistent with a memory response boosted by the exon skipped-dystrophin protein, suggests an adaptive immune response against dystrophin.

OPN-a induces muscle inflammation by increasing recruitment and activation of pro-inflammatory macrophages.

New Findings What is the central question of this study? What is the functional relevance of OPN isoform expression in muscle pathology? What is the main finding and its importance? The full‐length human OPN‐a isoform is the most pro‐inflammatory isoform in the muscle microenvironment, acting on macrophages and myoblasts in an RGD‐integrin‐dependent manner. OPN‐a upregulates expression of tenascin‐C (TNC), a known Toll‐like receptor 4 (TLR4) agonist. Blocking TLR4 signalling inhibits the pro‐inflammatory effects of OPN‐a, suggesting that a potential mechanism of OPN action is by promoting TNC–TLR4 signalling. Although osteopontin (OPN) is an important mediator of muscle remodelling in health and disease, functional differences in human spliced OPN variants in the muscle microenvironment have not been characterized. We thus sought to define the pro‐inflammatory activities of human OPN isoforms (OPN‐a, OPN‐b and OPN‐c) on cells present in regenerating muscle. OPN transcripts were quantified in normal and dystrophic human and dog muscle. Human macrophages and myoblasts were stimulated with recombinant human OPN protein isoforms, and cytokine mRNA and protein induction was assayed. OPN isoforms were greatly increased in dystrophic human (OPN‐a > OPN‐b > OPN‐c) and dog muscle (OPN‐a = OPN‐c). In healthy human muscle, mechanical loading also upregulated OPN‐a expression (eightfold; P < 0.01), but did not significantly upregulate OPN‐c expression (twofold; P > 0.05). In vitro, OPN‐a displayed the most pronounced pro‐inflammatory activity among isoforms, acting on both macrophages and myoblasts. In vitro and in vivo data revealed that OPN‐a upregulated tenascin‐C (TNC), a known Toll‐like receptor 4 (TLR4) agonist. Inhibition of TLR4 signalling attenuated OPN‐mediated macrophage cytokine production. In summary, OPN‐a is the most abundant and functionally active human spliced isoform in the skeletal muscle microenvironment. Here, OPN‐a promotes pro‐inflammatory signalling in both macrophages and myoblasts, possibly through induction of TNC–TLR4 signalling. Together, our findings suggest that specific targeting of OPN‐a and/or TNC signalling in the damaged muscle microenvironment may be of therapeutic relevance.

Levels of inflammation and oxidative stress, and a role for taurine in dystropathology of the Golden Retriever Muscular Dystrophy dog model for Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal skeletal muscle wasting disease presenting with excessive myofibre necrosis and increased inflammation and oxidative stress. In the mdx mouse model of DMD, homeostasis of the amino acid taurine is altered, and taurine administration drastically decreases muscle necrosis, dystropathology, inflammation and protein thiol oxidation. Since the severe pathology of the Golden Retriever Muscular Dystrophy (GRMD) dog model more closely resembles the human DMD condition, we aimed to assess the generation of oxidants by inflammatory cells and taurine metabolism in this species. In muscles of 8 month GRMD dogs there was an increase in the content of neutrophils and macrophages, and an associated increase in elevated myeloperoxidase, a protein secreted by neutrophils that catalyses production of the highly reactive hypochlorous acid (HOCl). There was also increased chlorination of tyrosines, a marker of HOCl generation, increased thiol oxidation of many proteins and irreversible oxidative protein damage. Taurine, which functions as an antioxidant by trapping HOCl, was reduced in GRMD plasma; however taurine was increased in GRMD muscle tissue, potentially due to increased muscle taurine transport and synthesis. These data indicate a role for HOCl generated by neutrophils in the severe dystropathology of GRMD dogs, which may be exacerbated by decreased availability of taurine in the blood. These novel data support continued research into the precise roles of oxidative stress and taurine in DMD and emphasise the value of the GRMD dogs as a suitable pre-clinical model for testing taurine as a therapeutic intervention for DMD boys.

