Altered muscle niche contributes to myogenic deficit in the D2-mdx model of severe DMD.

Lack of dystrophin expression is the underlying genetic basis for Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2-mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2-mdx muscles is associated with an enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports the excessive accumulation of fibroadipogenic progenitors (FAPs), leading to increased fibrosis. Unexpectedly, the extent of damage and degeneration in juvenile D2-mdx muscle is significantly reduced in adults, and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance regenerative myogenesis in the adult D2-mdx muscle, reaching levels comparable to the milder B10-mdx model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with juvenile D2-mdx FAPs reduces their fusion efficacy. Wild-type juvenile D2 mice also manifest regenerative myogenic deficit and glucocorticoid treatment improves their muscle regeneration. Our findings indicate that aberrant stromal cell responses contribute to poor regenerative myogenesis and greater muscle degeneration in juvenile D2-mdx muscles and reversal of this reduces pathology in adult D2-mdx muscle, identifying these responses as a potential therapeutic target for the treatment of DMD.

Altered muscle niche contributes to myogenic deficit in the D2- mdx model of severe DMD.

Lack of dystrophin is the genetic basis for the Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2- mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2- mdx muscles is associated with enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports excessive accumulation of fibroadipogenic progenitors (FAPs). Unexpectedly, the extent of damage and degeneration of juvenile D2- mdx muscle is reduced in adults and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance myogenesis in the adult D2- mdx muscle, reaching levels comparable to the milder (B10- mdx ) mouse model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with the juvenile D2- mdx FAPs reduced their fusion efficacy and in vivo glucocorticoid treatment of juvenile D2 mouse improved muscle regeneration. Our findings indicate that aberrant stromal cell response contributes to poor myogenesis and greater muscle degeneration in dystrophic juvenile D2- mdx muscles and reversal of this reduces pathology in adult D2- mdx mouse muscle, identifying these as therapeutic targets to treat dystrophic DMD muscles.

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

Enhancing Interrogation of Skeletal Muscle Samples for Informative Quantitative Data.

Careful quantitative analysis of histological preparations of muscle samples is crucial to accurate investigation of myopathies in man and of interpretation of data from animals subjected to experimental or potentially therapeutic treatments. Protocols for measuring cell numbers are subject to problems arising from biases associated with preparative and analytical techniques. Prominent among these is the effect of polarized structure of skeletal muscle on sampling bias. It is also common in this tissue to collect data as ratios to convenient reference dominators, the fundamental bases of which are ill-defined, or unrecognized or not accurately assessable. Use of such 'floating' denominators raises a barrier to estimation of the absolute values that assume practical importance in medical research, where accurate comparison between different scenarios in different species is essential to the aim of translating preclinical research findings in animal models to clinical utility in Homo sapiens.This review identifies some of the underappreciated problems with current morphometric practice, some of which are exacerbated in skeletal muscle, and evaluates the extent of their intrusiveness into the of building an objective, accurate, picture of the structure of the muscle sample. It also contains recommendations for eliminating or at least minimizing these problems. Principal among these, would be the use of stereological procedures to avoid the substantial counting biases arising from inter-procedure differences in object size and section thickness.Attention is also drawn to the distortions of interpretation arising from use of undefined or inappropriate denominators.

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies -Eteplirsen, Golodirsen, Viltolarsen, and Casimersen -for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

Effects of Chronic, Maximal Phosphorodiamidate Morpholino Oligomer (PMO) Dosing on Muscle Function and Dystrophin Restoration in a Mouse Model of Duchenne Muscular Dystrophy.

BACKGROUND: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used in clinical development to treat Duchenne muscular dystrophy (DMD), with four exon-skipping drugs achieving regulatory approval. Exon skipping elicits a truncated, but semi-functional dystrophin protein, similar to the truncated dystrophin expressed in patients with Becker Muscular dystrophy (BMD) where the disease phenotype is less severe than DMD. Despite promising results in both dystrophic animal models and DMD boys, restoration of dystrophin by exon skipping is highly variable, leading to contradictory functional outcomes in clinical trials. OBJECTIVE: To develop optimal PMO dosing protocols that result in increased dystrophin and improved outcome measures in preclinical models of DMD. METHODS: Tested effectiveness of multiple chronic, high dose PMO regimens using biochemical, histological, molecular, and imaging techniques in mdx mice. RESULTS: A chronic, monthly regimen of high dose PMO increased dystrophin rescue in mdx mice and improved specific force in the extensor digitorum longus (EDL) muscle. However, monthly high dose PMO administration still results in variable dystrophin expression localized throughout various muscles. CONCLUSIONS: High dose monthly PMO administration restores dystrophin expression and increases muscle force; however, the variability of dystrophin expression at both the inter-and intramuscular level remains. Additional strategies to optimize PMO uptake including increased dosing frequencies or combination treatments with other yet-to-be-defined therapies may be necessary to achieve uniform dystrophin restoration and increases in muscle function.

