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The dystrophin-associated glycoprotein complex (DGC) is found at the muscle fiber sarcolemma and forms an essential structural link between the basal lamina and internal cytoskeleton. In a set of muscular dystrophies known as the dystroglycanopathies, hypoglycosylation of the DGC component α-dystroglycan results in reduced binding to basal lamina components, a loss in structural stability, and repeated cycles of muscle fiber degeneration and regeneration. The satellite cells are the key stem cells responsible for muscle repair and reside between the basal lamina and sarcolemma. In this study, we aimed to determine whether pathological changes associated with the dystroglycanopathies affect satellite cell function. In the Largemyd mouse dystroglycanopathy model, satellite cells are present in significantly greater numbers but display reduced proliferation on their native muscle fibers in vitro, compared with wild type. However, when removed from their fiber, proliferation in culture is restored to that of wild type. Immunohistochemical analysis of Largemyd muscle reveals alterations to the basal lamina and interstitium, including marked disorganization of laminin, upregulation of fibronectin and collagens. Proliferation and differentiation of wild-type satellite cells is impaired when cultured on substrates such as collagen and fibronectin, compared with laminins. When engrafted into irradiated tibialis anterior muscles of mdx-nude mice, wild-type satellite cells expanded on laminin contribute significantly more to muscle regeneration than those expanded on fibronectin. These results suggest that defects in α-dystroglycan glycosylation are associated with an alteration in the satellite cell niche, and that regenerative potential in the dystroglycanopathies may be perturbed.

Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF and the consequent production of a shorter but functional dystrophin protein. This approach may lead to an effective disease modifying treatment for DMD and progress towards clinical application has been rapid. Less than a decade has passed between the first studies published in 1998 describing the use of AOs to modify the DMD gene in mice and the results of the first intramuscular proof of concept clinical trials. Whilst phase II and III trials are now underway, the heterogeneity of DMD mutations, efficient systemic delivery and targeting of AOs to cardiac muscle remain significant challenges. Here we review the current status of AO-mediated therapy for DMD, discussing the preclinical, clinical and regulatory hurdles and their possible solutions to expedite the translation of AO-mediated exon skipping therapy to clinic.

Stem cell transplantation is already in clinical practice for certain genetic diseases and is a promising therapy for dystrophic muscle. We used the mdx mouse model of Duchenne muscular dystrophy to investigate the effect of the host satellite cell niche on the contribution of donor muscle stem cells (satellite cells) to muscle regeneration. We found that incapacitation of the host satellite cells and preservation of the muscle niche promote donor satellite cell contribution to muscle regeneration and functional reconstitution of the satellite cell compartment. But, if the host niche is not promptly refilled, or is filled by competent host satellite cells, it becomes nonfunctional and donor engraftment is negligible. Application of this regimen to aged host muscles also promotes efficient regeneration from aged donor satellite cells. In contrast, if the niche is destroyed, yet host satellite cells remain proliferation-competent, donor-derived engraftment is trivial. Thus preservation of the satellite cell niche, concomitant with functional impairment of the majority of satellite cells within dystrophic human muscles, may improve the efficiency of stem cell therapy.

The 185th ENMC Workshop on stem/precursor cells as a therapeutic strategy for muscular dystrophies was held at Naarden, The Netherlands, on 3–5 June 2011. Sixteen participants from seven countries (United Kingdom, The Netherlands, France, Germany, Italy, Spain and Singapore) with expertise in satellite cells, muscle stem cells, muscle development, muscular dystrophies, clinical trials and patients’ perspectives, attended. Although skeletal muscle is largely a post-mitotic tissue, it regenerates efficiently following injury. Satellite cells, quiescent cells under the basal lamina of muscle fibres, become activated, proliferate to give a pool of myoblasts that can repair and regenerate muscle fibres [1]. In muscular dystrophies such as Duchenne muscular dystrophy (DMD), satellite cells contribute to muscle fibre regeneration, but soon become exhausted and muscle fibres are lost and replaced by fibrous connective tissue and fat (reviewed [2]). Stem/precursor cells have long been considered as a possible therapy of muscular dystrophies, since work in the late 1980s on myoblast transplantation in the dystrophindeficient mdx mouse model of DMD [3,4]. However, early clinical trials on intra-muscular myoblast transplantation were disappointing, the main problems being low efficiency of donor-derived muscle regeneration and lack of migration of donor cells within transplanted muscle [5]. Nevertheless, lessons learnt from these trials may be used as a basis of future trials. In planning a stem cell clinical trial, decisions have to be made about ethical implications, age of participants, scaling up of cell preparations, testing of cells and criteria used to assess safety and efficacy.

