The glucocorticoid receptor acts locally to protect dystrophic muscle and heart during disease.

Absence of dystrophin results in muscular weakness, chronic inflammation and cardiomyopathy in Duchenne muscular dystrophy (DMD). Pharmacological corticosteroids are the DMD standard of care; however, they have harsh side effects and unclear molecular benefits. It is uncertain whether signaling by physiological corticosteroids and their receptors plays a modifying role in the natural etiology of DMD. Here, we knocked out the glucocorticoid receptor (GR, encoded by Nr3c1) specifically in myofibers and cardiomyocytes within wild-type and mdx52 mice to dissect its role in muscular dystrophy. Double-knockout mice showed significantly worse phenotypes than mdx52 littermate controls in measures of grip strength, hang time, inflammatory pathology and gene expression. In the heart, GR deletion acted additively with dystrophin loss to exacerbate cardiomyopathy, resulting in enlarged hearts, pathological gene expression and systolic dysfunction, consistent with imbalanced mineralocorticoid signaling. The results show that physiological GR functions provide a protective role during muscular dystrophy, directly contrasting its degenerative role in other disease states. These data provide new insights into corticosteroids in disease pathophysiology and establish a new model to investigate cell-autonomous roles of nuclear receptors and mechanisms of pharmacological corticosteroids.

Vamorolone improves Becker muscular dystrophy and increases dystrophin protein in bmx model mice.

There is no approved therapy for Becker muscular dystrophy (BMD), a genetic muscle disease caused by in-frame dystrophin deletions. We previously developed the dissociative corticosteroid vamorolone for treatment of the allelic, dystrophin-null disease Duchenne muscular dystrophy. We hypothesize vamorolone can treat BMD by safely reducing inflammatory signaling in muscle and through a novel mechanism of increasing dystrophin protein via suppression of dystrophin-targeting miRNAs. Here, we test this in the bmx mouse model of BMD. Daily oral treatment with vamorolone or prednisolone improves bmx grip strength and hang time phenotypes. Both drugs reduce myofiber size and decrease the percentage of centrally nucleated fibers. Vamorolone shows improved safety versus prednisolone by avoiding or reducing key side effects to behavior and growth. Intriguingly, vamorolone increases dystrophin protein in both heart and skeletal muscle. These data indicate that vamorolone, nearing approval for Duchenne, shows efficacy in bmx mice and therefore warrants clinical investigation in BMD.

Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by the absence of dystrophin protein. One current DMD therapeutic strategy, exon skipping, produces a truncated dystrophin isoform using phosphorodiamidate morpholino oligomers (PMOs). However, the potential of exon skipping therapeutics has not been fully realized as increases in dystrophin protein have been minimal in clinical trials. Here, we investigate how miR-146a-5p, which is highly elevated in dystrophic muscle, impacts dystrophin protein levels. We find inflammation strongly induces miR-146a in dystrophic, but not wild-type myotubes. Bioinformatics analysis reveals that the dystrophin 3'UTR harbors a miR-146a binding site, and subsequent luciferase assays demonstrate miR-146a binding inhibits dystrophin translation. In dystrophin-null mdx52 mice, co-injection of miR-146a reduces dystrophin restoration by an exon 51 skipping PMO. To directly investigate how miR-146a impacts therapeutic dystrophin rescue, we generated mdx52 with body-wide miR-146a deletion (146aX). Administration of an exon skipping PMO via intramuscular or intravenous injection markedly increases dystrophin protein levels in 146aX versus mdx52 muscles; skipped dystrophin transcript levels are unchanged, suggesting a post-transcriptional mechanism-of-action. Together, these data show that miR-146a expression opposes therapeutic dystrophin restoration, suggesting miR-146a inhibition warrants further research as a potential DMD exon skipping co-therapy.

The X-linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45-47.

BACKGROUND: Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. METHODS: We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000 bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. RESULTS: Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P = 0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4-fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. CONCLUSIONS: The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

Multi-Omics Identifies Circulating miRNA and Protein Biomarkers for Facioscapulohumeral Dystrophy.

