Search

Accelerated approval based on a likely surrogate endpoint can be life-changing for patients suffering from a rare progressive disease with unmet medical need, as it substantially hastens access to potentially lifesaving therapies. In one such example, antisense morpholinos were approved to treat Duchenne muscular dystrophy (DMD) based on measurement of shortened dystrophin in skeletal muscle biopsies as a surrogate biomarker. New, promising therapeutics for DMD include AAV gene therapy to restore another form of dystrophin termed mini- or microdystrophin. AAV-microdystrophins are currently in clinical trials but have yet to be accepted by regulatory agencies as reasonably likely surrogate endpoints. To evaluate microdystrophin expression as a reasonably likely surrogate endpoint for DMD, this review highlights dystrophin biology in the context of functional and clinical benefit to support the argument that microdystrophin proteins have a high probability of providing clinical benefit based on their rational design. Unlike exon-skipping based strategies, the approach of rational design allows for functional capabilities (i.e. quality) of the protein to be maximized with every patient receiving the same optimized microdystrophin. Therefore, the presence of rationally designed microdystrophin in a muscle biopsy is likely to predict clinical benefit and is consequently a strong candidate for a surrogate endpoint analysis to support accelerated approval.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)–microdystrophin (μDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 10 13 vector genomes per kilogram (vg/kg), 1 × 10 14 vg/kg, and 2 × 10 14 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-μDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day −7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; μDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

Duchenne muscular dystrophy, like other muscular dystrophies, is a progressive disorder hallmarked by muscle degeneration, inflammation, and fibrosis. Latent transforming growth factor β (TGFβ) binding protein 4 (LTBP4) is an extracellular matrix protein found in muscle. LTBP4 sequesters and inhibits a precursor form of TGFβ. LTBP4 was originally identified from a genome-wide search for genetic modifiers of muscular dystrophy in mice, where there are two different alleles. The protective form of LTBP4, which contains an insertion of 12 amino acids in the protein’s hinge region, was linked to increased sequestration of latent TGFβ, enhanced muscle membrane stability, and reduced muscle fibrosis. The deleterious form of LTBP4 protein, lacking 12 amino acids, was more susceptible to proteolysis and promoted release of latent TGF-β, and together, these data underscored the functional role of LTBP4’s hinge. Here, we generated a monoclonal human anti-LTBP4 antibody directed toward LTBP4’s hinge region. In vitro, anti-LTBP4 bound LTBP4 protein and reduced LTBP4 proteolytic cleavage. In isolated myofibers, the LTBP4 antibody stabilized the sarcolemma from injury. In vivo, anti-LTBP4 treatment of dystrophic mice protected muscle against force loss induced by eccentric contraction. Anti-LTBP4 treatment also reduced muscle fibrosis and enhanced muscle force production, including in the diaphragm muscle, where respiratory function was improved. Moreover, the anti-LTBP4 in combination with prednisone, a standard of care for Duchenne muscular dystrophy, further enhanced muscle function and protected against injury in