We previously conducted a proof of principle; dose escalation study in Duchenne muscular dystrophy (DMD) patients using the morpholino splice-switching oligonucleotide AVI-4658 (eteplirsen) that induces skipping of dystrophin exon 51 in patients with relevant deletions, restores the open reading frame and induces dystrophin protein expression after intramuscular (i.m.) injection. We now show that this dystrophin expression was accompanied by an elevated expression of α-sarcoglycan, β-dystroglycan (BDG) and--in relevant cases--neuronal nitric oxide synthase (nNOS) at the sarcolemma, each of which is a component of a different subcomplex of the dystrophin-associated glycoprotein complex (DAPC). As expected, nNOS expression was relocalized to the sarcolemma in Duchenne patients in whom the dystrophin deletion left the nNOS-binding domain (exons 42-45) intact, whereas this did not occur in patients with deletions that involved this domain. Our results indicate that the novel internally deleted and shorter dystrophin induced by skipping exon 51 in patients with amenable deletions, can also restore the dystrophin-associated complex, further suggesting preserved functionality of the newly translated dystrophin.

Summary Background We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy. Method We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5–15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597. Findings 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1–10·6) to 16·4% (10·8–22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts. Interpretation The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy. Funding UK Medical Research Council; AVI BioPharma.

The muscular dystrophies are inherited disorders characterised by progressive muscle wasting and weakness. Stem cell therapy is considered to be one of the most promising strategies for treating muscular dystrophies. In this review, we first examine the evidence that a stem cell could be used to treat muscular dystrophies, and then discuss the criteria that an ideal stem cell should meet. We also highlight the importance of standard operation procedures to be followed for ensuring the consistent and reproducible efficacy of a particular stem cell. While at the moment the scientific community is looking for an ideal stem cell to treat muscular dystrophies, it is clear that in order for this field to benefit from therapeutic stem cell applications, additional careful investigations are required.

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.

Skeletal muscle is maintained and repaired by resident stem cells called muscle satellite cells, but there is a gradual failure of this process during the progressive skeletal muscle weakness and wasting that characterises muscular dystrophies. The pathogenic mutation causes muscle wasting, but in conditions including Duchenne muscular dystrophy, the mutant gene is not expressed in satellite cells, and so muscle maintenance/repair is not directly affected. The chronic muscle wasting, however, produces an increasingly hostile micro-environment in dystrophic muscle. This probably combines with excessive satellite cell use to eventually culminate in an indirect failure of satellite cell-mediated myofibre repair. By contrast, in disorders such as Emery-Dreifuss muscular dystrophy, the pathogenic mutation not only instigates muscle wasting, but could also directly compromise satellite cell function, leading to less effective muscle homeostasis. This may again combine with excessive use and a hostile environment to further compromise satellite cell performance. Whichever the mechanism, the ultimate consequence of perturbed satellite cell activity is a chronic failure of myofibre maintenance in dystrophic muscle. Here, we review whether the pathogenic mutation can directly contribute to satellite cell dysfunction in a number of muscular dystrophies.

The original version of this article contained an error in Fig. 3. In panel c, the labels ‘mdx’ and ‘mdx Ripk3-/-‘ were inadvertently inverted. This has now been corrected in the PDF and HTML versions of the Article.

Duchenne muscular dystrophy (DMD) is characterised by the absence of dystrophin in muscle biopsies, although residual dystrophin can be present, either as dystrophin-positive (revertant) fibres or traces. As restoration of dystrophin expression is the end point of clinical trials, such residual dystrophin is a key factor in recruitment of patients and may also confound the analysis of dystrophin restoration in treated patients, if, as previously observed in the mdx mouse, revertant fibres increase with age. In 62% of the diagnostic biopsies reports of 65 DMD patients studied, traces or revertants were recorded with no correlation between traces or revertants, the patients' performance, or corticosteroids response. In nine of these patients, there was no increase in traces or revertants in biopsies taken a mean of 8.23 years (5.8-10.4 years) after the original diagnostic biopsy. This information should help in the design and execution of clinical trials focused on dystrophin restoration strategies. (C) 2010 Elsevier B.V. All rights reserved.

Skeletal muscles of body and limb are derived from somites, but most head muscles originate from cranial mesoderm. The resident stem cells of muscle are satellite cells, which have the same embryonic origin as the muscle in which they reside. Here, we analysed satellite cells with a different ontology, comparing those of the extensor digitorum longus (EDL) of the limb with satellite cells from the masseter of the head. Satellite cell-derived myoblasts from MAS and EDL muscles had distinct gene expression profiles and masseter cells usually proliferated more and differentiated later than those from EDL. When transplanted, however, masseter-derived satellite cells regenerated limb muscles as efficiently as those from EDL. Clonal analysis showed that functional properties differed markedly between satellite cells: ranging from clones that proliferated extensively and gave rise to both differentiated and self-renewed progeny, to others that divided minimally before differentiating completely. Generally, masseter-derived clones were larger and took longer to differentiate than those from EDL. This distribution in cell properties was preserved in both EDL-derived and masseter-derived satellite cells from old mice, although clones were generally less proliferative. Satellite cells, therefore, are a functionally heterogeneous population, with many occupants of the niche exhibiting stem cell characteristics in both somite-derived and branchiomeric muscles.