Tadalafil Treatment Delays the Onset of Cardiomyopathy in Dystrophin-Deficient Hearts.

BackgroundCardiomyopathy is a leading cause of mortality among Duchenne muscular dystrophy patients and lacks effective therapies. Phosphodiesterase type 5 is implicated in dystrophic pathology, and the phosphodiesterase type 5 inhibitor tadalafil has recently been studied in a clinical trial for Duchenne muscular dystrophy.Methods and ResultsTadalafil was evaluated for the prevention of cardiomyopathy in the mdx mouse and golden retriever muscular dystrophy dog models of Duchenne muscular dystrophy. Tadalafil blunted the adrenergic response in mdx hearts during a 30‐minute dobutamine challenge, which coincided with cardioprotective signaling, reduced induction of μ‐calpain levels, and decreased sarcomeric protein proteolysis. Dogs with golden retriever muscular dystrophy began daily tadalafil treatment prior to detectable cardiomyopathy and demonstrated preserved cardiac function, as assessed by echocardiography and magnetic resonance imaging at ages 18, 21, and 25 months. Tadalafil treatment improved golden retriever muscular dystrophy histopathological features, decreased levels of the cation channel TRPC6, increased total threonine phosphorylation status of TRPC6, decreased m‐calpain levels and indicators of calpain target proteolysis, and elevated levels of utrophin. In addition, we showed that Duchenne muscular dystrophy patient myocardium exhibited increased TRPC6, m‐calpain, and calpain cleavage products compared with control human myocardium.ConclusionsProphylactic use of tadalafil delays the onset of dystrophic cardiomyopathy, which is likely attributed to modulation of TRPC6 levels and permeability and inhibition of protease content and activity. Consequently, phosphodiesterase type 5 inhibition is a candidate therapy for slowing the development of cardiomyopathy in Duchenne muscular dystrophy patients.

Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 (http://proteomecentral.proteomexchange.org/dataset/PXD001768).

Dystrophin Threshold Level Necessary for Normalization of Neuronal Nitric Oxide Synthase, Inducible Nitric Oxide Synthase, and Ryanodine Receptor-Calcium Release Channel Type 1 Nitrosylation in Golden Retriever Muscular Dystrophy Dystrophinopathy.

At present, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon-skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine the dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial has been conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger. Here, we exploited rAAV8-U7snRNA-transduced GRMD muscle samples, well characterized for their percentage of dystrophin-positive fibers, with the aim of defining the threshold of dystrophin rescue necessary for normalization of the status of neuronal nitric oxide synthase mu (nNOSμ), inducible nitric oxide synthase (iNOS), and ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for efficient contractile function. Results showed that restoration of dystrophin in 40% of muscle fibers is needed to decrease abnormal cytosolic nNOSμ expression and to reduce overexpression of iNOS, these two parameters leading to a reduction in the NO level in the muscle fibers. Furthermore, the same percentage of dystrophin-positive fibers of 40% was associated with the normalization of RyR1 nitrosylation status and with stabilization of the RyR1-calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology.

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Transplantation of Human Adipose Mesenchymal Stem Cells in Non-Immunosuppressed GRMD Dogs is a Safe Procedure.

The possibility to treat Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, through cell therapy with mesenchymal stromal cells (MSCs) has been widely investigated in different animal models. However, some crucial questions need to be addressed before starting human therapeutic trials, particularly regarding its use for genetic disorders. How safe is the procedure? Are there any side effects following mesenchymal stem cell transplantation? To address these questions for DMD the best model is the golden retriever muscular dystrophy dog (GRMD), which is the closest model to the human condition displaying a much longer lifespan than other models. Here we report the follow-up of 5 GRMD dogs, which were repeatedly transplanted with human adipose-derived mesenchymal stromal cells (hASC), derived from different donors. Xenogeneic cell transplantation, which was done without immunosuppression, was well tolerated in all animals with no apparent long-term adverse effect. In the present study, we show that repeated heterologous stem-cell injection is a safe procedure, which is fundamental before starting human clinical trials.

