• cell therapy;
  • gene therapy;
  • genome editing;
  • mRNA modifications;
  • muscle regeneration;
  • muscular dystrophy;
  • stem cells


  1. Top of page
  2. Abstract
  3. Introduction
  4. Repair: mutation-specific strategies
  5. Gene replacement
  6. Concluding remarks
  7. Acknowledgements
  8. References

Muscular dystrophies are genetic disorders characterized by skeletal muscle wasting and weakness. Although there is no effective therapy, a number of experimental strategies have been developed over recent years and some of them are undergoing clinical investigation. In this review, we highlight recent developments and key challenges for strategies based upon gene replacement and gene/expression repair, including exon-skipping, vector-mediated gene therapy and cell therapy. Therapeutic strategies for different forms of muscular dystrophy are discussed, with an emphasis on Duchenne muscular dystrophy, given the severity and the relatively advanced status of clinical studies for this disease.


methylphosphorothioate oligoribonucleotide


adeno-associated virus


antisense oligonucleotide


Becker's muscular dystrophy


dystrophin-associated glycoprotein complex


Duchenne muscular dystrophy


dystrophin human artificial chromosome


facioscapulohumeral muscular dystrophy


golden retriever muscular dystrophy


human artificial chromosome


muscle-derived stem cell






mesenchymal stem cell


oculo-pharyngeal muscular dystrophy


PW1+ interstitial cell


phosphorodiamidate morpholino oligomer


small nuclear RNA




  1. Top of page
  2. Abstract
  3. Introduction
  4. Repair: mutation-specific strategies
  5. Gene replacement
  6. Concluding remarks
  7. Acknowledgements
  8. References

Muscular dystrophies comprise a group of heterogeneous genetic myopathies that cause repeated skeletal muscle degeneration and regeneration [1]. The consequences of the molecular defect are apparent at the histological level, with a progressive degeneration of muscle fibres, ultimately resulting in tissue wasting. This becomes clinically evident in the form of weakness and a limitation of motor capacity, which gradually increases in severity over time. Among the various forms, X-linked Duchenne muscular dystrophy (DMD) is one of the most common (one in 3500 male births) and severe, with symptoms starting in early childhood. Muscle weakness is initially proximal and progressively leads to patients being confined to a wheelchair. Cardiac or respiratory failure are the most frequent cause of death by the third decade of life [2]. DMD is caused by mutations in the DMD gene encoding the dystrophin protein (2.4 Mb: the largest gene of the human genome), which stabilizes sarcolemma during muscle contractions, linking the cytoskeleton and extracellular matrix. Indeed, dystrophin N-terminal binds to the cytoskeletal actin, whereas the C-terminal end binds to the dystrophin-associated glycoprotein complex (DGC; also known as dystrophin-associated protein complex) at the sarcolemma [3]. An absence/reduction in dystrophin expression causes disruption of the DGC and sarcolemma fragility, leading to contraction-induced breakdown of muscle membrane, myofibre damage and tissue degeneration. Mutations in other members of DGC, such as those in genes encoding for sarcoglycans, give rise to a variety of muscular dystrophies, some of are discussed below.

Although corticosteroids delay loss of muscle function in the short term (6–24 months) [4], currently there is no effective therapy to stop the progression of muscular dystrophies. However, a significant number of experimental treatments are undergoing investigation. These strategies aim to repair the mutated gene or restore its expression. Alternatively, the mutated gene can be replaced by reintroducing a native or recombinant version using donor cells or different types of viral and nonviral vectors. Both approaches are undergoing clinical investigation and face major hurdles, such as, in many cases, a need to target all of the muscles in the body, as well as a potential immune response.

We review the current status of experimental strategies for muscular dystrophies based upon repair of the gene or its expression (mainly mutation specific) and gene replacement, focusing on the major pre-clinical and clinical work conducted in recent years. Although some of the strategies described in this review include the use of small molecules for the treatment of muscular dystrophies and may be applicable to dystrophic cardiomyopathies, we do not specifically review studies based upon anti-inflammatory and/or pro-regenerative drugs (e.g. nitric oxide, anti-myostatin antibodies, histone-deacetylase inhibitors), neither do we detail therapies aiming to counteract cardiac dysfunction or strategies based upon the up-regulation of homologue proteins (e.g. utrophin), for which other specific reviews are recommeded [5-9].

Repair: mutation-specific strategies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Repair: mutation-specific strategies
  5. Gene replacement
  6. Concluding remarks
  7. Acknowledgements
  8. References

In recent years, therapeutic approaches for repairing the genetic defect or the relative transcript, such as endonucleases, exon skipping and nonsense codon suppression, have been developed. In particular, both exon skipping and nonsense codon suppression strategies are undergoing clinical investigation.

Exon skipping and antisense oligonucleotide-mediated therapies

Approximately 70% of mutations responsible for DMD lead to a disrupted reading frame, resulting in a truncated nonfunctional dystrophin protein [3, 10]. The observation that there is a milder allelic variant caused by in-frame mutations, namely Becker's muscular dystrophy (BMD), which allows the translation of a smaller but partially functional dystrophin [11], provided a strong rationale for the application of the exon skipping strategy to DMD (with the aim of converting DMD into its milder BMD form). Although many different antisense oligonucleotide (AON) chemistries exist, such as locked nucleic acid and peptide nucleic acids, and others have been optimized recently, so far, two classes are under clinical experimentation: 2′O-methylphosphorothioate oligoribonucleotides (2′OMe) and phosphorodiamidate morpholino oligomers (PMOs). Both chemistries target specific exons, hiding them from the splicing machinery and causing their skipping during the splicing process. AONs have been indeed extensively and successfully tested in vitro [12] and, most importantly, in vivo [13-20]. Consequently, AON-based skipping of exon 51 underwent clinical experimentation and other trials are currently being developed, including skipping of exons other than exon 51 [21]. The first two clinical trials tested the intramuscular administration of 2′OMe (PRO051/GSK 2402968) and PMO (AVI-4658/Eteplirsen), respectively [22, 23]. Furthermore, systemic phase I/II studies have been completed [24, 25] demonstrating that exon skipping for DMD is so far safe, with dystrophin levels of up to 19% of normal controls detected in the PMO study. Although the outcome of randomized placebo-controlled studies for both AONs is expected in the near future, the use of either 2′OMe and PMO is still limited by the fact that they cannot be utilized for a significant number of DMD patients, in particular those with large deletions or with mutations in regulatory or N-/C-terminal regions of dystrophin [10]. Moreover, another hurdle to overcome is the relatively rapid clearance from the circulation, which means that repeated administrations (and probably a lifelong treatment) will be crucial to enable long-term therapeutic efficacy. To solve this problem, different groups have explored the possibility of the in situ production of AONs. Accordingly, chimeric small nuclear RNAs (snRNAs) have been designed to shuttle AONs that omit exon 51. Among these snRNAs, viral vector-mediated U7 and U1snRNA expression showed a long-lasting restoration of dystrophin in vitro [26, 27] and in vivo [28-31]. Moreover, recent work has focused on the optimization of U1snRNA constructs and on testing the feasibility of multiple exon skipping [32, 33]. This strategy requires a vector-mediated gene therapy approach, although it could offer the possibility of a long-lasting repair (because it will not require the repeated administration of AONs).

AONs are also becoming candidate therapeutics for other forms of muscular dystrophy, such as myotonic dystrophy type 1 [34-38], limb-girdle muscular dystrophy 2B [39] and Fukuyama congenital muscular dystrophy [40]. Further clinical translation from this work is expected in the near future.

Read-through strategies for nonsense mutation suppression

Read-through for nonsense mutations is based upon the administration of small molecules that are able to introduce a conformational change in the mRNA structure, thus allowing the ribosomal subunits to substitute a mutation-induced stop codon with a single amino acid. This results in an increased read-through of the premature stop and the production of a full-length protein [41]. To date, two drugs have undergone pre-clinical and clinical investigation: gentamicin and Ataluren (formerly known as PTC124; PTC Therapeutics, South Plainfield, NJ, USA) [41]. The results obtained after the administration of gentamicin in the mdx mouse (a model for DMD) provided the basis for a clinical trial in DMD patients [42]. After treatment, an increase in dystrophin production was found in most patients [43]. Gentamicin was found also to be effective in the treatment of congenital muscular dystrophy [44, 45]. On the other hand, several issues related to the use of gentamicin, such as the need for higher doses to further improve functional outcomes, as well as the limitation of intravenous delivery, highlighted the need for a safer and orally administered drug. Therefore, the oral administration of Ataluren in a pre-clinical mouse model resulted in the drug being well tolerated and effective in restoring dystrophin expression [46]. A subsequent phase I study in healthy volunteers also established the safety of Ataluren in humans [47]. Moreover, data from a phase IIa proof-of-concept study in DMD/BMD patients showing that an increased dystrophin production led to a randomized, double-blind, placebo-controlled dose-ranging phase IIb clinical trial. Although a good safety profile emerged from these studies, the preliminary results displayed no significant difference in functional tests ( NCT00592553) [41]. Finally, it was demonstrated that Ataluren is also able to induce read-through of a stop codon in cells derived from a LGMD2B patient with a R1905X mutation in the gene for dysferlin and to produce sufficient protein to rescue myotube membrane blebbing [48].

