Development of a Porcine Model of Duchenne Muscular Dystrophy

DESCRIPTION (provided by applicant): Project Summary/Abstract Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene with a prevalence of 1 in 3500 male births. The consequent loss of functional dystrophin results in the progressive degeneration of skeletal and cardiac muscle. Despite significant progress in our understanding of this disease and advances in the development of new therapeutic approaches, DMD remains a fatal disease. Much of what is known about DMD has come from studying dystrophin-deficient animals, particularly murine and canine models. While useful for mechanistic studies, dystrophic mice fail to develop the muscle weakness phenotype that is typical of DMD in patients. The canine models are more representative of human DMD, but are difficult to study due to extreme phenotypic variability. The canine models also suffer from a limited choice of mutations, significant expense, and social acceptance concerns. Therapeutic strategies that have shown promise in these models have failed to be translated to the clinic. An animal model that more accurately and consistently replicates the clinical manifestations of human DMD is sorely needed. Our objective is to create an improved model of DMD in thepig. We believe a porcine model offers several advantages over the existing models. Gene targeting is now available in pigs and would provide an opportunity to engineer patient-relevant mutations. Porcine cloning technology would allow the production of genetically identical dystrophic pigs and could yield reduced variability in phenotype severity. Also, pigs are less expensive to produce and easier to house than dogs and don't face the same ethical concerns as companion animals. The ultimate goal of this project is to develop and commercialize DMD-targeted pigs as a model of Duchenne muscular dystrophy. We intend to accomplish this by combining gene targeting and somatic cell nuclear transfer to create a porcine model harboring a common human DMD mutation.This proposal specifically outlines the development of porcine fibroblasts with a mutated DMD gene. Gene targeting vectors will be constructed to delete a portion of the endogenous porcine DMD gene in a region frequently mutated in patients. Porcine fetalfibroblasts will be infected with a virus carrying the DMD targeting vectors. Our plans for generating properly targeted cells are designed to maximize the frequency of homologous recombination, minimize random integration, and minimize the number of cellpassages before targeted cells are harvested. Subsequent work will use these cells for somatic cell nuclear transfer to produce DMD-targeted pigs, followed by phenotypic characterization and validation. This animal model will provide the academic and commercial research communities an opportunity to better understand DMD and to develop and test new therapeutic strategies PUBLIC HEALTH RELEVANCE: Project Narrative This proposal specifically outlines the development of porcine fibroblasts with mutated DMD alleles as a first step towards a new model of Duchenne muscular dystrophy. Subsequent work will use these cells for somatic cell nuclear transfer to produce dystrophic pigs that will then be characterized and validated as an appropriate model. This project is relevant to the NIH's mission because it will provide a resource to stimulate discovery, therapeutic application, and the development of new diagnostic tools.