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Duchenne muscular dystrophy is caused by lack of dystrophin expression in skeletal muscles and is characterized by progressive degeneration of muscle fibers. To be effective, gene therapy approaches to DMD need to target a large number of fibers in the muscle, and the distribution of dystrophin through the fiber length needs to be sufficient to prevent fiber degeneration. Plasmid-based gene therapies have been shown to be a valid approach to the treatment of a variety of disorders including DMD. Concerns, however, have been raised about the ability of extrachromosomal vectors to sustain gene expression for prolonged periods of time at levels that are therapeutic.

Non-viral approaches to gene therapy have often been limited by transient expression of delivered genes. Permanent integration of therapeutic genes into the host genome holds promise for lasting expression of the deficient or defective protein. Integration that is site-specific can limit the hazards of insertional mutagenesis. Here we demonstrate a novel non-viral approach to gene therapy for muscle disease. We used phage phiC31 integrase to catalyze unidirectional, site-specific integration of incoming plasmid DNA carrying the luciferase gene and an attB site into pseudo attP sites in the genomes of muscle fibers. In vivo bioluminescent imaging was used to assess the levels of luciferase expression, as a measure of gene transfer and integration into mouse leg skeletal muscle. in vivo imaging demonstrated that electroporation following intramuscular injection of plasmid DNA increased initial luciferase activity by 1-2 orders of magnitude. Intramuscular injection of the luciferase-attB plasmid together with a plasmid expressing the phiC31 integrase produced persistent levels of expression that were 5-10 fold higher than the levels seen without integrase. Genomic integration was verified by performing PCR on muscle DNA using primers spanning a known hotspot pseudo attP site for phiC31–mediated integration in mouse liver cells. Four out of four muscles that received integrase and the attB plasmid were positive for integration events at this site. Application of this site-specific integration technology to gene therapy for muscular dystrophies may provide long-term, sustained amelioration of the disease phenotype. Delivery of wild type dystrophin cDNA into dystrophin-deficient muscles using the phiC31 integrase system is currently under investigation.