Gene targeting and endonucleases

Gene targeting is an important tool for gene modification and is based on the use of endonucleases engineered to induce double-strand breaks in specific DNA sequences [49]. Endonucleases are divided into three main groups: meganucleases (MGNs) [50], zinc fingers [51, 52] and Tal effector nucleases [53]. As a consequence, double-strand breaks produced by endonucleases are spontaneously repaired using two systems: by homologous recombination or by nonhomologous end joining error-prone repair. Homologous recombination requires the presence of an identical, or almost identical, sequence to use as a template for repairing the breaks, whereas nonhomologous end joining frequently results in micro-insertions/deletions at the sites of the break, which eventually restore the reading frame. Indeed, it has been shown that both MGNs and zinc fingers can target the dystrophin gene producing insertions/deletions that restore the reading frame [54]. Recently, a canine micro-dystrophin was cloned into a plasmid together with a sequence containing a MGN target. When co-transfected with the micro-dystrophin plasmid into 293T cells, the MGN restored the normal reading frame and therefore dystrophin expression [55]. Moreover, MGNs were also able to restore micro-dystrophin expression in myoblasts and in muscle fibres [55].

Gene replacement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Repair: mutation-specific strategies
  5. Gene replacement
  6. Concluding remarks
  7. Acknowledgements
  8. References

Vector-mediated gene therapy

By contrast to the strategies described above, gene replacement is not mutation-specific. As a general scheme, an additional functional copy of a named gene is delivered into skeletal muscle with the aim of restoring muscle function using two main tools: viral or nonviral vectors. Safe and efficient gene delivery to muscle is challenging mainly because it is the most abundant tissue of the body and it is composed of nondividing fibres surrounded by layers of connective tissue. Additionally, dystrophin is the largest gene in the human genome (2.4 mb); as a result, it is particularly difficult to even clone its cDNA (14 kb) [56] into conventional gene delivery tools (for the pros and cons of the most utilized vectors for muscular dystrophy, see Table 1). We now discuss the various vectors currently used, with an emphasis on those that do not need to be shuttled to muscle by previous transfer into stem cells.

Table 1. Properties of the vectors utilized in gene and cell therapy studies for muscular dystrophy. Different viral and nonviral vectors utilized for gene transfer in muscular dystrophies are listed. Adapted from Edry et al. [190]. HD, helper-dependent; NA, not available.
Vector typeHost genome integrationMaximum transgene size (kb)Duration of expressionaImmune responseClinical studies
  1. aThis variable is highly dependent on the integration pattern and cell/tissue turnover. In the absence of integration into the host genome or without a stable maintenance, we consider the duration of expression as ‘short’. b Can rarely be integrated into host genome. c Can rarely be integrated into host genome. However, being limited to ex vivo gene correction, clones are normally screened by fluorescence in situ hybridization and PCR for integration. d Loss of efficiency with inserts > 6.5 kb but can be engineered to extend the clonal capacity > 10 kb.

PlasmidNob30ShortNo/minorDMD [106]
Herpes simplex virusNo150ShortYesNA
AdenovirusNo35VariableYes (reduced for HD)NA
AAVYes/No4.5LongVariableDMD [76, 77] LGMD2D [88, 191]
HACNocNo limitationLongNot expectedNA
Sleeping Beauty transposonYes10dLongNot expectedNA
Viral vectors

To engineer replication-defective viruses to efficiently deliver transgenes to muscle, many groups have exploited their natural ability to insert genetic sequences into cells. Several vectors, such as those based upon adeno-, lenti- and adeno-associated viruses (AAVs) have been studied for gene replacement strategies. However, with the exception of AAVs, the majority of them do not efficiently transduce muscle [57].