We tested the hypothesis that voluntary wheel running would complement microdystrophin gene therapy to improve muscle function in young mdx mice, a model of Duchenne muscular dystrophy. mdx mice injected with a single dose of AAV9-CK8-microdystrophin or vehicle at age 7 weeks were assigned to three groups: mdxRGT (run, gene therapy), mdxGT (no run, gene therapy), or mdx (no run, no gene therapy). Wild-type (WT) mice were assigned to WTR (run) and WT (no run) groups. WTR and mdxRGT performed voluntary wheel running for 21 weeks; remaining groups were cage active. Robust expression of microdystrophin occurred in heart and limb muscles of treated mice. mdxRGT versus mdxGT mice showed increased microdystrophin in quadriceps but decreased levels in diaphragm. mdx final treadmill fatigue time was depressed compared to all groups, improved in mdxGT, and highest in mdxRGT. Both weekly running distance (km) and final treadmill fatigue time for mdxRGT and WTR were similar. Remarkably, mdxRGT diaphragm power was only rescued to 60% of WT, suggesting a negative impact of running. However, potential changes in fiber type distribution in mdxRGT diaphragms could indicate an adaptation to trade power for endurance. Post-treatment in vivo maximal plantar flexor torque relative to baseline values was greater for mdxGT and mdxRGT versus all other groups. Mitochondrial respiration rates from red quadriceps fibers were significantly improved in mdxGT animals, but the greatest bioenergetic benefit was observed in the mdxRGT group. Additional assessments revealed partial to full functional restoration in mdxGT and mdxRGT muscles relative to WT. These data demonstrate that voluntary wheel running combined with microdystrophin gene therapy in young mdx mice improved whole-body performance, affected muscle function differentially, mitigated energetic deficits, but also revealed some detrimental effects of exercise. With microdystrophin gene therapy currently in clinical trials, these data may help us understand the potential impact of exercise in treated patients., Graphical abstract, We report that voluntary exercise complements microdystrophin gene therapy in the mdx mouse, a model of Duchenne muscular dystrophy (DMD), by increasing running endurance, muscle strength, and energy production. These and other findings may help us understand how exercise in DMD boys may influence microdystrophin gene therapy.

Aberrant expression of the cardiac gap junction protein connexin-43 (Cx43) has been suggested as playing a role in the development of cardiac disease in the mdx mouse model of Duchenne muscular dystrophy (DMD); however, a mechanistic understanding of this association is lacking. Here, we identified a reduction of phosphorylation of Cx43 serines S325/S328/S330 in human and mouse DMD hearts. We hypothesized that hypophosphorylation of Cx43 serine-triplet triggers pathological Cx43 redistribution to the lateral sides of cardiomyocytes (remodeling). Therefore, we generated knockin mdx mice in which the Cx43 serine-triplet was replaced with either phospho-mimicking glutamic acids (mdxS3E) or nonphosphorylatable alanines (mdxS3A). The mdxS3E, but not mdxS3A, mice were resistant to Cx43 remodeling, with a corresponding reduction of Cx43 hemichannel activity. MdxS3E cardiomyocytes displayed improved intracellular Ca2+ signaling and a reduction of NADPH oxidase 2 (NOX2)/ROS production. Furthermore, mdxS3E mice were protected against inducible arrhythmias, related lethality, and the development of cardiomyopathy. Inhibition of microtubule polymerization by colchicine reduced both NOX2/ROS and oxidized CaMKII, increased S325/S328/S330 phosphorylation, and prevented Cx43 remodeling in mdx hearts. Together, these results demonstrate a mechanism of dystrophic Cx43 remodeling and suggest that targeting Cx43 may be a therapeutic strategy for preventing heart dysfunction and arrhythmias in DMD patients.

The neuronal nitric-oxide synthase (nNOS) splice variant nNOSµ is essential for skeletal muscle function. Its localization is dependent on dystrophin, which stabilizes the dystrophin glycoprotein complex (DGC) at the sarcolemma of skeletal muscle fibers. In Duchenne muscular dystrophy (DMD) dystrophin is absent and sarcolemmal nNOS is lost. This leads to functional ischemia due to a decrease in contraction-induced vasodilation. In cardiomyocytes, nNOSµ is believed to be the predominant NOS isoform. However, the association of nNOS with the DGC in the heart is unclear. Here, we report nNOS localization at the intercalated discs (IDs) of cardiomyocytes, where utrophin is highly expressed. In mdx, mdx:utr, nNOSµ knock-out (KO), and mdx:nNOSµ KO mice, we observed a gradual reduction of nNOS at IDs and disrupted ID morphology, compared to wild-type. In mdx:nNOSµ KO mice, but not in mdx or nNOSµ KO mice, we also observed an early development of cardiac fibrosis. These findings suggest that nNOS localization in the heart may not depend exclusively on the presence of dystrophin. Additionally, the β1 subunit of soluble guanylyl cyclase (sGC), responsible for the production of cGMP through nitric oxide (NO) signaling, was also detected at the IDs. Together, our results suggest a new role of nNOS at the IDs for the cGMP-dependent NO pathway and the maintenance of ID morphology.

