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

Abstract

Duchenne muscular dystrophy is a severe life-threatening X-linked recessive disorder, caused by mutations in the dystrophin gene, for which currently there is no effective treatment. Because of the large size of the dystrophin cDNA (14 kb) this precluded it from being used in early adenovirus- or retrovirus-based gene therapy vectors. However, some therapeutic success has been achieved in mdx mice using adenovirus- and retrovirus-mediated transfer of a 6.3 kb recombinant mini-dystrophin cDNA. Despite this, problems with immunogenicity and inefficient transduction of mature myofibres make these vectors less than ideal for gene transfer to skeletal muscle. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems. However, AAV vectors can only accommodate <5 kb of foreign DNA. For this reason we have produced a micro-dystrophin cDNA gene construct that is <3.8 kb. This construct, driven by a CMV promoter, was introduced into the skeletal muscle of 12-day-old nude/mdx mice using an AAV vector, resulting in specific sarcolemmal expression of micro-dystrophin in >50% of myofibres up to 20 weeks of age, and effective restoration of the dystrophin-associated protein (DAP) complex components. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. We have therefore shown that the current micro-dystrophin gene delivered in vivo using an AAV vector is not only capable of restoring sarcolemmal DAP complexes, but can also ameliorate dystrophic pathology at the cellular level.

A canine minidystrophin is functional and therapeutic in mdx mice

“…Our laboratory and others have previously reported that AAV vector-mediated delivery of mini-or microdystrophins of human origin was effective and functional in improving muscle pathology and protected myofiber membrane integrity in the mdx mouse model. 4,[6][7][8][9] Although readily available and widely used, the mdx as a small rodent model has numerous limitations. The GRMD dog model, on the other hand, offers many advantages that the mdx mice lack, mainly the pathological similarity to those of DMD human patients, and a larger body mass for more accurate assessment of therapeutic vector dose.…”

“…These observations are in agreement with our previous human minidystrophin study 4 and studies from others. [6][7][8][9] Canine minidystrophin protects myofiber membrane integrity Plasma membrane damage and leakage in dystrophic muscle tissue is a major pathological cause in DMD patients. The dystrophic muscle cell membranes become damaged and leaky during contraction and relaxation.…”

“…The mdx mouse is a commonly used rodent model, which contains a premature stop codon in the dystrophin gene that results in the lack of dystrophin protein and the loss of the dystrophin-associated protein complex (DPC). We 4,5 and others [6][7][8][9] have previously reported that in the mdx mouse model, adeno-associated virus (AAV) vectors carrying miniaturized dystrophin genes can effectively protect the muscles from dystrophic pathologies, and improve muscle contractile force.…”

Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products

“…We and others have tried to design a short but functional microdystrophin gene that could be utilized as the therapeutic tool for DMD. [3][4][5][6] We constructed a series of rod-truncated microdystrophin cDNAs, 3 and generated transgenic mdx mice expressing each microdystrophin, and demonstrated that microdystrophin CS1 with four rod repeats and three hinges was a good candidate for therapeutic molecule. 7 We have also showed that the muscle-specific muscle creatine kinase (MCK) promoter in a rAAV drives longer transgene expression than the ubiquitous cytomegalovirus (CMV) promoter in mdx muscle.…”

Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies

“…For dystrophin, this has been carried out in the form of micro-dystrophin or of a modified U7 snRNA carrying an antisense sequence aimed at restoring the translational frame by inducing exon skipping and, for sarcoglycans, in the form of a normal version of the corresponding cDNA. [20][21][22][23][24][25] Before a therapeutic strategy can be implemented in a clinical trial, it is necessary to obtain objective indicators of therapeutic efficacy, especially since the approaches tested may have different outcomes and may correct some but not necessarily all the pathophysiological parameters. In the case of dystrophin or sarcoglycan deficiencies, a therapeutical benefit can be characterized by the restoration of the DGC at the sarcolemma, a correction of the dystrophic muscle histopathology or recovery of membrane integrity, and eventually by an increase in specific force and a decreased susceptibility to contraction-induced injury.…”