Adeno-associated viral vectors

AAVs have a limited cloning capacity (~ 4.5 kb) and this is a major hurdle for dystrophin cDNA. Accordingly, the discovery that, in humans, shorter dystrophins lead to a milder dystrophic phenotype [58] fuelled the idea that mini- (mDYS) or micro-dystrophins (μDYS) containing only some domains could be still functional. This idea was extensively demonstrated in mice [59-64]. This development, together with a high efficiency of transduction of skeletal and cardiac muscle, places AAVs among the best viral vectors for the gene therapy of muscular dystrophies [65-67]. The use of AAVs and shorter dystrophins has lead to good results being achieved in different large pre-clinical animals, even if further functional data are needed [68-71]. In rodents, AAV vectors carrying short dystrophins infrequently cause cellular immune responses against the capsid proteins or the transgene products, at variance with large animal models where different immunological outcomes have been detected [68, 71-74]. Immunological problems have also hampered the outcome of two recent clinical trials based on the intramuscular injection of AAV vectors. In the first case, this was a result of the presence of capsid-specific T cells [75], although, recently, some of these hurdles have been overcome by using novel optimized AAVs, which have already been tested in DMD patients [76]. In another clinical trial, mini-dystrophin-specific T lymphocytes were found in some of the treated patients. In this latter study, two out of six DMD patients presented a few fibres, which were positive for mDYS 42 days after injection but not after 3 months. Despite viral genomes being found in all of the treated muscles, the presence of mini-dystrophin-specific T lymphocytes was probably responsible for the lack of long-term expression of the dystrophin transgene [77]. These results raise the hypothesis of a possible antigenicity of dystrophin, suggesting the need for further studies and for monitoring of the immune response against therapeutic genes in autologous therapies.

Another recent approach consists of a series of dual-AAV vector systems that allow an efficient delivery of a larger mDYS. The dystrophin cDNA is split into two different parts and packaged separately into two AAV vectors, which are then reconstituted after co-infection. Although its delivery efficiency is still lower compared to a single AAV vector, pre-clinical studies with dystrophic animal models have been encouraging [78-80].

AAV-based gene therapy also has been adopted for facioscapulohumeral muscular dystrophy (FSHD; a dominant muscle disease) [1, 81] and for some LGMDs [82]. FSHD is the third most common form of muscular dystrophy and is associated with a typical pattern of muscle wasting/weakness. It is associated with epigenetic changes caused by a reduction in copy number of the D4Z4 macrosatellite repeat located at chromosome 4q35. A mouse model over-expressing FRG1, one of the candidate disease genes for FSHD, was generated [83] and Bortolanza et al. [84] demonstrated amelioration of its dystrophic phenotype upon FRG1 mRNA knockdown using AAV-mediated delivery of RNA interference [84]. In the case of LGMD2B (an autosomal recessive muscular dystrophy caused by a mutation in the gene for dysferlin), the large size of dysferlin (> 150 kb) represents a major issue for LGMD2B. To bypass this restriction, mini-dysferlin [85] and novel AAVs [86, 87] have been utilized. Autosomal recessive LGMD type 2D (LGMD2D) is caused by mutations in the gene encoding α-sarcoglycan, a member of the DGC [1]. In this case, a clinical trial showed the persistent expression of α-sarcoglycan in two out of three LGMD2D patients who received AAV-mediated gene transfer to the extensor digitorum brevis muscle using a muscle-specific promoter [88]. Overall, although the results obtained with AAV vectors are encouraging for the development of AAV-mediated gene therapy for muscular dystrophies, their effective clinical application will greatly benefit from further long-term studies on immunity, as well as improvements in delivery methods.

Adenoviruses and herpes simplex virus type 1-based vectors

Adenoviral vectors and herpes simplex virus type 1-based vectors can carry large cassettes and have been used to transfer the entire dystrophin cDNA in cells and muscle. However, their use is hampered by their immunogenicity, the stability of transgene expression in vivo and the physical impediment of the myofibre basal lamina [89-94]. More recently, capsid-modified helper-dependent adenoviral vectors have attracted some interest because of their enhanced safely profile and large cloning capacity. Indeed, dystrophic mice showed the stable expression of a marker gene together with improved motor performance and life span upon delivery of the full-length dystrophin cDNA [95, 96].

Lentiviral vectors

At variance with AAVs, lentiviral vectors have a relatively large size capacity (trangenes of up to 7.5 kb) and, although their direct injection into muscle also targets progenitor cells, the overall efficiency is limited [97]. Indeed, lentiviral vectors are currently being used to genetically modify myogenic stem cells ex vivo, which can then be transplanted into pre-clinical animal models of muscular dystrophy [98-104] or eventually into patients. Although the use of lentiviral vectors offers great potential, a possible risk of tumorigenicity as a result of insertional mutagenesis needs to be carefully assessed [105].

Nonviral vectors

Nonviral gene therapy is based mainly on the use of non-integrating gene delivery tools. Thus, one of the many advantages of this system over conventional viral vectors is to avoid any risk of immune response owing to viral capsids or other viral proteins and insertional mutagenesis [94, 105]. Among the non-integrating vectors, the most commonly used vectors for muscular dystrophy are plasmids, human artificial chromosomes (HACs) and transposons. Interestingly, almost all of them have a large cloning capacity (Table 1).


