Aberrant RhoA activation in macrophages increases senescence-associated secretory phenotypes and ectopic calcification in muscular dystrophic mice.

Duchenne Muscular Dystrophy (DMD) patients often suffer from both muscle wasting and osteoporosis. Our previous studies have revealed reduced regeneration potential in skeletal muscle and bone, concomitant with ectopic calcification of soft tissues in double knockout (dKO, dystrophin-/-; utrophin-/-) mice, a severe murine model for DMD. We found significant involvement of RhoA/ROCK (Rho-Associated Protein Kinase) signaling in mediating ectopic calcification of muscles in dKO mice. However, the cellular identity of these RhoA+ cells, and the role that RhoA plays in the chronic inflammation-associated pathologies has not been elucidated. Here, we report that CD68+ macrophages are highly prevalent at the sites of ectopic calcification of dKO mice, and that these macrophages highly express RhoA. Macrophages from dKO mice feature a shift towards a more pro-inflammatory M1 polarization and an increased expression of various senescence-associated secretory phenotype (SASP) factors that was reduced with the RhoA/ROCK inhibitor Y-27632. Further, systemic inhibition of RhoA activity in dKO mice led to reduced number of RhoA+/CD68+ cells, as well as a reduction in fibrosis and ectopic calcification. Together, these data revealed that RhoA signaling may be a key regulator of imbalanced mineralization in the dystrophic musculoskeletal system and consequently a therapeutic target for the treatment of DMD or other related muscle dystrophies.

Irradiation dependent inflammatory response may enhance satellite cell engraftment.

Skeletal muscle stem (satellite) cells transplanted into host mouse muscles contribute to muscle regeneration. Irradiation of host muscle enhances donor stem cell engraftment by promoting the proliferation of transplanted donor cells. We hypothesised that, similar to other systems, cells damaged by radiation might be effecting this donor cell proliferation. But we found no difference in the percentage of dying (TUNEL+) cells in immunodeficient dystrophic mouse muscles at the times after the irradiation dose that enhances donor cell engraftment. Similarly, irradiation did not significantly increase the number of TUNEL+ cells in non-dystrophic immunodeficient mouse muscles and it only slightly enhanced donor satellite cell engraftment in this mouse strain, suggesting either that the effector cells are present in greater numbers within dystrophic muscle, or that an innate immune response is required for effective donor cell engraftment. Donor cell engraftment within non-irradiated dystrophic host mouse muscles was not enhanced if they were transplanted with either satellite cells, or myofibres, derived from irradiated dystrophic mouse muscle. But a mixture of cells from irradiated muscle transplanted with donor satellite cells promoted donor cell engraftment in a few instances, suggesting that a rare, yet to be identified, cell type within irradiated dystrophic muscle enhances the donor stem cell-mediated regeneration. The mechanism by which cells within irradiated host muscle promote donor cell engraftment remains elusive.

Total Absence of Dystrophin Expression Exacerbates Ectopic Myofiber Calcification and Fibrosis and Alters Macrophage Infiltration Patterns.

Duchenne muscular dystrophy (DMD) causes severe disability and death of young men because of progressive muscle degeneration aggravated by sterile inflammation. DMD is also associated with cognitive and bone-function impairments. This complex phenotype results from the cumulative loss of a spectrum of dystrophin isoforms expressed from the largest human gene. Although there is evidence for the loss of shorter isoforms having impact in the central nervous system, their role in muscle is unclear. We found that at 8 weeks, the active phase of pathology in dystrophic mice, dystrophin-null mice (mdxßgeo) presented with a mildly exacerbated phenotype but without an earlier onset, increased serum creatine kinase levels, or decreased muscle strength. However, at 12 months, mdxßgeo diaphragm strength was lower, whereas fibrosis increased, compared with mdx. The most striking features of the dystrophin-null phenotype were increased ectopic myofiber calcification and altered macrophage infiltration patterns, particularly the close association of macrophages with calcified fibers. Ectopic calcification had the same temporal pattern of presentation and resolution in mdxßgeo and mdx muscles, despite significant intensity differences across muscle groups. Comparison of the rare dystrophin-null patients against those with mutations affecting full-length dystrophins may provide mechanistic insights for developing more effective treatments for DMD.

