Potential Therapy For Duchenne Muscular Dystrophy Research Articles

Assessing the Use of the sGC Stimulator BAY-747, as a Potential Treatment for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder, affecting one in 3500 to 5000 boys worldwide. The NO-sGC-cGMP pathway plays an important role in skeletal muscle function, primarily by improving blood flow and oxygen supply to the muscles during exercise. In fact, PDE5 inhibitors have previously been investigated as a potential therapy for DMD, however, a large-scale Phase III clinical trial did not meet its primary endpoint. Since the efficacy of PDE5i is dependent on sufficient endogenous NO production, which might be impaired in DMD, we investigated if NO-independent sGC stimulators, could have therapeutic benefits in a mouse model of DMD. Male mdx/mTRG2 mice aged six weeks were given food supplemented with the sGC stimulator, BAY-747 (150 mg/kg of food) or food alone (untreated) ad libitum for 16 weeks. Untreated C57BL6/J mice were used as wild type (WT) controls. Assessments of the four-limb hang, grip strength, running wheel and serum creatine kinase (CK) levels showed that mdx/mTRG2 mice had significantly reduced skeletal muscle function and severe muscle damage compared to WT mice. Treatment with BAY-747 improved grip strength and running speed, and these mice also had reduced CK levels compared to untreated mdx/mTRG2 mice. We also observed increased inflammation and fibrosis in the skeletal muscle of mdx/mTRG2 mice compared to WT. While gene expression of pro-inflammatory cytokines and some pro-fibrotic markers in the skeletal muscle was reduced following BAY-747 treatment, there was no reduction in infiltration of myeloid immune cells nor collagen deposition. In conclusion, treatment with BAY-747 significantly improves several functional and pathological parameters of the skeletal muscle in mdx/mTRG2 mice. However, the effect size was moderate and therefore, more studies are needed to fully understand the potential treatment benefit of sGC stimulators in DMD.

Abnormal Calcium Handling in Duchenne Muscular Dystrophy: Mechanisms and Potential Therapies.

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by the loss of dystrophin. DMD is associated with muscle degeneration, necrosis, inflammation, fatty replacement, and fibrosis, resulting in muscle weakness, respiratory and cardiac failure, and premature death. There is no curative treatment. Investigations on disease-causing mechanisms offer an opportunity to identify new therapeutic targets to treat DMD. An abnormal elevation of the intracellular calcium () concentration in the dystrophin-deficient muscle is a major secondary event, which contributes to disease progression in DMD. Emerging studies have suggested that targeting Ca2+-handling proteins and/or mechanisms could be a promising therapeutic strategy for DMD. Here, we provide an updated overview of the mechanistic roles the sarcolemma, sarcoplasmic/endoplasmic reticulum, and mitochondria play in the abnormal and sustained elevation of levels and their involvement in DMD pathogenesis. We also discuss current approaches aimed at restoring Ca2+ homeostasis as potential therapies for DMD.

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.219 Antisense oligonucleotide-mediated knockdown of IGFBP3 to increase IGF-1 signalling in dystrophic muscle

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by the absence of dystrophin protein. Upon exhaustion of the regenerative capacity of muscle, there is an imbalance between muscle damage and repair. Insulin-like growth factor 1 (IGF-1) plays an essential role in increasing muscle mass and strength, reducing degeneration, inhibiting chronic inflammation and increasing the proliferation potential of satellite cells. Although liver is the main source of IGF-1, it is also expressed in different tissues including skeletal muscle. Both in circulation and the extracellular matrix, IGF-1 is bound to one of its binding proteins (IGFBPs). The majority of IGF-1 transport is associated with IGFBP3. Other IGFBPs are also found in the circulation and complexes of these IGFBPs with IGF-1 can exit the circulation easily, making them more available to target tissues. By contrast, IGF-1+IGFBP3 complexes, unless cleaved by specific proteases, cannot cross vascular endothelium. IGFBP3 is thought to act by inhibiting the availability of IGF-1 to its receptor, which initiates most of the actions of IGF-1. More recently, studies revealed that IGFBP-3 can be inhibitory to myoblast proliferation even in the absence of IGF signalling. Therefore, decreasing IGFBP3 levels would increase IGF-1 action and prevent IGF-1 independent antiproliferative effect of IGFBP3, making it a potential therapy for DMD by improving muscle repair and growth. This can be achieved by downregulation of IGFBP3 using antisense oligonucleotides (AONs) that skip out of frame exon(s), thus preventing the expression of functional protein. In this study, two different AONs were designed to target exon 2 of Igfbp3 and transfected using lipofectamine in mouse C2C12 cell cultures. Cells were harvested after 24 hours and total RNA was isolated. Exon skipping was confirmed by RT-PCR on RNA level. In addition, time course and western blot experiments are ongoing. In vivo studies are being planned.

