The long range goal of this project is to develop viral vectors of gene therapy of muscle diseases. In transgenic mdx mice, a model for Duchenne muscular dystrophy (DMD), low-level expression of dystrophin mini-genes prevents dystrophy through at least years of age. Modified adenoviral vectors are being developed to test whether gene transfer can treat, rather than prevent, dystrophy in young and aging animals. Adenoviruses (Ad) efficiently infect muscle but they display a number of disadvantages preventing use in clinical trials for DMD. Ad vectors trigger a host immune response that prevents long term gene expression in vivo. Ad vectors have a cloning capacity of only approximately 8 kb, smaller than the 14 kb dystrophin cDNA. Finally. strong viral promoters typically used to driven transgene expression from Ad vectors are often shut-off in vivo. A new Ad vector is being developed that could overcome each of these problems. Our hypothesis is that Ad vector systems lacking all viral genes can support long-term gene expression. in muscle. Critical to this 'gutted' vector strategy is the development of a self-limiting helper virus system. Vector lacking viral genes can only be propagated in the presence of a helper virus that produces proteins needed for Ad replication and packaging. However, it the preparations of gutted vector contain significant levels of the helper virus, then the contaminating helper virus will trigger the same immune response that the gutted system is designed to avoid. Our strategy for perfecting the gutted vector system relies heavily on improvements to the helper viruses used for gutted vector propagation. The Cre-LoxP system will be used to disable packaging of helper DNA into virions. Modified Ad packaging cell lines will also be used to grow helper viruses lacking a subset of genes required for propagation and gene expression in vivo. Muscle specific promoters are also being developed to limit virally-delivered transgene expression to muscle tissues. Finally, a functional homologue of dystrophin [utrophin] will be tested for the ability to prevent an immune response against dystrophin. This gutted vector/helper virus system will be tested in vivo to identify any residual host immune responses that might be generated, and if needed a variety of vector modifications will be tested to attenuate this response. If successful, these studies could lead to a treatment for DMD and other muscle diseases.
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