The Danger Signal Extracellular ATP Is Involved in the Immunomediated Damage of α-Sarcoglycan-Deficient Muscular Dystrophy.

In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.

Biological scaffold-mediated delivery of myostatin inhibitor promotes a regenerative immune response in an animal model of Duchenne muscular dystrophy.

Recent studies have reported that the immune system significantly mediates skeletal muscle repair and regeneration. Additionally, biological scaffolds have been shown to play a role in polarizing the immune microenvironment toward pro-myogenic outcomes. Moreover, myostatin inhibitors are known to promote muscle regeneration and ameliorate fibrosis in animal models of Duchenne muscular dystrophy (DMD), a human disease characterized by chronic muscle degeneration. Biological scaffolds and myostatin inhibition can potentially influence immune-mediated regeneration in the dystrophic environment, but have not been evaluated together. Toward this end, here we created an injectable biological scaffold composed of hyaluronic acid and processed skeletal muscle extracellular matrix. This material formed a cytocompatible hydrogel at physiological temperatures in vitro When injected subfascially above the tibialis anterior muscles of both WT and dystrophic mdx-5Cv mice, a murine model of DMD, the hydrogel spreads across the entire muscle before completely degrading at 3 weeks in vivo We found that the hydrogel is associated with CD206+ pro-regenerative macrophage polarization and elevated anti-inflammatory cytokine expression in both WT and dystrophic mice. Co-injection of both hydrogel and myostatin inhibitor significantly increased FoxP3+ regulatory T cell modulation and Foxp3 gene expression in the scaffold immune microenvironment. Finally, delivery of myostatin inhibitor with the hydrogel increased its bioactivity in vivo, and transplantation of immortalized human myoblasts with the hydrogel promoted their survival in vivo This study identifies a key role for biological scaffolds and myostatin inhibitors in modulating a pro-regenerative immune microenvironment in dystrophic muscle.

Vascular Delivery of Allogeneic MuStem Cells in Dystrophic Dogs Requires Only Short-Term Immunosuppression to Avoid Host Immunity and Generate Clinical/Tissue Benefits.

Growing demonstrations of regenerative potential for some stem cells led recently to promising therapeutic proposals for neuromuscular diseases. We have shown that allogeneic MuStem cell transplantation into Golden Retriever muscular dystrophy (GRMD) dogs under continuous immunosuppression (IS) leads to persistent clinical stabilization and muscle repair. However, long-term IS in medical practice is associated with adverse effects raising safety concerns. Here, we investigate whether the IS removal or its restriction to the transplantation period could be considered. Dogs aged 4-5 months old received vascular infusions of allogeneic MuStem cells without IS (GRMDMU/no-IS) or under transient IS (GRMDMU/tr-IS). At 5 months post-infusion, persisting clinical status improvement of the GRMDMU/tr-IS dogs was observed while GRMDMU/no-IS dogs exhibited no benefit. Histologically, only 9-month-old GRMDMU/tr-IS dogs showed an increased muscle regenerative activity. A mixed cell reaction with the host peripheral blood mononucleated cells (PBMCs) and corresponding donor cells revealed undetectable to weak lymphocyte proliferation in GRMDMU/tr-IS dogs compared with a significant proliferation in GRMDMU/no-IS dogs. Importantly, any dog group showed neither cellular nor humoral anti-dystrophin responses. Our results show that transient IS is necessary and sufficient to sustain allogeneic MuStem cell transplantation benefits and prevent host immunity. These findings provide useful critical insight to designing therapeutic strategies.

Targeting early PKCθ-dependent T-cell infiltration of dystrophic muscle reduces disease severity in a mouse model of muscular dystrophy.

Chronic muscle inflammation is a critical feature of Duchenne muscular dystrophy and contributes to muscle fibre injury and disease progression. Although previous studies have implicated T cells in the development of muscle fibrosis, little is known about their role during the early stages of muscular dystrophy. Here, we show that T cells are among the first cells to infiltrate mdx mouse dystrophic muscle, prior to the onset of necrosis, suggesting an important role in early disease pathogenesis. Based on our comprehensive analysis of the kinetics of the immune response, we further identify the early pre-necrotic stage of muscular dystrophy as the relevant time frame for T-cell-based interventions. We focused on protein kinase C θ (PKCθ, encoded by Prkcq), a critical regulator of effector T-cell activation, as a potential target to inhibit T-cell activity in dystrophic muscle. Lack of PKCθ not only reduced the frequency and number of infiltrating T cells but also led to quantitative and qualitative changes in the innate immune cell infiltrate in mdx/Prkcq-/- muscle. These changes were due to the inhibition of T cells, since PKCθ was necessary for T-cell but not for myeloid cell infiltration of acutely injured muscle. Targeting T cells with a PKCθ inhibitor early in the disease process markedly diminished the size of the inflammatory cell infiltrate and resulted in reduced muscle damage. Moreover, diaphragm necrosis and fibrosis were also reduced following treatment. Overall, our findings identify the early T-cell infiltrate as a therapeutic target and highlight the potential of PKCθ inhibition as a therapeutic approach to muscular dystrophy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Co-delivery of indoleamine 2,3-dioxygenase prevents loss of expression of an antigenic transgene in dystrophic mouse muscles.

A significant problem affecting gene therapy approaches aiming at achieving long-term transgene expression is the immune response against the protein product of the therapeutic gene, which can reduce or eliminate the therapeutic effect. The problem is further exacerbated when therapy involves targeting an immunogenic tissue and/or one with a pre-existing inflammatory phenotype, such as dystrophic muscles. In this proof-of-principle study, we co-expressed a model antigen, bacterial ß-galactosidase, with an immunosuppressive factor, indoleamine 2,3-dioxygenase 1 (IDO1), in muscles of the mdx mouse model of Duchenne muscular dystrophy. This treatment prevented loss of expression of the transgene concomitant with significantly elevated expression of T-regulatory (Treg) markers in the IDO1-expressing muscles. Moreover, co-expression of IDO1 resulted in reduced serum levels of anti-ß-gal antibodies. These data indicate that co-expression of genes encoding immunomodulatory enzymes controlling kynurenine pathways provide a viable strategy for preventing loss of transgenes targeted into dystrophic muscles with pre-existing inflammation.

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.

Self-adjuvanted hyaluronate--antigenic peptide conjugate for transdermal treatment of muscular dystrophy.

Duchenne's muscular dystrophy (DMD) is a neuromuscular disorder accompanied with muscle weakness and wasting. Since myostatin was reported to be a key regulator of muscle wasting, myostatin inhibitors have been investigated as therapeutic candidates for the treatment of muscular diseases. Here, we report an antigenic peptide of myostatin fragment (MstnF) conjugated to hyaluronate (HA) with a low molecular weight (MW, 17 kDa) for transdermal immunotherapy of DMD. Facilitating the transdermal delivery, the low MW HA showed a boosting effect on the immunization of MstnF possibly by engaging both toll-like receptors and cluster of differentiation 44 (CD44). In vivo two-photon microscopy clearly visualized the effective transdermal penetration of HA-MstnF conjugates into deep intact skin layers. The transdermal immunization of mdx mice significantly increased antibody titers against myostatin. Furthermore, the mdx mice immunized with HA-MstnF conjugates resulted in statistically significant improvement in the biochemical and pathological status of skeletal musculature as well as functional behaviors.

Duchenne muscular dystrophy gene therapy in the canine model.

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disease caused by dystrophin deficiency. Gene therapy has significantly improved the outcome of dystrophin-deficient mice. Yet, clinical translation has not resulted in the expected benefits in human patients. This translational gap is largely because of the insufficient modeling of DMD in mice. Specifically, mice lacking dystrophin show minimum dystrophic symptoms, and they do not respond to the gene therapy vector in the same way as human patients do. Further, the size of a mouse is hundredfolds smaller than a boy, making it impossible to scale-up gene therapy in a mouse model. None of these limitations exist in the canine DMD (cDMD) model. For this reason, cDMD dogs have been considered a highly valuable platform to test experimental DMD gene therapy. Over the last three decades, a variety of gene therapy approaches have been evaluated in cDMD dogs using a number of nonviral and viral vectors. These studies have provided critical insight for the development of an effective gene therapy protocol in human patients. This review discusses the history, current status, and future directions of the DMD gene therapy in the canine model.

Intra-amniotic rAAV-mediated microdystrophin gene transfer improves canine X-linked muscular dystrophy and may induce immune tolerance.

Duchenne muscular dystrophy (DMD) is a severe congenital disease due to mutations in the dystrophin gene. Supplementation of dystrophin using recombinant adenoassociated virus vector has promise as a treatment of DMD, although therapeutic benefit of the truncated dystrophin still remains to be elucidated. Besides, host immune responses against the vector as well as transgene products have been denoted in the clinical gene therapy studies. Here, we transduced dystrophic dogs fetuses to investigate the therapeutic effects of an AAV vector expressing microdystrophin under conditions of immune tolerance. rAAV-CMV-microdystrophin and a rAAV-CAG-luciferase were injected into the amniotic fluid surrounding fetuses. We also reinjected rAAV9-CMV-microdystrophin into the jugular vein of an infant dystrophic dog to induce systemic expression of microdystrophin. Gait and cardiac function significantly improved in the rAAV-microdystrophin-injected dystrophic dog, suggesting that an adequate treatment of rAAV-microdystrophin with immune modulation induces successful long-term transgene expression to analyze improved dystrophic phenotype.

Regulatory T cells suppress muscle inflammation and injury in muscular dystrophy.

We examined the hypothesis that regulatory T cells (Tregs) modulate muscle injury and inflammation in the mdx mouse model of Duchenne muscular dystrophy (DMD). Although Tregs were largely absent in the muscle of wild-type mice and normal human muscle, they were present in necrotic lesions, displayed an activated phenotype, and showed increased expression of interleukin-10 (IL-10) in dystrophic muscle from mdx mice. Depletion of Tregs exacerbated muscle injury and the severity of muscle inflammation, which was characterized by an enhanced interferon-γ (IFN-γ) response and activation of M1 macrophages. To test the therapeutic value of targeting Tregs in muscular dystrophy, we treated mdx mice with IL-2/anti-IL-2 complexes and found that Tregs and IL-10 concentrations were increased in muscle, resulting in reduced expression of cyclooxygenase-2 and decreased myofiber injury. These findings suggest that Tregs modulate the progression of muscular dystrophy by suppressing type 1 inflammation in muscle associated with muscle fiber injury, and highlight the potential of Treg-modulating agents as therapeutics for DMD.

A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer.

Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy. Thus, we compared dystrophic mice devoid of α-sarcoglycan with healthy mice to characterize immune cell activation and cellular populations contributing to the loss of gene-modified myofibers. Following rAAV2/1 delivery of an immunogenic α-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates. Selective cell subset depletion models revealed that CD4+ T cells were essential for transgene rejection in both healthy and pathologic mice, but macrophages and CD8+ T cells additionally contributed as effector cells of transgene rejection only in dystrophic mice. Vectors restricting transgene expression in antigen-presenting cells showed that endogenous presentation of transgene products was the sole mechanism responsible for T-cell priming in normal mice, whereas additional and protracted antigenic presentation occurred in dystrophic animals, leading to secondary CD4+ T-cell activation and failure to maintain transgene expression. Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.

Immunoproteasome in animal models of Duchenne muscular dystrophy.

