Guy L Odom

Paul Gregorevic

Jeffrey S Chamberlain

Viral-mediated gene therapy for the muscular dystrophies: Successes, limitations and recent advances

Keywords: dystrophin, utrophin, adeno-associated virus, exon skipping, antisense oligonucleotide

Much progress has been made over the past decade elucidating the molecular basis for a variety of muscular dystrophies (MD's). Accordingly, there are examples of mouse models of MD whose disease progression has been halted in large part with the use of viral vector technology. Even so, we must acknowledge significant limitations of present vector systems that must be overcome prior to successful treatment of humans with such approaches. This review will present a variety of viral-mediated therapeutic strategies aimed at counteracting the muscle-wasting symptoms associated with muscular dystrophy. We include viral vector systems used for muscle gene transfer, with a particular emphasis on adeno-associated virus. Findings of several encouraging studies focusing on repair of the mutant dystrophin gene are also included. Lastly, we present a discussion of muscle compensatory therapeutics being considered that include pathways involved in the up-regulation of utrophin, promotion of cellular adhesion, enhancement of muscle mass, and antagonism of the inflammatory response.

Considering the complexity of the muscular dystrophies, it appears likely that a multilayered approach tailored to a patient sub-group may be warranted in order to effectively contest the progression of this devastating disease.

Introduction

The muscular dystrophies (MD's) are a heterogeneous group of inherited disorders characterized by progressive skeletal muscle degeneration. The clinical presentation of these disorders displays a broad spectrum of variability such as the degree and distribution of skeletal muscle involvement, cardiac/pulmonary involvement, age of onset, rate of progression, and mode of inheritance [1]. The diagnosis is often performed by examination of a muscle biopsy though in many cases genetic testing can be employed. Histologically, muscle biopsies may present with an increased number of myofibers with centrally located nuclei, an abnormally large myofiber size-both of which are characteristic of an enhanced degenerative and regenerative response, and in advanced cases replacement of myofibers with adipose, and connective tissue, in large part due to the loss of regenerative potential of dystrophic muscle fibers.

A variety of therapeutic options are being developed, particularly in small animal models, to counteract the muscle wasting associated with MD. These include autologous myoblast transplantation [START_REF] Partridge | Myoblast transplantation[END_REF], stem cell transfer [START_REF] Bachrach | Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle progenitor cells[END_REF][START_REF] Partridge | Stem cell route to neuromuscular therapies[END_REF], pharmacological intervention [START_REF] Khurana | Pharmacological strategies for muscular dystrophy[END_REF][START_REF] Badalamente | Delay of muscle degeneration and necrosis in mdx mice by calpain inhibition[END_REF][START_REF] Tidball | Calpains and muscular dystrophies[END_REF], and gene repair/replacement strategies [START_REF] Chamberlain | Gene therapy of muscular dystrophy[END_REF][START_REF] Goyenvalle | Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping[END_REF][START_REF] Gregorevic | Viral vectors for gene transfer to striated muscle[END_REF][START_REF] Van Deutekom | Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells[END_REF][START_REF] Van Deutekom | Advances in Duchenne muscular dystrophy gene therapy[END_REF][START_REF] Wang | Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model[END_REF]. These therapies are not without significant hurdles. For example, cell-based approaches are hindered by a lack of efficient methods to introduce and engraft adequate numbers of muscle precursor cells into the bulk tissue. Gene transfer approaches are limited by difficulties associated with producing sufficient quantities of vector, cellular tropism of the vectors, ectopic gene expression, the need for methods to achieve whole-body delivery of the vector, and inherent immunological challenges. Recently, great strides have been made in overcoming these hurdles, and this latter topic will be an emphasis of this review. Ultimately, a combinatorial approach involving different therapeutic strategies may be necessary to combat the mechanical, signaling, and immune related mechanisms that lead to dystrophy.

The dystrophinopathies are X-linked recessive disorders caused by dystrophin deficiency. Since the discovery of the dystrophin gene and protein over 18 years ago [START_REF] Koenig | Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals[END_REF][START_REF] Hoffman | Dystrophin: the protein product of the Duchenne muscular dystrophy locus[END_REF], more than 30 different forms of muscular dystrophy have been molecularly identified [START_REF] Cohn | Molecular basis of muscular dystrophies[END_REF][START_REF] Bornemann | Diagnostic protein expression in human muscle biopsies[END_REF]. The 427 kDa skeletal muscle specific isoform of dystrophin is a sarcolemmal protein that interacts with many other proteins to link the extracellular matrix to the cytoskeleton [START_REF] Yoshida | Glycoprotein complex anchoring dystrophin to sarcolemma[END_REF][START_REF] Ervasti | Membrane organization of the dystrophin-glycoprotein complex[END_REF][START_REF] Ervasti | A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin[END_REF]. Dystrophin is composed of four major structural domains (Figure 1) [START_REF] Straub | Muscular dystrophies and the dystrophin-glycoprotein complex[END_REF]. The N-terminal domain binds to Factin filaments in the subsarcolemal cytoskeleton. The central rod domain includes 24 triple-helical domains, often referred to as spectrin-like repeats, and 4 strategically placed hinges that together provide flexible, spring-like properties that are likely critical during muscle contraction. The cysteine-rich (CR) domain along with the distal C-

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The core skeletal muscle DGC is composed of dystrophin, the sarcoglycans (SG's) (α, ß, γ, and δ-SG), dystroglycans (DG's) (α and β-DG), sarcospan, and the syntrophins (Figure 2). Additionally, a number of extra-and intracellular proteins are less tightly associated with the DGC, such as neuronal nitric oxide synthase (nNOS) [START_REF] Brenman | Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy[END_REF],

dystrobrevin [START_REF] Grady | Role for alpha-dystrobrevin in the pathogenesis of dystrophin-dependent muscular dystrophies[END_REF][START_REF] Yoshida | Biochemical evidence for association of dystrobrevin with the sarcoglycan-sarcospan complex as a basis for understanding sarcoglycanopathy[END_REF], caveolin-3 [START_REF] Crosbie | Caveolin-3 is not an integral component of the dystrophin glycoprotein complex[END_REF] and laminin-2 [START_REF] Henry | Dystroglycan: an extracellular matrix receptor linked to the cytoskeleton[END_REF]. The observation that expression of several DGC members was abnormal in a variety of different types of MD subsequently led to the realization that the genes for these DGC members were mutated in other forms of MD [START_REF] Straub | Muscular dystrophies and the dystrophin-glycoprotein complex[END_REF][START_REF] Campbell | Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage[END_REF][START_REF] Ozawa | From dystrophinopathy to sarcoglycanopathy: evolution of a concept of muscular dystrophy[END_REF]. For the purpose of this review, Duchenne muscular dystrophy (DMD) will serve as the model for current viral-mediated gene transfer approaches being developed in animal models with the implicit assumption that these methods might also be applied to other forms of MD.

DMD is the most common inherited neuromuscular disorder, affecting 1 in 3500 newborn males worldwide [START_REF] Emery | Population frequencies of inherited neuromuscular diseases--a world survey[END_REF]. DMD results from mutations within the dystrophin gene [START_REF] Worton | Genetics of Duchenne muscular dystrophy[END_REF][START_REF] Dalkilic | Muscular dystrophies: genes to pathogenesis[END_REF]. This gene is ~2.5 MB and consists of 79 exons, including 7 promoters with 3 full-length isoforms (expressed in muscle, brain, and cerebellar Purkinje neurons), and an additional 4 truncated isoforms (Dp260; Dp140; Dp116; and Dp71) produced from separate promoters within the gene. The gene is the largest identified to date, which may account for the high frequency of mutation [START_REF] Coffey | Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach[END_REF]. Close to 30% of cases arise from spontaneous mutations [START_REF] Emery | Duchenne Muscular Dystrophy[END_REF]. Approximately 60% of mutations causing DMD are deletions of large segments of the gene usually including one or more exons [START_REF] Koenig | Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals[END_REF][START_REF] Forrest | Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies[END_REF][START_REF] Den Dunnen | Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications[END_REF], and a smaller ~5% of mutations are duplications of large segments of the gene [START_REF] Den Dunnen | Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications[END_REF]. Such mutations can result in mRNA instability, and/or bring about premature termination of translation leading to an unstable, incomplete protein that is nonfunctional and rapidly degraded. Some mutations in the dystrophin gene can lead to deletion of part of the coding sequence where the reading frame is maintained; such mutations result in a semifunctional, internally deleted dystrophin protein associated with a milder Becker muscular dystrophy (BMD) [START_REF] Koenig | Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals[END_REF]. Lastly, former studies in transgenic mice have demonstrated not only the prevention of abnormal mechanical properties, but also non-toxic side effects of the dystrophin protein [START_REF] Cox | Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity[END_REF], further suggesting that DMD can be amendable to a gene replacement strategy.

Model systems

The development of potential treatments has been greatly assisted by the discovery of a natural genetic model of DMD: the mdx mouse [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF]. The mdx mouse is dystrophin deficient owing to a point mutation that leads to a premature stop codon and the production of an unstable peptide [START_REF] Sicinski | The molecular basis of muscular dystrophy in the mdx mouse: a point mutation[END_REF]. Transgenic mdx mice expressing

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recombinant dystrophin cDNAs have revealed a wide therapeutic window, with prevention of pathology being achieved with dystrophin expression ranging from 0.2-times to 50-times normal endogenous levels [START_REF] Cox | Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity[END_REF][START_REF] Phelps | Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice[END_REF][START_REF] Wells | Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy[END_REF].