Stem cell therapy holds promise for treating muscle diseases. Although satellite cells regenerate skeletal muscle, they only have a local effect after intra-muscular transplantation. Alternative cell types, more easily obtainable and systemically-deliverable, were therefore sought. Human synovial stem cells (hSSCs) have been reported to regenerate muscle fibres and reconstitute the satellite cell pool. We therefore determined if these cells are able to regenerate skeletal muscle after intra-muscular injection into cryodamaged muscles of Rag2-/γ chain-/C5-mice. We found that hSSCs possess only limited capacity to undergo myogenic differentiation in vitro or to contribute to muscle regeneration in vivo. However, this is enhanced by over-expression of human MyoD1. Interestingly, hSSCs express extracellular matrix components laminin α2 and collagen VI within grafted muscles. Therefore, despite their limited capacity to regenerate skeletal muscle, hSSCs could play a role in treating muscular dystrophies secondary to defects in extracellular matrix proteins.

V. Arechavala-Gomeza, M. Kinali, L. Feng, S. C. Brown, C. Sewry, J. E. Morgan and F. Muntoni (2010) Neuropathology and Applied Neurobiology36, 265–274 Immunohistological intensity measurements as a tool to assess sarcolemma-associated protein expression Aims: The quantification of protein levels in muscle biopsies is of particular relevance in the diagnostic process of neuromuscular diseases, but is difficult to assess in cases of partial protein deficiency, particularly when information on protein localization is required. The combination of immunohistochemistry and Western blotting is often used in these cases, but is not always possible if the sample is scarce. We therefore sought to develop a method to quantify relative levels of sarcolemma-associated proteins using digitally captured images of immunolabelled sections of skeletal muscle. Methods: To validate our relative quantification method, we labelled dystrophin and other sarcolemmal proteins in transverse sections of muscle biopsies taken from Duchenne muscular dystrophy and Becker muscular dystrophy patients, a manifesting carrier of Duchenne muscular dystrophy and normal controls. Results: Using this method to quantify relative sarcolemmal protein abundance, we were able to accurately distinguish between the different patients on the basis of the relative amount of dystrophin present. Conclusions: This comparative method adds value to techniques that are already part of the diagnostic process and can be used with minimal variation of the standardized protocols, without using extra amounts of valuable biopsy samples. Comparative quantification of sarcolemmal proteins on immunostained muscle sections will be of use to establish both the abundance and localization of the protein. Moreover, it can be applied to assess the efficacy of experimental therapies where only partial restoration or upregulation of the protein may occur.

Changes that occur in the skeletal muscle environment with the progress of muscular dystrophies may affect stem cell function and result in impaired muscle regeneration. It has previously been suggested that the success of stem cell transplantation could therefore be dependent both on the properties of the cell itself and on the host muscle environment. Here we engrafted young and mature adult mdx-nude mice, which are the genetic homolog of Duchenne muscular dystrophy, with a small number of satellite cells freshly isolated from young, normal donor mice. We found that the donor satellite cells contributed to muscle regeneration and self-renewal as efficiently within mature adult, as in young, dystrophic host muscle. Donor-derived satellite cells also contributed to robust regeneration after further injury, showing that they were functional despite the more advanced dystrophic muscle environment. These findings provide evidence that muscle tissue in a later stage of dystrophy may be effectively treated by stem cells.

Summary Background Mutations that disrupt the open reading frame and prevent full translation of DMD , the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. Methods We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. Findings Two patients received 0·09 mg AVI-4658 in 900 μL (0·9%) saline and five patients received 0·9 mg AVI-4658 in 900 μL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44–79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26·4%), and the mean intensity was 17% (range 11–21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0·002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot. Interpretation Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD. Funding UK Department of Health.

Chromosomal correction of dystrophin gene mutations is a most desirable therapeutic solution for Duchenne muscular dystrophy, as it allows production of the full-length dystrophin under the control of locus-specific promoters. Here we explored gene targeting in conditionally immortal mouse dystrophin-deficient myoblasts. We constructed an adenoviral vector for the correction of the mdx mutation, containing 6.0 kb of sequence homologous to the target locus (partial intron 21 through to exon 24 with the normal sequence of exon 23) and a neomycin expression cassette inserted in intron 23. Adenovirus-based gene targeting was previously reported to be beneficial in mouse embryonic stem cells, resulting in one targeted integration per three integration events. However, we found no targeted integration events among 144 stably transduced G418-resistant myoblast clones, reflecting efficient random integration of the adenoviral vector in myogenic cells. We found that mouse myoblasts are capable of integrating recombinant adenoviral DNA with an efficiency approaching 1%. Interestingly, dermal fibroblasts integrate adenoviral DNA up to 100 times less efficiently than myoblasts from the same mice. We also show that the efficiency of recombinant adenoviral DNA integration is influenced by preinfection cell density, possibly indicating the importance of cellular DNA replication for adenoviral integration.

