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(The FASEB Journal. 2005;19:880-891.)
© 2005 FASEB

Molecular, cellular, and pharmacological therapies for Duchenne/Becker muscular dystrophies

Joe V. Chakkalakal*, Jennifer Thompson*, Robin J. Parks{dagger},{ddagger} and Bernard J. Jasmin*,{ddagger},1

* Department of Cellular and Molecular Medicine and Centre for Neuromuscular Disease, Faculty of Medicine,
{dagger} Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa; and
{ddagger} Ottawa Health Research Institute, Molecular Medicine Program, Ottawa Hospital, General Campus, Ottawa, Ontario, Canada

1Correspondence: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. E-mail: jasmin{at}uottawa.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 DMD AND DYSTROPHIN GENE...
 MOLECULAR THERAPIES TO TREAT...
 CELL-BASED THERAPIES
 PHARMACOLOGICAL APPROACHES
 CONCLUSIONS AND PERSPECTIVE
 REFERENCES
 
Although the molecular defect causing Duchenne/Becker muscular dystrophy (DMD/BMD) was identified nearly 20 years ago, the development of effective therapeutic strategies has nonetheless remained a daunting challenge. Over the years, a variety of different approaches have been explored in an effort to compensate for the lack of the DMD gene product called dystrophin. This review not only presents some of the most promising molecular, cellular, and pharmacological strategies but also highlights some issues that need to be addressed before considering their implementation. Specifically, we describe current strategies being developed to exogenously deliver healthy copies of the dystrophin gene to dystrophic muscles. We present the findings of several studies that have focused on repairing the mutant dystrophin gene using various approaches. We include a discussion of cell-based therapies that capitalize on the use of myoblast or stem cell transfer. Finally, we summarize the results of several studies that may eventually lead to the development of appropriate drug-based therapies. In this context, we review our current knowledge of the mechanisms regulating expression of utrophin, the autosomal homologue of dystrophin. Given the complexity associated with the dystrophic phenotype, it appears likely that a combinatorial approach involving different therapeutic strategies will be necessary for the appropriate management and eventual treatment of this devastating neuromuscular disease.—Chakkalakal, J. V., Thompson, J., Parks, R. J. Jasmin, B. J. Molecular, cellular, and pharmacological therapies for Duchenne/Becker muscular dystrophies.


Key Words: DMD • skeletal muscle • molecular therapy • muscle cell survival • dystrophin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
DMD AND DYSTROPHIN GENE...
 MOLECULAR THERAPIES TO TREAT...
 CELL-BASED THERAPIES
 PHARMACOLOGICAL APPROACHES
 CONCLUSIONS AND PERSPECTIVE
 REFERENCES
 
DUCHENNE MUSCULAR DYSTROPHY (DMD) is a severe degenerative disorder of skeletal and cardiac muscles that affects ~1 in 3500 male births (1) . DMD patients characteristically display progressive muscle weakness, which begins in early childhood (2) . Although DMD is present at birth, clinical symptoms are not evident until 3–5 years of age (3) . Initial symptoms include leg weakness, increasing convex curvature of the spine, and a waddle-like gait (3) . Continuous muscle wasting results in progressively weaker muscles, usually leaving DMD patients wheelchair bound by the age of 11 or 12 (4) . Affected individuals usually succumb to the disease by the second or third decade of life (4) . A similar, yet milder, dystrophy known as Becker muscular dystrophy (BMD) is more variable phenotypically and generally follows a less severe course than DMD (5) .

Although two decades have now past since the identification of the molecular defect involved in DMD, there are still no effective cures to significantly alter the relentless progression of the disease. The lack of an effective therapeutic regimen can in large part be explained by the inherent difficulties associated with replacing or repairing the diseased gene and the multifaceted nature of the symptoms that range from degeneration of muscle fibers to detrimental immune response. Collectively, these various difficulties and issues hinder the development of therapies aimed at correcting the molecular defect or promoting muscle cell survival. In this review, we will highlight several therapeutic strategies currently under intense investigation.


   DMD AND DYSTROPHIN GENE PRODUCTS
TOP
 ABSTRACT
 INTRODUCTION
 DMD AND DYSTROPHIN GENE...
MOLECULAR THERAPIES TO TREAT...
 CELL-BASED THERAPIES
 PHARMACOLOGICAL APPROACHES
 CONCLUSIONS AND PERSPECTIVE
 REFERENCES
 
Twenty years ago the genetic defect underlying DMD was mapped to chromosome Xp21 in humans (for a review, see refs 6 , 7 ). This gene is the largest identified to date, which could account for its relatively high frequency of mutation and incidence of DMD (8) . The DMD gene product is known as dystrophin (6 , 9 , 10) . The large DMD gene can accommodate production of several dystrophin isoforms through alternative promoter usage and splicing of pre-mRNA. The predominant dystrophin isoform found in skeletal and cardiac muscles is an ~427 kDa cytoskeletal protein predicted to contain 3685 amino acids. Full-length dystrophin is composed of four distinct structural domains: 1) an N-terminal "actin binding" domain; 2) a middle "rod" domain consisting of spectrin-like repeats (11) ; 3) a cysteine-rich domain; and 4) a carboxyl-terminal domain (10) (Fig. 1 A). Two additional isoforms are considered to be full-length and are expressed in the brain (12 , 13) . Four other variants contain unique first exons, giving rise to dystrophin proteins of 260 kDa (14) , 140 kDa (15) , 116 kDa (16) , and 71 kDa (17 , 18) . These shorter isoforms lack the actin binding domain, which suggests they may have other functions different from those ascribed to full-length dystrophin.