Dystrophin Expressing Chimeric (DEC) Human Cells Provide a Potential Therapy for Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a progressive and lethal disease caused by mutations of the dystrophin gene. Currently no cure exists. Stem cell therapies targeting DMD are challenged by limited engraftment and rejection despite the use of immunosuppression. There is an urgent need to introduce new stem cell-based therapies that exhibit low allogenic profiles and improved cell engraftment. In this proof-of-concept study, we develop and test a new human stem cell-based approach to increase engraftment, limit rejection, and restore dystrophin expression in the mdx/scid mouse model of DMD. We introduce two Dystrophin Expressing Chimeric (DEC) cell lines created by ex vivo fusion of human myoblasts (MB) derived from two normal donors (MBN1/MBN2), and normal and DMD donors (MBN/MBDMD). The efficacy of fusion was confirmed by flow cytometry and confocal microscopy based on donor cell fluorescent labeling (PKH26/PKH67). In vitro, DEC displayed phenotype and genotype of donor parent cells, expressed dystrophin, and maintained proliferation and myogenic differentiation. In vivo, local delivery of both DEC lines (0.5 × 106) restored dystrophin expression (17.27%±8.05—MBN1/MBN2 and 23.79%±3.82—MBN/MBDMD) which correlated with significant improvement of muscle force, contraction and tolerance to fatigue at 90 days after DEC transplant to the gastrocnemius muscles (GM) of dystrophin-deficient mdx/scid mice. This study establishes DEC as a potential therapy for DMD and other types of muscular dystrophies.

Innervation of dystrophic muscle after muscle stem cell therapy.

Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a potential therapy for DMD. It is unknown whether donor-derived muscle fibers are structurally innervated. Green fluorescent protein (GFP)-expressing MuSCs were transplanted into the tibials anterior of adult dystrophic mdx/mTR mice. Three weeks later the neuromuscular junction was labeled by immunohistochemistry. The percent overlap between pre- and postsynaptic immunolabeling was greater in donor-derived GFP(+) myofibers, and fewer GFP(+) myofibers were identified as denervated compared with control GFP(-) fibers (P = 0.001 and 0.03). GFP(+) fibers also demonstrated acetylcholine receptor fragmentation and expanded endplate area, indicators of muscle reinnervation (P = 0.008 and 0.033). It is unclear whether GFP(+) fibers are a result of de novo synthesis or fusion with damaged endogenous fibers. Either way, donor-derived fibers demonstrate clear histological innervation. Muscle Nerve 54: 763-768, 2016.

Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin-glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5-7 days of age to the mdx/utrn(-/-) mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn(-/-) mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD.

Taurine deficiency, synthesis and transport in the mdx mouse model for Duchenne Muscular Dystrophy