The application of plasmids coding for both truncated and full-length dystrophin is certainly attractive because they are produced more easily and faster than viral vectors. Indeed, previous studies based upon the direct intramuscular injection of plasmids have highlighted the feasibility of this approach, as well as the need to increase gene-transfer efficiency and to perform long-term analyses [61, 106]. Therefore, transvenous high pressure increases the efficiency when delivering plasmids carrying the dystrophin gene into rodents and primates [107, 108]. In addition, the safety of this technique has been recently tested using saline in dystrophic patients [109]. However, this method is less efficient than the systemic injection of AAV vectors and can only be used for certain muscles. An alternative system for improving plasmid transfer into muscle is electroporation [110, 111] and transgene expression by local plasmid electroporation into muscle has been demonstrated [99, 112]. However, in some cases, this can result in tissue damage and its use is hard to envisage in a large muscle [113].


HACs present several advantages as gene therapy tools, mainly owing to their peculiar structure (telomeres, centromere and replication origin), which, by mimicking endogenous chromosomes, allows stable episomal maintenance in host cells, together with an accurate copy number control. Importantly, HACs have unlimited cloning capacity, in contrast to other conventional vectors [114, 115]. Indeed, a HAC containing the entire 2.4-mb dystrophin locus, including its regulatory elements and its native promoters (DYS-HAC), has been generated [116]. The transfer of DYS-HAC leads to the genetic correction of dystrophin mutations in dystrophic-induced pluripotent [117] and mesoangioblasts stem cells [118]. In the latter case, HAC-corrected dystrophic mesoangioblasts have been transplanted into scid/mdx mice, with a significant amelioration of morphology and function of dystrophic muscles [118]. Recently, we also generated mesoangioblasts from DYS-HAC-corrected DMD induced pluripotent stem cells [104]. Moreover, to apply this strategy to primary human muscle-derived mesoangioblasts, these cells will require an extension of their proliferative capability (i.e. to ensure survival during clonal expansion after HAC transfer). To achieve this, we are currently developing a platform for engineering DMD mesoangioblasts with excisable lentiviral vectors expressing immortalizing genes (S. Benedetti, H. Hoshiya and F. S. Tedesco, unpublished results).


Finally, transposons might also be good candidate vectors for the future therapy of genetic diseases, particularly those based on the latest generation hyperactive transposases [119, 120]. Among these, Sleeping Beauty is a class II DNA transposon that uses a cut-and-paste transposition mechanism. This system leads to an almost random integration of the transgene into TA di-nucleotides within the host genome. Therefore, using Sleeping Beauty, the μDYS gene was stably integrated into the genome of a dystrophin-deficient murine cell line. Moreover, μDYS expression was detected in dystrophic mice transplanted with genetically corrected cells [121]. Furthermore, this technology could be employed to enforce the differentiation of stem cells into myogenic precursors [122] and/or to follow their fate in vivo [123].

Cell therapy: allogeneic and ex vivo-corrected stem cells

Muscle cell biology has remarkably progressed in the last few years and several myogenic stem/progenitor cells have been identified, characterized and used in animal transplantation experiments. Remarkably, some have already been or are undergoing clinical experimentation (Table 2). We now review key recent advancements of the field, emphasizing recent clinical and pre-clinical progress. Additional details have been provided in previous reviews [124, 125].

Table 2. List of stem cells with myogenic properties in vitro and in vivo. NA, not available
Cell typeDerivationDeliveryAnimal model (disease)Clinical trials
  1. a Past clinical trials have been summarized and reviewed previously [124, 125]. b Although this is the standard tissue to derive MSCs, there is heterogeneity with regard to the tissue from which the cells have been isolated in the quoted articles. c Retrospective analysis of a Duchenne patient receiving bone marrow transplantation [176]. d Model of spinal muscular atrophy. These mice carry a homozygous deletion of Smn exon 7 directed to skeletal muscle and present signs of muscular dystrophy.