Dystrophin expression can be restored in mdx mice by several routes, including drug administration, the use of antisense oligonucleotides targeted against the mutant exon, stem cell transplantation, and dystrophin gene transfer with viral or non-viral vectors. A related mouse model that more closely mimics the clinical picture in DMD patients is the dystrophin and utrophin double knock out (dko) mouse [START_REF] Deconinck | Postsynaptic abnormalities at the neuromuscular junctions of utrophin-deficient mice[END_REF]. A somewhat more attractive model because of its larger size and severe phenotype may be that of the canine x-linked muscular dystrophy (cxmd) model [START_REF] Cooper | The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs[END_REF][START_REF] Valentine | Canine X-linked muscular dystrophy as an animal model of Duchenne muscular dystrophy: a review[END_REF].

Adenovirus

Gene transfer vectors derived from adenovirus (Ad) have provided a wealth of knowledge that includes but is not limited to various stages of the viral life cycle, viral tropism, transduction efficiency, and the interplay between viral infection and the host immune response. In brief, adenoviruses are medium-sized (90-100 nm), nonenveloped icosohedral viruses containing a double-stranded DNA genome. There are >50 immunologically distinct serotypes (6 subgenera: A through F) that can cause human infections, most of which are benign and are targeted to the respiratory tract. These subgenera are distinguished from one another based on differential hemagglutination patterns, oncogenic potential, and DNA G+C content [START_REF] Shenk | Adenoviridae: the viruses and their replication[END_REF]. Subgroup C serotypes 2 or 5 are the major types used as viral vectors. The wild type adenovirus genome is approximately 35 kb [START_REF] Smith | Viral vectors in gene therapy[END_REF][START_REF] Verma | Gene therapy --promises, problems and prospects[END_REF]. There are four early transcriptional units (E1, E2, E3 & E4), which have regulatory functions, and a late transcript, which codes for structural proteins. Progenitor or first generation vectors have the E1 and often the E3 gene deleted, with the missing E1 genes being supplied in trans from an E1 region integrated into a producer cell such as the human fetal kidney cell line 293 [START_REF] Graham | Characteristics of a human cell line transformed by DNA from human adenovirus type 5[END_REF]. Second generation vectors additionally carry deletions of the E2b or E4 regions [START_REF] Armentano | Effect of the E4 region on the persistence of transgene expression from adenovirus vectors[END_REF][START_REF] Amalfitano | Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted[END_REF]. The most recent third generation or 'helper dependent' (hd) or gutted vectors contain only the inverted terminal repeats (ITRs) and a packaging sequence around the transgene with all of the necessary viral genes being provided in trans [START_REF] Chen | Persistence in muscle of an adenoviral vector that lacks all viral genes[END_REF][START_REF] Kumar-Singh | Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells[END_REF].

Attractive properties of Ad vectors included stable growth to very high titers (>10 11 particles /ml), a large transgene carrying capacity, very efficient transduction of target cells in vitro and in vivo, the ability to infect postmitotic cells such as myotubes, the relative ease of genome manipulation and the ability to generate high levels of transgene expression. A first-generation adenovirus, with a limited insert capacity of ~8 Kb, was the first viral

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vector to successfully deliver a human miniaturized dystrophin cDNA to mdx mice by intramuscular administration [START_REF] Ragot | Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice[END_REF]. The cDNA used in this study was deleted for segments encoded on exons 17 to 48 producing a ~200 kd protein that lacks most of the central rod domain, and was previously observed in a BMD patient [START_REF] England | Very mild muscular dystrophy associated with the deletion of 46% of dystrophin[END_REF]. In this study correct localization of a minidystrophin protein within the injected muscle was found in ~50% of the myofibers and persisted for up to 3 months. As with any viral vector there are distinct advantages and disadvantages to its use (Table 1). Several of these limitations have been overcome with use of the helper-dependent or gutted adenoviral vectors (hdAd) that are deleted for all of the viral protein coding sequences. The use of these vectors has led to a reduced immune response, improved transgene expression, and larger transgene carrying capacity [START_REF] Acsadi | Dystrophin expression in muscles of mdx mice after adenovirusmediated in vivo gene transfer[END_REF][START_REF] Parks | A helperdependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal[END_REF][START_REF] Dellorusso | Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin[END_REF]. The use of hdAd to deliver dystrophin to neonatal or adult mdx mice has demonstrated long-term expression, restoration of the dystroglycan complex at the sarcolemma, and amelioration of the physiological and pathological indices of muscle disease [START_REF] Dellorusso | Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin[END_REF][START_REF] Gilbert | Prolonged dystrophin expression and functional correction of mdx mouse muscle following gene transfer with a helper-dependent (gutted) adenovirus-encoding murine dystrophin[END_REF][START_REF] Dudley | Sustained improvement of muscle function one year after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector[END_REF]. Higher doses of vector were needed to treat adult mdx mice likely due to inefficient infection of mature muscle by Ad [START_REF] Dellorusso | Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin[END_REF].

Even with improvements in Ad vectorology, problems remain. The episomal Ad genomes are gradually lost from the transduced myofibers. High-titer neutralizing antibodies arise against Ad after the first administration of vector, preventing repetitive administration of Ad vector of the same serotype [START_REF] Morral | Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons[END_REF]. The administration of high doses of either first-generation or hdAd results in the induction of innate and cellular immune responses, that at very high doses are associated with direct toxicity that can be lethal [START_REF] Muruve | Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo[END_REF][START_REF] Muruve | Helper-dependent adenovirus vectors elicit intact innate but attenuated adaptive host immune responses in vivo[END_REF]. Further, systemic delivery of adenoviral vectors in mice leads to a significant drop in blood pressure likely due to the systemic activation of endothelial cells and increased production of nitric oxide, followed by destruction of Kupffer cells [START_REF] Schiedner | Selective depletion or blockade of Kupffer cells leads to enhanced and prolonged hepatic transgene expression using high-capacity adenoviral vectors[END_REF]. This acute toxicity is not prevented or reduced with hdAd. In the case of hdAd the capsid composition and structure are the same as in the first generation vectors and would not be expected to reduce the acute innate inflammatory response against the vector [START_REF] Stilwell | Development and characterization of novel empty adenovirus capsids and their impact on cellular gene expression[END_REF][START_REF] Brunetti-Pierri | Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into nonhuman primates[END_REF]. Several investigators are implementing methods to reduce the associated inflammation and toxicity.

For example, coating the virus with polyethylene glycol can serve to mask Ad epitopes from preexisting immune responses by reducing protein-protein interactions [START_REF] Mok | Evaluation of polyethylene glycol modification of first-generation and helper-dependent adenoviral vectors to reduce innate immune responses[END_REF]. The use of hdAd can limit the adaptive immune response against Ad proteins [START_REF] Muruve | Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo[END_REF], but generation of immunity to the transgene dystrophin is somewhat controversial in the context of human gene therapy. Many DMD patients have large deletions within the dystrophin gene that prevent expression of epitopes encoded by deleted exons such that exogenously delivered dystrophin could be perceived as a neoantigen [START_REF] Ohtsuka | Dystrophin acts as a transplantation rejection antigen in dystrophin-deficient mice: implication for gene therapy[END_REF]. In this regard, co-delivery of immunomodulatory molecules such as CTL4AIg [START_REF] Kay | Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration[END_REF] are being re-

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examined in the context of hdAd dystrophin delivery. Investigators have shown prolonged dystrophin expression in immuno-competent mice with systemic gene transfer utilizing T-cell costimulatory blockade [START_REF] Jiang | CTLA4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle[END_REF][START_REF] Jiang | Sustained muscle expression of dystrophin from a high-capacity adenoviral vector with systemic gene transfer of T cell costimulatory blockade[END_REF].

Lentivirus

An additional viral vector being tested for gene transfer to muscle is the HIV-derived lentiviral vector. This retroviral vector can be produced at moderate titers, can infect both dividing and non-dividing cells and be pseudotyped with a variety of different envelope glycoproteins [START_REF] Kootstra | Gene therapy with viral vectors[END_REF]. In contrast to results with Ad vectors, these vectors do not induce a cytotoxic T-cell lymphocyte (CTL) response. To address safety concerns, genetic engineering has produced a lentiviral vector system that is self-inactivating (SIN) [START_REF] Yu | Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells[END_REF]. These vectors have a large deletion in the 3' U3 region that contains the viral promoter and enhancer. This deletion is copied over to the 5' region during reverse transcription which in turn abolishes LTR-driven transcription in transduced cells [START_REF] Kootstra | Gene therapy with viral vectors[END_REF].

Furthermore, lentiviral vector systems can carry between 7.5 to 9 kb of cargo DNA, which is largely sufficient for cloning sizeable transgenes such as minidystrophin, selectable markers, and the promoters needed for expression.

This feature is important, especially when using large tissue-specific promoters. Finally, lentiviral vectors have been shown to permanently transduce and stably express transgenes in muscle cells and their precursors, making them the delivery system of choice for muscle progenitor cell based gene transfer [START_REF] Kafri | Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors[END_REF][START_REF] Mackenzie | Efficient transduction of liver and muscle after in utero injection of lentiviral vectors with different pseudotypes[END_REF][START_REF] Rubinson | A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference[END_REF]. In adult skeletal muscle, satellite cells are the primary stem cells that are responsible for postnatal muscle growth, hypertrophy, and regeneration.

These cells are normally quiescent but can activate to a proliferative state by extrinsic signaling mechanisms.

Activation of the satellite cell population is dramatically enhanced in response to muscle damage or degenerative disease where myoblast generation is necessary for repair and growth. Upon activation, there is a proliferative phase where progeny proceed down the myogenic lineage pathway to become fusion competent myoblasts [START_REF] Schultz | Skeletal muscle satellite cells[END_REF][START_REF] Hawke | Myogenic satellite cells: physiology to molecular biology[END_REF].