The amino acid taurine is essential for the function of skeletal muscle and administration is proposed as a treatment for Duchenne Muscular Dystrophy (DMD). Taurine homeostasis is dependent on multiple processes including absorption of taurine from food, endogenous synthesis from cysteine and reabsorption in the kidney. This study investigates the cause of reported taurine deficiency in the dystrophic mdx mouse model of DMD. Levels of metabolites (taurine, cysteine, cysteine sulfinate and hypotaurine) and proteins (taurine transporter [TauT], cysteine deoxygenase and cysteine sulfinate dehydrogenase) were quantified in juvenile control C57 and dystrophic mdx mice aged 18 days, 4 and 6 weeks. In C57 mice, taurine content was much higher in both liver and plasma at 18 days, and both cysteine and cysteine deoxygenase were increased. As taurine levels decreased in maturing C57 mice, there was increased transport (reabsorption) of taurine in the kidney and muscle. In mdx mice, taurine and cysteine levels were much lower in liver and plasma at 18 days, and in muscle cysteine was low at 18 days, whereas taurine was lower at 4: these changes were associated with perturbations in taurine transport in liver, kidney and muscle and altered metabolism in liver and kidney. These data suggest that the maintenance of adequate body taurine relies on sufficient dietary intake of taurine and cysteine availability and metabolism, as well as retention of taurine by the kidney. This research indicates dystrophin deficiency not only perturbs taurine metabolism in the muscle but also affects taurine metabolism in the liver and kidney, and supports targeting cysteine and taurine deficiency as a potential therapy for DMD.

G.P.90: Effects of S48168/Arm210, a new rycal® compound, on pathology related signs of exercised dystrophic mdx mouse

The progressive myofiber degeneration in Duchenne muscular dystrophy (DMD) is caused by the complex cascade of events triggered by the absence of dystrophin. Studies in the mdx mouse model of DMD have shown that pathology-related post-translational modifications of the Ryanodine receptor subtype 1 (RyR1) result in the dissociation of the stabilizing subunit calstabin1, leading to leaky channels and altered Ca2+homeostasis, a key event in DMD (Bellinger, Nat Med 2009). S48168, also known as ARM210, is a small molecule stabilizer of the calstabin-RyR channel complex that prevents Ca 2+ leak without altering RyR1 post-translational modifications. We assessed the effects of 4weeks oral administration of S48168 (10 and 50mg/kg/day) on treadmill-exercised mdx mice, starting at 4–5weeks of age. A functional improvement was observed in vivo in S48168 -treated mdx mice, with a 50% recovery score of maximal forelimb force at 50mg/kg. Both doses prevented the decline in running performance, observed in vehicle-treated mdx mice, as assessed in an exhaustion test. Ex vivo assessment showed a dose-dependent improvement of diaphragm specific force, with a significant 30% increase of maximal force (100–140Hz) at 50mg/kg, while minor, if any, effects were observed in hind-limb EDL muscle. However, and consistently with the S48168 mechanism of action, the mechanical threshold EDL myofibers, an index of calcium handling during contraction, was significantly shifted toward the wild type (wt) values at the highest dose. In parallel, the resting cytosolic calcium level in FDB myofibers was significantly reduced by 25% in the S48168-treated mice. S48168 did not lower CK or LDH plasma levels. However a reduction of damaged area was detected in diaphragm and in gastrocnemius muscle of 50mg/kg treated mdx mice. Dose-proportional increases in plasma and muscle drug content were also observed, without any signs of toxicity. These results support S48168 as a potential therapy for DMD.

Regulation of the ubiquitin proteasome and calpain systems in a dog model of duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an incurable and lethal genetic disorder characterized by progressive muscle wasting caused by the loss of functional dystrophin. Activation of both calpain and the proteasome degradation systems has been reported in the pathogenesis of DMD in skeletal muscle. In fact, calpain and proteasome inhibitors have been used as therapies in mouse models. Here we investigated specific calpain and proteasome activities of 5 skeletal muscles and the left ventricle (LV) in a more severe canine model of DMD (golden retriever muscular dystrophy [GRMD]). The expression of ubiquitin proteasome system components and calpain 1 and 2 were increased in GRMD skeletal muscle generally, but decreased in the LV. We identified significant increases in all three proteasome activities (trypsin‐, chymotrypsin‐, and caspase‐like) in only 1 GRMD muscle and no activation of any proteasome activities in the LV. Calpain 1&2 activities were increased in 2 GRMD skeletal muscles and the LV compared to controls. Furthermore, protease activity of most GRMD skeletal muscles studied correlate with the degree of atrophy and hypertrophy found in the individual muscles. As proteasome and calpain inhibitors continue to be considered as potential therapies for DMD, this work provides evidence for the variable activation of these systems in different skeletal muscles and the heart. Supported by NIH R01HL104129.

