Muscle-mediated gene therapy

Transfer of therapeutic genes into muscle tissue holds promise for the treatment of a variety of muscular dystrophies, serum protein deficiencies and vascular proliferative disorders. Recent progress achieved in development of improved vectors allowed prolonged and efficient expression of genes encoding therapeutic proteins in muscle cells. The most important advances include: novel plasmid DNA vectors and methods for their efficient transfection in vivo, helper-dependent adenoviral vectors, allowing long-term gene expression and effective readministration in immunocompetent hosts and adeno-associated vectors. On the other hand, recent progress in this field has been facilitated by development of systems that enable regulated therapeutic gene expression in muscle tissue.     

Molecular biology of distal muscular dystrophies--sarcomeric proteins on top

During the last 10 years several muscular dystrophies within the group of distal myopathies have been clarified as to the molecular genetic cause of the disease. Currently, the next steps are carried out to identify the molecular pathogenesis downstream of the gene defects. Some early ideas on what is going on in the muscle cells based on the defect proteins are emerging. However, in no single distal muscular dystrophy these efforts have yet reached the point where direct trials for therapy would have been launched, and in many distal dystrophies the causative gene is still lacking. When comparing the gene defects in the distal dystrophies with the more common proximal muscular dystrophies such as dystrophinopathies or limb-girdle muscular dystrophies, there is a striking difference: the genes for distal dystrophies encode sarcomere proteins whereas the genes for proximal dystrophies more often encode sarcolemmal proteins.     

Molecular biology of distal muscular dystrophies—Sarcomeric proteins on top

During the last 10 years several muscular dystrophies within the group of distal myopathies have been clarified as to the molecular genetic cause of the disease. Currently, the next steps are carried out to identify the molecular pathogenesis downstream of the gene defects. Some early ideas on what is going on in the muscle cells based on the defect proteins are emerging. However, in no single distal muscular dystrophy these efforts have yet reached the point where direct trials for therapy would have been launched, and in many distal dystrophies the causative gene is still lacking. When comparing the gene defects in the distal dystrophies with the more common proximal muscular dystrophies such as dystrophinopathies or limb-girdle muscular dystrophies, there is a striking difference: the genes for distal dystrophies encode sarcomere proteins whereas the genes for proximal dystrophies more often encode sarcolemmal proteins.     

Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases.     

Congenital muscular dystrophies: new aspects of an expanding group of disorders

The congenital muscular dystrophies comprise a genetically and clinically heterogeneous group of disorders characterized by early onset of progressive muscle weakness and often involvement of other organ systems such as the brain and eyes. During the last decade, significant progress has been made to further characterize various forms of congenital muscular dystrophies based on their specific genetic and clinical appearance. This review represents an overview of the recent accomplishments as they relate to clinical, diagnostic, pathogenetic and therapeutic aspects of congenital muscular dystrophies.     

Genome- and cell-based strategies in therapy of muscular dystrophies

Muscular dystrophies are a group of heterogeneous genetic disorders characterized by progressive loss of skeletal muscle mass. Depending on the muscular dystrophy, the muscle weakness varies in degree of severity. The majority of myopathies are due to genetic events leading to a loss of function of key genes involved in muscle function. Although there is until now no curative treatment to stop the progression of most myopathies, a significant number of experimental gene- and cell-based strategies and approaches have been and are being tested in vitro and in animal models, aiming to restore gene function. Genome editing using programmable endonucleases is a powerful tool for modifying target genome sequences and has been extensively used over the last decade to correct in vitro genetic defects of many single-gene diseases. By inducing double-strand breaks (DSBs), the engineered endonucleases specifically target chosen sequences. These DSBs are spontaneously repaired either by homologous recombination in the presence of a sequence template, or by nonhomologous-end joining error prone repair. In this review, we highlight recent developments and challenges for genome-editing based strategies that hold great promise for muscular dystrophies and regenerative medicine.     