However, the transduction of satellite cells using lentiviral vectors would provide an attractive therapeutic option, particularly when robust isolation methodologies in the absence of activation and homing mechanisms become further identified. A large advantage of this therapy is the potential use of the patient's own cells and the inherent avoidance of the immune response. As with many ex vivo approaches there are challenges and this includes efficient isolation of satellite cells, development of optimal expansion conditions, enhancement of efficient muscle homing mechanisms, and minimization of insertional oncogenesis within active genes. Numerous other stem cells

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are also being studied for potential use in ex vivo gene therapy strategies for DMD, including mesangioblasts, SP cells and MAPC [START_REF] Bachrach | Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle progenitor cells[END_REF][START_REF] Sampaolesi | Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts[END_REF][START_REF] Li | Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin[END_REF].

Adeno-associated virus

Among the viral vector systems being utilized today, one that has generated much optimism towards eventual treatment of muscular dystrophy is adeno-associated virus (AAV). AAV is classified as a Parvoviridae family member being of the genus Dependovirus. Eleven strains of AAV (1-11) have been identified from primates thus far, and more than one hundred sequences representing novel clades of AAV have been reported [START_REF] Rutledge | Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2[END_REF][START_REF] Xiao | Adenoassociated virus as a vector for liver-directed gene therapy[END_REF][START_REF] Gao | Clades of Adeno-associated viruses are widely disseminated in human tissues[END_REF][START_REF] Gao | Novel adenoassociated viruses from rhesus monkeys as vectors for human gene therapy[END_REF].

AAV is a single stranded DNA virus (~4.7 kb) whose genome is flanked by inverted terminal repeats (ITRs) that are ~150 nucleotides in length. The ITRs are critical for stability and priming of genome replication through a "rolling hair pin" mechanism [START_REF] Tattersall | Rolling hairpin model for replication of parvovirus and linear chromosomal DNA[END_REF]. These elements contain all the cis-active elements necessary for replication and encapsidation of AAV genomes [START_REF] Xiao | A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle[END_REF]. The recombinant-AAVs (rAAV) in use today are deleted of all viral genes, specifically the rep and cap genes that encode the nonstructural and structural proteins respectively. Viral packaging constraints limit the size of the transgene that may be included to the size of the wild type genome. rAAV for gene transfer has been developed using a pseudotyping approach that consists of a recombinant genome framed by the ITR of AAV serotype 2 and a capsid based on any one of the cloned AAV serotypes [START_REF] Flotte | Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors[END_REF]. Recombinant vectors based on these variants demonstrate significantly different tropism for tissues such as cardiac muscle, skeletal muscle, liver, and lung as well as transduction differences in vitro versus in vivo [START_REF] Grimm | From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy[END_REF][START_REF] Duan | Enhancement of muscle gene delivery with pseudotyped adeno-associated virus type 5 correlates with myoblast differentiation[END_REF][START_REF] Halbert | Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration[END_REF].

For the expression of protein using rAAV there is the requirement of flanking ITR's, a promoter, transgene, and polyadenylation signal. The production of virions has vastly improved over the years. Refinement has allowed the use of a two plasmid system, where the Ad functions and Rep/Cap ORF's are supplied on a single plasmid [START_REF] Grimm | Novel tools for production and purification of recombinant adenoassociated virus vectors[END_REF][START_REF] Grimm | Progress in adeno-associated virus type 2 vector production: promises and prospects for clinical use[END_REF]. The virions may be purified and concentrated from the cellular lysate by density gradient centrifugation, ion exchange or affinity chromatography [START_REF] Halbert | Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors[END_REF][START_REF] Davidoff | Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock[END_REF][START_REF] Sommer | Quantification of adeno-associated virus particles and empty capsids by optical density measurement[END_REF]. In the case of large-scale production, column chromatography has a distinct advantage in terms of logistics. However, one must be aware that this procedure will not distinguish between virions that contain viral genomes from those that do not (empty capsids) as density gradient centrifugation allows. Several serotypes, for example, AAV2 and AAV6 have the capacity to bind heparin with a relatively high affinity thus allowing purification by heparin chromatography [START_REF] Halbert | Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors[END_REF].

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Many researchers have focused on the ability to systemically deliver rAAV carrying truncated versions of dystrophin to achieve body-wide transduction of the musculature. Though this is no small accomplishment, we must further address safety concerns of not only the viral vector, but also the transgene with more rigorous testing prior to proceeding to clinical trials. Irrespective to the transgene, the use of rAAV vectors is likely several years away from being a practical clinical therapy for DMD. However, there are several characteristics suggesting it as an attractive candidate for gene-replacement/corrective therapy. These include the nonpathogenic nature of AAV, the availability of alternative serotypes with muscle tropism, the ability to generate vectors at relatively high titer, a relatively low but not absent immunogenicity, and an ability to achieve long-term transgene expression. Further improvements in vector design with the goal of strictly limiting gene expression to muscle will undoubtedly add to the safety of AAV vectors on several levels. Optimization of methods to produce enough rAAV vector for routine use in patients, and the ability to develop techniques that allow transduction of enough of the musculature to provide a therapeutic benefit while minimizing toxicity will favorably influence the realization of therapeutic success in the clinic. In the future, with combined improvements in production and delivery approaches, these vectors likely will become more internally customized to a particular disease. Many of these promising technologies are new and likely to advance rapidly in the coming years. Analysis of specific and non-specific toxic effects will have to be a prominent aspect of future trials, and these will undoubtedly be balanced against the benefit of this type of treatment.

Gene Targeting

Gene targeting is the replacement of an endogenous DNA segment with a homologous, exogenous segment of DNA by homologous recombination (HR). Homologous recombination technology has rapidly advanced the field of molecular genetics, but initially fell short of realization in terms of a gene repair strategy. This was primarily because vectors used for HR typically inserted into the host genome at random locations with a frequency greater than that of HR [START_REF] Roth | Relative rates of homologous and nonhomologous recombination in transfected DNA[END_REF]. However, recently this area of research has been revitalized thanks to the efforts of several laboratories offering hope toward efficient chromosomal manipulation. A major advantage of the HR gene targeting approach is that the risk of random integration as is presented with retroviral mediated gene replacement [START_REF] Hacein-Bey-Abina | LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1[END_REF] can be avoided or minimized. In one study HR was accomplished by using hdAd as a vector platform [START_REF] Ohbayashi | Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors[END_REF].

The frequency of HR using this system with an 18.6 kb region of homology reached ~2x10 13 , while detection of

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ACCEPTED MANUSCRIPT 10 random integration events was found to be ~5% per transduced mouse embryonic stem cell at a multiplicity of infection (moi) of 10. This is a vast improvement from the 10 -5 to 10 -7 per infected cell that was previously demonstrated using E1-deleted replication incompetent Ad to target the single-copy Fgr locus in mammalian cells [START_REF] Mitani | Gene targeting in mouse embryonic stem cells with an adenoviral vector Somat[END_REF]. It was also shown that HR could be accomplished on a relatively small number of cells, which would be of benefit in the stem cell field. Similarly, a recombinant adeno-associated virus (rAAV) vector has been used to correct dominant mutations in mesenchymal stem cells (MSC's) from patients with osteogenesis imperfecta [START_REF] Chamberlain | Gene targeting in stem cells from individuals with osteogenesis imperfecta[END_REF],

demonstrating that gene targeting by HR is possible for non-embryonic stem cells. In this study 6x10 5 to 2.3x10 4 MSC's underwent gene targeting. This strategy has an advantage of minimizing the possibility of insertional mutagenesis, and there is the further advantage that MSC's are typically non-immunogenic.

Recently, an exciting technical development has emerged with the use of chimeric zinc finger nucleases (ZFN's) where a FokI endonuclease domain is combined with a zinc finger protein. When designed properly, this system can exhibit exquisite sequence specificity and targeted DNA cleavage [START_REF] Urnov | Highly efficient endogenous human gene correction using designed zinc-finger nucleases[END_REF]. This study demonstrated a remarkable 18% level of gene-modified human cells without selection. DNA cleavage is followed mechanistically by stimulation of HR between the chromosome and the extrachromosomal DNA donor. By altering the specificity of the zinc finger domain one can alter the target site specificity of the ZFN, making this a highly attractive methodology. The ability to engineer ZFN's to target essentially any sequence clearly establishes the utility of ZFNdriven genome editing. The same concerns of present-day gene therapy strategies exist, namely delivery method, associated toxicity, and a host immune response.