Human muscle stem cells are refractory to aging.

Age-related loss of muscle mass and strength is widely attributed to limitation in the capacity of muscle resident satellite cells to perform their myogenic function. This idea contains two notions that have not been comprehensively evaluated by experiment. First, it entails the idea that we damage and lose substantial amounts of muscle in the course of our normal daily activities. Second, it suggests that mechanisms of muscle repair are in some way exhausted, thus limiting muscle regeneration. A third potential option is that the aged environment becomes inimical to the conduct of muscle regeneration. In the present study, we used our established model of human muscle xenografting to test whether muscle samples taken from cadavers, of a range of ages, maintained their myogenic potential after being transplanted into immunodeficient mice. We find no measurable difference in regeneration across the range of ages investigated up to 78 years of age. Moreover, we report that satellite cells maintained their myogenic capacity even when muscles were grafted 11 days postmortem in our model. We conclude that the loss of muscle mass with increasing age is not attributable to any intrinsic loss of myogenicity and is most likely a reflection of progressive and detrimental changes in the muscle microenvironment such as to disfavor the myogenic function of these cells.

A long-read RNA-seq approach to identify novel transcripts of very large genes.

RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap: 5 kb), large (Neb: 22 kb), and very large (Ttn: 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type-specific differential expression of these novel transcripts. The improved transcript identification and quantification shown by our approach removes previous impediments to studies aimed at quantitative differential expression of ultralong transcripts.

TGF-ß-driven muscle degeneration and failed regeneration underlie disease onset in a DMD mouse model.

Duchenne muscular dystrophy (DMD) is a chronic muscle disease characterized by poor myogenesis and replacement of muscle by extracellular matrix. Despite the shared genetic basis, severity of these deficits varies among patients. One source of these variations is the genetic modifier that leads to increased TGF-ß activity. While anti-TGF-ß therapies are being developed to target muscle fibrosis, their effect on the myogenic deficit is underexplored. Our analysis of in vivo myogenesis in mild (C57BL/10ScSn-mdx/J and C57BL/6J-mdxΔ52) and severe DBA/2J-mdx (D2-mdx) dystrophic models reveals no defects in developmental myogenesis in these mice. However, muscle damage at the onset of disease pathology, or by experimental injury, drives up TGF-ß activity in the severe, but not in the mild, dystrophic models. Increased TGF-ß activity is accompanied by increased accumulation of fibroadipogenic progenitors (FAPs) leading to fibro-calcification of muscle, together with failure of regenerative myogenesis. Inhibition of TGF-ß signaling reduces muscle degeneration by blocking FAP accumulation without rescuing regenerative myogenesis. These findings provide in vivo evidence of early-stage deficit in regenerative myogenesis in D2-mdx mice and implicates TGF-ß as a major component of a pathogenic positive feedback loop in this model, identifying this feedback loop as a therapeutic target.

Skeletal muscle in health and disease.

Skeletal muscle fibres are multinucleated cells that contain postmitotic nuclei (i.e. they are no longer able to divide) and perform muscle contraction. They are formed by fusion of muscle precursor cells, and grow into elongating myofibres by the addition of further precursor cells, called satellite cells, which are also responsible for regeneration following injury. Skeletal muscle regeneration occurs in most muscular dystrophies in response to necrosis of muscle fibres. However, the complex environment within dystrophic skeletal muscle, which includes inflammatory cells, fibroblasts and fibro-adipogenic cells, together with the genetic background of the in vivo model and the muscle being studied, complicates the interpretation of laboratory studies on muscular dystrophies. Many genes are expressed in satellite cells and in other tissues, which makes it difficult to determine the molecular cause of various types of muscular dystrophies. Here, and in the accompanying poster, we discuss our current knowledge of the cellular mechanisms that govern the growth and regeneration of skeletal muscle, and highlight the defects in satellite cell function that give rise to muscular dystrophies.

Vamorolone, a dissociative steroidal compound, reduces collagen antibody-induced joint damage and inflammation when administered after disease onset.