Muscular dystrophy of mink: a new animal model.

Muscular dystrophies comprise an important group of inherited disorders of man. Although the disease has been studied extensively, little is known about the underlying primary pathomechanisms. Consequently, treatment of patients is difficult and prognosis is poor. An animal model of muscular dystrophy is a useful research tool for approaching the basic problems of pathogenesis in muscle diseases. An inherited progressive muscular dystrophy of mink which resembles the amyotonic forms of human muscular dystrophy is currently under study. Clinically, the earliest sign is progressive muscular weakness and atrophy. Muscle enzyme activities in serum are usually elevated to pathologic levels. Urinary creatine/creatinine ratio is elevated. Pathologic changes are limited to skeletal muscle and are typical of those seen in amyotonic forms of human muscular dystrophy. These changes include variation in diameter size of muscle fibers, centralized nuclei, floccular and hyaline degeneration of scattered muscle fibers, increase in connective tissue in endomysial and perimysial areas, and regenerative attempts. Both type I and type II muscle fibers are involved in the disease process. Genetic studies indicate an autosomal recessive mode of inheritance. Although the primary defect in muscular dystrophy is traditionally thought to reside in skeletal muscle, recent studies have produced theories of primary involvement of other tissues and organ systems. These theories are presented and relationships to the traditional theory are discussed.     

Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology

We used expression profiling to define the pathophysiological cascades involved in the progression of two muscular dystrophies with known primary biochemical defects, dystrophin deficiency (Duchenne muscular dystrophy) and alpha-sarcoglycan deficiency (a dystrophin-associated protein). We employed a novel protocol for expression profiling in human tissues using mixed samples of multiple patients and iterative comparisons of duplicate datasets. We found evidence for both incomplete differentiation of patient muscle, and for dedifferentiation of myofibers to alternative lineages with advancing age. One developmentally regulated gene characterized in detail, alpha-cardiac actin, showed abnormal persistent expression after birth in 60% of Duchenne dystrophy myofibers. The majority of myofibers ( approximately 80%) remained strongly positive for this protein throughout the course of the disease. Other developmentally regulated genes that showed widespread overexpression in these muscular dystrophies included embryonic myosin heavy chain, versican, acetylcholine receptor alpha-1, secreted protein, acidic and rich in cysteine/osteonectin, and thrombospondin 4. We hypothesize that the abnormal Ca(2)+ influx in dystrophin- and alpha-sarcoglycan-deficient myofibers leads to altered developmental programming of developing and regenerating myofibers. The finding of upregulation of HLA-DR and factor XIIIa led to the novel identification of activated dendritic cell infiltration in dystrophic muscle; these cells mediate immune responses and likely induce microenvironmental changes in muscle. We also document a general metabolic crisis in dystrophic muscle, with large scale downregulation of nuclear-encoded mitochondrial gene expression. Finally, our expression profiling results show that primary genetic defects can be identified by a reduction in the corresponding RNA.     

Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector

Limb-girdle muscular dystrophies 2C-F represent a family of autosomal recessive diseases caused by defects in sarcoglycan genes. The cardiomyopathic hamster is a naturally occurring model for limb-girdle muscular dystrophy caused by a primary deficiency in delta-sarcoglycan. We show here that acute sarcolemmal disruption occurs in this animal model during forceful muscle contraction. A recombinant adeno-associated virus vector encoding human delta-sarcoglycan conferred efficient and stable genetic reconstitution in the adult cardiomyopathic hamster when injected directly into muscle. A quantitative assay demonstrated that vector-transduced muscle fibers are stably protected from sarcolemmal disruption; there was no associated inflammation or immunologic response to the vector-encoded protein. Efficient gene transduction with rescue of the sarcoglycan complex in muscle fibers of the distal hindlimb was also obtained after infusion of recombinant adeno-associated virus into the femoral artery in conjunction with histamine-induced endothelial permeabilization. This study provides a strong rationale for the development of gene therapy for limb-girdle muscular dystrophy.     