Muscle gene transfer

rAAV vectors support stable, long-term gene expression following infection of muscle cells, where they persist as an episome [START_REF] Rabinowitz | Adeno-associated virus expression systems for gene transfer[END_REF]. Serotypes that have demonstrated a particular tropism for the musculature include AAV1, 5, 6, and 8 [START_REF] Duan | Enhancement of muscle gene delivery with pseudotyped adeno-associated virus type 5 correlates with myoblast differentiation[END_REF][START_REF] Chao | Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors[END_REF][START_REF] Scott | Viral vectors for gene transfer of micro-, mini-, or full-length dystrophin[END_REF][START_REF] Auricchio | Noninvasive gene transfer to the lung for systemic delivery of therapeutic proteins[END_REF][START_REF] Zhu | Sustained whole-body functional rescue in congestive heart failure and muscular dystrophy hamsters by systemic gene transfer[END_REF]. These serotypes have demonstrated a transduction efficiency of up to 500 times greater than that of AAV2 [START_REF] Gao | Novel adenoassociated viruses from rhesus monkeys as vectors for human gene therapy[END_REF][START_REF] Blankinship | Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6[END_REF][START_REF] Mah | A new method for recombinant adeno-associated virus vector delivery to murine diaphragm[END_REF][START_REF] Chao | Sustained and complete phenotype correction of hemophilia B mice following intramuscular injection of AAV1 serotype vectors[END_REF]. Although wild type AAV is capable of integration into the host genome in the presence of Rep, integration of a rAAV genome into myonuclei has not been detected [START_REF] Schnepp | Genetic fate of recombinant adeno-associated virus vector genomes in muscle[END_REF]. Long-term transgene expression of rAAV has been observed for over 5 years in dogs and rhesus macaques, and for 2 years in mice [START_REF] Fisher | Recombinant adeno-associated virus for muscle directed gene therapy[END_REF][START_REF] Herzog | Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector[END_REF][START_REF] Song | Sustained secretion of human alpha-1antitrypsin from murine muscle transduced with adeno-associated virus vectors[END_REF][START_REF] Xiao | Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector[END_REF][START_REF] Manno | AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia[END_REF]. Vectors pseudotyped with capsids from AAV1, 6, and 8 display peak expression several weeks

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prior to that of AAV2 [START_REF] Blankinship | Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6[END_REF][START_REF] Grimm | Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy[END_REF][START_REF] Thomas | Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors[END_REF]. This delay in transduction is quite significant for a disease such as DMD where there is a rapid turnover of muscle fibers [START_REF] Harper | Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy[END_REF].

The route of administration of rAAV is a major consideration for the treatment of DMD, taking into account that body wide distribution would likely be necessary for an effective therapy. Previous efforts to target organs such as the diaphragm or heart, whose failure accounts for the majority of patient deaths, have utilized invasive surgical procedures for intravenous (IV) vector delivery using potentially toxic cofactors to induce vascular permability [START_REF] Greelish | Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector[END_REF]. Although such studies yielded beneficial results, they underscore the need to deliver a therapeutic vector in a noninvasive manner. In another approach, researchers administered viral vector to rodents intra-arterially using high pressure injections [START_REF] Gonin | Femoral intra-arterial injection: a tool to deliver and assess recombinant AAV constructs in rodents whole hind limb[END_REF]. More recently, the ability to transduce the entire cardiac and skeletal musculature of a mouse has been accomplished with a single low pressure intravenous administration of rAAV6 [START_REF] Gregorevic | Systemic delivery of genes to striated muscles using adeno-associated viral vectors[END_REF]. The inclusion of vascular endothelial growth factor (VEGF) was shown to dramatically increase the efficiency of this method at lower vector doses [START_REF] Gregorevic | Systemic delivery of genes to striated muscles using adeno-associated viral vectors[END_REF]. This response to VEGF was not observed at higher vector doses (>10 12 vg). Importantly, IV administration of rAAV6 demonstrated efficient transduction of the diaphragm and intercostal muscles that are vital to respiration [START_REF] Gregorevic | Systemic delivery of genes to striated muscles using adeno-associated viral vectors[END_REF]. In a follow up study in which rAAV6 encoding human alkaline phosphatase was delivered by IV administration, persistence of transgene expression was demonstrated out to 20 months in mice (Figure 3). Since this initial demonstration there have been numerous reports using various animal models that follow along a similar vein [START_REF] Zhu | Sustained whole-body functional rescue in congestive heart failure and muscular dystrophy hamsters by systemic gene transfer[END_REF][START_REF] Noro | Adeno-associated viral vector-mediated expression of endostatin inhibits tumor growth and metastasis in an orthotropic pancreatic cancer model in hamsters[END_REF][START_REF] Sandalon | Secretion of a TNFR:Fc fusion protein following pulmonary administration of pseudotyped adeno-associated virus vectors[END_REF][START_REF] Wang | Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart[END_REF][START_REF] Streck | Adeno-associated virus vector-mediated systemic delivery of IFN-beta combined with low-dose cyclophosphamide affects tumor regression in murine neuroblastoma models[END_REF][START_REF] Liu | Inhibition of atherogenesis in LDLR knockout mice by systemic delivery of adeno-associated virus type 2-hIL-10[END_REF][START_REF] Britton | Functional characterization of novel alternatively spliced ClC-2 chloride channel variants in the heart[END_REF][START_REF] Muller | Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors[END_REF][START_REF] Cardone | Correction of Hunter syndrome in the MPSII mouse model by AAV2/8mediated gene delivery[END_REF]. Additional optimization is warranted in order to allow for a lower effective dose in larger animal models, providing for a decreased potential for toxicity and a safer outcome. The low immune response elicited by rAAV vectors in animal studies suggests that this vector may have a distinct advantage over others such as Ad [START_REF] Manno | AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia[END_REF]149]. To date there is little evidence for rAAV vectors causing direct toxicity in either animal studies or human clinical trials. Doses up to 10 12 vg/kg of rAAV have been well tolerated in human studies with no toxicity [START_REF] Manno | AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia[END_REF][START_REF] Moss | Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebocontrolled trial[END_REF][START_REF] Wagner | A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies[END_REF]. However, evaluation of clinical samples from a recent hemophilia trial revealed a cytotoxic T-cell (CTL) mediated immune response that was likely generated by memory CD8 + T cells against the AAV capsid. This immune response resulted in a decline of transgene (Factor IX) expression and hepatic injury supported by an elevation of alanine and aspartate transaminases [START_REF] Manno | Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response[END_REF]. While this data is from a relatively small sample size, it should be considered carefully when proceeding to human clinical trials given the frailty of older DMD patients.

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Gene repair strategies

It has recently been reported by the International Human Genome Sequencing Consortium (IHGSC) that the human genome contains ~ 25,000 gene loci covering ~1.7 Gb, with 231,667 protein-encoding exons. The tremendous size of the DMD gene represents an enormous target for genetic mutations. Approximately 20% of the mutations leading to DMD are point mutations or small insertions or deletions that effect one or a few exons [START_REF] Buzin | Mutation rates in the dystrophin gene: a hotspot of mutation at a CpG dinucleotide[END_REF].

With that in mind, nature has provided us with a clue for an alternative gene therapy approach to the gene replacement strategy. DMD patients occasionally have rare, dystrophin positive fibers (revertant fibers) that likely originate from exon skipping in the dystrophin gene and which generate a truncated transcript with a restored open reading frame [START_REF] Lu | Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion[END_REF]. Exon skipping therapy is based on the reading frame rule [START_REF] Monaco | An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus[END_REF] which states that frameshifting mutations in the dystrophin gene result in DMD, while frame-conserving ones primarily lead to a milder Becker muscular dystrophy (BMD). Thus, the ability to induce exon skipping has the potential to convert a DMD phenotype to a much less severe BMD phenotype. Several laboratories have demonstrated induction of exon skipping [START_REF] Van Deutekom | Advances in Duchenne muscular dystrophy gene therapy[END_REF] using synthetic antisense oligonucleotides (AONs) [START_REF] Goyenvalle | Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping[END_REF][START_REF] Lu | Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles[END_REF][START_REF] Denti | Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model[END_REF]. These AONs bind to specific sequences in the pre-mRNA and thus disturb exon inclusion signals such as splice sites, intronic branch point sequences, or exonic splicing enhancer elements. In turn, this leads to removal of the targeted exon by inhibiting its inclusion within the processed mRNA.

One model to test the induction of exon skipping is through the targeted skipping of exon 23 in the mdx mouse. This exon contains a nonsense mutation that results in dystrophin synthesis being aborted prematurely. The researchers used an AON that targets the 5'splice-site of this exon, in combination with a drug carrier called F127 [START_REF] Lu | Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles[END_REF] (a block copolymer belonging to the group of amphiphillic pluronics used in the pharmaceutical industry).

This drug carrier enhances the circulation time, metabolic stability, and transport of AONs across the cell membrane. It was demonstrated that AON-induced skipping of exon 23 could be therapeutic as up to ~20% of the muscle fibers in the mdx mouse were converted to a dystrophin positive state with positive functional consequences.

The distribution of the dystrophin positive fibers was highly variable which was attributed to the regeneration process in individual mdx muscle fibers that led to differential uptake of the AONs. After repeated administration, In a more efficient and persistent strategy, rAAV was used to deliver AONs as part of either U1 [START_REF] Denti | Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model[END_REF] or U7 small nuclear RNAs [START_REF] Goyenvalle | Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping[END_REF]. The latter study utilized a previously described antisense-plasmid system (U7mOPT) [START_REF] Suter | Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations[END_REF] that contains a modified U7snRNA gene (U7mOPT). This RNA, as a part of the U7 ribonucleoprotein particle, is involved in the processing of the 3' end of histone pre-mRNAs within the nucleus through an antisense mechanism. Within the U7mOPT, the original U7 antisense sequence was replaced with two antisense sequences.

These sequences target the branch point sequence in intron 22 and the 5' splice site of exon 23. After intramuscular injection of the rAAV-U7-AON vector in mdx mice up to 77% of dystrophin positive fibers were observed, and 3 months following administration a 50% level was reported. Taking this approach one step further by using the systemic delivery capability of rAAV, it was demonstrated that AAV-mediated antisense-U1 small nuclear RNA expression resulted in effective dystrophin production through the hind limb of an mdx mouse, significant recovery of the functional properties in vivo, and lower serum creatine kinase levels, suggesting an overall decrease in muscle wasting [START_REF] Denti | Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model[END_REF].