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  		Figure 1. Schematic diagram of dystrophin and dystrophin-related proteins. A) Shown are the four main domains of full-length dystrophin: the actin binding domain, rod domain (26 spectrin-like repeats), cysteine-rich domain and C terminus domain. The cysteine-rich domain and carboxyl terminus are found in all the isoforms. Name and molecular mass are indicated for each dystrophin isoform. B) Comparison of dystrophin and utrophin. Note that all domains of dystrophin are present in utrophin.

In skeletal muscle fibers, full-length dystrophin accumulates predominantly at the cytoplasmic face of the sarcolemma (19 , 20) . The distribution of dystrophin along muscle fibers is rather homogeneous, thereby creating a submembranous mesh of dystrophin molecules. Mutations and/or deletions in the dystrophin gene, as seen in DMD patients, prevent the production of dystrophin and lead to its complete absence in muscle fibers (9) . In BMD, however, the nature of the mutations/deletions are such that they allow for synthesis of a truncated yet partly functional dystrophin molecule, likely explaining the milder phenotype observed for BMD patients (5) .

At the sarcolemma, dystrophin is part of a macromolecular group of proteins collectively referred to as the dystrophin-associated protein complex (DAP) (21) (Fig. 2 ). Several key components of this complex have been well studied in recent years, and their interaction patterns and organization support the notion that dystrophin serves to link the intracellular microfilament network of actin to the extracellular matrix (21 , 22) . The absence of dystrophin at the sarcolemma results in a dramatic change in the level and localization of the dystrophin complex. For example, studies have reported marked reductions in the overall levels of DAPs in dystrophin-deficient skeletal muscle from DMD patients and animal models such as the mdx mouse (23 , 24) . These data indicate that the absence of dystrophin ensues the loss of DAPs at the sarcolemma. The absence of this physical link between the interior and exterior of the muscle cell renders the sarcolemma fragile, making muscle fibers susceptible to degeneration during repeated cycles of muscle contraction and relaxation. Support for this model stems primarily from observations showing an increased sensitivity of mdx muscle fibers to mechanical stress (25 , 26) . Mutations in the genes that encode some DAPs also cause various forms of muscular dystrophies (for a review, see refs 27 , 28 ). Therefore, dystrophin and its associated proteins assume specific functions at the sarcolemma that are crucial to the survival of muscle fibers.



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  		Figure 2. Schematic representation of dystrophin localization in muscle fibers. Dystrophin is found throughout the sarcolemma of adult muscle fibers as well as at the troughs of neuromuscular junctions. Dystrophin is thought to link cytoskeletal actin filaments to the extracellular matrix via a complex comprised of DAP. This complex consists of dystroglycans ({alpha}, ß), sarcoglycans ({alpha}, ß, {delta}, {gamma}, {varepsilon}), sarcospan, syntrophins ({alpha}, ß), dystrobrevins, and NOS.

Nonstructural roles have been described for dystrophin, making it a multifunctional protein. The localization of dystrophin and its associated components at the sarcolemma places them at an appropriate position to serve as a signaling scaffold that is responsive to extracellular stressors. For example, binding of the extracellular matrix ligand laminin to {alpha}-dystroglycan results in the recruitment of signaling molecules involved in actin remodelling, such as Rac1 (29) . Several other signaling molecules interact with components of the dystrophin complex, including NOS, Grb2, and diacylglycerol kinase (30 31 32) . The mounting evidence pointing to a role for the dystrophin complex in localizing signaling mediators argues in favor of the notion that this function may be important in stimulating appropriate downstream cascades in response to various extracellular stimuli. Naturally, these signaling events would not function correctly in a dystrophin-deficient muscle, which likely contributes to the disease pathology.

Dystrophin has been proposed to play a role in calcium homeostasis. In mdx mice and DMD patients, intracellular calcium levels are elevated relative to normal controls (33 , 34) . In cultured DMD and mdx myotubes, increased leak channel activity, affecting the calcium permeability of the sarcolemma, has been reported (33 , 35 , 36) . Young mdx diaphragm muscles isolated before the onset of significant pathology show enhanced calcium influx through calcium/stretch-activated channels, resulting in the aberrant hyperactivation of signaling cascades involved in the inflammatory response (37) . Elevated expression of inflammatory mediators and chemoattractants has been observed to occur in dystrophin-deficient muscles prior to the onset of major disease symptoms (38 39 40) . This apparent sensitivity of dystrophic muscle in triggering inflammation due to aberrant calcium homeostasis may be detrimental to muscle cell survival and to the potential introduction of therapeutics.