The development of therapeutics for muscle diseases such as facioscapulohumeral dystrophy (FSHD) is impeded by a lack of objective, minimally invasive biomarkers. Here we identify circulating miRNAs and proteins that are dysregulated in early-onset FSHD patients to develop blood-based molecular biomarkers. Plasma samples from clinically characterized individuals with early-onset FSHD provide a discovery group and are compared to healthy control volunteers. Low-density quantitative polymerase chain reaction (PCR)-based arrays identify 19 candidate miRNAs, while mass spectrometry proteomic analysis identifies 13 candidate proteins. Bioinformatic analysis of chromatin immunoprecipitation (ChIP)-seq data shows that the FSHD-dysregulated DUX4 transcription factor binds to regulatory regions of several candidate miRNAs. This panel of miRNAs also shows ChIP signatures consistent with regulation by additional transcription factors which are up-regulated in FSHD (FOS, EGR1, MYC, and YY1). Validation studies in a separate group of patients with FSHD show consistent up-regulation of miR-100, miR-103, miR-146b, miR-29b, miR-34a, miR-454, miR-505, and miR-576. An increase in the expression of S100A8 protein, an inflammatory regulatory factor and subunit of calprotectin, is validated by Enzyme-Linked Immunosorbent Assay (ELISA). Bioinformatic analyses of proteomics and miRNA data further support a model of calprotectin and toll-like receptor 4 (TLR4) pathway dysregulation in FSHD. Moving forward, this panel of miRNAs, along with S100A8 and calprotectin, merit further investigation as monitoring and pharmacodynamic biomarkers for FSHD.

Serum miRNAs Are Pharmacodynamic Biomarkers Associated With Therapeutic Response in Pediatric Inflammatory Bowel Disease.

BACKGROUND: We sought to identify microRNAs (miRNAs) associated with response to anti-TNF-α or glucocorticoids in children with inflammatory bowel disease (IBD) to generate candidate pharmacodynamic and monitoring biomarkers. METHODS: Clinical response was assessed by Pediatric Crohn's Disease Activity Index and Pediatric Ulcerative Colitis Activity Index. Quantitative real-time polymerase chain reaction via Taqman Low-Density Array cards were used to identify miRNAs in a discovery cohort of responders (n = 11) and nonresponders (n = 8). Seven serum miRNAs associated with clinical response to treatment, along with 4 previously identified (miR-146a, miR-146b, miR-320a, miR-486), were selected for further study. Candidates were assessed in a validation cohort of serum samples from IBD patients pre- and post-treatment and from healthy controls. Expression of miRNA was also analyzed in inflamed mucosal biopsies from IBD patients and non-IBD controls. RESULTS: Discovery cohort analysis identified 7 miRNAs associated with therapeutic response: 5 that decreased (miR-126, miR-454, miR-26b, miR-26a, let-7c) and 2 that increased (miR-636, miR-193b). In the validation cohort, 7 of 11 candidate miRNAs changed in the same direction with response to anti-TNF-α therapies, glucocorticoids, or both. In mucosal biopsies, 7 out of 11 miRNAs were significantly increased in IBD vs healthy controls. CONCLUSIONS: Five candidate miRNAs associated with clinical response and mucosal inflammation in pediatric IBD patients were identified (miR-126, let-7c, miR-146a, miR-146b, and miR-320a). These miRNAs may be further developed as pharmacodynamic and response monitoring biomarkers for use in clinical care and trials.