Increased proteasome activity has been implicated in the atrophy and deterioration associated with dystrophic muscles of Duchenne muscular dystrophy (DMD). While proteasome inhibitors show promise in the attenuation of muscle degeneration, proteasome inhibition-induced toxicity was a major drawback of this therapeutic strategy. Inhibitors that selectively target the proteasome subtype that is responsible for the loss in muscle mass and quality would reduce side effects and be less toxic. This study examined proteasome activity and subtype populations, along with muscle function, morphology and damage in wild-type (WT) mice and two murine models of DMD, dystrophin-deficient (MDX) and dystrophin- and utrophin-double-knockout (DKO) mice. We found that immunoproteasome content was increased in dystrophic muscles while the total proteasome content was unchanged among the three genotypes of mice. Proteasome proteolytic activity was elevated in dystrophic muscles, especially in DKO mice. These mice also exhibited more severe muscle atrophy than either WT or MDX mice. Muscle damage and regeneration, characterized by the activity of muscle creatine kinase in the blood and the percentage of central nuclei were equally increased in dystrophic mice. Accordingly, the overall muscle function was similarly reduced in both dystrophic mice compared with WT. These data demonstrated that there was transformation of standard proteasomes to immunoproteasomes in dystrophic muscles. In addition, DKO that showed greatest increase in proteasome activities also demonstrated more severe atrophy compared with MDX and WT. These results suggest a putative role for the immunoproteasome in muscle deterioration associated with DMD and provide a potential target for therapeutic intervention.

Selective activation of α7 nicotinic acetylcholine receptor (nAChRα7) inhibits muscular degeneration in mdx dystrophic mice.

Amount evidence indicates that α7 nicotinic acetylcholine receptor (nAChRα7) activation reduces production of inflammatory mediators. This work aimed to verify the influence of endogenous nAChRα7 activation on the regulation of full-blown muscular inflammation in mdx mouse with Duchenne muscular dystrophy. We used mdx mice with 3 weeks-old at the height myonecrosis, and C57 nAChRα7(+/+) wild-type and nAChRα7(-/-) knockout mice with muscular injury induced with 60µL 0.5% bupivacaine (bp) in the gastrocnemius muscle. Pharmacological treatment included selective nAChRα7 agonist PNU282987 (0.3mg/kg and 1.0mg/kg) and the antagonist methyllycaconitine (MLA at 1.0mg/kg) injected intraperitoneally for 7 days. Selective nAChRα7 activation of mdx mice with PNU282987 reduced circulating levels of lactate dehydrogenase (LDH, a marker of cell death by necrosis) and the area of perivascular inflammatory infiltrate, and production of inflammatory mediators TNFα and metalloprotease MMP-9 activity. Conversely, PNU282987 treatment increased MMP-2 activity, an indication of muscular tissue remodeling associated with regeneration, in both mdx mice and WTα7 mice with bp-induced muscular lesion. Treatment with PNU282987 had no effect on α7KO, and MLA abolished the nAChRα7 agonist-induced anti-inflammatory effect in both mdx and WT. In conclusion, nAChRα7 activation inhibits muscular inflammation and activates tissue remodeling by increasing muscular regeneration. These effects were not accompanied with fibrosis and/or deposition of non-functional collagen. The nAChRα7 activation may be considered as a potential target for pharmacological strategies to reduce inflammation and activate mechanisms of muscular regeneration.

Characterization of 65 epitope-specific dystrophin monoclonal antibodies in canine and murine models of duchenne muscular dystrophy by immunostaining and western blot.

Epitope-specific monoclonal antibodies can provide unique insights for studying cellular proteins. Dystrophin is one of the largest cytoskeleton proteins encoded by 79 exons. The absence of dystrophin results in Duchenne muscular dystrophy (DMD). Over the last two decades, dozens of exon-specific human dystrophin monoclonal antibodies have been developed and successfully used for DMD diagnosis. Unfortunately, the majority of these antibodies have not been thoroughly characterized in dystrophin-deficient dogs, an outstanding large animal model for translational research. To fill the gap, we performed a comprehensive study on 65 dystrophin monoclonal antibodies in normal and dystrophic dogs (heart and skeletal muscle) by immunofluorescence staining and western blot. For comparison, we also included striated muscles from normal BL10 and dystrophin-null mdx mice. Our analysis revealed distinctive species, tissue and assay-dependent recognition patterns of different antibodies. Importantly, we identified 15 antibodies that can consistently detect full-length canine dystrophin in both immunostaining and western blot. Our results will serve as an important reference for studying DMD in the canine model.

Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model.