Duchenne muscular dystrophy (DMD) associated cardiomyopathy remains incurable. Connexin 43 (Cx43) is upregulated and remodeled in the hearts of mdx mice, a mouse model of DMD. Hearts from Wild Type, mdx, and mdx:Cx43(+/-) mice were studied before (4-6 months) and after (10-15 months) the onset of cardiomyopathy to assess the impact of decreasing Cx43 levels on cardiac pathology in dystrophic mice. Increased connexin 43 protein levels in mdx hearts were not observed in mdx:Cx43(+/-) hearts. Cx43 remodeling in mdx hearts was attenuated in mdx:Cx43(+/-) hearts. At time-point 4-6 months, isolated cardiomyocytes from mdx hearts displayed enhanced ethidium bromide uptake, augmented intracellular calcium signals and increased production of reactive oxygen species. These pathological features were improved in mdx:Cx43(+/-) cardiomyocytes. Isoproterenol-challenged mdx:Cx43(+/-) mice did not show arrhythmias or acute lethality observed in mdx mice. Likewise, isoproterenol-challenged mdx:Cx43(+/-) isolated hearts were also protected from arrhythmogenesis. At time-point 10-15 months, mdx:Cx43(+/-) mice showed decreased cardiac fibrosis and improved ventricular function, relative to mdx mice. These results suggest that normalization of connexin 43 protein levels in mdx mice reduces overall cardiac pathology.

Inhibitor of DNA binding (Id) proteins play important roles in regulating cardiac development via paracrine signaling. Id1/Id3 knockout mice die at mid-gestation with multiple cardiac defects. Single Id knockout studies have not reported cardiomyopathies. To bypass embryonic lethality we used Tie2CRE-mediated recombination to conditionally delete Id1 against global Id3 ablation (Id cDKOs), which develops adult-onset dilated cardiomyopathy. We confirm upregulation of thrombospondin-1 (TSP1) in Id cDKO hearts. Colocalization studies reveal increased TSP1 expression in the vicinity of endothelial cells and near regions of endocardial fibrosis/disruption. Downstream fibrotic molecules were upregulated. Endocardial capillary density was reduced with evidence of vascular distention. Treatment of Id cDKO cardiac explants with LSKL, a peptide antagonist of TSP1 activation of TGFβ, reversed the increased expression of fibrotic molecules. We conducted bone marrow transplant experiments in which we transferred bone marrow cells from Id cDKO mice into lethally irradiated WT mice. The majority of WT recipients of Id cDKO bone marrow cells phenocopied Id cDKO cardiac fibrosis 4 months post-transplantation. Injection of LSKL into adult Id cDKO mice led to downregulation of fibrotic molecules. The results prompt caution when bone marrow transfers from individuals potentially carrying mutations in the Id axis are applied in clinical settings.

Duchenne muscular dystrophy (DMD) is caused by an X-linked mutation that leads to the absence of dystrophin, resulting in life-threatening arrhythmogenesis and associated heart failure. The downstream events that lead to cardiomyopathy are largely unexplored. We unveiled a novel role for the gap junction protein connexin43 (Cx43) in maintaining cardiac conduction and ultimately in preventing heart failure from occurring in DMD. In mild mdx and severe mdx:utr mouse models of DMD, and human DMD tissues, Cx43 was found to be aberrantly upregulated and mislocalized to lateral sides of cardiomyocytes, with an abnormal phosphorylation pattern. We found that a triplet of serine residues, which plays a key role in a variety of cardiac pathologies, was hypophosphorylated in mdx relative to wild-type hearts. Electrocardiography on isoproterenol (Iso) challenged mice showed that both DMD mouse models developed arrhythmias and died within 24 hours, while wild-type mice were free of pathology. Administering peptide mimetic inhibitors of lateralized Cx43 channels prior to Iso challenge protected mdx mice from arrhythmogenesis and death. Cx43 copy number reduction in mdx:Cx43(+/-) mice not only ameliorated acute but also chronic, long-term symptoms of cardiomyopathy in mdx mice. Overall, our findings suggest that Cx43 lateralization contributes significantly to DMD cardiomyopathy and that selective inhibition of misplaced, upregulated channels may provide substantial benefit.

Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin–positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present.

The Inhibitor of DNA Binding (Id) proteins play a crucial role in regulating hematopoiesis and are known to interact with E proteins and the bHLH family of transcription factors. Current efforts seek to elucidate the individual roles of Id members in regulating hematopoietic development and specification. However, the nature of their functional redundancies remains elusive since ablation of multiple Id genes is embryonically lethal. We developed a model to test this compensation in the adult. We report that global Id3 ablation with Tie2Cre-mediated conditional ablation of Id1 in both hematopoietic and endothelial cells (Id cDKO) extends viability to 1 year but leads to multi-lineage hematopoietic defects including the emergence of anemia associated with defective erythroid development, a novel phenotype unreported in prior single Id knockout studies. We observe decreased cell counts in the bone marrow and splenomegaly to dimensions beyond what is seen in single Id knockout models. Transcriptional dysregulation of hematopoietic regulators observed in bone marrow cells is also magnified in the spleen. E47 protein levels were elevated in Id cDKO bone marrow cell isolates, but decreased in the erythroid lineage. Chromatin immunoprecipitation (ChIP) studies reveal increased occupancy of E47 and GATA1 at the promoter regions of β-globin and E2A. Bone marrow transplantation studies highlight the importance of intrinsic Id signals in maintaining hematopoietic homeostasis while revealing a strong extrinsic influence in the development of anemia. Together, these findings demonstrate that loss of Id compensation leads to dysregulation of the hematopoietic transcriptional network and multiple defects in erythropoietic development in adult mice.

Duchenne muscular dystrophy (DMD) is caused by an X-linked mutation that leads to the absence of dystrophin, resulting in life-threatening arrhythmogenesis and associated heart failure. We targeted the gap junction protein connexin43 (Cx43) responsible for maintaining cardiac conduction. In mild mdx and severe mdx:utr mouse models of DMD and human DMD tissues, Cx43 was found to be pathologically mislocalized to lateral sides of cardiomyocytes. In addition, overall Cx43 protein levels were markedly increased in mouse and human DMD heart tissues examined. Electrocardiography on isoproterenol challenged mice showed that both models developed arrhythmias and died within 24 hours, while wild-type mice were free of pathology. Administering peptide mimetics to inhibit lateralized Cx43 function prior to challenge protected mdx mice from arrhythmogenesis and death, while mdx:utr mice displayed markedly improved ECG scores. These findings suggest that Cx43 lateralization contributes significantly to DMD arrhythmogenesis and that selective inhibition may provide substantial benefit.

According to the endosymbiotic hypothesis, the precursor of mitochondria invaded the precursor of eukaryotic cells, a process that began roughly 2 billion years ago. Since then, the majority of the genetic material translocated from the mitochondria to the nucleus, where now almost all mitochondrial proteins are expressed. Only a tiny amount of DNA remained in the mitochondria, known as mitochondrial DNA (mtDNA). In this study, we report that the transfer of mtDNA fragments to the nucleus of pluripotent stem cells is still ongoing. We show by in situ hybridization and agarose DNA two-dimensional gel technique that induced pluripotent stem (iPS) cells contain high levels of mtDNA in the nucleus. We found that a large proportion of the accumulated mtDNA sequences appear to be extrachromosomal. Accumulation of mtDNA in the nucleus is present not only in the iPS cells, but also in embryonic stem (ES) cells. However upon differentiation, the level of mtDNA in the nuclei of iPS and ES cells is substantially reduced. This reversible accumulation of mtDNA in the nucleus supports the notion that the nuclear copy number of mtDNA sequences may provide a novel mechanism by which chromosomal DNA is dynamically regulated in pluripotent stem cells.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies.