Muscular dystrophies are a group of inherited disorders characterized by progressive muscle weakness and degeneration. Among these, Duchenne muscular dystrophy (DMD) is an X-linked wasting disease that affects skeletal and cardiac muscles leading to loss of motor functions and premature death. Severe form of DMD affects 1 in 3,500 newborn males and is caused by mutations in the dystrophin (DYST) gene, resulting in the lack or reduction of the protein DYST. Dystrophin is the major component of the dystrophin-glycoprotein complex (DG-C) and constitutes a link between the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. It is also responsible for the maintenance of cell integrity and muscle cell function (Matsumura et al., 1993). As a consequence of DG-C inefficiency, muscle fragility is lost, leading to contraction-induced damage, necrosis and inflammation. The molecular mechanisms of the disease have been extensively investigated since the discovery of the gene in 1986 and there are, currently, no effective therapies to stop the progression of the disease. However, several promising experimental strategies are currently under investigation.
As DMD is a recessively inherited disorder, the prospect of gene replacement is a conceptually simple approach to therapy. Significant progress has been made with gene therapy, which aims to introduce a functional recombinant version of the DYST gene using viral vectors or to modify the DYST pre-mRNA, a technique commonly referred to as exon skipping. These strategies face major requirements such as the targeting of different muscles in the body, the long-term expression of the protein, and the absence of an immune response. Three types of viral vectors have primarily been used by researchers studying muscular dystrophy: adenoviral, lentiviral, and adeno-associated viral (AAV) vectors. All three vectors have shown some success in transduction and stable expression of striated muscles. Adenoviruses are attractive for gene transfer because they have a large packaging capacity (~30 kb) and can be produced in high titers. The development of these vectors for DYST gene transfer was set back following reports of acute cytotoxic responses and toxicity. Lentiviral vectors are a class of retroviral vectors that stably integrate transgenes into the genomes of quiescent and non-quiescent cells. Unfortunately, transduction efficiency is too low for direct therapeutic applications. Only AAV vectors have progressed to clinical trials. However, the limited packaging capacity of the AAV vectors (~5kb), compared with the size of the DYST gene, which encodes a 14kb mRNA, implies that truncated versions of dystrophin need to be used. Despite significant advances in viral vector engineering, safety concerns such as genotoxic effects and potential malignant cell transformation are the biggest challenges.
In addition to viral gene therapy, several non-viral replacement and repair approaches have been studied for the treatment of DMD such as the delivery of unencapsidated plasmids, the modification of mRNA splicing, and the ribosomal read-through of premature stop codons. Efforts are underway to characterize the genomic integration locus in single clones with the goal of reducing the risk of adverse effects. Matrix attachment regions (MARs) are sequences in the DNA of eukaryotic chromosomes where the nuclear matrix attaches. Plasmids containing human MAR elements were shown to promote plasmid integration in the host cell genome by homologous recombination and increase transgene transcription efficiency by tenfold.
Van Zwieten and colleagues from University of Lausanne and University College of London transfected mesoangioblasts with eGFP plasmid containing MARs. Using probes prepared with Enzo’s
Nick Translation DNA labeling system and
Orange 552 dUTP, they determined the number of transgene integration sites by fluorescent
in situ hybridization (FISH). The transfected clones were capable of expressing dystrophin following transplantation into the muscle of dystrophic mice. Using this approach, they were able to obtain full-length DYST expression from stable plasmid transfection of muscle precursor cells and incorporation of these cells in muscle fibers
in vivo. This new strategy offers possible treatment of muscular dystrophies using genetically-corrected cells.
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