Purpose of review The satellite cell is the principal muscle stem cell. Recent research, however, has highlighted new stem cell sources that, once activated in the muscle tissue, can participate in muscle regeneration. This article reviews the state of research on stem cell populations that have potential for treatment of muscular dystrophies. Recent findings Despite recent findings about the stem cell character of satellite cells and their in-vivo myogenic potential, limitations related to muscle precursor cell transfer therapy have encouraged the investigation of stem cell sources other than satellite cells. Current research is focused on identifying the best stem cell in the endothelial compartment, which is able to be systemically delivered to reach all the muscles and to contribute to widespread muscle regeneration within these muscles. Summary Current results highlight many possible stem cell sources for stem cell therapy of muscle diseases, and work is ongoing to identify the most effective candidate that is able to robustly regenerate muscle tissue and to functionally repopulate the muscle stem cell compartment.

Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation.

Duchenne muscular dystrophy (DMD) is a common X-linked disease resulting from the absence of dystrophin in muscle. Affected boys suffer from incurable progressive muscle weakness, leading to premature death. Stem cell transplantation may be curative, but is hampered by the need for systemic delivery and immune rejection. To address these barriers to stem cell therapy in DMD, we investigated a fetal-to-fetal transplantation strategy. We investigated intramuscular, intravascular, and intraperitoneal delivery of human fetal mesenchymal stem cells (hfMSCs) into embryonic day (E) 14–16 MF1 mice to determine the most appropriate route for systemic delivery. Intramuscular injections resulted in local engraftment, whereas both intraperitoneal and intravascular delivery led to systemic spread. However, intravascular delivery led to unexpected demise of transplanted mice. Transplantation of hfMSCs into E14–16 mdx mice resulted in widespread long-term engraftment (19 weeks) in multiple organs, with a predilection for muscle compared with nonmuscle tissues (0.71% vs. 0.15%, p < .01), and evidence of myogenic differentiation of hfMSCs in skeletal and myocardial muscle. This is the first report of intrauterine transplantation of ontologically relevant hfMSCs into fully immunocompetent dystrophic fetal mice, with systemic spread across endothelial barriers leading to widespread long-term engraftment in multiple organ compartments. Although the low-level of chimerism achieved is not curative for DMD, this approach may be useful in other severe mesenchymal or enzyme deficiency syndromes, where low-level protein expression may ameliorate disease pathology. Disclosure of potential conflicts of interest is found at the end of this article.

Cell therapy for degenerative muscle diseases such as the muscular dystrophies requires a source of cells with the capacity to participate in the formation of new muscle fibers. We investigated the myogenic potential of human fetal mesenchymal stem cells (hfMSCs) using a variety of stimuli. The use of 5-azacytidine or steroids did not produce skeletal muscle differentiation, whereas myoblast-conditioned medium resulted in only 1%-2% of hfMSCs undergoing muscle differentiation. However, in the presence of galectin-1, 66.1% +/- 5.7% of hfMSCs, but not adult bone marrow-derived mesenchymal stem cells, assumed a muscle phenotype, forming long, multinucleated fibers expressing both desmin and sarcomeric myosin via activation of muscle regulatory factors. Continuous exposure to galectin-1 resulted in more efficient muscle differentiation than pulsed exposure (62.3% vs. 39.1%; p < .001). When transplanted into regenerating murine muscle, galectin-1-exposed hfMSCs formed fourfold more human muscle fibers than nonstimulated hfMSCs (p = .008), with similar results obtained in a scid/mdx dystrophic mouse model. These data suggest that hfMSCs readily undergo muscle differentiation in response to galectin-1 through a stepwise progression similar to that which occurs during embryonic myogenesis. The high degree of myogenic conversion achieved by this method has relevance for the development of therapies for muscular dystrophies.

Locus control regions (LCRs) are defined by their ability to confer reproducible physiological levels of transgene expression in mice and therefore thought to possess the ability to generate dominantly a transcriptionally active chromatin structure. We report the first characterization of a muscle-cell-specific LCR, which is linked to the human desmin gene (DES). The DES LCR consists of five regions of muscle-specific DNase I hypersensitivity (HS) localized between −9 and −18 kb 5′ of DES and reproducibly drives full physiological levels of expression in all muscle cell types. The DES LCR DNase I HS regions are highly conserved between humans and other mammals and can potentially bind a broad range of muscle-specific and ubiquitous transcription factors. Bioinformatics and direct molecular analysis show that the DES locus consists of three muscle-specific (DES) or muscle preferentially expressed genes (APEG1 and SPEG, the human orthologue of murine striated-muscle-specific serine/threonine protein kinase, Speg). The DES LCR may therefore regulate expression of SPEG and APEG1 as well as DES.

Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.