503. Adeno-Associated Virus Vector (AAV) Microdystrophin Gene Therapy Prolongs Survival and Restores Muscle Function in the Canine Model of Duchenne Muscular Dystrophy (DMD)

Duchenne Muscular Dystrophy (DMD) is a X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5,000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterized by systemic, progressive, irreversible and severe loss of muscle function. Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno-Associated Virus vectors (rAAV) hold great promise and allow very efficient transduction of skeletal and cardiac muscles. However, full-length dystrophin cDNA exceeds the packaging capacity for a single rAAV gene-delivery cassette. Therefore, truncated versions namely micro-dystrophins have been designed and optimized to contain few clinically important regions of the dystrophin protein. We have tested a rAAV2/8 vector encoding a sequence optimised canine micro-dystrophin transgene, driven by a muscle-synthetic Spc512 promoter (rAAV2/8-Spc512-µDys) in a total of 12 Golden Retriever Muscular Dystrophy (GRMD) dogs, the canine model of DMD. Isolated limb perfusion studies using a single administration of vector induced high levels of micro-dystrophin expression in the treated limb (up to 90% dystrophin positive fibres) with significant normalisation of histological, NMR imaging and spectroscopy parameters and muscle strength, without deleterious immune responses. Similarly, single-dose intravascular delivery of the same rAAV2/8-Spc512-µDys, in absence of immunosuppression, led to long-term transduction of distant muscle groups and extended lifespan (up to 2 years). Profound improvement of multiple clinical features was observed, including gait and respiratory parameters and no toxicity or deleterious humoral and/or cell-mediated immune responses were observed. This study demonstrates the safety and long term efficacy of rAAV2/8-Spc5.12-µDys gene therapy in a relevant large-animal models of DMD and paves the way towards human clinical gene therapy using systemic peripheral vein administration of vector, and applicable to all DMD patients regardless of their genotype.

388. Hypersensitivity of MYH16 Fibers (M Fibers) to Myodegeneration in Canine Muscular Dystrophy

Golden Retriever Muscular Dystrophy (GRMD) dogs frequently develop severe restriction to mouth opening, a condition known as trismus, as one of their earliest physical signs of the disease. Trismus is generally due to fibrosis and/or tonic contraction of the muscles of mastication, the temporalis and the masseter muscle. These muscles are primarily composed of unique myofibers exhibiting a masticatory myosin heavy chain encoded by the MYH 16 gene (M fibers). M fibers have been shown to produce the highest specific force among mammalian myofibers, based on their atypical crossbridge kinetics. Here, we show for a first time a morphological analysis of M fibers for the purposes of their classification, distribution and proportion in the masticatory muscles in normal and GRMD dogs based on immunofluorescence staining by a highly specific anti-MYH16 antibody. Our data demonstrate that in both temporalis and masseter muscles, the predominant M fibers are larger in size when compared to any other striated myofiber. A comparative histological analysis of these and other muscles in GRMD dogs demonstrates a significant relative increase in the proportion of injured, hypercontracted M fibers, a highly significant relative abundance of regenerating fibers expressing the embryonic myosin isoform encoded by the MYH3 gene, and prominent fibrosis when compared to the other affected striated muscles. These results suggest that the higher specific force developed by M fibers, due to the MYH16 encoded MyHC isoform, accelerates the fibers’ rate of degeneration in response to myonecrotizing sarcolemmal injury in the absence of dystrophin. Trismus is not seen in Duchenne MD, suggesting that the deletional frameshift in the human MYH16 gene (Nature 428: 373-4, 415-8) dramatically reduces the relative rate of sarcolemmal injury. These observations may offer novel insights into the physiological role of dystrophin in striated muscles, and provide an especially sensitive test for use in experimental therapeutics in the canine models for DMD.

