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954. Electrotransfer of the Full Length Dog Dystrophin in Mouse and Dystrophic Dog Muscles
MUSCLE AND CONNECTIVE TISSUE - MUSCLE DISEASE DIRECT GENE THERAPY tissues resulting in high expression of therapeutic genes, which can effectively correct symptoms of genetic disorders. Because of the natural virus tropism, the liver has been a common target organ for adenoviral gene therapy approaches. However, targeting the liver by intravenous administration leads to dose-dependent acute toxicity making adenovirus-based gene replacement a suboptimal therapy approach. Therefore, skeletal muscle tissue seems to be a better target since no toxicity is associated with intramuscular administration. However, adenovirus 5 only poorly transduces mature skeletal muscle tissue. Furthermore, it is questionable if long term expression can be achieved and if expressed proteins can be secreted from muscle cells. We hypothesized that retargeting of adenovirus 5 based vectors by means of genetically engineered capsid modifications increases transduction of myotubes in vitro and mature skeletal muscle tissue in vivo resulting in prolonged gene expression. In vitro, myotubes infected with Ad5/3luc1, a serotype 5 based adenovirus featuring serotype 3 knob, and Ad5lucRGD, which features an RGD motif in the HI loop of the knob, displayed up to 15 fold higher marker gene expression compared to wild type Ad5luc1 infected cells. In vivo in hind leg muscles of immunocompetent C57 mice, transduction efficiency of intramuscularly injected Ad5/3luc1 was significantly higher compared to Ad5luc1 while Ad5lucRGD resulted in lower gene transfer levels compared to Ad5luc1. Moreover, Ad5/3luc1 displayed robust marker gene expression in C57 mice over 3 months as determined by non invasive bioluminescence imaging. To assess whether intramuscularly injected adenovirus might leak to the liver, we analyzed marker gene expression from liver tissue ex vivo at different time points. While low levels were seen on day 2 and 14, no marker gene expression could be detected at 4 weeks. In conclusion, we show here that 5/3 capsid modification on adenoviruses significantly increases transduction to adult skeletal muscle tissue. Moreover, while expression from virus that had leaked to the liver was shut off after 4 weeks, robust long term gene expression was seen in muscle tissue of immunocompetent mice, although first generation vectors were used. We are currently evaluating if skeletal muscle gene transfer with 5/3 adenoviruses can also result in long term secretion of a clinically relevant protein.
this large transgene, 293T cells were transfected with this plasmid. The expression of the dog dystrophin was successfully detected in these cells by western blot analysis. After this first positive result, this plasmid was electrotransferred into immunodeficient mouse muscles and two weeks later, the expression of the dog dystrophin was observed in electrotransferred muscles.
The same experiment was thus repeated in the muscle of a dystrophic dog and after two weeks, the full length dog dystrophin was also expressed in the electrotransferred muscle.
954. Electrotransfer of the Full Length Dog Dystrophin in Mouse and Dystrophic Dog Muscles
Christophe Pichavant,1 Pierre Chapdelaine,1 João C. S. Bizario,2 Jacques P. Tremblay.1 1 CHUL, Quebec, Canada; 2AADM/UNAERP, Ribeirão Preto, Brazil.
Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by the absence of dystrophin in the muscle. This large protein of 427 kDa is needed to insure mechanical stress resistance during muscle contraction. The lack of dystrophin weakens the sarcolemma and thus makes fibers less resistant to stress. In an attempt to eventually restore the phenotype of DMD patients and since the phenotype of the dystrophic dog is closest to the human phenotype, we have decided to study the delivery of the full length dog dystrophin transgene in the dog muscle. As already mentioned, the full length dystrophin cDNA is large, which brings certain complexity to introduce this plasmid in muscle fibers. Indeed, there is currently no viral vector able to introduce a gene of this size into muscle fibers safely. Since the muscle fibers have a long lifespan due to their post-mitotic stage, this allows a prolonged expression of a plasmid introduced in these fibers. Nevertheless, a simple intramuscular injection of plasmid coding for dystrophin does not allow a high expression of this protein in muscle. A non viral method, the electrotransfer, allows via an electric field to introduce in the fibers a plasmid previously injected into the muscle. A plasmid containing the dog full length dystrophin (∼17kb) was cloned in three consecutive steps. To verify whether our construct is able to express S364
This is the first study demonstrating a local restoration of the full length dog dystrophin in dystrophic dog muscles. In the aim to restore the muscle strength and thus to correct the phenotype of the dystrophic dog, the next step will be to improve the efficiency of the delivery method and to study the introduction of the dystrophin throughout a large dog muscle.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
this large transgene, 293T cells were transfected with this plasmid. The expression of the dog dystrophin was successfully detected in these cells by western blot analysis. After this first positive result, this plasmid was electrotransferred into immunodeficient mouse muscles and two weeks later, the expression of the dog dystrophin was observed in electrotransferred muscles.
The same experiment was thus repeated in the muscle of a dystrophic dog and after two weeks, the full length dog dystrophin was also expressed in the electrotransferred muscle.
954. Electrotransfer of the Full Length Dog Dystrophin in Mouse and Dystrophic Dog Muscles
Christophe Pichavant,1 Pierre Chapdelaine,1 João C. S. Bizario,2 Jacques P. Tremblay.1 1 CHUL, Quebec, Canada; 2AADM/UNAERP, Ribeirão Preto, Brazil.
Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by the absence of dystrophin in the muscle. This large protein of 427 kDa is needed to insure mechanical stress resistance during muscle contraction. The lack of dystrophin weakens the sarcolemma and thus makes fibers less resistant to stress. In an attempt to eventually restore the phenotype of DMD patients and since the phenotype of the dystrophic dog is closest to the human phenotype, we have decided to study the delivery of the full length dog dystrophin transgene in the dog muscle. As already mentioned, the full length dystrophin cDNA is large, which brings certain complexity to introduce this plasmid in muscle fibers. Indeed, there is currently no viral vector able to introduce a gene of this size into muscle fibers safely. Since the muscle fibers have a long lifespan due to their post-mitotic stage, this allows a prolonged expression of a plasmid introduced in these fibers. Nevertheless, a simple intramuscular injection of plasmid coding for dystrophin does not allow a high expression of this protein in muscle. A non viral method, the electrotransfer, allows via an electric field to introduce in the fibers a plasmid previously injected into the muscle. A plasmid containing the dog full length dystrophin (∼17kb) was cloned in three consecutive steps. To verify whether our construct is able to express S364
This is the first study demonstrating a local restoration of the full length dog dystrophin in dystrophic dog muscles. In the aim to restore the muscle strength and thus to correct the phenotype of the dystrophic dog, the next step will be to improve the efficiency of the delivery method and to study the introduction of the dystrophin throughout a large dog muscle.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy