Regulation of DMD pathology by an ankyrin-encoded miRNA

<p>Abstract</p> <p>Background</p> <p>Duchenne muscular dystrophy (DMD) is an X-linked myopathy resulting from the production of a nonfunctional dystrophin protein. MicroRNA (miRNA) are small 21- to 24-nucleotide RNA that can regulate both individual genes and entire cell signaling pathways. Previously, we identified several mRNA, both muscle-enriched and inflammation-induced, that are dysregulated in the skeletal muscles of DMD patients. One particularly muscle-enriched miRNA, miR-486, is significantly downregulated in dystrophin-deficient mouse and human skeletal muscles. miR-486 is embedded within the <it>ANKYRIN1(ANK1) </it>gene locus, which is transcribed as either a long (erythroid-enriched) or a short (heart muscle- and skeletal muscle-enriched) isoform, depending on the cell and tissue types.</p> <p>Results</p> <p>Inhibition of miR-486 in normal muscle myoblasts results in inhibited migration and failure to repair a wound in primary myoblast cell cultures. Conversely, overexpression of miR-486 in primary myoblast cell cultures results in increased proliferation with no changes in cellular apoptosis. Using bioinformatics and miRNA reporter assays, we have identified platelet-derived growth factor receptor β, along with several other downstream targets of the phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN/AKT) pathway, as being modulated by miR-486. The generation of muscle-specific transgenic mice that overexpress miR-486 revealed that miR-486 alters the cell cycle kinetics of regenerated myofibers <it>in vivo</it>, as these mice had impaired muscle regeneration.</p> <p>Conclusions</p> <p>These studies demonstrate a link for miR-486 as a regulator of the PTEN/AKT pathway in dystrophin-deficient muscle and an important factor in the regulation of DMD muscle pathology.</p

Restoring Dystrophin Expression in Duchenne Muscular Dystrophy: Current Status of Therapeutic Approaches

Duchenne muscular dystrophy (DMD), a rare genetic disorder characterized by progressive muscle weakness, is caused by the absence or a decreased amount of the muscle cytoskeletal protein dystrophin. Currently, several therapeutic approaches to cure DMD are being investigated, which can be categorized into two groups: therapies that aim to restore dystrophin expression, and those that aim to compensate for the lack of dystrophin. Therapies that restore dystrophin expression include read-through therapy, exon skipping, vector-mediated gene therapy, and cell therapy. Of these approaches, the most advanced are the read-through and exon skipping therapies. In 2014, ataluren, a drug that can promote ribosomal read-through of mRNA containing a premature stop codon, was conditionally approved in Europe. In 2016, eteplirsen, a morpholino-based chemical capable of skipping exon 51 in premature mRNA, received conditional approval in the USA. Clinical trials on vector-mediated gene therapy carrying micro- and mini- dystrophin are underway. More innovative therapeutic approaches include CRISPR/Cas9-based genome editing and stem cell-based cell therapies. Here we review the current status of therapeutic approaches for DMD, focusing on therapeutic approaches that can restore dystrophin

Exon skipping for Duchenne muscular dystrophy: a systematic review and meta-analysis

Abstract Background Exon skipping has been considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Eteplirsen received conditional approval in the United States in 2016. To date, no systematic reviews or meta-analyses of randomized controlled trials (RCTs) of exon skipping drugs have been published to determine the pooled estimates for the effect of exon skipping in treating DMD. Methods A systematic review and meta-analysis of double-blind RCTs comparing exon-skipping drugs with placebo in DMD was performed. Trials were identified by searching published and unpublished studies from electronically available databases and clinical trial registries through October 2017. The primary outcomes were changes in the 6-min walk test (6MWT) distance, North Star Ambulatory Assessment (NSAA) scores, and adverse events. Random-effects meta-analysis and assessment of risk of bias were performed. This systematic review was registered at PROSPERO (CRD42016037504). Results Five studies involving 322 participants were included, investigating eteplirsen in one and drisapersen in four studies. There were no changes in 6MWT distance (mean difference [MD] − 9.16, 95% confidence interval [CI] − 21.94 to 3.62) or NSAA scores (MD 1.20, 95% CI − 2.35 to 4.75) after 24 weeks of treatment in the exon-skipping group compared with placebo. Subgroup analysis for a 6 mg/kg weekly injection of drisapersen showed significant changes in the 6MWT, favoring drisapersen after 24 weeks (MD − 20.24; 95% CI − 39.59 to − 0.89). However, drisapersen resulted in a significant increase in injection site reactions (risk ratio [RR] 3.67, 95% CI 1.96 to 6.89, p < 0.0001) and renal toxicity (RR 1.81, 95% CI 1.11 to 2.94, p = 0.02). Risk of bias was high in two of the five studies, including the eteplirsen and one drisapersen study. Conclusions Current available data do not show evidence that exon-skipping drugs are effective in DMD. Despite potential effectiveness when used at a specific dose, significant side effects were reported with drisapersen. The small number of RCTs with relatively small numbers of participants indicate the difficulty in conducting sufficiently powered studies of DMD. Prospectively planned meta-analysis and utilization of the real-world data may provide a more precise estimate of the effect of exon skipping in this disease

Transgene Insertion in Proximity to thec-myb Gene Disrupts Erythroid-Megakaryocytic Lineage Bifurcation

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival, and differentiation of hematopoietic cells. We established three lines of erythropoietin receptor-transgenic mice and found that one of them exhibited anemia, thrombocythemia, and splenomegaly. These abnormalities were independent of the function of the transgenic erythropoietin receptor and were observed exclusively in mice harboring the transgene homozygously, suggesting transgenic disruption of a certain gene. The transgene was inserted 77 kb upstream of the c-myb gene, and c-Myb expression was markedly decreased in megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs) of the homozygous mutant mice. In the bone marrows and spleens of the mutant mice, numbers of megakaryocytes were increased and numbers of erythroid progenitors were decreased. These abnormalities were reproducible in vitro in a coculture assay of MEPs with OP9 cells but eliminated by the retroviral expression of c-Myb in MEPs. The erythroid/megakaryocytic abnormalities were reconstituted in mice in vivo by transplantation of mutant mouse bone marrow cells. These results demonstrate that the transgene insertion into the c-myb gene far upstream regulatory region affects the gene expression at the stage of MEPs, leading to an imbalance between erythroid and megakaryocytic cells, and suggest that c-Myb is an essential regulator of the erythroid-megakaryocytic lineage bifurcation

<i>Annexin A1</i> (<i>Anxa1</i>) is up-regulated after treatment with corticosterone.

<p>(A, B) Female <i>mdx</i> myotubes were incubated for 20 hours with vehicle, corticosterone or corticosterone and mifepristone. After 1 hour of incubation with 100% medium or 50% hypo-osmotic shock medium, relative dead cells were calculated as Trypan blue-positive cells (red arrows) per total cells (n = 3). Bar (200 μm). (C) mRNA levels of <i>Inta7</i>, <i>MG53</i>, <i>Anxa1</i>, and <i>Serca1</i> in vehicle or corticosterone-treated wild-type or <i>mdx</i> myotubes were quantified with real-time qPCR (n = 3). <i>β-Actin</i> was used for internal control. (D) Female <i>mdx</i> myotubes were incubated for 20 hours with vehicle or corticosterone, and stained for MHC (green) and annexin A1 (red). DAPI (blue) denotes all nuclei. Bar (50 μm).</p

Decreased EBD uptake and increased annexin A1 up-regulation in the diaphragm of male <i>mdx</i> mice after injection with corticosterone or PD2 female <i>mdx</i> mice.

<p>EBD-positive area per total diaphragm section area was calculated after injecting male (A and B) and female (C) <i>mdx</i> mice with vehicle, corticosterone, estradiol, or progesterone for 2 days. Vehicle, n = 8 (male) and n = 4 (female); corticosterone, n = 9 (male) and n = 5 (female); estradiol, n = 9 (male) and n = 5 (female); progesterone, n = 9 (male) and n = 5 (female). Bar (200 μm). (D) mRNA levels of <i>Anxa1</i> in vehicle or corticosterone-treated mice, or virgin or PD2 female mice were quantified with real-time qPCR. Vehicle, n = 4 (male) and n = 6 (female); corticosterone, n = 6 (male) and n = 6 (female); n = 7 (virgin) and n = 6 (PD2). <i>GAPDH</i> was used for internal control.</p

Pregnancy-Induced Amelioration of Muscular Dystrophy Phenotype in <i>mdx</i> Mice via Muscle Membrane Stabilization Effect of Glucocorticoid

<div><p>Duchenne muscular dystrophy (DMD), the most common and severe type of dystrophinopathy, is an X-linked recessive genetic disease caused by the absence of dystrophin, which leads to fragility and vulnerability of the sarcolemma to mechanical stretching with increased membrane permeability. Currently, glucocorticoids such as prednisolone are the only medication available for DMD. However, molecular pathways responsible for this effect are still unclear. In addition, it remains unclear whether sex-related factors, including pregnancy and the postpartum period, affect the phenotype of dystrophinopathy. Here, we report the amelioration of muscle membrane permeability in the diaphragm muscle of pregnant and postpartum, but not in nulliparous, <i>mdx</i> mice, an animal model for DMD, during the physiological surge of corticosterone, the most abundant glucocorticoid in rodents. Cultures of single muscle fibers and myotubes isolated from <i>mdx</i> mouse diaphragm demonstrate resistance to hypo-osmotic shock when treated with corticosterone but not with estradiol or progesterone. This corticosterone-mediated resistance was diminished by an antagonist of corticosterone, indicating that the glucocorticoid-glucocorticoid receptor axis plays a role in this membrane stabilization effect on muscle. Moreover, subcutaneous injection of corticosterone into <i>mdx</i> mice showed decreased membrane permeability. This is the first report to demonstrate that pregnancy-related resistance to muscle fiber damage in <i>mdx</i> mice due to the membrane stabilization effect of corticosterone. We also propose that this membrane stabilization effect is exerted through annexin A1 up-regulation as the molecular mechanisms of glucocorticoid effects on DMD muscle. Furthermore, single muscle fiber culture studies provide a sensitive chemical screening platform for muscular dystrophies.</p></div

Muscle fiber characterizations in the diaphragm from virgin, pregnant and postpartum <i>mdx</i> mice.

<p>(A) Diaphragm from 2.5-month-old virgin and pregnant female <i>mdx</i> mice with EBD injection. Gestation day 7.5 (GD7.5) and GD14.5 mean 7 and 14 days after the recognition of vaginal plug in mated female mice, respectively. Postpartum day 2 (PD2), PD7 and PD14 mean 2, 7 and 14 days after parturition, respectively. Bar (2 mm) (B) Diaphragm sections examined by fluorescence microscopy. Bars (200 μm). (C) The EBD uptake at GD14.5 (n = 3), PD2 (n = 4), PD7 (n = 3) and the virgin female <i>mdx</i> mice (n = 5). (D) Fiber size (μm) distribution in virgin (n = 4) and PD2 <i>mdx</i> mice (n = 4). (E) Histogram of number of fibers with centrally located nuclei (CLN) in virgin (n = 9), PD2 (n = 7) and PD7 (n = 5) <i>mdx</i> mice.</p