In order to test the effectiveness of the AON strategy in the context of human dystrophin a hDMD transgenic mouse was developed with the full-length human DMD gene [START_REF] Bremmer-Bout | Targeted exon skipping in transgenic hDMD mice: A model for direct preclinical screening of humanspecific antisense oligonucleotides[END_REF]. This model demonstrated that the targeting of human exons with AONs can induce their specific removal from the human DMD transcript. Further,

showing exquisite specificity, no detectable effects in the homologous mouse dystrophin transcript were detected.

Immune evasion strategies

Any viral-mediated gene delivery method can potentially lead to a destructive host immune response against vector proteins. This result can occur by cell transduction and antigen presentation in the context of the major histocompatibility complex, resulting in elicitation of a CTL response that destroys transduced cells. It must also be considered that DMD patients, whose muscles do not express dystrophin, may be more likely to develop an immune response to vector-delivered dystrophin than would patients who express low levels or truncated forms of dystrophin. Therefore, patients with different mutations may respond differently to gene transfer. Understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to develop approaches to prevent induction of an immune response is of utmost importance. Various immunosuppressive

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ACCEPTED MANUSCRIPT 14 protocols have been shown to potentially circumvent this dilemma. For example, transient depletion of CD4+ T cells during the initial exposure to rAAV allowed successful readministration of AAV vectors to skeletal muscle [START_REF] Manning | Transient immunosuppression allows transgene expression following readministration of adenoassociated viral vectors[END_REF]. Similarly, the use of anti-CD40L antibodies, which block T cell activation of B cells, allowed prolonged gene expression in mouse lung [START_REF] Halbert | Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure[END_REF]. These treatments would be expected to inhibit the initiation and expansion of a primary immune response and promote viral delivery of a transgene [START_REF] Manning | Transient immunosuppression allows transgene expression following readministration of adenoassociated viral vectors[END_REF][START_REF] Kay | Transient immunomodulation with anti-CD40 ligand antibody and CTLA4Ig enhances persistence and secondary adenovirus-mediated gene transfer into mouse liver[END_REF]. However, these approaches would be less effective in humans that possess an arsenal of pre-existing neutralizing antibodies that could block readministration of the viral vector.

Recently a large (>10 6 ) AAV-2 capsid library was generated with randomly dispersed capsid mutations in a process similar to DNA shuffling [START_REF] Maheshri | Directed evolution of adenoassociated virus yields enhanced gene delivery vectors[END_REF]. This approach was applied to a clinically relevant problem of AAV:

preexisting immunity in the human population. In this case the authors applied repeated amplification of those AAV2 mutants that retained infectivity in the presence of antisera with increasing titer of neutralizing antibody [START_REF] Maheshri | Directed evolution of adenoassociated virus yields enhanced gene delivery vectors[END_REF]. The results were impressive: numerous antibody-evading mutants were obtained, one which showed the ability to moderately transduce cells while in the presence of a 1:2 antiserum dilution. When neutralizing antibodies to other AAV serotypes as well as crystal structures become available an AAV capsid regional immunogenic profile should become possible. These data have the potential to bring a wealth of knowledge to our current understanding of antigenicity of the AAV capsid and when combined with the knowledge of the altered tropism capacity of AAV serotypes [START_REF] Muzyczka | Custom adeno-associated virus capsids: the next generation of recombinant vectors with novel tropism[END_REF] provides a powerful tool for therapeutic applications.

The promoter is an important consideration in determining both the level of expression and tissue specificity of rAAV-mediated gene transfer. Tissue specific gene expression within skeletal muscle has been achieved using the synthetic C5-12 promoter [START_REF] Liu | Therapeutic levels of factor IX expression using a musclespecific promoter and adeno-associated virus serotype 1 vector[END_REF], the muscle creatine kinase (MCK) promoter [START_REF] Yoshimura | AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype[END_REF] and the desmin promoter [START_REF] Zhang | Intraarterial delivery of naked plasmid DNA expressing fulllength mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy[END_REF]. However, even when tissue-specific promoters are employed clearance of the vector from the bloodstream can occur from high levels of neutralizing antibody to the viral capsid or transgene product. It would be of great interest to specifically tailor transgene cassettes so that the gene expression could be prevented in undesireable cell types such as immune effector cells. Restricting expression with the use of tissue-specific promoters may also limit the potential efficacy of gene transfer by constricting the population of cells expressing the transgene.

Recently, micro RNA (miRNA) regulation was demonstrated within a lentiviral system to prevent transgene expression in hematopoietic lineages while permitting high expression in nonhematopoietic cells [START_REF] Brown | Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer[END_REF].

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15 miRNAs are a family of small (~21mer) RNAs that, at least for those that have characterized targets, negativelyregulate gene expression at the post-transcriptional level [START_REF] Ambros | MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing[END_REF][START_REF] Chen | MicroRNAs modulate hematopoietic lineage differentiation[END_REF][START_REF] Lewis | Prediction of mammalian microRNA targets[END_REF][START_REF] Lai | microRNAs: runts of the genome assert themselves[END_REF]. The researchers incorporated four tandemly positioned copies of a 23-bp sequence (mirT) within the 3' untranslated region of green fluorescent protein. This design provides complementary target sequence to multiple miRNAs with the intention of optimizing repression of the transgene. The selection of targets was based upon emerging constraints of miRNA mediated regulation [START_REF] Bartel | Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs[END_REF][START_REF] Doench | siRNAs can function as miRNAs[END_REF]. This system was found to be an improved means for preventing expression in hematopoietic lineage cells when compared to a hepatocyte-specific albumin promoter which may have also better prevented cross-priming of the T-cell population to professional APC's. This improved ability to exclude expression was suggested to occur because post-transcriptional regulation can trump off-target promoter activity. The engineering of miRNA-mediated regulation into a viral vector could potentially allow for an exquisite layer of control over transgene expression at the level of transcription irregardless of the relative strength of the promoter, and as such potentially allowing avoidance of the host immune response.

Addressing the rAAV carrying capacity limitation

One of the most challenging aspects of developing rAAV for the treatment of DMD is the limited DNA packaging capacity of the capsid. The upper limit capacity of rAAV for foreign DNA is considered to be ~5 kb [START_REF] Dong | Quantitative analysis of the packaging capacity of recombinant adeno-associated virus[END_REF] while the coding region of dystrophin covers 11.2 kb of cDNA sequence. Three approaches have explored the ability to circumvent this size constraint, namely the minigene, trans-splicing, and recombination vector approaches.

Patients with mild phenotypes of muscular dystrophy have been described that arise from large deletions within the dystrophin gene [START_REF] England | Very mild muscular dystrophy associated with the deletion of 46% of dystrophin[END_REF]. It was concluded that large portions of the gene are not vital for function (Figure 1). Several transgenic mdx mice were engineered to carry different deletions throughout the four dystrophin domains in order to map the regions that are crucial for function. Mild phenotypes were associated with N-terminal domain deletions, indicating that this region is important but not essential for attachment to actin and the cytoskeleton [START_REF] Corrado | Transgenic mdx mice expressing dystrophin with a deletion in the actinbinding domain display a "mild Becker" phenotype[END_REF]. The evaluation of a series of large deletions within the central rod domain indicated that as an entirety this structure is indispensable but the number of spectrin repeats can be markedly reduced [START_REF] Wang | Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model[END_REF][START_REF] Harper | Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy[END_REF][START_REF] Sakamoto | Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene[END_REF]. Also the proper positioning of the repeats and the presence and configuration of the hinge regions is crucial. Deletions in the cysteine-rich domain cause severe dystrophy, also resulting in disrupted expression of the entire DGC [START_REF] Rafael | Forced expression of dystrophin deletion constructs reveals structure-function correlations[END_REF]. The Cterminal domain was found not to be required for assembly of the full DGC [START_REF] Rafael | Forced expression of dystrophin deletion constructs reveals structure-function correlations[END_REF][START_REF] Crawford | Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain[END_REF]. Engineered to mimic and

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16 improve upon the exon 17-48 deletion in a BMD patient [START_REF] England | Very mild muscular dystrophy associated with the deletion of 46% of dystrophin[END_REF], the 6.2 kb minidystrophin construct (∆H2-R19) contains 8 repeats and hinge regions 1, 3 and 4. This construct was found to be completely functional, in that mdx mice that expressed this mini-dystrophin demonstrated normal muscle morphology and force generation. Several microdystrophin constructs (3.6-4.2 kb) have also been developed that display a highly effective ability to support near normal muscle structure and function in mice [START_REF] Harper | Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy[END_REF][START_REF] Sakamoto | Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene[END_REF][START_REF] Fabb | Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice[END_REF]. Truncated dystrophins carrying 4-7 spectrin-like repeats have been delivered to mouse muscle by rAAV. These constructs have been shown to ameliorate many of the dystrophic pathophysiological effects in the mdx mouse such as increased susceptibility to eccentric contractioninduced injury, high proportions of centrally-nucleated myofibers, and elevated levels of serum creatine kinase [START_REF] Wang | Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model[END_REF][START_REF] Harper | Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy[END_REF][START_REF] Gregorevic | Systemic delivery of genes to striated muscles using adeno-associated viral vectors[END_REF][START_REF] Yoshimura | AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype[END_REF][START_REF] Liu | Adenoassociated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury[END_REF][START_REF] Watchko | Adeno-associated virus vector-mediated minidystrophin gene therapy improves dystrophic muscle contractile function in mdx mice[END_REF]. The micro-dystrophin found to have optimal characteristics of effectiveness and small size was the construct ∆R4-R23/∆CT, which spans 3.6 kb of DNA and carried 4 spectrin-like repeats and hinges 1, 2 and 4 while lacking the C-terminal domain. Although most microdystrophins vastly improve the phenotype of mdx mice, restoration of the muscles to complete parity with those of wild type mice is not accomplished. There is not a full restoration of absolute or specific force generation, and the degree of correction is both age and muscle dependent [START_REF] Yoshimura | AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype[END_REF][START_REF] Liu | Adenoassociated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury[END_REF]. Importantly, in young and old mdx mice, microdystrophin mediated correction of the abnormal properties of the diaphragm is significantly more effective than in limb muscles. This observation is noteworthy considering the phenotype in the diaphragm of the mdx mouse much more closely mimics the pathology found in human muscle and many patients succumb to respiratory failure. Even with these encouraging results it is possible that other more effective microdystrophins could be engineered.