OBJECTIVE AND DESIGN: The objective of this study was to assess the effect of vamorolone, a first-in-class dissociative steroidal compound, to inhibit inflammation when administered after disease onset in the murine collagen antibody-induced arthritis model of arthritis. ANIMALS: 84 DBA1/J mice were used in this study (n = 12 per treatment group). TREATMENT: Vamorolone or prednisolone was administered orally after disease onset for a duration of 7 days. METHODS: Disease score and bone erosion were assessed using previously described scoring systems. Cytokines were measured in joints via immunoassay, and joint cathepsin B activity (marker of inflammation) was assessed using optical imaging of joints on live mice. RESULTS: We found that vamorolone treatment led to a reduction of several disease parameters including disease score, joint inflammation, and the presence of pro-inflammatory mediators to a degree similar of that observed with prednisolone treatment. More importantly, histopathological analysis of affected joints showed that vamorolone treatment significantly reduced the degree of bone erosion while this bone-sparing property was not observed with prednisolone treatment at any of the tested doses. CONCLUSIONS: While many intervention regimens in other studies are administered prior to disease onset in animal models, the current study involves delivery of the potential therapeutic after disease onset. Based on the findings, vamorolone may offer an efficacious, yet safer alternative to conventional steroidal compounds in the treatment of rheumatoid arthritis and other inflammatory diseases.

Fibroadipogenic progenitors are responsible for muscle loss in limb girdle muscular dystrophy 2B.

Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients.

Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice.

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded 'revertant' dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

Shorter Phosphorodiamidate Morpholino Splice-Switching Oligonucleotides May Increase Exon-Skipping Efficacy in DMD.

Duchenne muscular dystrophy is a fatal muscle disease, caused by mutations in DMD, leading to loss of dystrophin expression. Phosphorodiamidate morpholino splice-switching oligonucleotides (PMO-SSOs) have been used to elicit the restoration of a partially functional truncated dystrophin by excluding disruptive exons from the DMD messenger. The 30-mer PMO eteplirsen (EXONDYS51) developed for exon 51 skipping is the first dystrophin-restoring, conditionally FDA-approved drug in history. Clinical trials had shown a dose-dependent variable and patchy dystrophin restoration. The main obstacle for efficient dystrophin restoration is the inadequate uptake of PMOs into skeletal muscle fibers at low doses. The excessive cost of longer PMOs has limited the utilization of higher dosing. We designed shorter 25-mer PMOs directed to the same eteplirsen-targeted region of exon 51 and compared their efficacies in vitro and in vivo in the mdx52 murine model. Our results showed that skipped-dystrophin induction was comparable between the 30-mer PMO sequence of eteplirsen and one of the shorter PMOs, while the other 25-mer PMOs showed lower exon-skipping efficacies. Shorter PMOs would make higher doses economically feasible, and high dosing would result in better drug uptake into muscle, induce higher levels of dystrophin restoration in DMD muscle, and, ultimately, increase the clinical efficacy.

Greater Colo-Rectal Activation Phenotype in Exercised mdx Mice.

INTRODUCTION: Duchenne Muscular Dystrophy is a genetic disease that is caused by a deficiency of dystrophin protein. Both Duchenne Muscular Dystrophy patients and dystrophic mice suffer from intestinal dysfunction. METHODS: The present study arose from a chance observation of differences in fecal output of dystrophic vs. normal mice during 20-minutes of forced continuous treadmill exercise. Here, we report on the effects of exercise on fecal output in two different dystrophic mutants and their normal background control strains. All fecal materials evacuated during exercise were counted, dried and weighed. RESULTS: Mice of both mutant dystrophic strains produced significantly more fecal material during the exercise bout than the relevant control strains. ISCUSSION: We propose that exercise--induced Colo--Rectal Activation Phenotype test could be used as a simple, highly sensitive, non-invasive biomarker to determine efficacy of dystrophin replacement therapies.

Author Correction: Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle.

The originally published version of this Article contained an error in Figure 6. In panel b, the top graph (BrdU 21-24d) and the bottom graph (BrdU 28-31d) were inadvertently swapped. This error has now been corrected in both the PDF and HTML versions of the Article.

Author Correction: Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle.

In the original version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support from the CRI Light Microscopy and Image Analysis Core.

Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle.

Exon skipping is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), employing morpholino antisense oligonucleotides (PMO-AO) to exclude disruptive exons from the mutant DMD transcript and elicit production of truncated dystrophin protein. Clinical trials for PMO show variable and sporadic dystrophin rescue. Here, we show that robust PMO uptake and efficient production of dystrophin following PMO administration coincide with areas of myofiber regeneration and inflammation. PMO localization is sustained in inflammatory foci where it enters macrophages, actively differentiating myoblasts and newly forming myotubes. We conclude that efficient PMO delivery into muscle requires two concomitant events: first, accumulation and retention of PMO within inflammatory foci associated with dystrophic lesions, and second, fusion of PMO-loaded myoblasts into repairing myofibers. Identification of these factors accounts for the variability in clinical trials and suggests strategies to improve this therapeutic approach to DMD.Exon skipping is a strategy for the treatment of Duchenne muscular dystrophy, but has variable efficacy. Here, the authors show that dystrophin restoration occurs preferentially in areas of myofiber regeneration, where antisense oligonucleotides are stored in macrophages and delivered to myoblasts and newly formed myotubes.