Satellite cells/myoblastsSkeletal muscleLocalmdx mice (DMD) [132]Phase II: completed (DMD) [192]a Phase II: on-going (OPMD)
MDSCsSkeletal muscleLocalmdx mice (DMD) [143, 144, 146]; mdx nude mice (DMD) [147]; GRMD dogs (DMD) [148]NA
CD133+ cellsBlood and skeletal muscleSystemic/localscid/mdx mice (DMD) [149, 150]Phase I: completed (DMD) [151]; Phase II: Recruiting (DMD)
PICsSkeletal muscleLocalInjured nude mice [152]NA
Perycites/mesoangioblastsVessels/skeletal muscleSystemic/localscid/mdx mice (DMD) [118, 158]; sgca-null mice (LMG2D) [154, 155, 157]; scid/BIAJ mice (LMG2B) [156]; GRDM dogs (DMD) [98]Phase I/II: on-going (DMD)
MSCsBone marrow vesselsbSystemic/localInjured rats and mdx nude mice (DMD) [166]; injured NOD/scid and scid/mdx mice (DMD) [167]; injured Rag2−/−ϒc−/−/C5- mice [172]; injured nude mice [171]; mdx mice (DMD) [168, 169, 171]NAc
Haematopoietic stem cellsBone marrow and bloodSystemic/localmdx mice (DMD) [144]; injured scid/beige mice [174]; mdx4cv mice (DMD) [175]; injured mice [178]NA
Amniotic fluid stem cellsAmniotic fluidSystemicHSA-Cre, SmnF7/F7 mice [165]d>NA
Embryonic stem cell-derived progenitorsEmbryoSystemic/localInjured scid/beige mice [181]; injured mdx mice (DMD) [182, 184, 185]; injured nude mice [183]; injured Rag2−/−ϒc−/− mice [184]; injured NSG mice and NSG-mdx4Cv (DMD) [188]; Rag2−/−/mdx (DMD) and injured Rag2−/−mice [189];NA
Induced pluripotent stem cell-derived progenitorsDermis and other tissuesSystemic/localsgca-null/scid/beige mice (LMG2D) [104]; sgca-null mice (LMG2D) [123]; injured NSG mice and NSG-mdx4Cv (DMD) [188]; Rag2−/−/mdx (DMD) and injured Rag2−/−mice [189]NA
Satellite cells and myoblasts

Starting from the pioneering observation of Alexander Mauro in the early 1960s [126], it was possible to identify the resident stem cells of skeletal muscle, called satellite cells, as representing a key player in muscle growth and regeneration. Satellite cells originate from embryonic somites and express a panel of characteristic but not unique markers, which vary in response to muscle injury, when satellite cells become activated and are named myoblasts [124]. At this stage, asymmetric cell division takes place, generating a committed myoblast ready for expansion and a satellite cell that maintains the pool of quiescent stem cells [127-131]. Because of their ability to generate/regenerate skeletal muscle, myoblasts were considered as an ideal candidate for cell and gene therapy of muscular dystrophies. Indeed, after encouraging experiments performed in mdx mice [132], some clinical trials showed occasional dystrophin production but with no beneficial effect, probably as a result of poor survival, limited migration and immune rejection of transplanted cells [124, 133]. Consequently, a number of studies have focused on improving the knowledge of satellite cell biology, as well as on solving the issues related to intramuscular transplantation [134, 135]. Moreover, the idea that satellite and other muscle stem cells are depleted or dysfunctional in muscular dystrophies [136] is now supported by additional evidence [104, 137-140]. On the other hand, the limited migration of myoblasts could not be an obstacle for disorders such as oculo-pharyngeal muscular dystrophy (OPMD) that affect only specific and limited muscle groups [1]. Notably, myoblasts isolated from OPMD-affected muscles show defects that are not detectable in non-affected muscles of the same patient, suggesting a possible autologous graft [141]. Indeed, a phase II clinical trial based on autologous transplantation of myoblasts is now on-going ( NCT00773227).

Other myogenic stem/progenitor cells from skeletal muscle

The results obtained in myoblast-based clinical trials have highlighted the need for further studies and new alternative strategies regarding the treatment of muscular dystrophies. As a result, attention has focused on identifying other cell populations that are able to play an active role during muscle regeneration.

Muscle-derived and CD133+ stem cells

Muscle-derived stem cells (MDSCs) have been isolated from skeletal muscle tissue using different methods. They share some markers with satellite, haematopoietic and pericyte/mesoangioblast cells, and have been transplanted into dystrophic models with variable outcomes [142-147]. Notably, similarly to mesoangioblasts (see below), the systemic delivery of MDSCs induced long-term muscle repair and clinical efficacy in golden retriever muscular dystrophy (GRMD) dogs [148].

After the discovery that CD133+ human circulating cells were able to contribute to muscle regeneration in a dystrophic mouse model [149], Benchaouir et al. [150] identified CD133+ cells expressing myogenic markers, as well as CD45 associated with skeletal muscle vessels [150]. When derived from both blood and muscle of DMD patients, CD133+ cells were genetically corrected with a lentivirus expressing U7snRNA and differentiated in vivo [150]. Moreover, a double-blind phase I clinical trial in eight DMD patients based upon local intramuscular autologous CD133+ cell transplantation confirmed their safety. No adverse events were reported and the patients who were treated showed an increased number of capillaries per muscle fibre [151].