IGF‐I improves excitation‐contraction coupling in skeletal muscle fibers of dystrophic mdx mice

The absence of dystrophin in Duchenne muscular dystrophy (DMD) causes progressive muscle wasting and possibly aberrant intracellular signaling including abnormal excitation-contraction coupling (ECC) in skeletal muscle. Insulin-like growth factor-I (IGF-I) is a potential therapy for DMD, and can alter ECC. We tested the hypothesis that muscle specific over-expression of IGF-I could ameliorate disruptions in ECC in muscle fibers from transgenic mdx (IGF-mdx) mice. Mechanically-skinned single fibers were prepared from the EDL muscles of mdx, IGF-mdx, and wild type mice. The number of depolarization-induced contractions (DICR) before a 50% reduction in amplitude was lower in mdx fibres (7 ± 1, n=15 fibers) than control (16 ± 2, n=12, P<0.05). There was no difference in SR Ca2+ properties. Rundown of DICR was improved significantly in IGF-mdx (14 ± 2, n=14) compared with mdx mice (7 ± 1, n=15, P<0.05). SR Ca2+ release was 300% higher in IGF-I-mdx mice, possibly due to a decrease in SR Ca2+ leak rate [mdx, 19.0 ± 3.5% of loaded SR (n=13) vs. IGF-mdx, 7.2 ± 3.0% (n=14)]. These results demonstrate that muscle specific over-expression of IGF-I in mdx mice can attenuate abnormal E-C coupling. Supported by the Muscular Dystrophy Association (USA).

Alternative dystrophin gene transcripts in golden retriever muscular dystrophy.

Golden retriever muscular dystrophy (GRMD), the canine model of Duchenne muscular dystrophy (DMD), is caused by a splice site mutation in the dystrophin gene. This mutation predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. Western blot analysis of skeletal muscle from GRMD dogs reveals a slightly truncated 390-kD protein that is approximately 91% the size of normal dystrophin. This 390-kD dystrophin suggests that GRMD dogs, like some DMD patients, employ a mechanism to overcome their predicted frameshift. Reverse-transcriptase polymerase chain reaction on GRMD muscle has revealed two in-frame dystrophin transcripts which lack either exons 3-9 or exons 5-12. Both transcripts could be translated into a dystrophin protein of approximately 390 kD. An understanding of how truncated dystrophin is produced in GRMD may allow this mechanism to be manipulated toward a potential therapy for DMD.

Transplantation of Dermal Fibroblasts Expressing MyoD1 in Mouse Muscles

Transplantation of normal myoblasts into dystrophic muscles is a potential treatment for Duchenne muscular dystrophy (DMD). However, the success of such grafts is limited by the immune system responses. To avoid rejection problems, autologous transplantation of the patient's corrected myoblasts has been proposed. Regretfully, the low proliferative capacity of DMD myoblasts in culture (due to their premature senescence) limits such procedure. On the other hand, modification of dermal fibroblasts leading to the myogenic pathway using a master regulatory gene for myogenesis is an interesting alternative approach. In this study, the retrovirally encoded MyoD1 cDNA was introduced in dermal fibroblasts of TnI LacZ mice to provoke their conversion into myoblast-like cells.In vitroandin vivoassays were done and the results were compared to those obtained with uninfected fibroblasts and myoblasts. Some MyoD1-expressing fibroblasts were able to fuse and to express β-galactosidase (under the transcriptionnal control of the Troponin I promoter), dystrophin and desminin vitro.Thirty days following implantation of these modified fibroblasts in muscles of mdx mice, an average of 7 β-Gal+/Dys− muscle fibers were observed. No β-Gal+ fibers were observed after the transplantation of uninfected fibroblasts. Our results indicate that the successful implantation of myoblasts obtained from genetically modified fibroblasts is indeed feasible. However, thein vitroconversion rate and thein vivofusion of genetically modified fibroblasts must be largely increased to consider this approach as a potential therapy for DMD and other myopathies.