We previously demonstrated that direct intramuscular injection of rAAV2 or rAAV6 in wild-type dogs resulted in robust T-cell responses to viral capsid proteins, and others have shown that cellular immunity to adeno-associated virus (AAV) capsid proteins coincided with liver toxicity and elimination of transgene expression in a human trial of hemophilia B. Here, we show that the heparin-binding ability of a given AAV serotype does not determine the induction of T-cell responses following intramuscular injection in dogs, and identify multiple epitopes in the AAV capsid protein that are recognized by T cells elicited by AAV injection. We also demonstrate that noninvasive magnetic resonance imaging (MRI) can accurately detect local inflammatory responses following intramuscular rAAV injection in dogs. These studies suggest that pseudotyping rAAV vectors to remove heparin-binding activity will not be sufficient to abrogate immunogenicity, and validate the utility of enzyme-linked immunosorbent spot (ELISpot) assay and MRI for monitoring immune and inflammatory responses following intramuscular injection of rAAV vectors in preclinical studies in dogs. These assays should be incorporated into future human clinical trials of AAV gene therapy to monitor immune responses.

To explore whether stable transduction of myogenic stem cells using lentiviral vectors could be of benefit for treating dystrophic muscles, we generated vectors expressing a functional microdystrophin/enhanced green fluorescence protein fusion (microDys/eGFP) gene. Lentiviral vector injection into neonatal mdx(4cv) muscles resulted in widespread and stable expression of dystrophin for at least 2 years. This expression resulted in a significant amelioration of muscle pathophysiology as assessed by a variety of histological and functional assays. To assess whether this long-term expression was accompanied by stable transduction of satellite cells, we harvested muscle mononuclear cells 1 year after vector injection. Up to 20% of the cultured myoblast colonies expressed the microDys/eGFP transgene following myotube formation. Furthermore, transplantation of the muscle mononuclear cells into secondary mdx(4cv) recipients showed their ability to regenerate dystrophin-expressing myofibers in vivo. The ability to isolate myogenic cells able to form dystrophin-positive myotubes or myofibers in vitro and in vivo >1 year postinjection indicates that the vectors stably transduced muscle satellite cells, or a progenitor of such cells, in neonatal mdx(4cv) muscles. These studies suggest that integrating lentiviral vectors have potential utility for gene therapy of muscular dystrophy.

The muscular dystrophies are a heterogeneous group of over 40 disorders that are characterised by muscle weakness and wasting. The most common are Duchenne muscular dystrophy and Becker muscular dystrophy, which result from mutations within the gene encoding dystrophin; myotonic dystrophy type 1, which results from an expanded trinucleotide repeat in the myotonic dystrophy protein kinase gene; and facioscapulohumeral dystrophy, which is associated with contractions in the subtelomeric region of human chromosome 1. Currently the only treatments involve clinical management of symptoms, although several promising experimental strategies are emerging. These include gene therapy using adeno-associated viral, lentiviral and adenoviral vectors and nonviral vectors, such as plasmid DNA. Exon-skipping and cell-based therapies have also shown promise in the effective treatment and regeneration of dystrophic muscle. The availability of numerous animal models for Duchenne muscular dystrophy has enabled extensive testing of a wide range of therapeutic approaches for this type of disorder. Consequently, we focus here on the therapeutic developments for Duchenne muscular dystrophy as a model of the types of approaches being considered for various types of dystrophy. We discuss the advantages and limitations of each therapeutic strategy, as well as prospects and recent successes in the context of future clinical applications.

A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow,(ii) exsanguination via compression bandage, and (iii) vector "dwell" time of <20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans.Molecular Therapy (2009); doi:10.1038/mt.2009.116.

Research into therapeutic approaches for both recessive and dominant neuromuscular disorders has made great progress over the past few years. In the field of gene therapy, antisense-mediated exon skipping is being applied to bypass deleterious mutations in the dystrophin gene and restore dystrophin expression in animal models of muscular dystrophy. Approaches for the dominant genetic muscle diseases have turned toward elimination of the mutant gene product with anti-sense oligonucleotide therapy and RNA interference techniques. Refinements of adeno-associated viral vectors and strategies for their delivery are also leading towards future clinical trials. The discovery of new, multipotent cell lineages, some of which possess the ability to successfully engraft muscle following vascular delivery, presents exciting prospects for the field of stem cell therapy. These discoveries represent steady progress towards the development of effective therapies for a wide range of neuromuscular disorders.