The aim of this review is to highlight advances in the field of skeletal muscle regeneration that have been made in the last year.Studies have increased our understanding of the activation of satellite cells within their niche on the muscle fibre, the contribution of satellite cell-derived muscle precursor cells to skeletal muscle regeneration and the reduction of satellite cell function in old muscle. Although other stem cells, either bone marrow derived or present within skeletal muscle or other tissues, do contribute to muscle regeneration, recent studies have highlighted that this is at best minimal compared with the ability of satellite cells to regenerate skeletal muscle. The effect of the host muscle environment has been shown to have a profound effect on skeletal muscle regeneration. Age and denervation have a detrimental effect and certain types of muscle injury a positive effect. Work continues on the effect of growth factors on muscle cell lines in vitro and muscle regeneration in vivo.Recent work has focused on the contribution of satellite-cell derived muscle precursor cells and other stem cells to skeletal muscle regeneration. The muscle environment has a profound effect on the regenerative capacity of resident and implanted cells. Muscle regeneration may be optimized by using the best stem cell population and by modifying the host muscle environment.

The myogenic regulatory factor Myf5 is integral to the initiation and control of skeletal muscle formation. In adult muscle, Myf5 is expressed in satellite cells, stem cells of mature muscle, but not in the myonuclei that sustain the myofibre. Using the Myf5nlacZ/+ mouse, we now show that Myf5 is also constitutively expressed in muscle spindles-stretch-sensitive mechanoreceptors, while muscle denervation induces extensive reactivation of the Myf5 gene in myonuclei. To identify the elements involved in the regulation of Myf5 in adult muscle, we analysed reporter gene expression in a transgenic bacterial artificial chromosome (BAC) deletion series of the Mrf4/Myf5 locus. A BAC carrying 140 kb upstream of the Myf5 transcription start site was sufficient to drive all aspects of Myf5 expression in adult muscle. In contrast, BACs carrying 88 and 59 kb upstream were unable to drive consistent expression in satellite cells, although expression in muscle spindles and reactivation of the locus in myonuclei were retained. Therefore, as during development, multiple enhancers are required to generate the full expression pattern of Myf5 in the adult. Together, these observations show that elements controlling adult Myf5 expression are genetically separable and possibly distinct from those that control Myf5 during development. These studies are a first step towards identifying cognate transcription factors involved in muscle stem cell regulation.

Myoblasts transplanted into muscles of recipient mice mostly die, only a minor stem cell-like subpopulation surviving and participating in muscle regeneration. To investigate this phenomenon further, we used a retrovirus expressing β-galactosidase to provide a unique marker for satellite-cell-derived muscle precursor cells, before transplanting them into myopathic mdx nu/nu mouse muscle. We employed inverse polymerase chain reaction to identify viral integrations, to follow the fate of clones present within the injected cells.Mass-infected cultures contained many marked clones, some of which contributed disproportionately to muscle regeneration. Although no particular clones showed overall predominance, some were present in more than one injected muscle, an eventuality unlikely to arise by chance. Conversely, in grafts of muscle precursor cells that had either been labelled as sparse satellite-cell derived cultures, or had been cloned, all clones were shown to be able to survive and form muscle in vivo. Moreover, all clones contributed to further generations of new-formed muscle fibres following a series of injuries administered to injected muscles, demonstrating that some cells of each clone had been retained as stem-cell-like muscle precursors. Furthermore, retrovirally marked satellite-cell-derived clones were derived from muscles that had been injected with marked muscle precursor cells. These cells formed muscle following their transplantation into a new host mouse, confirming their stem cell properties.

Skeletal muscle satellite cells are quiescent mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally-differentiated muscle fibres. These are normally quiescent in adult muscle, but act as a reserve population of cells, able to proliferate in response to injury and give rise to regenerated muscle and to more satellite cells. The recent discovery of a number of markers expressed by satellite cells has provided evidence that satellite cells, which had long been presumed to be a homogeneous population of muscle stem cells, may not be equivalent. It is possible that a sub-population of satellite cells may be derived from a more primitive stem cell. Satellite cell-derived muscle precursor cells may be used to repair and regenerate damaged or myopathic skeletal muscle, or to act as vectors for gene therapy. CELL FACTS: (1) Number of cells in body: 2 x 10(7) to 3 x 10(7) myonuclei/g, 20-25 kg muscle in average man; 2 x 10(5) to 10 x 10(5) satellite cells/g, i.e. approximately 1 x 10(10) to 2 x 10(10) satellite cells per person. (2) Main functions: repair and maintenance of skeletal muscle. (3) Turnover rate: close to zero in non-traumatic conditions-high in disease or severe trauma.

Objective: Skeletal myoblast transplantation is a promising alternative to treat heart failure. A single fiber, the minimal functional unit of skeletal muscle, retains skeletal myoblasts beneath the basal lamina. When surrounding muscle is injured, myoblasts migrate from the fiber into the damaged area to regenerate muscle. We hypothesized that such isolated fibers could be used as an efficient vehicle to deliver myoblasts into damaged myocardium, resulting in improved cardiac function. Methods: Living single fibers of rat skeletal muscle were isolated, and their behavior was characterized in vitro. Single fibers were injected into the myocardium (at 4 sites, each receiving a single fiber) of rats in 2 models of heart failure induced either by means of doxorubicin administration or left coronary artery occlusion. Results: Skeletal myoblasts dissociated from an isolated single fiber, proliferated, and differentiated into multinucleated myotubes in vitro. Within 3 days after grafting in vivo, original fibers provided putative myoblasts and disappeared. At 4 weeks, discrete loci consisting of several multinucleated myotubes were observed. Furthermore, single-fiber transplantation significantly improved cardiac function compared with the control treatment in either doxorubicin-treated hearts (maximum dP/dt, 4013.9 ± 96.1 vs 3603.1 ± 102.3 mm Hg/s; minimum dP/dt, −2313.7 ± 75.1 vs −2057.1 ± 52.4 mm Hg/s) or ischemic hearts (maximum dP/dt, 3905.6 ± 103.0 vs 3572.6 ± 109.7 mm Hg/s; minimum dP/dt, −2336.1 ± 69.7 vs −2106.4 ± 74.2 mm Hg/s). Conclusion: Single-fiber transplantation acts as a vehicle for delivering putative skeletal myoblasts that appear to differentiate into myotubes within the myocardium. This was associated with improved function of failing hearts, suggesting its efficacy as a novel graft for cellular cardiomyoplasty. J Thorac Cardiovasc Surg 2002;123:984-92

There are over 30 mouse models with mutations or inactivations in the dystrophin-associated protein complex. This complex is thought to play a crucial role in the functioning of muscle, as both a shock absorber and signalling centre, although its role in the pathogenesis of muscular dystrophy is not fully understood. The first mouse model of muscular dystrophy to be identified with a mutation in a component of the dystrophin-associated complex (dystrophin) was the mdx mouse in 1984. Here, we evaluate the key characteristics of the mdx in comparison with other mouse mutants with inactivations in DAPC components, along with key modifiers of the disease phenotype. By discussing the differences between the individual phenotypes, we show that the functioning of the DAPC and consequently its role in the pathogenesis is more complicated than perhaps currently appreciated.

Importance: In Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)–mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping. / Objective: To quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping. / Design, Setting, and Participants: Retrospective study of nondystrophic controls (n = 2), patients with DMD (n = 5), patients with intermediate muscular dystrophy (n = 3), and patients with BMD (n = 13) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype. / Main Outcomes and Measures: Dystrophin mRNA and protein expression. / Results: Patients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes). / Conclusions and Relevance: Exon 44 or 45 skipping will likely yield lower levels of dystrophin than exon 51 skipping, although the resulting protein is functional enough to often maintain a mild BMD phenotype. Dystrophin transcript stability is an important indicator of dystrophin expression, and transcript instability in DMD compared with BMD should be explored as a potential biomarker of response to AOs. This study is beneficial for the planning, execution, and analysis of clinical trials for exon 44 and 45 skipping.

Extracellular microRNAs (miRNAs) are promising biomarkers of the inherited muscle wasting condition Duchenne muscular dystrophy, as they allow non-invasive monitoring of either disease progression or response to therapy. In this study, serum miRNA profiling reveals a distinct extracellular miRNA signature in dystrophin-deficient mdx mice, which shows profound dose-responsive restoration following dystrophin rescue. Extracellular dystrophy-associated miRNAs (dystromiRs) show dynamic patterns of expression that mirror the progression of muscle pathology in mdx mice. Expression of the myogenic miRNA, miR-206 and the myogenic transcription factor myogenin in the tibialis anterior muscle were found to positively correlate with serum dystromiR levels, suggesting that extracellular miRNAs are indicators of the regenerative status of the musculature. Similarly, extracellular dystromiRs were elevated following experimentally-induced skeletal muscle injury and regeneration in non-dystrophic mice. Only a minority of serum dystromiRs were found in extracellular vesicles, whereas the majority were protected from serum nucleases by association with protein/lipoprotein complexes. In conclusion, extracellular miRNAs are dynamic indices of pathophysiological processes in skeletal muscle.

Myoblasts, the precursors of skeletal muscle fibers, can be induced to withdraw from the cell cycle and differentiate in vitro. Recent studies have also identified undifferentiated subpopulations that can self-renew and generate myogenic cells (Baroffio, A., M. Hamann, L. Bernheim, M.-L. Bochaton-Pillat, G. Gabbiani, and C.R. Bader. 1996. Differentiation. 60:47–57; Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, and Y. Nabeshima. 1998. J. Cell Sci. 111:769–779). Cultured myoblasts can also differentiate and contribute to repair and new muscle formation in vivo, a capacity exploited in attempts to develop myoblast transplantation (MT) for genetic modification of adult muscle. Our studies of the dynamics of MT demonstrate that cultures of myoblasts contain distinct subpopulations defined by their behavior in vitro and divergent responses to grafting. By comparing a genomic and a semiconserved marker, we have followed the fate of myoblasts transplanted into muscles of dystrophic mice, finding that the majority of the grafted cells quickly die and only a minority are responsible for new muscle formation. This minority is behaviorally distinct, slowly dividing in tissue culture, but rapidly proliferative after grafting, suggesting a subpopulation with stem cell–like characteristics.

Muscle of donor origin was formed after implantation of H-2Kb-tsA58 muscle precursor cells (mpc) into irradiated mdx nu/nu mouse muscles. A series of injections of the myotoxin, notexin, which destroys mature muscle fibers but spares muscle precursor cells and other tissues, was made into the mpc-injected muscles, leaving time for regeneration to occur between each injection. New muscle fibers of donor origin were formed after up to four notexin treatments, providing evidence that some of the implanted mpc reentered an undifferentiated, quiescent, stem cell-like state and were capable of myogenesis after further injuries to the muscle. A similar model could be used to assay whether preparations of human mpc contain long-lasting precursor cells, prior to their implantation into patients. In control mdx muscles, which had been irradiated, injected with tissue culture medium, and given three notexin injections, regeneration also occurred, indicating that radiation-resistant mpc were present, presumably within the treated muscle. © 1999 John Wiley & Sons, Inc. Muscle Nerve 22: 174–185, 1999

Despite being largely a post-mitotic tissue, adult skeletal muscle exhibits a remarkable capacity for regeneration. A new study has shown that cells derived from mural cells (pericytes) from blood vessels in postnatal human skeletal muscle contribute to robust skeletal muscle regeneration after intra-arterial delivery into a dystrophic mouse model.

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin expression in patient muscle cells. Restoration of dystrophin expression could alleviate the symptoms of DMD, although the large size of full-length dystrophin cDNA (11.1kb) and the restricted packaging capacity of viral vectors limit the application of standard gene transfer methods.Retroviral vectors have a pseudo-diploid RNA genome which can co-package two distinct RNA genomes to form a heterozygous virion. Template-switching between co-packaged strands is a common event during HIV-1 reverse transcription, which can lead to recombination and the production of chimeric proviruses. Template-switching can be influenced by a number of cis and trans-acting factors such as structures in the viral packaging sequences and mutations of reverse transcriptase.In this work, the recombinogenic characteristics of HIV-1 based lentiviral vectors have been exploited for the purpose of reconstituting full-length dystrophin cDNA from overlapping 5’ and 3’ portions of the full-length coding sequence. The vector has been initially optimised using a small bicistronic cassette to evaluate the recombination efficiency in standard lentiviral vectors compared with those harbouring mutations in the HIV-1 packaging sequence and reverse transcriptase.Interestingly, the results suggested that a standard lentivirus vector could deliver a full-length, recombined provirus with greater efficiency than vectors harbouring mutations to cis and trans factors. A multiplicity of infection (MOI) of 40 heterozygous vector particles per target cell was sufficient to obtain a reconstituted full-length Neomycin-IRES-GFP provirus in 40% of transduced HeLa cells. When the viral RNA components were further modified to render functional provirus synthesis dependent on recombination (schematic displayed in figure 1), the vector was capable of reconstituting a full-length dystrophin-gfp fusion protein in HEK293t cells. Flow cytometry analysis showed that heterozygous vectors produced 0.1% GFP-positive cells following a MOI50 vector dose. Furthermore, genomic DNA extracted from these cells contained a full-length dystrophin provirus, which was detected by nested PCR and sequenced by clonal analysis.This work reveals that retroviral recombination offers a means to circumvent retroviral packaging limitations and may be of use in dystrophin gene therapy following further optimisation. View Large Image | Download PowerPoint Slide

Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle disease characterized by a lack of dystrophin expression. Myoblast transplantation and gene therapy have the potential of restoring dystrophin, thus decreasing the muscle weakness associated with this disease. In this study we present data on the myoblast mediated ex vivo gene transfer of full-length dystrophin to mdx (dystrophin deficient) mouse muscle as a model for autologous myoblast transfer. Both isogenic primary mdx myoblasts and an immortalized mdx cell line were transduced with an adenoviral vector that has all viral coding sequences deleted and encodes beta-galactosidase and full-length dystrophin. Subsequently, these transduced myoblasts were injected into dystrophic mdx muscle, where the injected cells restored dystrophin, as well as dystrophin-associated proteins. A greater amount of dystrophin replacement occurred in mdx muscle following transplantation of mdx myoblasts isolated from a transgenic mouse overexpressing dystrophin suggesting that engineering autologous myoblasts to express high amounts of dystrophin might be beneficial. The ex vivo approach possesses attributes that make it useful for gene transfer to skeletal muscle including: (1) creating a reservoir of myoblasts capable of regenerating and restoring dystrophin to dystrophic muscle; and (2) achieving a higher level of gene transfer to dystrophic muscle compared with adenovirus-mediated direct gene delivery. However, as observed in direct gene transfer studies, the ex vivo approach also triggers a cellular immune response which limits the duration of trans-gene expression.

