Duchenne muscular dystrophy (DMD) is a common inheritable human disease, caused by mutations in the dystrophin gene, the longest known human gene (~2.5Mb long), located on the short arm of X chromosome. DMD is clinically characterized by progressive muscle weakness and wasting in affected boys, causing wheelchair bound status at around 12 years of age and death due to loss of function of vital muscles, such as intercostal muscles, and consequent complications in late teenage and early twenties. Dystrophin protein (427KDa), the product of dystrophin gene in skeletal muscles, is absent from DMD myofibres. This long multi-domain protein links cytoskeletal actin at one end to the extra cellular matrix at the other, acting as a structural stabilizer in mechanically active myofibres. Dystrophin has also developed highly complex interactions with some other structural/signaling proteins called dystrophin associated proteins (DAPs) in myofibres, which are also implicated in other muscular dystrophies such as limb girdle muscular dystrophies. These complex interactions suggest a complicated molecular pathology for DMD. RNAi technology allows scientists to knock down the expression of genes of interest and to look for the direct results of this process without directly affecting the gene itself. Upon the introduction of siRNA to the cell, it conducts the specific destruction of the mRNA to which it exactly matches. We have successfully targeted dystrophin using RNAi technology by transient transfection of myogenic primary cultures with synthetic siRNAs. We extended the RNAi to in vivo studies by expressing shRNAs from an rAAV2/6 vector. This system potently knocked down dystrophin in vivo and established a very long half-life for naturally-expressed dystrophin. This new model provides the opportunity of studying the molecular pathology of dystrophin deficiency from new angles, and may help elucidate the complex processes involved in this disease. We have also used our in vitro model to study the expression profile in primary cultures in which dystrophin had been knocked down by RNAi.

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