Animal and human gene therapy studies utilizing AAV vectors have shown that immune responses to AAV capsid proteins can severely limit transgene expression. The main source of capsid antigen is that associated with the AAV vectors, which can be reduced by stringent vector purification. A second source of AAV capsid proteins is that expressed from cap genes aberrantly packaged into AAV virions during vector production. This antigen source can be eliminated by the use of a cap gene that is too large to be incorporated into an AAV capsid, such as a cap gene containing a large intron (captron gene). Here, we investigated the effects of elimination of cap gene transfer and of vector purification by CsCl gradient centrifugation on AAV vector immunogenicity and expression following intramuscular injection in dogs. We found that both approaches reduced vector immunogenicity and that combining the two produced the lowest immune responses and highest transgene expression. This combined approach enabled the use of a relatively mild immunosuppressive regimen to promote robust micro-dystrophin gene expression in Duchenne muscular dystrophy-affected dogs. Our study shows the importance of minimizing AAV cap gene impurities and indicates that this improvement in AAV vector production may benefit human applications.

Distinct roles of TRAF6 at early and late stages of muscle pathology in the mdx model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by loss of functional dystrophin protein. Accumulating evidence suggests that the deficiency of dystrophin leads to aberrant activation of many signaling pathways which contribute to disease progression. However, the proximal signaling events leading to the activation of various pathological cascades in dystrophic muscle remain less clear. TNF receptor-associated factor 6 (TRAF6) is an adaptor protein which acts as a signaling intermediate for several receptor-mediated signaling events leading to the context-dependent activation of a number of signaling pathways. TRAF6 is also an E3 ubiquitin ligase and an important regulator of autophagy. However, the role of TRAF6 in pathogenesis of DMD remains unknown. Here, we demonstrate that the levels and activity of TRAF6 are increased in skeletal muscle of mdx (a mouse model of DMD) mice. Targeted deletion of TRAF6 improves muscle strength and reduces fiber necrosis, infiltration of macrophages and the activation of proinflammatory transcription factor nuclear factor-kappa B (NF-κB) in 7-week-old mdx mice. Ablation of TRAF6 also increases satellite cells proliferation and myofiber regeneration in young mdx mice. Intriguingly, ablation of TRAF6 exacerbates muscle injury and increases fibrosis in 9-month-old mdx mice. TRAF6 inhibition reduces the markers of autophagy and Akt signaling in dystrophic muscle of mdx mice. Collectively, our study suggests that while the inhibition of TRAF6 improves muscle structure and function in young mdx mice, its continued inhibition causes more severe myopathy at later stages of disease progression potentially through repressing autophagy.

Prolonged gene expression in muscle is achieved without active immune tolerance using microrRNA 142.3p-regulated rAAV gene transfer.

Gene transfer efficacy is limited by unwanted immunization against transgene products. In some models, immunization may be avoided by regulating transgene expression with mir142.3p target sequences. Yet, it is unclear if such a strategy controls T-cell responses following recombinant adeno-associated viral vector (rAAV)-mediated gene transfer, particularly in muscle. In mice, intramuscular rAAV1 gene delivery of a tagged human sarcoglycan muscle protein is robustly immunogenic and leads to muscle destruction. In this model, the simple insertion of mir142.3p-target sequences in the transgene expression cassette modifies the outcome of gene transfer, providing high and persistent levels of muscle transduction in C57Bl/6 mice. Such regulated vector fails to prime specific CD4 and CD8 T cells; although, transgene tolerance seems to result from ignorance and could be broken by a robust antigenic challenge. While effective in normal mice, the mir142.3p-regulated transgene remains immunogenic in sarcoglycan-deficient dystrophic mice. In these mice, transgene expression is only prolonged but does not persist as effector CD4 and CD8 T-cell responses develop. Thus, using a mir142.3p-regulated transgene can improve rAAV muscle gene transfer results, but the level of efficacy depends on the context of application. In normal muscle, this strategy is sufficient to prevent immunization and functions even more effectively than tissue-specific promoters. In dystrophic models, additional strategies are required to fully control T-cell responses.