Two such strategies have come to light that may allow larger minidystrophins to be expressed by rAAV delivery. The first is gene expression following trans-splicing. This approach utilizes two rAAV vectors with the transgene split between them. The 5' gene portion is followed by a splice donor sequence from a natural dystrophin intron without a polyadenylation signal while the 3' gene portion is preceded by a splice acceptor from the same intron, followed by a polyadenylation signal [START_REF] Liu | Adenoassociated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury[END_REF]. Expression of the large protein occurs in co-infected cells,

where the viral genomes concatamerize, and cell mediated splicing generates an mRNA for a minidystrophin. RNA processing thus represents a crucial barrier to the efficiency of cellular transduction using this vector system. The sequence environment of these splice sites and their location within the split gene can have profound influences on transcription and splicing efficiencies [START_REF] El | Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers[END_REF][START_REF] Cartegni | Listening to silence and understanding nonsense: exonic mutations that affect splicing[END_REF].

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Another system that utilizes a dual vector strategy is often referred to as recombination or overlapping rAAV vectors. In essence, two rAAV vectors containing overlapping fragments of a larger gene are delivered simultaneously to cells resulting in homologous recombination at the overlap such that the larger gene is reconstituted [START_REF] Ghosh | Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle[END_REF][187][START_REF] Halbert | Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene[END_REF]. The efficiency with which transduction occurs using the recombination system can be influenced by the AAV serotype, desired tissue target, and the specific transgene overlap composition. In mouse lung it was demonstrated that rAAV6-Alkaline phosphatase (AP) was superior to that of rAAV2-AP, where the degree and distribution of transduction of the former recombinant vectors was similar to the parental intact vector.

Similarly, rAAV6 recombination vectors have been found to be more efficient in skeletal muscle than rAAV2 counterparts [START_REF] Ghosh | Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle[END_REF]. Further, this study demonstrated that the transgene overlap can influence the recombination kinetics. This observation implies that sequence characteristics of the overlap region such as degree of secondary structure, and GC content should be taken into account when designing such a system.

Compensatory Therapeutics

Utrophin

The spectrin superfamily of proteins includes dystrophin, utrophin (formerly called dystrophin-related protein, or DRP), DRP2, and dystrobrevin [START_REF] Love | An autosomal transcript in skeletal muscle with homology to dystrophin[END_REF][START_REF] Roberts | Characterization of DRP2, a novel human dystrophin homologue[END_REF][START_REF] Blake | Utrophin: a structural and functional comparison to dystrophin[END_REF]. There is a high degree of sequence similarity between dystrophin and utrophin at both the DNA and protein levels [START_REF] Blake | Utrophin: a structural and functional comparison to dystrophin[END_REF][START_REF] Blake | Function and genetics of dystrophin and dystrophin-related proteins in muscle[END_REF]. Similar to dystrophin, utrophin is encoded by many small exons over a relatively large genomic region (~1 Mb) [START_REF] Pearce | The utrophin and dystrophin genes share similarities in genomic structure[END_REF]. Additionally, transcription is carried out from several promoters [START_REF] Herzog | Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector[END_REF], and several truncated isoforms have been identified [START_REF] Greelish | Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector[END_REF][START_REF] Blake | G-utrophin, the autosomal homologue of dystrophin Dp116, is expressed in sensory ganglia and brain[END_REF][START_REF] Lumeng | Characterization of dystrophin and utrophin diversity in the mouse[END_REF] . In normal skeletal muscle utrophin is found concentrated at the myotendinous and neuromuscular junctions, whereas it is localized under the entire sarcolemma in regenerating fibers present in mdx skeletal muscle [START_REF] Bewick | Different distributions of dystrophin and related proteins at nerve-muscle junctions[END_REF][START_REF] Law | Talin, vinculin and DRP (utrophin) concentrations are increased at mdx myotendinous junctions following onset of necrosis[END_REF]. In skeletal muscle, utrophin is detectable in early fetal development over the sarcolemma where during development it is gradually replaced by dystrophin [START_REF] Takemitsu | Dystrophin-related protein in the fetal and denervated skeletal muscles of normal and mdx mice[END_REF]. This difference in developmental expression has resulted in the proposal that utrophin may be the embryonic/neonatal form of dystrophin [START_REF] Clerk | Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle[END_REF], implying that utrophin provides dystrophinlike function in embryonic and developing skeletal muscle. Recent evidence has come to light that may partially begin to explain some of these modest differences in terms of the structural/functional relationships of these two quite similar proteins. Alternative molecular mechanisms of beta-dystroglycan [START_REF] Ishikawa-Sakurai | ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to betadystroglycan[END_REF] and actin filament interactions [START_REF] Rybakova | Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle[END_REF][START_REF] Rybakova | Identification of spectrin-like repeats required for high affinity utrophin-actin interaction[END_REF][START_REF] Rybakova | Dystrophin and utrophin bind actin through distinct modes of contact[END_REF] for utrophin when compared to dystrophin have been put forth. It may be that subtle differences or

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undisclosed areas within or adjacent to the N-terminal or cysteine-rich domains may impart a more significant contribution to binding affinity than previously considered. This would be of obvious importance when considering the development of micro or mini genes for therapeutic potential. In contrast to dystrophin, whose actin binding domain resides within the amino terminus and the central rod domain (Figure 1); utrophin actin binding capacity resides in the continuous segment that includes the amino terminus and the first ten spectrin-like repeats of the rod domain [START_REF] Rybakova | Identification of spectrin-like repeats required for high affinity utrophin-actin interaction[END_REF]. Given this arrangement, and the degree of developmental expression, utrophin has been proposed to act as a stabilizer of newly formed actin filaments during costamere development [START_REF] Rybakova | Dystrophin and utrophin bind actin through distinct modes of contact[END_REF]. Additionally, given the non-competitive nature of dystrophin/utrophin binding the overlapping expression of these two proteins may provide for an effective changing of the muscle fiber guard during the period shortly after birth of utrophin downregulation and dystrophin upregulation.

This expression pattern led to the hypothesis that utrophin may be able to functionally compensate for dystrophin in DMD patients [204]. This idea was supported by studies in mdx mice where it was found that elevation of utrophin levels in dystrophic muscle fibers restored sarcolemmal expression of DGC members, and alleviated the dystrophic pathology. The concept of upregulating utrophin to allow for a replacement of dysfunctional or absent dystrophin provides promising therapeutic possibilities for DMD. For example, adenovirus delivery of utrophin in muscle has shown phenotypic improvement in mdx and double knock-out (dko) mice that are deficient for both utrophin and dystrophin [START_REF] Gilbert | Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles[END_REF][START_REF] Wakefield | Prevention of the dystrophic phenotype in dystrophin/utrophin-deficient muscle following adenovirus-mediated transfer of a utrophin minigene[END_REF]. In the dko mice administered the utrophin minigene neonatally, expression was detected 30 days post-injection in ~95% of myofibers within the limb muscle, providing protection from subsequent dystrophic damage in dystrophin deficient muscle. Further, transgenic mdx mice that expressed full-length utrophin mRNA had a more improved outcome in preventing dystrophy even with relatively low levels of expression [START_REF] Tinsley | Expression of full-length utrophin prevents muscular dystrophy in mdx mice[END_REF]. Thus, experimental evidence suggests that a gene transfer approach focusing on the exogenous delivery of utrophin to DMD muscle fibers is a viable treatment option for this disease. As well, there is an added benefit to this approach; the lack of neoantigen production and subsequent immune response against the transgene that might result with the delivery of dystrophin [START_REF] Ebihara | Differential effects of dystrophin and utrophin gene transfer in immunocompetent muscular dystrophy (mdx) mice[END_REF].

Further evidence that utrophin can functionally compensate for the lack of dystrophin in muscle was provided by a transgenic mdx mouse line that expressed a mini-utrophin in muscle [START_REF] Dennis | Molecular and functional analysis of the utrophin promoter[END_REF]. The results were striking: histochemical analysis demonstrated a dramatic decrease in fibrosis, necrosis, serum creatine kinase levels, and the frequency of centrally nucleated myotubes. It is noteworthy that no toxicity was associated with high-level

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expression of utrophin, even in non-muscle tissues [START_REF] Rybakova | Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle[END_REF][START_REF] Tinsley | Expression of full-length utrophin prevents muscular dystrophy in mdx mice[END_REF][START_REF] Fisher | Non-toxic ubiquitous over-expression of utrophin in the mdx mouse[END_REF]. Subtle structural differences between dystrophin and utrophin are known. For example, there is a short extension in the N-terminal domain that is absent from dystrophin that results in a greater binding affinity for actin. Also these two proteins do not appear to compete for binding sites on actin [START_REF] Rybakova | Dystrophin and utrophin bind actin through distinct modes of contact[END_REF][START_REF] Moores | Biochemical characterisation of the actin-binding properties of utrophin[END_REF][START_REF] Moores | Structure of the utrophin actin-binding domain bound to F-actin reveals binding by an induced fit mechanism[END_REF]. Furthermore, a difference can be found when comparing the rod domain of these two proteins though it is thought that this region has been under less selective pressure [START_REF] England | Very mild muscular dystrophy associated with the deletion of 46% of dystrophin[END_REF]. Obvious differences include a less defined first hinge following the N-terminal domain, two fewer spectrin-like repeats, and the lack of basically charged spectrin-like repeats that provide a second actin-binding domain in dystrophin [START_REF] Amann | Utrophin lacks the rod domain actin binding activity of dystrophin[END_REF].

These differences are in contrast to the cysteine-rich and C-terminal domains, which display ~80% amino acid sequence identity between dystrophin and utrophin [START_REF] Tinsley | Primary structure of dystrophinrelated protein[END_REF].

Endogenous utrophin up-regulation

A number of labs have investigated the possibility of stimulating endogenous utrophin expression by pharmacological intervention. A challenge with this approach is achieving increased expression of utrophin along the entire length of myofibers to act as a dystrophin surrogate. In order to accomplish this one must begin to understand what motifs within the DNA are responsible for restricting utrophin expression at the neuromuscular junction and for allowing more widespread expression in developing and regenerating myofibers. Several labs are using such analyses to develop small-targeted molecules that might enable upregualtion of the gene in all myonuclei.

A more thorough understanding of trans-acting factors and their appropriate signaling pathways would be of great benefit towards the design and delivery of specific molecules to induce targeted transcriptional activation. Recently, the delivery of a small peptide region of the heregulin ectodomain to mdx mice increased utrophin expression and ameliorated the dystrophic phenotype [START_REF] Krag | Heregulin ameliorates the dystrophic phenotype in mdx mice[END_REF]. The improvements appeared to be a result of utrophin up-regulation as the same treatment in the dko model failed to improve muscle pathology. However, the benefits obtained were not as dramatic (~2-3 fold) as those obtained previously by somatic gene transfer of mini-utrophin genes [START_REF] Gilbert | Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles[END_REF].

In addition to transcriptional regulatory mechanisms, utrophin appears to be regulated post-transcriptionally [START_REF] Gramolini | Distinct regions in the 3' untranslated region are responsible for targeting and stabilizing utrophin transcripts in skeletal muscle cells[END_REF][START_REF] Gramolini | Regulation and functional significance of utrophin expression at the mammalian neuromuscular synapse[END_REF][START_REF] Gramolini | Discordant expression of utrophin and its transcript in human and mouse skeletal muscles[END_REF][START_REF] Gramolini | Increased expression of utrophin in a slow vs. a fast muscle involves posttranscriptional events[END_REF]. Regions within the 3' untranslated region have been found to be responsible for targeting and stabilizing utrophin transcripts, allowing a potential means of sustaining increased levels in muscle. Studies in vivo have demonstrated that post-transcriptional processing plays a contributory role towards increased utrophin transcript stability in slow versus fast twitch muscle [START_REF] Gramolini | Increased expression of utrophin in a slow vs. a fast muscle involves posttranscriptional events[END_REF]. It has been proposed that this may be regulated by calcineurin

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ACCEPTED MANUSCRIPT 20 levels [START_REF] Chakkalakal | Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling[END_REF]. This study indicated that transgenic mice overexpressing calcineurin showed increased utrophin mRNA levels, thought to be mediated through NFAT signaling, an effector of calcineurin. Inhibition of calcineurin resulted in a ~80% decrease in utrophin mRNA levels. An internal ribosomal entry site (IRES) has also been identified within the 5' untranslated region of utrophin. This IRES has been shown to regulate the expression of utrophin during muscle regeneration [START_REF] Miura | The utrophin A 5'untranslated region confers internal ribosome entry site-mediated translational control during regeneration of skeletal muscle fibers[END_REF]. Further, post-translational mechanisms have been implicated in utrophin expression such as the involvement of calpain-mediated proteolysis [START_REF] Waheed | Factors associated with induced chronic inflammation in mdx skeletal muscle cause posttranslational stabilization and augmentation of extrasynaptic sarcolemmal utrophin[END_REF], and the GTPase RhoA was found to stabilize and aid in localization of utrophin protein in muscle cell cultures [START_REF] Bonet-Kerrache | The GTPase RhoA increases utrophin expression and stability, as well as its localization at the plasma membrane[END_REF]. All together, each of these regulatory events represent potential targets for the development of strategies designed to increase endogenous utrophin in dystrophin deficient myofibers.

Muscle supportive therapy

In the absence of utrophin-based therapy, successful treatment of DMD will likely require gene replacement or correction of the genetic defect. However, given the complexity of the symptoms associated with this progressive disease it is to be expected that therapies may need to be tailored to a particular stage of the disease process. In addition to dystrophin family members, a variety of genes or compounds have been put forth as therapeutic candidates in an effort to address the varied arms of DMD pathogenesis. Most of these approaches are aimed at promoting cellular adhesion, reducing inflammation and eventual necrosis, and sustaining the regenerative capacity; all of which appear to aid in muscle stability. We will present a highlight of potential candidates that can be delivered to muscle using viral vectors many of which may even provide a synergistic effect in combination with dystrophin/utrophin delivery.

Promotion of cellular adhesion

Within muscle there are two major cell adhesion complexes that provide muscle fiber attachment to laminin in the basal lamina, the DGC and α7ß1 integrin. In transgenic mdx:utrn-/-dko mice modest (2-3 fold) overexpression of integrin α7 was sufficient to significantly extend lifespan [START_REF] Burkin | Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice[END_REF]. Further, the heterodimeric partner ß1D was also restored, and the transgenic mice displayed reduced kyphosis and muscle disease pathology.

Additionally, delivering α7 integrin with rAAV to muscle, as with utrophin, would have the advantage of not producing an exogenous protein that could be recognized as foreign by the immune system. An additional protein

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ACCEPTED MANUSCRIPT 21 found to be involved in cellular adhesion is a component of the DGC, namely α-dystroglycan (α-Dgn). α-Dgn binds to several extracellular ligands, including laminin, agrin and perlecan in muscle (Figure 2) [START_REF] Bowe | Identification and purification of an agrin receptor from Torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans[END_REF][START_REF] Campanelli | A role for dystrophinassociated glycoproteins and utrophin in agrin-induced AChR clustering[END_REF][START_REF] Gee | Dystroglycan-alpha, a dystrophin-associated glycoprotein, is a functional agrin receptor[END_REF][START_REF] Sugiyama | Dystroglycan binds nerve and muscle agrin[END_REF]. Recent reports have shown that this interaction is strictly dependent on the glycosylation status of α-dystroglycan [START_REF] Michele | Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies[END_REF][START_REF] Michele | Dystrophin-glycoprotein complex: post-translational processing and dystroglycan function[END_REF][START_REF] Muntoni | Defective glycosylation in congenital muscular dystrophies[END_REF]. In one study a dramatic beneficial effect on degeneration and histopathology was observed in mdx mice that overexpressed N-acetyl galactosamine [START_REF] Nguyen | Overexpression of the cytotoxic T cell GalNAc transferase in skeletal muscle inhibits muscular dystrophy in mdx mice[END_REF]. This result was suggested to strengthen the link to laminin in the extracellular matrix. Along that same line, overexpression of the glycoslytransferase LARGE was demonstrated to functionally bypass α-dystroglycan glycosylation abnormalities in a model of congenital muscular dystrophy [START_REF] Barresi | LARGE can functionally bypass alpha-dystroglycan glycosylation defects in distinct congenital muscular dystrophies[END_REF].

It is possible that additional forms of post-translational modifications may serve as potential therapeutic targets to support muscle in multiple forms of muscular dystrophy.

Intracellular signaling and inflammation

ERK's act as an integrative point for multiple biochemical signals, and are involved in a wide variety of cellular processes. One such process is mechanical stress of muscle fibers where a strong correlation between peak tension and ERK phosphorylation in situ suggests these MAP kinases are particularly responsive to muscle damage [START_REF] Martineau | Insight into skeletal muscle mechanotransduction: MAPK activation is quantitatively related to tension[END_REF]. Considering that myofibers in vivo are continually subjected to mechanical forces, disregulation of this pathway and others in response to mechanical stress could impart a considerable role on the progression of muscle pathogenesis. Another MAP kinase, JNK is also activated in mdx mice, and be involved in pathogenesis [START_REF] Kolodziejczyk | Activation of JNK1 contributes to dystrophic muscle pathogenesis[END_REF].

Nitric oxide, the product of nitric oxide synthase, functions as the major endothelial-derived relaxing factor.

Skeletal-muscle-derived NO functions as an important vasodilator [START_REF] Thomas | Impaired metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle[END_REF]. The activity and expression of nNOS are regulated by both myogenic and neurogenic factors while the association of nNOS with the sarcolemma is mediated by the DGC (Figure 2). More specifically, nNOS is linked to the DGC through alpha-1-syntrophin PDZ-PDZ domain interactions where production is stimulated by muscle contraction. By a yet to be resolved mechanism, NO opposes alpha-adrenergic vasoconstriction. As a by-product of NO, increased production of cGMP may contribute to the inhibition of receptor-mediated Ca ++ influx in vascular smooth muscle. In the case of DMD and the absence of dystrophin, nNOS is not properly localized to the sarcolemma [START_REF] Brenman | Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy[END_REF]. In this circumstance nNOS protein is inappropriately localized within the cytosol, and the absence of nNOS from the sarcolemma can impede NOmediated regulation of vasodilation [START_REF] Sander | Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy[END_REF][START_REF] Victor | Presynaptic modulation of cortical synaptic activity by calcineurin[END_REF]. DMD muscle was shown to have abnormal vasoconstriction during mild exercise, resulting in muscle ischemia induced by inadequate blood flow to actively contracting muscle [START_REF] Thomas | Impaired metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle[END_REF].