Stem cell transplantation is being tested as a potential therapy for a number of diseases. Stem cells isolated directly from tissue specimens or generated via reprogramming of differentiated cells require rigorous testing for both safety and efficacy in preclinical models. The availability of mice with immune-deficient background that carry additional mutations in specific genes facilitates testing the efficacy of cell transplantation in disease models. The muscular dystrophies are a heterogeneous group of disorders, of which Duchenne muscular dystrophy is the most severe and common type. Cell-based therapy for muscular dystrophy has been under investigation for several decades, with a wide selection of cell types being studied, including tissue-specific stem cells and reprogrammed stem cells. Several immune-deficient mouse models of muscular dystrophy have been generated, in which human cells obtained from various sources are injected to assess their preclinical potential. After transplantation, the presence of engrafted human cells is detected via immunofluorescence staining, using antibodies that recognize human, but not mouse, proteins. Here we show that one antibody specific to human spectrin, which is commonly used to evaluate the efficacy of transplanted human cells in mouse muscle, detects myofibers in muscles of NOD/Rag1(null)mdx(5cv), NOD/LtSz-scid IL2Rγ(null) mice, or mdx nude mice, irrespective of whether they were injected with human cells. These "reactive" clusters are regenerating myofibers, which are normally present in dystrophic tissue and the spectrin antibody is likely recognizing utrophin, which contains spectrin-like repeats. Therefore, caution should be used in interpreting data based on detection of single human-specific proteins, and evaluation of human stem cell engraftment should be performed using multiple human-specific labeling strategies.

Skeletal muscle tissue has a remarkable capability of regenerating in pathological conditions or after injury. The principal muscle stem cells, satellite cells, are responsible for this prompt and efficient process. Normally quiescent in their niches underneath the basal lamina of each muscle fiber, satellite cells become activated to repair or form new fibers. Ideally, healthy donor stem cells could be transplanted to regenerate the skeletal muscle tissue to repair a genetic defect. However, to be efficient, cell grafting requires modulation of the host muscle environment to allow homing of, and regeneration by, donor satellite cells. Here, we provide methods to modulate the host mouse muscle environment in order to destroy or preserve the muscle niche before transplanting donor satellite cells. We also describe methods to investigate donor-derived muscle regeneration and self-renewal.

Maintenance, repair and regeneration of adult skeletal muscle are mediated by stem or precursor cells within the muscle. In addition to the satellite cell, which is the archetypal muscle stem cell, there are other stem cells within skeletal muscle that can contribute to muscle regeneration under experimental conditions. We describe these different cells within skeletal muscle and evaluate the experimental evidence for them being skeletal muscle stem cells. Further studies will be needed to determine the roles of different skeletal muscle resident cells to repair, maintain and regenerate skeletal muscle.

Skeletal muscle tissue has a remarkable capability of regenerating in pathological conditions or after injury. The principal muscle stem cells, satellite cells, are responsible for this prompt and efficient process. Normally quiescent in their niches underneath the basal lamina of each muscle fiber, satellite cells become activated to repair or form new fibers. Ideally, healthy donor stem cells could be transplanted to regenerate the skeletal muscle tissue to repair a genetic defect. However, to be efficient, cell grafting requires modulation of the host muscle environment to allow homing of, and regeneration by, donor satellite cells. Here, we provide methods to modulate the host mouse muscle environment in order to destroy or preserve the muscle niche before transplanting donor satellite cells. We also describe methods to investigate donor-derived muscle regeneration and self-renewal.

Background Dilated cardiomyopathy ( DCM ) is a leading cause of chronic morbidity and mortality in muscular dystrophy ( MD ) patients. Current pharmacological treatments are not yet able to counteract chronic myocardial wastage, thus novel therapies are being intensely explored. Micro RNA s have been implicated as fine regulators of cardiomyopathic progression. Previously, miR‐669a downregulation has been linked to the severe DCM progression displayed by Sgcb ‐null dystrophic mice. However, the impact of long‐term overexpression of miR‐669a on muscle structure and functionality of the dystrophic heart is yet unknown. Methods and Results Here, we demonstrate that intraventricular delivery of adeno‐associated viral (AAV) vectors induces long‐term (18 months) miR‐669a overexpression and improves survival of Sgcb ‐null mice. Treated hearts display significant decrease in hypertrophic remodeling, fibrosis, and cardiomyocyte apoptosis. Moreover, miR‐669a treatment increases sarcomere organization, reduces ventricular atrial natriuretic peptide (ANP) levels, and ameliorates gene/mi RNA profile of DCM markers. Furthermore, long‐term miR‐669a overexpression significantly reduces adverse remodeling and enhances systolic fractional shortening of the left ventricle in treated dystrophic mice, without significant detrimental consequences on skeletal muscle wastage. Conclusions Our findings provide the first evidence of long‐term beneficial impact of AAV ‐mediated mi RNA therapy in a transgenic model of severe, chronic MD ‐associated DCM .