   MOLECULAR THERAPIES TO TREAT DMD
TOP
 ABSTRACT
 INTRODUCTION
 DMD AND DYSTROPHIN GENE...
 MOLECULAR THERAPIES TO TREAT...
CELL-BASED THERAPIES
 PHARMACOLOGICAL APPROACHES
 CONCLUSIONS AND PERSPECTIVE
 REFERENCES
 
DMD is a monogenic disorder in which the absence of full-length dystrophin leads to the characteristic features of the disease. To assess whether expression of dystrophin in a dystrophic model could reverse the disease symptoms, transgenic mdx mice bearing a full or minidystrophin transgene were generated (41 42 43) . These mice do not display the characteristic dystrophic pathology. Transgenic mdx mice expressing full-length dystrophin show no sign of the muscle pathology even when dystrophin is expressed at 70% of normal levels (42 , 44) . Even low level of dystrophin expression can result in some disease correction, suggesting that partial restoration of dystrophin levels leads to significant improvements (42 , 45) . Collectively, these results indicate that the reintroduction of dystrophin into dystrophin-deficient muscles can restore proper muscle function. Over the last several years, a number of strategies have been examined to achieve this.

Dystrophin gene delivery
Perhaps the most direct method for introducing a functional dystrophin gene into dystrophin-deficient muscle is through gene replacement or "gene therapy." The simplest form of gene therapy consists in injecting plasmid DNA encoding dystrophin directly into muscle (46) . Although such gene delivery is typically not efficient, it bypasses the disadvantages linked to viral vectors that are notorious for triggering a strong anti-vector immune response. Intramuscular or intravenous/arterial injection of a therapeutic plasmid into mdx mice can result in expression of dystrophin in up to 10% of the muscle fibers, concurrent with the correct localization of the protein (and at least some of the DAPs), and an increase in peripheral nuclei (47 48 49 50) . In one plasmid-based study, dystrophin could be detected for at least 6 months after a single administration, although an antibody response, but no cellular response, was developed against the newly synthesized dystrophin protein (50) . These encouraging results led to the initiation of a phase 1 clinical trial involving plasmid-mediated delivery of dystrophin cDNA to the radial muscle (51) . In this trial, a low level of dystrophin expression was detected for up to 3 wk in muscle fibers of six of nine patients (52) . No adverse effects were noted. Although a limited amount of dystrophin was detected in the injected muscles, this study remains an important first step in moving dystrophin gene replacement therapy into the clinic.

An alternative gene therapy approach for delivering the dystrophin gene to dystrophic fibers is through the use of viral vectors. To date, most studies have used either adenovirus or adeno-associated viral vectors for delivery of dystrophin, each system having distinct advantages or disadvantages. Adenovirus (Ad) vectors have a fairly large cloning capacity (~8 kb in traditional early region 1 (E1) -deleted vectors), and were the first viral vector system to successfully deliver a truncated dystrophin cDNA (~6.3 kb) to mdx mice (53) . Ad-mediated delivery of dystrophin yielded promising results, including restoration of the dystrophin complex and improvements in several physiological parameters. However, these vectors have inherent limitations, including activation of undesirable cellular and humoral immune responses, transient gene expression, and the inability to carry large genes, which together limit their usefulness for applications requiring long-term correction. Several of these limitations have been overcome in a newer generation of Ad vector termed helper-dependent Ad (hdAd), which are deleted of all viral protein coding sequences. hdAd have some important advantages over E1-deleted Ad such as a reduced immune response, improved transgene expression, and larger insert carrying capacity (up to 36 kb; refs 54 , 55 ). Thus, hdAd are very promising vectors for gene delivery, and are being evaluated in a variety of animal models of human disease, including DMD.

Studies using hdAd to deliver dystrophin to mdx mice have shown the restoration of the dystrophin complex at the sarcolemma, long-term expression of dystrophin and correction of pathological and physiological indices of muscle disease, particularly in neonatal mice (45 , 56 , 57) . Successful treatment of adult mdx mice with E1-deleted Ad or hdAd vectors has been more problematic, due in part to inefficient infection of mature muscle by Ad. The natural cell surface receptor for Ad, the coxsackie-adenovirus receptor (CAR), is developmentally down-regulated on muscle cells, preventing Ad from efficiently attaching to, and infecting, mature muscles (58) . Redirecting virus binding to alternative receptors, such as heparan sulfate proteoglycans, which are prevalent on many cell types including muscle, can enhance Ad transduction (59) . However, infection of juvenile animals whose muscles have already endured major cycles of degeneration and regeneration resulted in minimal improvements in pathological indices when compared with neonatal animals (59) , suggesting that, to be successful, gene therapy for DMD must be initiated early, before the onset of major disease symptoms.