PW1+ interstitial cells

Recently, cells located in the skeletal muscle interstitium and expressing the stress mediator marker PW1 (PICs) have been identified in the mouse and characterized as myogenic progenitors involved in muscle regeneration at levels similar to those observed for satellite cells [152]. Moreover, using a lineage tracing approach, it has been demonstrated that PICs do not derive from satellite cells [152]. On the other hand, further studies will be necessary to identify the human counterpart of these cells, which will be important for the evaluation of their therapeutic potential.


Mesoangioblasts are vessel-associated stem/progenitor cells isolated from the skeletal muscle vasculature of different species and are able to differentiate into skeletal myofibres and to cross the vessel wall, a feature that enables them to be delivered through the bloodstream [153, 154]. Accordingly, intra-arterial transplantation of mesoangioblasts ameliorated the dystrophic phenotype of different pre-clinical models of muscular dystrophy [98, 154-157]. Similar cells have also been isolated from human adult skeletal muscle and, at variance with mouse, human mesoangioblasts express pericyte markers, including alkaline phosphatase (AP), CD146 and NG2 [158]. Importantly, lineage-tracing experiment showed that AP+ pericytes naturally contribute to postnatal skeletal muscle development [159]. In addition, it was shown that the number of AP+ muscle pericytes varies significantly in certain myopathies [104, 160]. This evidence has prompted us to suggest that, in the skeletal muscle, mesoangioblasts might be related to pericytes in the same way as myoblasts are related to satellite cells: they may represent a different functional/activated state of the same cell type. Although intriguing, more evidence is necessary to support this model and to determine the relationship between pericytes and satellite cells in vivo. Indeed, although recent evidence indicates that, upon satellite cell ablation, no other cell type regenerates skeletal muscle [161], the lineage-tracing experiment described above clearly shows that muscle pericytes do participate in muscle growth and regeneration [159].

Recently, we have also reported the first evidence of an efficacious stem cell-mediated gene replacement therapy using the DYS-HAC in combination with mesoangioblast transplantation [118]. Finally, similarly to mesenchymal stem cells (MSCs), English et al. [162] recently reported the immunomodulatory potential of human adult mesoangioblasts. Overall, these results support a phase I/II clinical trial based upon the intra-arterial allogeneic transplantation of mesoangioblasts, which is currently on-going in five DMD patients at San Raffaele Hospital (Milan, Italy; EudraCT no. 2011-000176-33). That study is mainly designed to address the safety of the medicinal product (the cells) and three out of five patients have completed the four serial cell infusions. To be able to assess any change in their motor capacity and force of contraction, the five enrolled patients, plus another 23 DMD children, have been monitored for 18 months before the start of the study [163] and they will be tested both during and after the trial. More results are expected during 2013 after all of the patients have completed the clinical protocol.

Myogenic progenitors from unconventional sources

In the last 10 years, there have been a variety of studies demonstrating the existence of progenitors that are able to differentiate (to different extents) into cell types that do not correlate with their embryological origin. All of these findings have challenged the dogma of irreversibly committed cell fates, introducing the concept of tissue plasticity [164]. We now focus our attention on mesenchymal, haematopoietic and pluripotent stem cells, although cells capable of contributing to muscle regeneration have been described in various other tissues (e.g. amniotic fluid stem cells [165]).

Mesenchymal and haematopoietic stem cells

MSCs from various sources have been shown to be capable of some skeletal myogenesis [166, 167]. Nevertheless, work from independent groups demonstrated that, although they were able to engraft skeletal muscle, MSCs do not ameliorate the phenotype of mdx mice [168, 169]; however, it was proposed that they could be beneficial by reducing inflammation [170]. Human myogenic MSCs have also been derived from the synovial membrane [171]. Another study showed that the in vivo contribution of these cells to muscle regeneration is limited. However, donor-derived extracellular matrix was found, suggesting an alternative role for these cells in treating secondary defects in muscular dystrophies [172]. The definition of bona fide MSCs applies to cells that reside in a sub-endothelial niche in the bone marrow and are capable of generating a heterotopic bone (an ossicle containing bone marrow) in vivo [173]; thus, their therapeutic potential in muscle regeneration still needs to be elucidated.

The myogenic potential of haematopoietic stem cells was investigated in the late 1990s, demonstrating that bone marrow contains progenitors that can be recruited and participate in muscle regeneration [174], although with a very low frequency and with no amelioration of the dystrophic phenotype [144, 175]. A retrospective analysis in a bone marrow-transplanted DMD patient showed donor-derived skeletal muscle cells over many years, again at a very low frequency [176]. Subsequent studies identified the haematopoietic origin of these cells [177, 178].