623. Dystrophin Exon 52-Deleted Pigs as a New Animal Model of Duchenne Muscular Dystrophy: Its Characterization and Potential as a Tool for Developing Exon Skipping Therapy

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder primarily affecting boys, which is caused by mutations in the dystrophin (DMD) gene and the subsequent lack of dystrophin protein. Patients exhibit progressive degeneration and weakness in bodywide muscles, leading to death due to respiratory or cardiac failure. Currently, a most promising therapeutic approach is exon skipping with antisense oligonucleotides (AOs), which can correct the reading frame of mutant dystrophin mRNA and restore truncated-yet-functional dystrophin protein. Dystrophic mouse and dog models have been widely used for developing exon skipping therapies, as well as for improving scientific understanding of DMD pathogenesis; however, currently available animal models have a few limitations: First, they do not always share similar disease phenotypes with DMD patients (e.g., milder symptoms). Second, their available mutation patterns are limited, reducing applicability for research (importantly, AO drugs for exon skipping need to be developed according to mutation patterns). Lastly, mice and dogs are far from humans in terms of their anatomy, physiology, and genetics, which could prove a hurdle to interpreting and extrapolating treatment effects from animal to human. Although the wide variety of currently available animal models may partially compensate for some drawbacks, a more suitable animal model is most desirable to overcome them. Here, we generated a new DMD animal model of miniature pigs. Exon 52 deletion, one of the most common mutation patterns in the human DMD gene, was created in the pig dystrophin gene using a combination technique involving somatic cell nuclear transfer and gene targeting. The pig model systemically produced out-of-frame dystrophin mRNA transcripts lacking exon 52. Accordingly, no expression of dystrophin protein was observed in bodywide muscles, including the heart. Serum creatine kinase levels were dramatically increased, accompanied with histological deterioration in the muscles as observed in patients with DMD. We also tested AO-mediated exon skipping targeting exon 51 or 53 in primary skeletal muscle cells derived from the dystrophic pig model. The exon 52 deletion mutation is correctable by either exon 51 or 53 skipping which approach is theoretically applicable to the largest proportion of DMD patients (14% and 10%, respectively). AO sequences for skipping porcine exon 51 or 53 were designed as analogs of human AO sequences identified with our in silico AO design tool (Echigoya et al, PLoS One 2015 Mar 27;10(3):e0120058) and current antisense drugs under clinical trials. We successfully induced exon 51 or 53-excised transcripts in vitro with primary skeletal muscle cells derived from the exon 52-deleted transgenic pigs. We are currently planning in vivo exon skipping in the new pig model. Therapeutic outcomes derived from the DMD pig model could be more reliably extrapolated to human patients, facilitating development of novel AO drugs and translation into human clinical trials.

628. Transposons Expressing Full-Length Human Dystrophin Enable Genetic Correction of Dystrophic Mesoangioblasts and iPS-Derived Mesoangioblast-Like Cells

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. Alternatively, dystrophic mesoangioblast-like cells were generated from iPS of DMD patients. These iPS-derived mesoangioblasts, constitute an essentially unlimited supply of stem/progenitor cells that could be genetically corrected using PB transposons expressing dystrophin. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.

85. AAV9-Utrophin Prevents Myonecrosis in Dystrophic Mice and Dogs without Immunosuppression

The majority of mutations causing Duchenne muscular dystrophy (DMD) are multi-exon, frameshifting deletions, complicating therapy with recombinant dystrophin because of the potential for chronic immune recognition of the “non-self” protein. The paralogous protein utrophin is ubiquitously expressed at levels insufficient to prevent myonecrosis in animal models for DMD, but may confer central immunological tolerance through early developmental expression in the thymus. Here we show for a first time histological evidence for the complete prevention of myonecrosis in dystrophin-deficient striated muscles following systemic administration of an AAV9 vector carrying a 3.5 kb synthetic utrophin transgene (AAVµU). The cDNA was miniaturized by removal of domains least conserved in a comprehensive evolutionary comparison, and further optimized for maximal expression in striated muscle by using the codon bias of mammalian genes encoding contractile proteins. Administration of 1015 AAVµU vector genomes (vg) per kg to neonatal mice prevented centronucleation and saturated global recovery of the sarcoglycan complex, despite a subsequent tenfold increase in striated muscle mass with growth. In neonatal dystrophic dogs, intravenous injection of 1013.5 AAVµU vg/kg without immunosuppression restored sarcoglycan levels and normalized the myofiber size-distribution following a fourfold increase in muscle mass. Interferon-gamma ELISpot assays using utrophin-derived peptides revealed no reactivity in injected dogs, consistent with central immunological tolerance. These findings provide a rationale for high dose, neonatal gene therapy using utrophin as a “self” protein to forestall disability and mortality in DMD, while minimizing the risk of chronic immunotoxicity.

552. AAV9-Utrophin Restores Muscle Function in Dystrophic Mice

The majority of mutations causing Duchenne muscular dystrophy (DMD) are multi-exon, frameshifting deletions, complicating therapy with recombinant dystrophin because of the potential for chronic immune recognition of the “non-self” protein. The paralogous protein utrophin is ubiquitously expressed at levels insufficient to prevent myonecrosis in animal models for DMD, but may confer central immunological tolerance through early developmental expression in the thymus. Here we show for a first time histological evidence for the complete prevention of myonecrosis in dystrophin-deficient striated muscles following systemic administration of an AAV9 vector carrying a 3.5 kb synthetic utrophin transgene (AAVµU). The cDNA was miniaturized by removal of domains least conserved in a comprehensive evolutionary comparison, and further optimized for maximal expression in striated muscle by using the codon bias of mammalian genes encoding contractile proteins. Administration of 1015 AAVµU vector genomes (vg) per kg to neonatal mice prevented centronucleation and saturated global recovery of the sarcoglycan complex, despite a subsequent tenfold increase in striated muscle mass with growth. In neonatal dystrophic dogs, intravenous injection of 1013.5 AAVµU vg/kg without immunosuppression restored sarcoglycan levels and normalized the myofiber size-distribution following a fourfold increase in muscle mass. Interferon-gamma ELISpot assays using utrophin-derived peptides revealed no reactivity in injected dogs, consistent with central immunological tolerance. Here we present for the first time results of a complex approach for the evaluation of functional rescue in mdx mice using non-invasive tests relevant to the clinically relevant symptoms of DMD through an open field cage system. The comparison between congenic wild type (C57Bl10) and mdx mice indicates similarity in parameters like rest time and the number of times entering and initiating use of the running wheel per day, but very significant differences with regards to running wheel associated locomotor activity characteristics including velocity, time, distance per run, and total distance per day. These data correlate with the differences in testing results between the two mouse strains observed in the force grip evaluation and serum levels of CK. Finally, we have shown significant enhancement of physiological performance using an open field running wheel system, and normalization of serum creatine kinase (CK) for the first time ever in treated dystrophic mice. These mice also demonstrated, improvements in both in vivo and ex vivo muscle strength, when compared with untreated mdx. The combination of these findings provide a rationale for high dose, neonatal gene therapy using utrophin as a “self” protein to forestall disability and mortality in DMD, while minimizing the risk of chronic immunotoxicity. These results also support the use of AAVµU as an experimental therapeutic for DMD based on a high level of functional reversal of muscular dystrophy in mdx mice. They demonstrate strong evidence in support of the running wheel open field system as a reliable and reproducible approach to assess the therapeutic efficacy in the mouse model of DMD. This approach also provides a platform for dissecting the physiological roles of dystrophin in supporting precisely measurable volitional activities in unrestricted animals, thereby offering potential improvements in the predictive power of preclinical studies of therapeutic efficacy to inform clinical trials.

Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contractures.

BackgroundMyostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials.MethodsTo address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn+/−) whippets. A total of four GRippets (dystrophic and Mstn+/−), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated.ResultsMyostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no differential expression between GRMD and GRippet dogs. Satellite cell exhaustion was not observed in GRippets up to 3 years of age.ConclusionsPartial myostatin loss may exaggerate selective muscle hypertrophy or atrophy/hypoplasia in GRMD dogs and worsen contractures. While muscle imbalance is not a feature of myostatin inhibition in mdx mice, findings in a larger animal model could translate to human experience with myostatin inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0085-7) contains supplementary material, which is available to authorized users.