Despite the improvements in Ad vector design, significant problems still remain. For example, injection of high doses of E1-deleted or hdAd leads to the induction of innate and inflammatory immune responses and, at very high doses, direct toxicity, which can be lethal (60 , 61) . Many researchers are investigating methods to reduce Ad-induced inflammation and toxicity, such as coating the virus with polyethylene glycol, which serves to mask Ad epitopes from the immune system (62) . Although the use of hdAd does reduce the adaptive immune response to the viral vector (60) , generation of immunity to the delivered, therapeutic protein in the dystrophin-naive mice remains a concern, and will likely be a confounding factor as these therapies proceed into the clinic. Early gene therapy studies involving codelivery of immune-modulatory molecules such as CTL4AIg, which blocks costimulatory signals between T cells and antigen presenting cells (63) , have been re-examined in the context of hdAd-mediated dystrophin delivery to muscle and can prolong dystrophin expression in immunocompetent mice (64 , 65) . It is hoped that through these varied approaches, immune responses to the Ad vector and therapeutic protein can be minimized, or eliminated, resulting in a greater therapeutic index.

The second class of viral vector that has received considerable attention of late is adeno-associated virus (AAV). AAV vectors are characterized by reduced immunological and inflammatory responses in vivo compared with Ad. Unfortunately, AAV vectors have a cloning capacity of only ~4 kb, and cannot accommodate a full-length dystrophin cDNA. However, recent advances in the creation of mini- and microdystrophin genes have resulted in dystrophin expression cassettes that can be packaged in the AAV capsid (66 67 68) . Delivery of AAV-microdystrophin to mdx mice can result in widespread transduction and improved muscle function. Indeed, a recent study showed that a single systemic injection of an AAV-dystrophin resulted in transduction of virtually every skeletal and cardiac muscle groups of an adult mouse (68) . However, it has yet to be determined if these minimal dystrophin proteins fully compensate for the lack of full-length dystrophin, as these proteins may only serve to convert a DMD patient into a phenotypic BMD patient.

Taken together, these studies show that gene therapy through the use of viral vectors appears promising in the treatment of DMD. Over the next few years, improvements in vector design and/or delivery will hopefully result in more efficient gene replacement therapies. Already, the use of viral vectors, in combination with other therapies designed to aid in the correction of mutated dystrophin leading to the production of functional protein, has shown some rather promising results (see below).

Correction of mutant dystrophin
Chimeric RNA/DNA oligonucleotides can be used to direct the correction of a mutation by inducing preferential gene conversion from a mutant to a functional allele (69 70 71) . These oligonucleotides are homologous to a targeted gene, yet include one mismatched base (72) . Binding of the chimeric oligonucleotide to the mutant gene stimulates the cell to "repair" the defective gene based on the correct sequence contained in the oligonucleotide. Injection of chimeric oligonucleotides designed to correct the point mutation underlying the genetic defect seen in mdx mice, resulted in the expression of dystrophin in muscle fibers surrounding the site of injection (73) . Further characterization of this treatment showed that the resultant dystrophin protein expressed in these mice was full-length and included all the coding exons surrounding the mutation site (73) . A more recent study confirmed these earlier results in vitro as well as in vivo (74) , and determined that gene conversion had indeed occurred at the target DNA site in cultured cells.

Although chimeraplast-mediated therapy was initially designed to correct point mutations, which constitute 40% of DMD mutations (75) , a recent study has extended the utility of this approach to correct mutations that disrupt the translational reading frame, which account for a majority of DMD defects (76) . In this study, restoration of the translational reading frame involved targeting the exon/intron boundaries of the mutated exon 23 in mdx mice, resulting in skipping/removal of this exon and production of a truncated yet, functional protein. The rationale for inducing exon skipping stems from two important observations. First, whereas muscle fibers from DMD patients contain essentially no functional dystrophin protein, fibers from BMD patients possess partially functional dystrophin that results from in-frame deletions (5) . Second, although muscles from DMD patients and mdx mice basically contain no dystrophin, a few "revertant" fibers that express dystrophin are occasionally seen, thought to result from frame-restoring exon-skipping (77 , 78) .

Despite these advances, several aspects need to be resolved before chimeric oligonucleotides may be considered in a clinical setting. Since the frequency of gene conversion is rather modest and varies among different cell types, it becomes imperative to establish the beneficial dosage of chimeric oligonucleotides and whether multiple injections are required to obtain therapeutic levels of gene conversion. In addition, although this approach demonstrates some degree of molecular correction, more data are required concerning functional aspects of the correction in vivo.

An alternative to this chimeraplast-mediated approach involves inducing exon skipping through the use of antisense oligonucleotides (AOs) that target transcribed RNA molecules. In a recent study, direct intramuscular injection of AOs resulted in a significant increase in the number of dystrophin-positive fibers (~20%) in mdx mouse muscle (79) . In this case, the newly expressed dystrophin contained both N- and C-terminal domains, which therefore led to the restoration of the dystrophin complex (79) . The expression of dystrophin persisted for up to 2 months after the initial injection and resulted in improved force production. This therapy was not associated with autoimmune or humoral immune responses to the AOs.

Recently, a systemic delivery and direct injection of a single dose of AAVs carrying modified AOs linked to small nuclear RNAs to improve correction of targeted mRNAs, was shown to result in widespread expression of functional dystrophin in 50–80% adult mdx muscle fibers (44) . This widespread expression of dystrophin was shown to persist for > 3 months and led to improved muscle morphology, correct localization of some DAPs, and increased resistance to forced lengthening contractions. Based on these encouraging results, the use of AOs in combination with AAVs holds potential as a systemically delivered therapy that can correct the primary defect seen in DMD. One distinct drawback to this approach is that it would only be useful to those patients that contain mutations primarily in regions of the gene that, if excised (or "skipped"), would not severely compromise the function of the resulting dystrophin protein. Thus, oligonucleotide- and antisense-based therapies may only be applicable to a subset of DMD patients.


   CELL-BASED THERAPIES
TOP
 ABSTRACT
 INTRODUCTION
 DMD AND DYSTROPHIN GENE...
 MOLECULAR THERAPIES TO TREAT...
 CELL-BASED THERAPIES
PHARMACOLOGICAL APPROACHES
 CONCLUSIONS AND PERSPECTIVE
 REFERENCES
 
Cell-based therapies involve the delivery of normal cells to the dystrophic muscle, with the hope that the delivered cells will fuse or repopulate the dystrophic muscle, thereby improving muscle pathology and function. The first attempts at a cell-based therapy consisted of grafting a normal muscle into a dystrophic recipient muscle bed (80 , 81) . An initial study using this approach showed nearly normal contractile properties in adult dystrophic hosts after implantation of a muscle graft, suggesting that muscle transplantation may indeed be a viable treatment (81) . However, some ethical issues make this form of treatment difficult to pursue particularly the required use of newborn muscle in order to overcome problems seen with adult tissues, including appropriate reinnervation and revascularization.

A second, more promising cell-based approach is myoblast transfer, a procedure that involves injecting or transplanting donor muscle precursor cells (myoblasts) into a dystrophic host. The goal of myoblast transfer is to allow fusion of donor, dystrophin-positive myoblasts with host, dystrophin-deficient muscle fibers, thereby inducing expression of dystrophin. Injected myoblasts can indeed fuse into host mdx myofibers and can result in dystrophin expression at 30–40% of normal levels. Despite some promising results, myoblast transfer has many obstacles to overcome before it becomes a suitable therapeutic approach. These obstacles include attaining sufficient distribution and fusion of donor cells with host muscle fibers, extending the donor myoblast survival period (since many cells die soon after transplantation), and eliminating the immune response to donor myoblasts or newly synthesized dystrophin protein (82) . Human clinical studies using myoblast transfer were performed on DMD patients many years ago, despite a lack of evidence fully supporting the effectiveness of this treatment. Even with multiple injection sites, the efficiency of myoblast transfer was very low and failed to improve muscle strength in the patient group (83 84 85) . Skuk and colleagues (86 87 88) have been performing similar studies of nonhuman primates with the goal of improving the efficiency of myoblast engraftment. Several different parameters have been examined in an attempt to increase the efficiency of myoblast transfer including the use of various immunosuppressive agents, myotoxins and multiple injection protocols.

A recent strategy that has been explored to improve the efficiency of myoblast transfer and/or gene delivery, is the use of genetically modified myoblasts in ex vivo based therapies. In this approach, myoblasts from DMD patients are isolated, modified genetically, then reintroduced into the patient. Genetic modification can include introduction of the dystrophin gene, or antisense-based methodologies designed to enhance the proliferative capacities of myoblasts (89 , 90) . Ex vivo based approaches, although displaying some capacity to improve myoblast transfer, still require further optimization particularly to circumvent host immune responses to the delivered cells and to develop more efficient injection protocols.

An alternative cell-based method to myoblast transfer is the systemic delivery of precursor cells with myogenic potential. The feasibility of this approach came with the discovery of a population of cells identified via FACS analysis and based on their ability to exclude the fluorescent dye Hoescht (91) . These multipotential cells, referred to as side population (SP) cell, can be derived from different tissues including bone marrow and muscle. SP cells demonstrate a clear plasticity to myogenic and hematopoietic lineages (92) . The discovery of SP cells suggested the existence of progenitor cells that could be systemically delivered to dystrophic recipients to subsequently give rise to muscle cells in a myogenic environment. In initial studies, intravenous injection of isolated SP cells from marrow and muscle into mdx mice, led to the incorporation of donor nuclei within existing muscle fibers and to the expression of dystrophin (92) . Unfortunately, the frequency of incorporation of donor nuclei and the level of dystrophin expression were rather low and did not result in major therapeutic benefits. In a related study, examination of muscle biopsies from a DMD patient who had received a bone marrow transplant revealed the presence of donor-derived nuclei and dystrophin-positive fibers 13 years after the bone marrow transplant (93) . Although the dystrophin present in this case was a truncated version, and the source of expression remains to be determined, these results are clearly encouraging as they showed that fusion of donor-stem cells and dystrophin expression can persist for many years (93) . To improve on the persistence and modest level of dystrophin expression, SP cells have been exploited for use as a systemic carrier in ex vivo-based therapies. This property was recently tested through the use of SP cells transduced with lentiviral vectors containing microdystrophin (94) . These experiments demonstrated low levels of incorporation of the modified cells into dystrophic myofibers and successful expression of the transgene (94) .

An issue that remains to be resolved is the controversy surrounding the potential plasticity of SP cells. Recently, Asakura et al. demonstrated an intrinsic lack of myogenicity of SP cells despite containing markers that could specify potential lineage commitment toward myeloid or myogenic fates (95) . However, these cells were able to undergo myogenic differentiation when cocultured with myoblasts or upon injection into regenerating muscle (95) , suggesting that for SP cells to undergo myogenic commitment, they require the environment provided by myogenic cells. To further examine the issue of SP cell plasticity, Camargo et al. purified SP cells with markers indicative of hematopoietic and myogenic plasticity. They also observed that regeneration and inflammation were required for the incorporation of these cells into muscle fibers (96) . Using different morphological and functional parameters, this study suggested that the incorporation of donor bone marrow and SP cells into regenerating, inflamed myofibers may in fact stem from spontaneous fusion of myeloid progeny with existing fibers, and their subsequent transformation to the myogenic lineage is caused by the presence of regulatory factors from neighboring myonuclei (96) . Spontaneous myogenic differentiation of cells derived from different tissues including bone marrow has been previously observed and deemed to be inefficient as a therapeutic strategy for DMD treatment (reviewed in ref 97 ). Although the idea of isolating multipotent progenitor cells that can give rise to myogenic progeny is quite appealing, a great deal of work is still required to further characterize these cells before clinical trials can be envisioned.


   PHARMACOLOGICAL APPROACHES
TOP
 ABSTRACT
 INTRODUCTION
 DMD AND DYSTROPHIN GENE...
 MOLECULAR THERAPIES TO TREAT...
 CELL-BASED THERAPIES
 PHARMACOLOGICAL APPROACHES
CONCLUSIONS AND PERSPECTIVE
 REFERENCES
 
Over the years, several pharmacological interventions have been proposed as potential treatments for DMD/BMD. Various compounds such as allupurinol (98) , vitamin E (99) and selenium as well as mazindol, a growth hormone inhibitor (100 , 101) , were deemed ineffective or not beneficial against the progression of DMD. Despite these drawbacks, there has been a continued focus in trying to identify compounds that can improve functional and morphological aspects of DMD muscle. Concurrent with improved morphology of existing fibers, the identification of prophylactics that can improve the regenerative process may be effective in delaying the onset of DMD pathology.

Myostatin and glucocorticoids
Studies aimed at the manipulation of satellite cell function have identified potential drug-based therapies. Satellite cells are quiescent myogenic precursor cells residing at the interface between the basal lamina and sarcolemma. Satellite cells have the capacity to give rise to myoblasts that partake in the normal development of myofibers. These normally quiescent cells can give rise to myoblasts that can migrate to sites of muscle injury and fuse with existing myofibers to maintain muscle mass. Unlike satellite cells purified from normal muscle fibers, DMD satellite cells display an intrinsic defect in proliferative capacity that is exasperated with age, thus partially explaining the progressiveness of muscle wasting seen as DMD patients mature (102) .

One paradigm that may improve the proliferative capacity of DMD satellite cells is blocking the function of myostatin (MSTN). Myostatin (GDF-8) is a TGF-ß family member that is a negative regulator of skeletal muscle growth, as evidenced by the increased musculature of animals with inactivating mutations in this gene (103 104 105) . Although the mechanism whereby MSTN mutations result in enhanced muscle mass remains to be fully characterized, a recent report attributed this muscle gain to delayed differentiation coupled with enhanced activation, renewal, and proliferative capacities of MSTN–/– satellite cells (106) . Myostatin blockade in mdx mice results in increased body weight, muscle mass, and size, as well as in an increase in muscle strength (107) . A significant reduction in muscle fiber degeneration and serum creatine kinase levels was observed in treated animals suggesting that some degree of functional improvements had occurred (107) . Similarly, mdx/MSTN–/– double mutant mice display an improved phenotype (108) . The mechanism whereby MSTN improves muscle function is not known but appears not to involve an increase in the expression of the dystrophin homologue called utrophin (107) . Although it seems paradoxical to increase the amount of dystrophic muscle in DMD patients, these preclinical results showing improvements in mdx mice clearly lend support to investigating further the usefulness of this approach.

Glucocorticoids, such as prednisone, are effective in slowing the progression of DMD (109 110 111) . Despite the promise of prednisone as an effective therapy to combat DMD pathology, the potential for side effects has limited the widespread use of this drug. In contrast, the prednisone-derivative deflazacort appears to provide an effective alternative with less severe side effects (112 , 113) . Treatment of DMD boys with deflazacort resulted in improved pulmonary function, suggesting an improvement in the function of respiratory muscles (114) . Deflazacort treatment of mdx diaphragms was shown to improve the growth and repair process of this respiratory muscle (115) . Some of the beneficial effects of deflazacort are due to its ability to promote laminin expression, myoblast fusion, and myogenic differentiation in mdx mice, all suggestive of improved satellite cell function (115 116 117) . Subsequent studies correlated the beneficial effects of deflazacort with the elevated expression of the muscle-specific isoform of nitric oxide synthase. Consistent with these observations, treatment of mdx mice with deflazacort and the NO donor L-arginine resulted in greater morphological improvements (117) . Recent studies have provided new mechanistic insights into how deflazacort may mediate these effects (see below).