The muscular dystrophies are a group of genetically and phenotypically heterogeneously inherited diseases characterized by progressive muscle wasting, which can lead to premature death in severe forms such as Duchenne muscular dystrophy (DMD). In many cases they are caused by the absence of proteins that are critical components of the dystrophin-glycoprotein complex, which links the cytoskeleton and the basal lamina. There is no effective treatment for these disorders at present, but several novel strategies for replacing or repairing the defective gene are in development, with early encouraging results from animal models. We review these strategies, which include the use of stem cells of different tissue origins, gene replacement therapies mediated by various viral vectors, and transcript repair treatments using exon skipping strategies. We comment on their advantages and on limitations that must be overcome before successful application to human patients. Our focus is on studies in a clinically relevant large canine model of DMD. Recent advances in the field suggest that effective therapies for muscular dystrophies are on the horizon. Because of the complex nature of these diseases, it may be necessary to combine multiple approaches to achieve a successful treatment.

Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated alpha-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.

Angiotensin-converting enzyme (ACE) 2 is a recently identified homologue of ACE. There is great interest in the therapeutic benefit for ACE2 overexpression in the heart. However, the role of ACE2 in the regulation of cardiac structure and function, as well as maintenance of systemic blood pressure, remains poorly understood. In cell culture, ACE2 overexpression led to markedly increased myocyte volume, assessed in primary rabbit myocytes. To assess ACE2 function in vivo, we used a recombinant adeno-associated virus 6 delivery system to provide 11-week overexpression of ACE2 in the myocardium of stroke-prone spontaneously hypertensive rats. ACE2, as well as the ACE inhibitor enalapril, significantly reduced systolic blood pressure. However, in the heart, ACE2 overexpression resulted in cardiac fibrosis, as assessed by histological analysis with concomitant deficits in ejection fraction and fractional shortening measured by echocardiography. Furthermore, global gene expression profiling demonstrated the activation of profibrotic pathways in the heart mediated by ACE2 gene delivery. This study demonstrates that sustained overexpression of ACE2 in the heart in vivo leads to the onset of severe fibrosis.

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy. There is no effective treatment and patients typically die in approximately the third decade. DMD is an X-linked recessive disease caused by mutations in the dystrophin gene. There are three mammalian models of DMD that have been used to understand better the pathogenesis of disease and develop therapeutic strategies. The mdx mouse is the most widely used model of DMD that displays some features of muscle degeneration, but the pathogenesis of disease is comparatively mild. The severity of disease in mice lacking both dystrophin and utrophin is similar to DMD, but one has to account for the discrete functions of utrophin. Canine X-linked muscular dystrophy (cxmd) is the best representation of DMD, but the phenotype of the most widely used golden retriever (GRMD) model is variable, making functional endpoints difficult to ascertain. Although each mammalian model has its limitations, together they have been essential for the development of several treatment strategies for DMD that target dystrophin replacement, disease progression, and muscle regeneration.

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and structural defects in the neuromuscular synapse that are caused by mutations in dystrophin. Whether aberrant neuromuscular synapse structure is an indirect consequence of muscle degeneration or a direct result of loss of dystrophin function is not known. Rational design of truncated dystrophins has enabled the design of expression cassettes highly effective at preventing muscle degeneration in mouse models of DMD using gene therapy. Here we examined the functional capacity of a minidystrophin (minidysGFP) and a microdystrophin (microdystrophin(DeltaR4-R23)) transgene on the maturation and maintenance of neuromuscular junctions (NMJ) in mdx mice. We found that minidysGFP prevents fragmentation and the loss of postsynaptic folds at the NMJ. In contrast, microdystrophin (DeltaR4-R23) was unable to prevent synapse fragmentation in the limb muscles despite preventing muscle degeneration, although fragmentation was observed to temporally correlate with the formation of ringed fibers. Surprisingly, microdystrophin(DeltaR4-R23) increased the length of synaptic folds in the diaphragm muscles of mdx mice independent of muscle degeneration or the formation of ringed fibers. We also demonstrate that the number and depth of synaptic folds influences the density of voltage-gated sodium channels at the neuromuscular synapse in mdx, microdystrophin(DeltaR4-R23)/mdx and mdx:utrophin double knockout mice. Together, these data suggest that maintenance of the neuromuscular synapse is governed through its lateral association with the muscle cytoskeleton, and that dystrophin has a direct role in promoting the maturation of synaptic folds to allow more sodium channels into the junction.