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.

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.

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.

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.

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.

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 vs. mdx52 muscles while skipped dystrophin transcript levels are unchanged supporting 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 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.

Motor neuron rescue in spinal muscular atrophy mice demonstrates that sensory-motor defects are a consequence, not a cause, of motor neuron dysfunction.

The loss of motor neurons (MNs) is a hallmark of the neuromuscular disease spinal muscular atrophy (SMA); however, it is unclear whether this phenotype autonomously originates within the MN. To address this question, we developed an inducible mouse model of severe SMA that has perinatal lethality, decreased motor function, motor unit pathology, and hyperexcitable MNs. Using an Hb9-Cre allele, we increased Smn levels autonomously within MNs and demonstrate that MN rescue significantly improves all phenotypes and pathologies commonly described in SMA mice. MN rescue also corrects hyperexcitability in SMA motor neurons and prevents sensory-motor synaptic stripping. Survival in MN-rescued SMA mice is extended by only 5 d, due in part to failed autonomic innervation of the heart. Collectively, this work demonstrates that the SMA phenotype autonomously originates in MNs and that sensory-motor synapse loss is a consequence, not a cause, of MN dysfunction.

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.

Arrhythmia and cardiac defects are a feature of spinal muscular atrophy model mice.

Proximal spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. Traditionally, SMA has been described as a motor neuron disease; however, there is a growing body of evidence that arrhythmia and/or cardiomyopathy may present in SMA patients at an increased frequency. Here, we ask whether SMA model mice possess such phenotypes. We find SMA mice suffer from severe bradyarrhythmia characterized by progressive heart block and impaired ventricular depolarization. Echocardiography further confirms functional cardiac deficits in SMA mice. Additional investigations show evidence of both sympathetic innervation defects and dilated cardiomyopathy at late stages of disease. Based upon these data, we propose a model in which decreased sympathetic innervation causes autonomic imbalance. Such imbalance would be characterized by a relative increase in the level of vagal tone controlling heart rate, which is consistent with bradyarrhythmia and progressive heart block. Finally, treatment with the histone deacetylase inhibitor trichostatin A, a drug known to benefit phenotypes of SMA model mice, produces prolonged maturation of the SMA heartbeat and an increase in cardiac size. Treated mice maintain measures of motor function throughout extended survival though they ultimately reach death endpoints in association with a progression of bradyarrhythmia. These data represent the novel identification of cardiac arrhythmia as an early and progressive feature of murine SMA while providing several new, quantitative indices of mouse health. Together with clinical cases that report similar symptoms, this reveals a new area of investigation that will be important to address as we move SMA therapeutics towards clinical success.

Translational readthrough by the aminoglycoside geneticin (G418) modulates SMN stability in vitro and improves motor function in SMA mice in vivo.

Proximal spinal muscular atrophy (SMA) is a neuromuscular disorder for which there is no available therapy. SMA is caused by loss or mutation of the survival motor neuron 1 gene, SMN1, with retention of a nearly identical copy gene, SMN2. In contrast to SMN1, most SMN2 transcripts lack exon 7. This alternatively spliced transcript, Delta7-SMN, encodes a truncated protein that is rapidly degraded. Inhibiting this degradation may be of therapeutic value for the treatment of SMA. Recently aminoglycosides, which decrease translational fidelity to promote readthrough of termination codons, were shown to increase SMN levels in patient cell lines. Amid uncertainty concerning the role of SMN's C-terminus, the potential of translational readthrough as a therapeutic mechanism for SMA is unclear. Here, we used stable cell lines to demonstrate the SMN C-terminus modulates protein stability in a sequence-independent manner that is reproducible by translational readthrough. Geneticin (G418) was then identified as a potent inducer of the Delta7-SMN target sequence in vitro through a novel quantitative assay amenable to high throughput screens. Subsequent treatment of patient cell lines demonstrated that G418 increases SMN levels and is a potential lead compound. Furthermore, treatment of SMA mice with G418 increased both SMN protein and mouse motor function. Chronic administration, however, was associated with toxicity that may have prevented the detection of a survival benefit. Collectively, these results substantiate a sequence independent role of SMN's C-terminus in protein stability and provide the first in vivo evidence supporting translational readthrough as a therapeutic strategy for the treatment of SMA.

Development of electrocardiogram intervals during growth of FVB/N neonate mice.

BACKGROUND: Electrocardiography remains the best diagnostic tool and therapeutic biomarker for a spectrum of pediatric diseases involving cardiac or autonomic nervous system defects. As genetic links to these disorders are established and transgenic mouse models produced in efforts to understand and treat them, there is a surprising lack of information on electrocardiograms (ECGs) and ECG abnormalities in neonate mice. This is likely due to the trauma and anaesthesia required of many legacy approaches to ECG recording in mice, exacerbated by the fragility of many mutant neonates. Here, we use a non-invasive system to characterize development of the heart rate and electrocardiogram throughout the growth of conscious neonate FVB/N mice. RESULTS: We examine ECG waveforms as early as two days after birth. At this point males and females demonstrate comparable heart rates that are 50% lower than adult mice. Neonatal mice exhibit very low heart rate variability. Within 12 days of birth PR, QRS and QTc interval durations are near adult values while heart rate continues to increase until weaning. Upon weaning FVB/N females quickly develop slower heart rates than males, though PR intervals are comparable between sexes until a later age. This suggests separate developmental events may contribute to these gender differences in electrocardiography. CONCLUSIONS: We provide insight with a new level of detail to the natural course of heart rate establishment in neonate mice. ECG can now be conveniently and repeatedly used in neonatal mice. This should serve to be of broad utility, facilitating further investigations into development of a diverse group of diseases and therapeutics in preclinical mouse studies.

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.

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.

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.

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.

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.

SMN transcript stability: could modulation of messenger RNA degradation provide a novel therapy for spinal muscular atrophy?

Proximal spinal muscular atrophy is caused by deletion or mutation of the survival motor neuron 1 gene, SMN1. Rentention of a nearly identical copy gene, SMN2, enables survival but is unable to fully compensate for the loss of SMN1. The SMN1 and SMN2 genes differ by a single nucleotide that results in alternative splicing of SMN2 exon 7 due to the disruption of a binding site for an essential splicing factor. This alternatively spliced form encodes a partially functional truncated protein. Because SMN2 is present in patients with spinal muscular atrophy, it is an ideal therapeutic target. Some of the current approaches to increase SMN protein levels are aimed at increasing the transcription from SMN2 or at preventing exon 7 skipping. One area that has yet to be investigated is the stability of messenger ribonucleic acid (RNA) transcripts produced from SMN2. We postulated that transcripts derived from SMN2 may be less stable because alternative splicing, recruitment of RNA-binding proteins, and alteration of stop codons have been associated with changes in rates of messenger RNA decay; these features are all characteristic of SMN2. Accordingly, transcript degradation was examined within primary fibroblast cells that exclusively contained SMN1 or SMN2 by treating cultures with a transcriptional inhibitor to observe messenger RNA stability. The results indicate that SMN transcript instability does not play a role in the disease mechanism, suggesting that therapeutic modulation of messenger RNA degradation would not target a molecular defect in patients with spinal muscular atrophy, although it could provide general benefits by increasing total pools of SMN2 transcripts.