Treatment with antibiotics
Some mutations causing DMD are due to the formation of a premature stop codon within the coding sequence of dystrophin, and a recent study examined whether inducing stop codon read-through could result in restoration of dystrophin expression (118) . Aminoglycoside treatment of cultured cells can cause suppression of stop codons by extensive misreading of RNA codes (118 , 119) , and insertion of alternative amino acids in place of the stop codon. Since the defect in the mdx mouse is caused by a mutation that introduces a premature stop codon, Sweeney and colleagues sought to determine whether treatment of these mice with gentamicin, an aminoglycoside antibiotic, could lead to the synthesis of dystrophin in muscle fibers (118) . Indeed, muscles of mdx mice treated with this antibiotic showed increased sarcolemmal expression of dystrophin and restoration of some components of the dystrophin complex together with improved resistance to lengthening contractions (118) . However, dystrophin levels were ~10–20% of normal mouse muscle, suggesting that misreading of RNA codes through this approach cannot completely restore dystrophin expression (118) . Nonetheless, this level of dystrophin expression was sufficient to protect the muscles from contraction-induced damage.

Due to the negative reports from human trials using gentamicin (120) , a recent study tried to replicate some of the results from the initial animal study (121) . In contrast to earlier findings, no increase in dystrophin expression was observed. The authors of this study proposed that differences in the treatment protocol may account for this divergence. Clearly, additional experimentation is warranted to determine the effectiveness of this approach for treating DMD.

Up-regulation of a dystrophin-related protein
The aforementioned gene-based treatments share a variety of constraints stemming from immunological responses triggered by the induction of dystrophin expression. The proposed pharmacological treatments suffer from potential adverse side effects, and lack of a clear understanding of the mechanisms whereby some of these interventions result in functional benefits. An alternative approach that could evade the negative impact of the immunological reaction, and provide a defined target, involves up-regulating a protein of therapeutic benefit that is expressed endogenously in dystrophic muscle. The premise of this approach is that the up-regulated protein could compensate for the lack of dystrophin by assuming some, or even all, of its functional roles. One such protein is the nonmuscle dystrophin isoform Dp71. However, expression of this dystrophin isoform in mdx mouse muscle failed to alleviate the dystrophic phenotype, indicating that Dp71 cannot compensate for the lack of full-length dystrophin (122 , 123) . These results indicate that C-terminal isoforms cannot replace dystrophin or mitigate the dystrophic pathology. These data indicate that the amino and carboxyl terminals of dystrophin are both required for its function, and homologous regions must be contained in any dystrophin-related protein that is considered for DMD treatment.

The only protein containing both homologous terminals to dystrophin is the dystrophin-related protein 1, now referred to as utrophin (Fig. 1) . The overall structure of dystrophin and utrophin are indeed similar, with the C and N termini displaying the greatest homology, and greater divergence of sequence in the central, rod domain (124) . In addition to this significant homology, utrophin can associate with DAP, thereby serving as a link between the actin filament network and the extracellular matrix (125) . Despite these structural similarities, the pattern of expression of dystrophin and utrophin in skeletal muscle differs markedly. Whereas dystrophin is found along the entire length of the sarcolemma, utrophin preferentially accumulates at the neuromuscular (126 127 128) and myotendinous junctions (127) .

Due to the genetic and functional similarities between these two proteins, a number of studies have tested the hypothesis that utrophin overexpression along the length of dystrophin-deficient muscles may correct dystrophic symptoms. Accordingly, the expression of full-length or truncated utrophin in muscles from transgenic mdx mice can lead to significant improvements in mechanical functions and prevention of the dystrophic pathology (129 , 130) . In theory, using utrophin as opposed to dystrophin would be more advantageous since utrophin is normally expressed in mdx and DMD muscles, and no anti-transgene immune response should be induced. Overexpression of utrophin using Ad vectors in muscles of mdx mice and dystrophin-deficient dogs led to a homogeneous distribution of utrophin throughout transduced muscle fibers, as well as to the restoration of DAP (131 , 132) without any detectable immune response to the introduced transgene (132) . Taken together, these studies clearly support the hypothesis that up-regulation of utrophin in the extrajunctional compartment of dystrophic muscle fibers is a plausible therapeutic avenue. Accordingly, it becomes imperative to elucidate the mechanisms presiding over utrophin expression in muscle.