Estrogen Receptor, Inflammatory, and FOXO Transcription Factors Regulate Expression of Myasthenia Gravis-Associated Circulating microRNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate important intracellular biological processes. In myasthenia gravis (MG), a disease-specific pattern of elevated circulating miRNAs has been found, and proposed as potential biomarkers. These elevated miRNAs include miR-150-5p, miR-21-5p, and miR-30e-5p in acetylcholine receptor antibody seropositive (AChR+) MG and miR-151a-3p, miR-423-5p, let-7a-5p, and let-7f-5p in muscle-specific tyrosine kinase antibody seropositive (MuSK+) MG. In this study, we examined the regulation of each of these miRNAs using chromatin immunoprecipitation sequencing (ChIP-seq) data from the Encyclopedia of DNA Elements (ENCODE) to gain insight into the transcription factor pathways that drive their expression in MG. Our aim was to look at the transcription factors that regulate miRNAs and then validate some of those in vivo with cell lines that have sufficient expression of these transcription factors This analysis revealed several transcription factor families that regulate MG-specific miRNAs including the Forkhead box or the FOXO proteins (FoxA1, FoxA2, FoxM1, FoxP2), AP-1, interferon regulatory factors (IRF1, IRF3, IRF4), and signal transducer and activator of transcription proteins (Stat1, Stat3, Stat5a). We also found binding sites for nuclear factor of activated T-cells (NFATC1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), early growth response factor (EGR1), and the estrogen receptor 1 (ESR1). AChR+ MG miRNAs showed a stronger overall regulation by the FOXO transcription factors, and of this group, miR-21-5p, let-7a, and let 7f were found to possess ESR1 binding sites. Using a murine macrophage cell line, we found activation of NF-κB -mediated inflammation by LPS induced expression of miR-21-5p, miR-30e-5p, miR-423-5p, let-7a, and let-7f. Pre-treatment of cells with the anti-inflammatory drugs prednisone or deflazacort attenuated induction of inflammation-induced miRNAs. Interestingly, the activation of inflammation induced packaging of the AChR+-specific miRNAs miR-21-5p and miR-30e-5p into exosomes, suggesting a possible mechanism for the elevation of these miRNAs in MG patient serum. In conclusion, our study summarizes the regulatory transcription factors that drive expression of AChR+ and MuSK+ MG-associated miRNAs. Our findings of elevated miR-21-5p and miR-30e-5p expression in immune cells upon inflammatory stimulation and the suppressive effect of corticosteroids strengthens the putative role of these miRNAs in the MG autoimmune response.

Muscle Weakness in Myositis: MicroRNA-Mediated Dystrophin Reduction in a Myositis Mouse Model and Human Muscle Biopsies.

OBJECTIVE: Muscle inflammation is a feature in myositis and Duchenne muscular dystrophy (DMD). Autoimmune mechanisms are thought to contribute to muscle weakness in patients with myositis. However, a lack of correlation between the extent of inflammatory cell infiltration and muscle weakness indicates that nonimmune pathologic mechanisms may play a role. The present study focused on 2 microRNA (miRNA) sets previously identified as being elevated in the muscle of patients with DMD-an "inflammatory" miRNA set that is dampened with glucocorticoids, and a "dystrophin-targeting" miRNA set that inhibits dystrophin translation-to test the hypothesis that these miRNAs are similarly dysregulated in the muscle of patients with myositis, and could contribute to muscle weakness and disease severity. METHODS: A major histocompatibility complex class I-transgenic mouse model of myositis was utilized to study gene and miRNA expression and histologic features in the muscle tissue, with the findings validated in human muscle biopsy tissue from 6 patients with myositis. Mice were classified as having mild or severe myositis based on transgene expression, body weight, histologic disease severity, and muscle strength/weakness. RESULTS: In mice with severe myositis, muscle tissue showed mononuclear cell infiltration along with elevated expression of type I interferon and NF-κB-regulated genes, including Tlr7 (3.8-fold increase, P < 0.05). Furthermore, mice with severe myositis showed elevated expression of inflammatory miRNAs (miR-146a, miR-142-3p, miR-142-5p, miR-455-3p, and miR-455-5p; ~3-40-fold increase, P < 0.05) and dystrophin-targeting miRNAs (miR-146a, miR-146b, miR-31, and miR-223; ~3-38-fold increase, P < 0.05). Bioinformatics analyses of chromatin immunoprecipitation sequencing (ChIP-seq) data identified at least one NF-κB consensus element within the promoter/enhancer regions of these miRNAs. Western blotting and immunofluorescence analyses of the muscle tissue from mice with severe myositis demonstrated reduced levels of dystrophin. In addition, elevated levels of NF-κB-regulated genes, TLR7, and miRNAs along with reduced dystrophin levels were observed in muscle biopsy tissue from patients with histologically severe myositis. CONCLUSION: These data demonstrate that an acquired dystrophin deficiency may occur through NF-κB-regulated miRNAs in myositis, thereby suggesting a unifying theme in which muscle injury, inflammation, and weakness are perpetuated both in myositis and in DMD.

