Muscular Dystrophy

The muscular dystrophies are a clinically and genetically heterogeneous group of disorders that show myofiber degeneration and regeneration, and are characterized by progressive muscle wasting and weakness of variable distribution and severity. They are associated with mutations in genes encoding several classes of proteins ranging from extracellular matrix and integral membrane proteins to cytoskeletal proteins, but also include a heterogeneous group of proteins including proteases, nuclear proteins, and signaling molecules [for a recent review, see (223)].

The most common myopathy in children, Duchenne muscular dystrophy (DMD), is a severe X-linked neuromuscular disease that affects approximately 1 of every 3500 males born and is caused by recessive mutations in the gene for the muscle protein dystrophin. Affected boys begin manifesting signs of disease early in life, cease walking at the beginning of the second decade, and often die due to cardiac arrest or respiratory insufficiency by age 20 years. The most common mutation in the dystrophin gene that causes the severe DMD pheno-type is a deletion that results in a premature stop codon. The mutant protein is unable to bind to dystrophin-associated proteins at the muscle membrane. This lack of a functional dys-trophin protein in DMD results in loss of muscle fiber integrity by disrupting the physical linkage between the actin cytoskele-ton within the muscle fiber and the extracellular matrix (224).

Attempts to develop gene therapy for DMD have been complicated by the enormous size of the dystrophin gene, which is the largest known gene with a full-length cDNA that is 14 kb in size. However, dystrophin can retain significant function even when missing large portions of its sequence. For example, large, in-frame deletions in the central-rod domain often lead to the milder Becker muscular dystrophy (225). The mdx mouse is a naturally occurring murine model that has a premature stop codon generated by a point mutation in exon 23 of the dystrophin gene (226). Functional analysis of dystrophin structural domains in transgenic dystrophin-de-ficient mdx mice revealed multiple regions of the protein that can be deleted in various combinations to generate potentially highly functional minidystrophin genes (227). Three groups have generated functional miniature versions of the human dystrophin gene that can be readily packaged into AAV vec tors (228-230). When injected into the muscle of mdx mice, efficient and stable expression was noted in a majority of myofibers, and the missing dystrophin and dystrophin-associ-ated protein complexes were restored onto the plasma membrane. This treatment ameliorated dystrophic pathology in the mdx muscle and led to normal myofiber morphology, histology, and cell membrane integrity.

The limb girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited autosomal recessive neuromuscular diseases characterized by proximal muscular weakness and variable progression of symptoms. LGMD disease is caused by mutations in a number of genes, including 1 of the 4 small (cDNA < 2 kb)-muscle sarcoglycan genes (a,p,^,8) expressed predominantly in striated muscle. These transmembrane glycoproteins associate with each other in equal stoichi-ometry to form the sarcoglycan complex, and a deficiency of one component typically leads to partial or complete absence of all the other sarcoglycan proteins on the sarcolemma. The Bio 14.6 cardiomyopathy hamster is a naturally occurring LGMD model due to a deletion in the 8-sarcoglycan gene. Administration of AAV 8-sarcoglycan vectors to these animals either by intramuscular or intravascular administration led to genetic, biochemical, histological, and functional rescue of relatively large regions of muscle (231-234). Mice that are null mutants for ^-sarcoglycan exhibit severe muscle pathology that can be partly corrected if treated at less than 3 weeks of age by intramuscular injections of an AAV vector expressing the ^-sarcoglycan gene from a muscle-specific promoter (235). Also, AAV a- or p-sarcoglycan vectors can rescue an a- or p-sarcoglycan defect in the corresponding knockout mouse model. Interestingly, while the p-sarcoglycan vector showed long-term sustained expression for more than 21 months and led to widespread biochemical and histological rescue of the dystrophic muscle, transduction of myofibers by the a-sarcoglycan vector was transient and correlated with induction of significant immune response (236). The transience of the latter vector was attributed to cytotoxicity resulting from overexpression, by more than 100-fold over normal levels of the protein rather than due to an immune response to the transgene. A phase I clinical trial of AAV to deliver sar-coglycan genes was initiated, but it enrolled and treated only 1 patient (12).

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