Muscular degeneration in the absence of dystrophin is a calcium-dependent process.

Duchenne muscular dystrophy (DMD) is a progressive degenerative muscular disease that is due to mutations in the dystrophin gene. Neither the function of dystrophin nor the physiopathology of the disease have been clearly established yet. Several groups have reported elevated calcium concentrations in the mdx mouse model of DMD, but the effect of calcium levels on the progression of the disease continues to be a matter of debate. Here, we show that, in Caenorhabditis elegans, a gain-of-function mutation in the egl-19 calcium channel gene dramatically increases muscle degeneration in dystrophin mutants. Conversely, RNAi-mediated inhibition of egl-19 function reduces muscle degeneration by half. Therefore, our results demonstrate that calcium channel activity is a critical factor in the progression of dystrophin-dependent muscle degeneration.     

The role of free radicals in the pathophysiology of muscular dystrophy.

Null mutation of any one of several members of the dystrophin protein complex can cause progressive, and possibly fatal, muscle wasting. Although these muscular dystrophies arise from mutation of a single gene that is expressed primarily in muscle, the resulting pathology is complex and multisystemic, which shows a broader disruption of homeostasis than would be predicted by deletion of a single-gene product. Before the identification of the deficient proteins that underlie muscular dystrophies, such as Duchenne muscular dystrophy (DMD), oxidative stress was proposed as a major cause of the disease. Now, current knowledge supports the likelihood that interactions between the primary genetic defect and disruptions in the normal production of free radicals contribute to the pathophysiology of muscular dystrophies. In this review, we focus on the pathophysiology that results from dystrophin deficiency in humans with DMD and the mdx mouse model of DMD. Current evidence indicates three general routes through which free radical production can be disrupted in dystrophin deficiency to contribute to the ensuing pathology. First, constitutive differences in free radical production can disrupt signaling processes in muscle and other tissues and thereby exacerbate pathology. Second, tissue responses to the presence of pathology can cause a shift in free radical production that can promote cellular injury and dysfunction. Finally, behavioral differences in the affected individual can cause further changes in the production and stoichiometry of free radicals and thereby contribute to disease. Unfortunately, the complexity of the free radical-mediated processes that are perturbed in complex pathologies such as DMD will make it difficult to develop therapeutic approaches founded on systemic administration of antioxidants. More mechanistic knowledge of the specific disruptions of free radicals that underlie major features of muscular dystrophy is needed to develop more targeted and successful therapeutic approaches.     

Therapeutic gene transfer to dystrophic diaphragm by an adenoviral vector deleted of all viral genes

Duchenne muscular dystrophy is caused by defects in the dystrophin gene, and the mdx mouse is the most frequently employed genetic model of this disease. It is well known that different muscle groups do not respond in the same way to dystrophin deficiency. In particular, the mdx mouse diaphragm exhibits severe morphological and functional changes not found in other mdx muscles. Use of early generation adenoviral vectors to deliver genes to the diaphragm in immunocompetent mdx mice has been associated with substantial functional toxicity and a rapid loss of transgene expression. Here we determined the response to dystrophin gene replacement in the mdx diaphragm using a "gutted" adenoviral vector that contains the coding sequence of two full-length dystrophin genes and is deleted of most viral DNA sequences. At 1 wk postdelivery of the vector, 23.6 +/- 4% of total fibers in the injected diaphragm bundle expressed dystrophin at the sarcolemma, which remained stable over the study duration of 30 days without the need for continuous immunosuppression. Treated diaphragms showed a significantly improved resistance to the abnormal force deficits induced by high-stress muscle contractions, the latter being a functional hallmark of dystrophin-deficient muscle. This functional amelioration was achieved despite the presence of mildly increased inflammation (CD4+ and CD8+ lymphocytes) within the vector-treated diaphragms. To our knowledge, this is the first demonstration that a viral vector can achieve reversal of functional abnormalities in the dystrophic diaphragm via therapeutic dystrophin gene transfer without the need for sustained immunosuppressive therapy.     

Oxidative stress and muscular dystrophy

Oxidative stress may be the fundamental basis of many of the structural, functional and biochemical changes characteristic of the inherited muscular dystrophies in animals and humans. The presence of by-products of oxidative damage, and the compensatory increases in cellular antioxidants, both indicate oxidative stress may be occurring in dystrophic muscle. Changes in the proportions and metabolism of cellular lipids, abnormal functions of cellular membranes, altered activity of membrane-bound enzymes such as the SR Ca2+-ATPase, disturbances in cellular protein turnover and energy production and a variety of other changes all indicate that these inherited muscular dystrophies appear more like the results of oxidative stress to muscle than any other type of underlying muscle disturbance. Particular details of these altered characteristics of dystrophic muscle, in combination with current knowledge on the processes of oxidative damage to cells, may provide some insight into the underlying biochemical defect responsible for the disease, as well as direct research towards the ultimate goal of an effective treatment.     

Mouse models of fukutin-related protein mutations show a wide range of disease phenotypes

Dystroglycanopathies are characterized by a reduction in the glycosylation of alpha-dystroglycan (α-DG). A common cause for this subset of muscular dystrophies is mutations in the gene of fukutin-related protein (FKRP). FKRP mutations have been associated with a wide spectrum of clinical severity from severe Walker–Warburg syndrome and muscle–eye–brain disease with brain and eye defects to mild limb–girdle muscular dystrophy 2I with myopathy only. To examine the affects of FKRP mutations on the severity of the disease, we have generated homozygous and compound heterozygous mouse models with human mutations in the murine FKRP gene. P448Lneo+ and E310delneo+ mutations result in severe dystrophic and embryonic lethal phenotypes, respectively. P448Lneo+/E310delneo+ compound heterozygotes exhibit brain defects and severe muscular dystrophies with near absence of α-DG glycosylation. Removal of the Neo^r cassette from the P448Lneo+ homozygous mice eliminates overt brain and eye defects, and reduces severity of dystrophic phenotypes. Furthermore, introduction of the common L276I mutation to generate transgenic L276Ineo+ homozygous and L276Ineo+/P448Lneo+ and L276Ineo+/E310delneo+ compound heterozygotes results in mice displaying milder dystrophies with reduced α-DG glycosylation and no apparent brain defects. Limited sampling and variation in functionally glycosylated α-DG levels between and within muscles may explain the difficulties in correlating FKRP expression levels with phenotype in clinics. The nature of individual mutations, expression levels and status of muscle differentiation all contribute to the phenotypic manifestation. These mutant FKRP mice are useful models for the study of disease mechanism(s) and experimental therapies.     

Direct effects of the pathogenic mutation on satellite cell function in muscular dystrophy

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

Grafting of a Single Donor Myofibre Promotes Hypertrophy in Dystrophic Mouse Muscle

Skeletal muscle has a remarkable capability of regeneration following injury. Satellite cells, the principal muscle stem cells, are responsible for this process. However, this regenerative capacity is reduced in muscular dystrophies or in old age: in both these situations, there is a net loss of muscle fibres. Promoting skeletal muscle muscle hypertrophy could therefore have potential applications for treating muscular dystrophies or sarcopenia. Here, we observed that muscles of dystrophic mdx nude host mice that had been acutely injured by myotoxin and grafted with a single myofibre derived from a normal donor mouse exhibited increased muscle area. Transplantation experiments revealed that the hypertrophic effect is mediated by the grafted fibre and does not require either an imposed injury to the host muscle, or the contribution of donor cells to the host muscle. These results suggest the presence of a crucial cross-talk between the donor fibre and the host muscle environment.     

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles ¹ and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies ². Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.