Embryonic and induced pluripotent stem cells

Embryonic stem (ES) cells are pluripotent cells derived from the early embryo that are capable of differentiating into all tissues of the three germ layers [179, 180]. Myoblast- or satellite-like cells have been derived from mouse and human ES cells with the aim of regenerating injured muscles [181-183]. Similarly, Darabi et al. [184, 185] showed the generation and transplantation of PDGFRα+Flk myogenic cells from murine ES cells.

However, the generation of ES cells involves ethical concerns related to the destruction of human blastocysts. In this scenario, the reprogramming of adult somatic cells to an ES cell-like state (induced pluripotent stem, iPS cells) revolutionized the field of cell therapy and regenerative medicine [186]. Indeed, the possibility of deriving patient-specific iPS cells for autologous cell therapies is one of the most promising strategies for future personalized medicine [187]. Recent work has described the generation of myogenic progenitors from iPS cells able that are able to play an active role during muscle regeneration in pre-clinical models for both DMD and LGMD2D [104, 123, 188, 189]. Among these studies, Darabi et al. [188] have demonstrated dystrophin restoration, improved contractility and contribution to the satellite cell pool upon transplantation of healthy donor human ES/iPS-derived myogenic progenitors into a novel immunodeficient mdx mouse model. In parallel, our group reported evidence that human iPS cells generated from patients with LGMD2D give rise to myogenic stem/progenitor cells that can be genetically corrected ex vivo, restoring α-sarcoglycan expression upon xenotransplantation into a novel immunodeficient α-sarcoglycan-null mouse model [104]. Notably, we also showed functional phenotype amelioration and re-establishment of progenitors upon intra-specific transplantation, together with an extension of this strategy to DMD using HACs [104].

Deriving patient-specific iPS cells and expanding their differentiated progeny provides a unique tool for gene and cell therapies, even if further safety studies and improvements in protocols might be necessary to avoid any potential risk that could hamper the translation of these promising strategies into future clinical trials (e.g. transgene expression, genome stability and immunological studies).

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Repair: mutation-specific strategies
  5. Gene replacement
  6. Concluding remarks
  7. Acknowledgements
  8. References

Considerable progress has been made in the identification and characterization of novel classes of drugs, vectors and muscle stem cells. Almost all of them have been shown to produce some beneficial effect in dystrophic muscles using animal models. However, although a number of clinical trials have been completed and others have started, more basic studies will be necessary to continuously feed into the translational chain of research that led to these encouraging findings (Fig. 1). Almost all of the strategies described in the present review could benefit from this: from the refinement of AON chemistry aiming to improve efficacy, to the reduction of viral vector immunogenicity and insertional mutagenesis; from the identification of the best culture conditions to preserve stemness of the different cell populations with myogenic potential, to the studies addressing safety and efficacy in human muscle stem cells of novel vectors such as HACs and transposons.


Figure 1. The translational status of gene and cell therapy strategies for muscular dystrophy. An overview is provided of the current translational status of the therapeutic strategies described in this review. Blue arrows refer to ‘repair’ strategies and green arrows refer to ‘replacement’ strategies. In the ‘in vivo/pre-clinical studies’ column, the strategies indicated in bold are those that underwent efficacy/functional tests. In the ‘clinical trials’ column, bold refers to recent/on-going clinical trials. Strategies have been listed regardless of the outcomes. The artwork was produced using servier medical art ( HD, helper-dependent.

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Importantly, the final success of these experimental therapies will also depend on the continuous cooperation of scientists and clinicians with regulatory agencies. Indeed, most of these treatments could not be simply compared to drugs (e.g. vectors, cells) and their development into medicinal product requires ad hoc procedures that inevitably decelerate their clinical development. Interventions aimed at improving this ring of the translational chain will became critical in the next decade.

What might be the best therapeutic strategy to pursue? DMD may offer a clear paradigm for this: exon-skipping for ‘skippable’ mutations, read-through for stop codons, gene and/or cell therapy for the other mutations (e.g. large deletions). Indeed, it is unlikely that a universal one-size-fits-all approach will be the final answer for our ‘repair or replace?’ question. Rather, we believe that the toolbox of the future neuromuscular physician-scientists needs to include a portfolio of different treatments to address specifically the peculiar aspects (e.g. mutations) of the various disorders in a patient-specific fashion.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Repair: mutation-specific strategies
  5. Gene replacement
  6. Concluding remarks
  7. Acknowledgements
  8. References

The authors thank Giulio Cossu, Sharon Boast and Sara Maffioletti for helpful discussion. The present study is supported by the UK Medical Research Council, European Research Council, European Community 7th Framework project Optistem and the Italian Duchenne Parent Project.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Repair: mutation-specific strategies
  5. Gene replacement
  6. Concluding remarks
  7. Acknowledgements
  8. References
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