Vamorolone targets dual nuclear receptors to treat inflammation and dystrophic cardiomyopathy.

Cardiomyopathy is a leading cause of death for Duchenne muscular dystrophy. Here, we find that the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) can share common ligands but play distinct roles in dystrophic heart and skeletal muscle pathophysiology. Comparisons of their ligand structures indicate that the Δ9,11 modification of the first-in-class drug vamorolone enables it to avoid interaction with a conserved receptor residue (N770/N564), which would otherwise activate transcription factor properties of both receptors. Reporter assays show that vamorolone and eplerenone are MR antagonists, whereas prednisolone is an MR agonist. Macrophages, cardiomyocytes, and CRISPR knockout myoblasts show vamorolone is also a dissociative GR ligand that inhibits inflammation with improved safety over prednisone and GR-specific deflazacort. In mice, hyperaldosteronism activates MR-driven hypertension and kidney phenotypes. We find that genetic dystrophin loss provides a second hit for MR-mediated cardiomyopathy in Duchenne muscular dystrophy model mice, as aldosterone worsens fibrosis, mass and dysfunction phenotypes. Vamorolone successfully prevents MR-activated phenotypes, whereas prednisolone activates negative MR and GR effects. In conclusion, vamorolone targets dual nuclear receptors to treat inflammation and cardiomyopathy with improved safety.

Muscle miRNAome shows suppression of chronic inflammatory miRNAs with both prednisone and vamorolone.

Corticosteroids are highly prescribed and effective anti-inflammatory drugs but the burden of side effects with chronic use significantly detracts from patient quality of life, particularly in children. Developing safer steroids amenable to long-term use is an important goal for treatment of chronic inflammatory diseases such as Duchenne muscular dystrophy (DMD). We have developed vamorolone (VBP15), a first-in-class dissociative glucocorticoid receptor (GR) ligand that shows the anti-inflammatory efficacy of corticosteroids without key steroid side effects in animal models. miRNAs are increasingly recognized as key regulators of inflammatory responses. To define effects of prednisolone and vamorolone on the muscle miRNAome, we performed a preclinical discovery study in the mdx mouse model of DMD. miRNAs associated with inflammation were highly elevated in mdx muscle. Both vamorolone and prednisolone returned these toward wild-type levels (miR-142-5p, miR-142-3p, miR-146a, miR-301a, miR-324-3p, miR-455-5p, miR-455-3p, miR-497, miR-652). Effects of vamorolone were largely limited to reduction of proinflammatory miRNAs. In contrast, prednisolone activated a separate group of miRNAs associated with steroid side effects and a noncoding RNA cluster homologous to human chromosome 14q32. Effects were validated for inflammatory miRNAs in a second, independent preclinical study. For the anti-inflammatory miRNA signature, bioinformatic analyses showed all of these miRNAs are directly regulated by, or in turn activate, the inflammatory transcription factor NF-κB. Moving forward miR-146a and miR-142 are of particular interest as biomarkers or novel drug targets. These data validate NF-κB signaling as a target of dissociative GR-ligand efficacy in vivo and provide new insight into miRNA signaling in chronic inflammation.

Identification of Pathway-Specific Serum Biomarkers of Response to Glucocorticoid and Infliximab Treatment in Children with Inflammatory Bowel Disease.

OBJECTIVE: Serum biomarkers may serve to predict early response to therapy, identify relapse, and facilitate drug development in inflammatory bowel disease (IBD). Biomarkers are particularly important in children, in whom achieving early remission and minimizing procedures are especially beneficial. METHODS: We profiled protein and micro RNA (miRNA) in serum from patients pre- and post-therapy, to identify molecular markers of pharmacodynamic effect. Serum was obtained from children with IBD before and after treatment with either corticosteroids (prednisone; n=12) or anti-tumor necrosis factor-α biologic (infliximab; n=7). Over 1,100 serum proteins were assayed using aptamer-based SOMAscan proteomics, and 22 miRNAs analyzed by quantitative real time PCR. Concordance of longitudinal changes between the groups was used to identify markers responsive to treatment. Bioinformatic analysis was used to build insight into mechanisms of changes in response to treatment. RESULTS: We identified 18 proteins and three miRNAs responsive to both prednisone and infliximab. Eight markers that decreased are associated with inflammation and have gene promoters regulated by nuclear factor (NF)-κB. Several that increased are associated with resolving inflammation and tissue damage. We also identified six markers that appear to be steroid-specific, three of which have glucocorticoid receptor binding elements in their promoter region. CONCLUSIONS: Serum markers regulated by the inflammatory transcription factor NF-κB are potential candidates for pharmacodynamic biomarkers that, if correlated with later outcomes like endoscopic or histologic healing, could be used to monitor treatment, optimize dosing, and enhance drug development. The pharmacodynamic biomarkers identified here hold potential to improve both clinical care and drug development. Further studies are warranted to investigate these markers as early predictors of response, or possibly surrogate outcomes.

Serum pharmacodynamic biomarkers for chronic corticosteroid treatment of children.

Corticosteroids are extensively used in pediatrics, yet the burden of side effects is significant. Availability of a simple, fast, and reliable biochemical read out of steroidal drug pharmacodynamics could enable a rapid and objective assessment of safety and efficacy of corticosteroids and aid development of corticosteroid replacement drugs. To identify potential corticosteroid responsive biomarkers we performed proteome profiling of serum samples from DMD and IBD patients with and without corticosteroid treatment using SOMAscan aptamer panel testing 1,129 proteins in <0.1 cc of sera. Ten pro-inflammatory proteins were elevated in untreated patients and suppressed by corticosteroids (MMP12, IL22RA2, CCL22, IGFBP2, FCER2, LY9, ITGa1/b1, LTa1/b2, ANGPT2 and FGG). These are candidate biomarkers for anti-inflammatory efficacy of corticosteroids. Known safety concerns were validated, including elevated non-fasting insulin (insulin resistance), and elevated angiotensinogen (salt retention). These were extended by new candidates for metabolism disturbances (leptin, afamin), stunting of growth (growth hormone binding protein), and connective tissue remodeling (MMP3). Significant suppression of multiple adrenal steroid hormones was also seen in treated children (reductions of 17-hydroxyprogesterone, corticosterone, 11-deoxycortisol and testosterone). A panel of new pharmacodynamic biomarkers for corticosteroids in children was defined. Future studies will need to bridge specific biomarkers to mechanism of drug action, and specific clinical outcomes.

VBP15, a novel dissociative steroid compound, reduces NFκB-induced expression of inflammatory cytokines in vitro and symptoms of murine trinitrobenzene sulfonic acid-induced colitis.

OBJECTIVE AND DESIGN: The goal of this study was to assess the capacity of VBP15, a dissociative steroidal compound, to reduce pro-inflammatory cytokine expression in vitro, to reduce symptoms of colitis in the trinitrobenzene sulfonic acid-induced murine model, and to assess the effect of VBP15 on growth stunting in juvenile mice. MATERIALS: In vitro studies were performed in primary human intestinal epithelial cells. Colitis was induced in mice by administering trinitrobenzene sulfonic acid. Growth stunting studies were performed in wild type outbred mice. TREATMENT: Cells were treated with VBP15 or prednisolone (10 µM) for 24 h. Mice were subjected to 3 days of VBP15 (30 mg/kg) or prednisolone (30 mg/kg) in the colitis study. In the growth stunting study, mice were subjected to VBP15 (10, 30, 45 mg/kg) or prednisolone (10 mg/kg) for 5 weeks. METHODS: Cytokines were measured by PCR and via Luminex. Colitis symptoms were evaluated by assessing weight loss, intestinal blood, and stool consistency. Growth stunting was assessed using an electronic caliper. RESULTS: VBP15 significantly reduced the in vitro production of CCL5 (p < 0.001) IL-6 (p < 0.001), IL-8 (p < 0.05) and reduced colitis symptoms (p < 0.05). VBP15 caused less growth stunting than prednisolone (p < 0.001) in juvenile mice. CONCLUSION: VBP15 may reduce symptoms of IBD, while decreasing or avoiding detrimental side effects.

TNF-α-Induced microRNAs Control Dystrophin Expression in Becker Muscular Dystrophy.

The amount and distribution of dystrophin protein in myofibers and muscle is highly variable in Becker muscular dystrophy and in exon-skipping trials for Duchenne muscular dystrophy. Here, we investigate a molecular basis for this variability. In muscle from Becker patients sharing the same exon 45-47 in-frame deletion, dystrophin levels negatively correlate with microRNAs predicted to target dystrophin. Seven microRNAs inhibit dystrophin expression in vitro, and three are validated in vivo (miR-146b/miR-374a/miR-31). microRNAs are expressed in dystrophic myofibers and increase with age and disease severity. In exon-skipping-treated mdx mice, microRNAs are significantly higher in muscles with low dystrophin rescue. TNF-α increases microRNA levels in vitro whereas NFκB inhibition blocks this in vitro and in vivo. Collectively, these data show that microRNAs contribute to variable dystrophin levels in muscular dystrophy. Our findings suggest a model where chronic inflammation in distinct microenvironments induces pathological microRNAs, initiating a self-sustaining feedback loop that exacerbates disease progression.

ECG in neonate mice with spinal muscular atrophy allows assessment of drug efficacy.

Molecular technologies have produced diverse arrays of animal models for studying genetic diseases and potential therapeutics. Many have neonatal phenotypes. Spinal muscular atrophy (SMA) is a neuromuscular disorder primarily affecting children, and is of great interest in translational medicine. The most widely used SMA mouse models require all phenotyping to be performed in neonates since they do not survive much past weaning. Pre-clinical studies in neonate mice can be hindered by toxicity and a lack of quality phenotyping assays, since many assays are invalid in pups or require subjective scoring with poor inter-rater variability. We find, however, that passive electrocardiography (ECG) recording in conscious 11-day old SMA mice provides sensitive outcome measures, detecting large differences in heart rate, cardiac conduction, and autonomic control resulting from disease. We find significant drug benefits upon treatment with G418, an aminoglycoside targeting the underlying protein deficiency, even in the absence of overt effects on growth and survival. These findings provide several quantitative physiological biomarkers for SMA preclinical studies, and will be of utility to diverse disease models featuring neonatal cardiac arrhythmias.

VBP15, a novel anti-inflammatory, is effective at reducing the severity of murine experimental autoimmune encephalomyelitis.

Multiple sclerosis is a chronic disease of the central nervous system characterized by an autoimmune inflammatory reaction that leads to axonal demyelination and tissue damage. Glucocorticoids, such as prednisolone, are effective in the treatment of multiple sclerosis in large part due to their ability to inhibit pro-inflammatory pathways (e.g., NFκB). However, despite their effectiveness, long-term treatment is limited by adverse side effects. VBP15 is a recently described compound synthesized based on the lazeroid steroidal backbone that shows activity in acute and chronic inflammatory conditions, yet displays a much-reduced side effect profile compared to traditional glucocorticoids. The purpose of this study was to determine the effectiveness of VBP15 in inhibiting inflammation and disease progression in experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of multiple sclerosis. Our data show that VBP15 is effective at reducing both disease onset and severity. In parallel studies, we observed that VBP15 was able to inhibit the production of NFκB-regulated pro-inflammatory transcripts in human macrophages. Furthermore, treatment with prednisolone-but not VBP15-increased expression of genes associated with bone loss and muscle atrophy, suggesting lack of side effects of VBP15. These findings suggest that VBP15 may represent a potentially safer alternative to traditional glucocorticoids in the treatment of multiple sclerosis and other inflammatory diseases.

Non-invasive MRI and spectroscopy of mdx mice reveal temporal changes in dystrophic muscle imaging and in energy deficits.