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The sarcoglycan (SG) complex is an integral subcomplex of the DGC, and in muscle consists of α, ß, γ, and δ-SG (Figure 2) all of which are glycosylated [START_REF] Ozawa | From dystrophinopathy to sarcoglycanopathy: evolution of a concept of muscular dystrophy[END_REF]. Evidence that the ß/d-sarcoglycan core can associate with the C-terminus of dystrophin independent of dystroglycan or syntrophin was recently demonstrated [START_REF] Chen | Identification of functional domains in sarcoglycans essential for their interaction and plasma membrane targeting[END_REF]. It was further suggested that the interaction between the sarcoglycans and dystrophin is dependent upon the conformation of the sarcoglycan complex. This conformational change may also have an affect on membrane targeting or localization as well as post-translational modification events. The intracellular regions of α, ß, and γ-SG have tyrosine phosphorylation sites and it has been shown in cell culture that adhesion leads to phosphorylation of each of these SGs [START_REF] Yoshida | Bidirectional signaling between sarcoglycans and the integrin adhesion system in cultured L6 myocytes[END_REF]. In vitro, these SG's are phosphorylated by Src kinase [START_REF] Yoshida | Bidirectional signaling between sarcoglycans and the integrin adhesion system in cultured L6 myocytes[END_REF], similar to the phosphorylation of focal adhesion proteins by integrin-activated kinases (FAK and Src). Recently it has been shown that the SG subcomplex plays a role in mechanical signal transduction in skeletal muscle, where changes in the phosphorylation status of ERK was demonstrated in muscles from γ-SG null (gsg -/-) mice when compared to mdx muscles [START_REF] Barton | Impact of sarcoglycan complex on mechanical signal transduction in murine skeletal muscle[END_REF]. It would be interesting to determine if aberrant ERK phosphorylation that arises in these mice is associated with impaired nNOS signaling. Because the SG subcomplex has been suggested to interact with α-dystrobrevin [START_REF] Yoshida | Biochemical evidence for association of dystrobrevin with the sarcoglycan-sarcospan complex as a basis for understanding sarcoglycanopathy[END_REF], it may be that the absence of the SGs affects the manner in which nNOS is activated.

The use of antibodies to block the action of tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine also involved in signaling, resulted in reduction in the breakdown of dystrophic muscle in mdx mice [START_REF] Grounds | Anti-TNFalpha (Remicade) therapy protects dystrophic skeletal muscle from necrosis[END_REF].

This result may be tied to NFκB activation [START_REF] Reid | Tumor necrosis factor-alpha and muscle wasting: a cellular perspective[END_REF][START_REF] Porreca | Haemostatic abnormalities, cardiac involvement and serum tumor necrosis factor levels in X-linked dystrophic patients[END_REF] which appears to play an important role in muscle wasting [START_REF] Kumar | Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma[END_REF]. The use of rAAV to deliver the TNF-α soluble receptor in conjunction with microdystrophin may be of benefit in more advanced cases of DMD.

The sarcolemma is also susceptible to microrupture in dystrophic muscles [START_REF] Clarke | Loss of cytoplasmic basic fibroblast growth factor from physiologically wounded myofibers of normal and dystrophic muscle[END_REF] which leads to increased levels of intracellular Ca 2+ and by extension altered Ca 2+ signaling, and calpain-mediated proteolysis resulting in muscle degeneration [START_REF] Turner | Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice[END_REF]. This idea is consistent with the original formulation of the calcium hypothesis of muscular dystrophy [START_REF] Duncan | Role of intracellular calcium in promoting muscle damage: a strategy for controlling the dystrophic condition[END_REF]. Interestingly, a recent subproteonomics analysis revealed that a major luminal Ca 2+ binding protein, calsequestrin and the previously implicated Ca 2+ shuttle element, sarcalumenin [START_REF] Dowling | Drastic reduction of sarcalumenin in Dp427 (dystrophin of 427 kDa)-deficient fibres indicates that abnormal calcium handling plays a key role in muscular dystrophy[END_REF] are greatly reduced in mdx skeletal muscles [START_REF] Doran | Subproteomics analysis of Ca+-binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle[END_REF]. By extension, Ca 2+ exchangers, Ca 2+ pumps, and Ca 2+ -binding proteins may represent viable therapeutic targets for preventing muscle fiber degeneration. Alteration of Ca 2+ cycling may prove

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useful in avoiding Ca 2+ -related proteolytic processes, and future studies may address whether this can be combined with other muscle supportive therapies in order to improve long-term muscle mass and strength.

The identification of contributing factors to the dystrophic process, for example disregulation of signaling pathways, provides additional targets that may modify the disease process. Admittedly, the normal localization of signaling proteins to the sarcolemma likely will not be restored in the absence of an intact DGC, but promotion of muscle cell integrity and survival may be accomplished by exogenous regulation of their downstream effectors. It may be that a combination of agents to simultaneously provide mechanical muscle support, and to fortuitously regulate perturbed pathways could have a more significant benefit.

Myostatin and IGF-1

Two potential therapies have been considered for increasing muscle mass. The first is by delivery of insulin-like growth factor-1 (IGF-1). IGF-1 is a signaling peptide involved in muscle myogenesis, which works by enhancing myoblast proliferation, differentiation and survival. Moderate overexpression of IGF-1 in mdx muscles yielded increased muscle mass and strength with decreased necrosis and fibrosis [START_REF] Barton-Davis | Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function[END_REF][START_REF] Musaro | Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle[END_REF]. Similarly, delivery of IGF-1 using rAAV has been shown to result in phenotypic improvement of dystrophic muscles [START_REF] Musaro | Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle[END_REF][START_REF] Abmayr | Phenotypic improvement of dystrophic muscles by rAAV/microdystrophin vectors is augmented by Igf1 codelivery[END_REF]. One study suggested an increase in cellular survival was promoted in the absence of dystrophin by showing a dramatic increase in the phosphorylation status of the anti-apoptotic protein akt in mdx mice that received IGF-1 [START_REF] Barton | Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice[END_REF].

Another target candidate is the protein myostatin, a TGF-ß family member which is a negative regulator of both muscle fiber number and size [START_REF] Lee | Regulation of myostatin activity and muscle growth[END_REF]. Although the precise mechanism by which myostatin inhibition results in increased muscle mass has not been fully elucidated, one report has proposed delayed differentiation coupled with enhanced activation, renewal, and proliferation of satellite cells as a likely mechanism [START_REF] Mccroskery | Myostatin negatively regulates satellite cell activation and self-renewal[END_REF]. Whatever the mechanism, it likely does not involve compensation by utrophin [START_REF] Bogdanovich | Functional improvement of dystrophic muscle by myostatin blockade[END_REF]. A number of studies have shown that there is indeed a benefit to dystrophic muscles from the blockade of myostatin. However, what the precise benefit is remains debatable. In one study, mice null for both dystrophin and myostatin were generated. As expected, there was increased muscle mass, though in contrast to a study that used neutralizing antibody to myostatin on young mdx mice [START_REF] Bogdanovich | Functional improvement of dystrophic muscle by myostatin blockade[END_REF], a significant reduction in necrosis and inflammation was not observed [START_REF] Wagner | Loss of myostatin attenuates severity of muscular dystrophy in mdx mice[END_REF]. The mdx mouse may not be the best model considering that muscles in these mice do not have a robust regenerative capacity. This approach to therapy holds much promise particularly in combination with a dystrophin or utrophin replacement approach as A C C E P T E D M A N U S C R I P T

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24 there are several myostatin inhibitors that could be co-delivered systemically with rAAV, such as follistatin and the myostatin prodomain [START_REF] Lee | Regulation of myostatin activity and muscle growth[END_REF][START_REF] Hill | The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum[END_REF]. This approach would allow for an increase in the intergrity of the sarcolemma membrane via restoration of the DGC, as well as added muscle mass and strength that may be necessary to provide a better clinical outcome. In addition, as we develop a better understanding of muscle biology and other biological processes such as aging and atrophy there may be signaling pathways that are revealed whose mediators can become candidates for additional vector-based modulation of muscle.

Conclusion

Conceptually the replacement of dystrophin by gene transfer represents an appealing and simplistic approach for the treatment of DMD. However, even though technological advances have allowed small successes in a variety of diseases and animal models, the ability to optimally integrate these advances into a therapy for a human disease that is as complicated as DMD likely requires a more complete understanding of the disease process.

Understanding the underlying mechanisms of the degenerative process in the dystrophic muscle fiber is still preliminary and requires consideration of multiple mechanisms and approaches for analysis. Various processes that occur in normal and dystrophic muscle development should be considered in order to shed more light on how muscles degenerate. Finally, the effects on the entire musculature must also be considered because the challenge to retain functionality long-term in proper balance related to movement and posture of the patient will undoubtedly surface.

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  dystrophin levels were shown to be 1-5% of normal. Absence of dystrophin expression in the heart and the increased dystrophin in older mdx mice compared to younger mice further indicate a role of the regenerative process A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 13 on AON uptake. The lack of expression in cardiac tissue is a concern considering more than 30% of DMDassociated deaths occur as a result of cardiomyopathy [158].

Figure 1 AFigure 2 AFigure 3

 123 Figure1

Acknowledgments:

We thank James Allen, Luke Judge and Glen Banks for helpful discussions, and Miki Haraguchi for artwork. Supported by grant # AR44533 from the National Institutes of Health (to JSC). G.O. was supported by NIH grant T32 HL07828.

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