Serum Osteopontin as a Novel Biomarker for Muscle Regeneration in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is a lethal X-linked muscle disorder. We have already reported that osteopontin (OPN), an inflammatory cytokine and myogenic factor, is expressed in the early dystrophic phase in canine X-linked muscular dystrophy in Japan, a dystrophic dog model. To further explore the possibility of OPN as a new biomarker for disease activity in Duchenne muscular dystrophy, we monitored serum OPN levels in dystrophic and wild-type dogs at different ages and compared the levels to other serum markers, such as serum creatine kinase, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1. Serum OPN levels in the dystrophic dogs were significantly elevated compared with those in wild-type dogs before and 1 hour after a cesarean section birth and at the age of 3 months. The serum OPN level was significantly correlated with the phenotypic severity of dystrophic dogs at the period corresponding to the onset of muscle weakness, whereas other serum markers including creatine kinase were not. Immunohistologically, OPN was up-regulated in infiltrating macrophages and developmental myosin heavy chain-positive regenerating muscle fibers in the dystrophic dogs, whereas serum OPN was highly elevated. OPN expression was also observed during the synergic muscle regeneration process induced by cardiotoxin injection. In conclusion, OPN is a promising biomarker for muscle regeneration in dystrophic dogs and can be applicable to boys with Duchenne muscular dystrophy.

Increased collagen cross-linking is a signature of dystrophin-deficient muscle.

ABSTRACTIntroductionCollagen cross‐linking is a key parameter in extracellular matrix (ECM) maturation, turnover, and stiffness. We examined aspects of collagen cross‐linking in dystrophin‐deficient murine, canine, and human skeletal muscle.MethodsDMD patient biopsies and samples from mdx mice and golden retriever muscular dystrophy dog samples (with appropriate controls) were analyzed. Collagen cross‐linking was evaluated using solubility and hydroxyproline assays. Expression of the cross‐linking enzyme lysyl oxidase (LOX) was determined by real‐time polymerase chain reaction, immunoblotting, and immunofluorescence.ResultsLOX protein levels are increased in dystrophic muscle from all species evaluated. Dystrophic mice and dogs had significantly higher cross‐linked collagen than controls, especially in the diaphragm. Distribution of intramuscular LOX was heterogeneous in all samples, but it increased in frequency and intensity in dystrophic muscle.ConclusionThese findings implicate elevated collagen cross‐linking as an important component of the disrupted ECM in dystrophic muscles, and heightened cross‐linking is evident in mouse, dog, and man. Muscle Nerve 54: 71–78, 2016

PiggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.

Translating golden retriever muscular dystrophy microarray findings to novel biomarkers for cardiac/skeletal muscle function in Duchenne muscular dystrophy.

BackgroundIn Duchenne muscular dystrophy (DMD), abnormal cardiac function is typically preceded by a decade of skeletal muscle disease. Molecular reasons for differences in onset and progression of these muscle groups are unknown. Human biomarkers are lacking.MethodsWe analyzed cardiac and skeletal muscle microarrays from normal and golden retriever muscular dystrophy (GRMD) dogs (ages 6, 12, or 47+ months) to gain insight into muscle dysfunction and to identify putative DMD biomarkers. These biomarkers were then measured using human DMD blood samples.ResultsWe identified GRMD candidate genes that might contribute to the disparity between cardiac and skeletal muscle disease, focusing on brain-derived neurotropic factor (BDNF) and osteopontin (OPN/SPP1). BDNF was elevated in cardiac muscle of younger GRMD but was unaltered in skeletal muscle, while SPP1 was increased only in GRMD skeletal muscle. In human DMD, circulating levels of BDNF were inversely correlated with ventricular function and fibrosis, while SPP1 levels correlated with skeletal muscle function.ConclusionThese results highlight gene expression patterns that could account for differences in cardiac and skeletal disease in GRMD. Most notably, animal model-derived data were translated to DMD and support use of BDNF and SPP1 as biomarkers for cardiac and skeletal muscle involvement, respectively.