There are two different full-length utrophin isoforms, termed A and B (133 , 134) . A recent study established that these isoforms are expressed in a tissue-specific manner, with the A protein being the abundant isoform in skeletal muscle (133 , 134) . Although little is known about the mechanisms regulating expression of utrophin B, some events regulating expression of utrophin A have been identified. Results from several studies led to the idea that the synaptic accumulation of utrophin in muscle occurs as a result of the release of neurally secreted factors such as agrin and heregulin, whose coordinate activities appear to stimulate transcription of the utrophin gene in myonuclei located in the vicinity of the postsynaptic apparatus (135 136 137) . Downstream mediators of agrin and heregulin signaling likely include members of the ras-MAP kinase pathway that can phosphorylate the ets-related transcription factor GABP. GABP is then able to bind to the N-box motif located in the utrophin A promoter, thereby stimulating local transcription (135 136 137 138 ; for a review, see ref 139 ) (Fig. 3 ). Introduction of small peptides designed to stimulate heregulin-based signaling in dystrophic mdx diaphragms has been shown to result in significant improvements in the dystrophic pathology (140) . The positive effects of stimulated heregulin signaling on mdx muscles were specifically attributed to utrophin induction, since mice deficient in dystrophin and utrophin maintained their diseased phenotype (140) .



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  		Figure 3. Schematic diagram depicting the regulatory mechanisms mediating utrophin expression in synaptic and extrasynaptic regions of muscle fibers. Several studies suggest that local utrophin transcription is influenced by nerve-derived trophic factors such as heregulin and agrin. Heregulin binds ErbB receptors and activates the MAPK signaling cascade. This pathway leads to phosphorylation of Ets-related transcription factor GABP in synaptic nuclei. Once phosphorylated, GABP binds the N-box region of the utrophin promoter and activates transcription. Agrin increases utrophin expression via the N-box motif. In addition to this neural influence of utrophin expression in synaptic regions more recent studies suggest the involvement of calcineurin/NFAT signaling in influencing the extrasynaptic expression of utrophin found in slower, more oxidative fibers. The mechanisms whereby calcineurin influences utrophin expression extrasynaptically involve transcriptional and possibly post-transcriptional mechanisms.

Recently it was shown that stimulation of calcineurin/NFAT signaling can lead to increased extrasynaptic expression of utrophin A through transcriptional, and possibly post-transcriptional, mechanisms (141 , 142) (Fig. 3) . Overexpression of a constitutively active form of calcineurin in muscles of mdx mice increased utrophin A expression and led to restoration of DAP at the sarcolemma and an attenuation of the dystrophic pathology (143) . In agreement with these findings, treating mdx mice with the calcineurin inhibitor and immunosuppressant cyclosporine A can in fact accelerate the development of dystrophic pathology (144) . Treatment of mdx mice with NO donors also results in enhanced expression and the sarcolemmal appearance of utrophin with concomitant morphological corrections of muscle (145) . As mentioned, deflazacort has been demonstrated to improve muscle morphology and enhance the activity of the NO pathway. Recently, deflazacort has been shown to stimulate calcineurin/NFAT signaling and utrophin expression, with correlated improvements in dystrophic pathology (146) . Collectively, these studies suggest that an increased understanding of the ability of deflazacort to stimulate gene expression has relevance for the design of therapies that can improve intrinsic defects in DMD muscle development and increase expression of utrophin to functionally compensate for dystrophin.


   CONCLUSIONS AND PERSPECTIVE
TOP
 ABSTRACT
 INTRODUCTION
 DMD AND DYSTROPHIN GENE...
 MOLECULAR THERAPIES TO TREAT...
 CELL-BASED THERAPIES
 PHARMACOLOGICAL APPROACHES
 CONCLUSIONS AND PERSPECTIVE
REFERENCES
 
Initially it was thought that the characterization of the molecular defects causing DMD and the identification of dystrophin would soon result in a cure for this progressive neuromuscular disorders. Almost 20 years have passed since the discovery of the gene and, unfortunately, we have yet to find an effective therapy that could mitigate the dystrophic process. Although numerous approaches are currently being explored, many suffer from a variety of drawbacks. It is only through additional research that these barriers will be overcome and ultimately lead to the development of therapeutic strategies effective in stemming the progressive and multifactorial nature of DMD. A clear possibility is that the effective management and therapy of DMD may only be achieved through a combination of approaches. Thus, it is imperative to initiate studies assessing the potential beneficial impact of multiple strategies combined into a single preclinical trial.


   ACKNOWLEDGMENTS
 
We thank two anonymous reviewers for helpful comments. Research in the Jasmin and Parks laboratories was supported by the Canadian Institutes of Health Research (CIHR), CIHR/Muscular Dystrophy Association of Canada/Amyotrophic Lateral Sclerosis Society of Canada Partnership Grant (R.J.P.), Premier’s Research Excellence Award (B.J.J., R.J.P.), a CIHR Institute Strategic Initiative (B.J.J., R.J.P.), Muscular Dystrophy Association of America (B.J.J.), the Association Française contre les Myopathies (B.J.J.), and the Jesse Davidson Foundation for Gene and Cell Therapy (R.J.P.). J.V.C. is the recipient of a Studentship from CIHR. B.J.J. is supported by a CIHR Investigator Award and R.J.P. by a CIHR New Investigator Award.

Received for publication September 7, 2004. Accepted for publication February 17, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 DMD AND DYSTROPHIN GENE...
 MOLECULAR THERAPIES TO TREAT...
 CELL-BASED THERAPIES
 PHARMACOLOGICAL APPROACHES
 CONCLUSIONS AND PERSPECTIVE
 REFERENCES
 

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