In Duchenne muscular dystrophy (DMD), a genetic disruption of dystrophin protein expression results in repeated muscle injury and chronic inflammation. Magnetic resonance imaging shows promise as a surrogate outcome measure in both DMD and rehabilitation medicine that is capable of predicting clinical benefit years in advance of functional outcome measures. The mdx mouse reproduces the dystrophin deficiency that causes DMD and is routinely used for preclinical drug testing. There is a need to develop sensitive, non-invasive outcome measures in the mdx model that can be readily translatable to human clinical trials. Here we report the use of magnetic resonance imaging and spectroscopy techniques for the non-invasive monitoring of muscle damage in mdx mice. Using these techniques, we studied dystrophic mdx muscle in mice from 6 to 12 weeks of age, examining both the peak disease phase and natural recovery phase of the mdx disease course. T2 and fat-suppressed imaging revealed significant levels of tissue with elevated signal intensity in mdx hindlimb muscles at all ages; spectroscopy revealed a significant deficiency of energy metabolites in 6-week-old mdx mice. As the mdx mice progressed from the peak disease stage to the recovery stage of disease, each of these phenotypes was either eliminated or reduced, and the cross-sectional area of the mdx muscle was significantly increased when compared to that of wild-type mice. Histology indicates that hyper-intense MRI foci correspond to areas of dystrophic lesions containing inflammation as well as regenerating, degenerating and hypertrophied myofibers. Statistical sample size calculations provide several robust measures with the ability to detect intervention effects using small numbers of animals. These data establish a framework for further imaging or preclinical studies, and they support the development of MRI as a sensitive, non-invasive outcome measure for muscular dystrophy.

VBP15, a novel anti-inflammatory and membrane-stabilizer, improves muscular dystrophy without side effects.

Absence of dystrophin makes skeletal muscle more susceptible to injury, resulting in breaches of the plasma membrane and chronic inflammation in Duchenne muscular dystrophy (DMD). Current management by glucocorticoids has unclear molecular benefits and harsh side effects. It is uncertain whether therapies that avoid hormonal stunting of growth and development, and/or immunosuppression, would be more or less beneficial. Here, we discover an oral drug with mechanisms that provide efficacy through anti-inflammatory signaling and membrane-stabilizing pathways, independent of hormonal or immunosuppressive effects. We find VBP15 protects and promotes efficient repair of skeletal muscle cells upon laser injury, in opposition to prednisolone. Potent inhibition of NF-κB is mediated through protein interactions of the glucocorticoid receptor, however VBP15 shows significantly reduced hormonal receptor transcriptional activity. The translation of these drug mechanisms into DMD model mice improves muscle strength, live-imaging and pathology through both preventive and post-onset intervention regimens. These data demonstrate successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.

The DcpS inhibitor RG3039 improves survival, function and motor unit pathologies in two SMA mouse models.

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein due to the functional loss of the SMN1 gene and the inability of its paralog, SMN2, to fully compensate due to reduced exon 7 splicing efficiency. Since SMA patients have at least one copy of SMN2, drug discovery campaigns have sought to identify SMN2 inducers. C5-substituted quinazolines increase SMN2 promoter activity in cell-based assays and a derivative, RG3039, has progressed to clinical testing. It is orally bioavailable, brain-penetrant and has been shown to be an inhibitor of the mRNA decapping enzyme, DcpS. Our pharmacological characterization of RG3039, reported here, demonstrates that RG3039 can extend survival and improve function in two SMA mouse models of varying disease severity (Taiwanese 5058 Hemi and 2B/- SMA mice), and positively impacts neuromuscular pathologies. In 2B/- SMA mice, RG3039 provided a >600% survival benefit (median 18 days to >112 days) when dosing began at P4, highlighting the importance of early intervention. We determined the minimum effective dose and the associated pharmacokinetic (PK) and exposure relationship of RG3039 and DcpS inhibition ex vivo. These data support the long PK half-life with extended pharmacodynamic outcome of RG3039 in 2B/- SMA mice. In motor neurons, RG3039 significantly increased both the average number of cells with gems and average number of gems per cell, which is used as an indirect measure of SMN levels. These studies contribute to dose selection and exposure estimates for the first studies with RG3039 in human subjects.

Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice.

The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2K(b) -tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective glucocorticoid receptor modulation by compound A represents a potential therapeutic strategy to improve dystrophic pathology.