? ? Inherited diseases, occurring in solid tissues, are often caused by simple recessive mutations derived from two carrier parents with a normal phenotype. In X-linked diseases, hemizygous males with a mutant allele will be affected while the female carrier is not. Gene therapy for recessive diseases has typically centered on a DNA-based gene system to deliver the normal allele in a mammalian expression vector or via an allogenic cell transplant. Numerous problems have been identified with these gene complementing systems arising from the lack of proper gene regulation, poor distribution of the transfecting vector, antigenic response from host immune system and the oncogenic potential of viral integration. These adverse events coupled with our long-standing interest in using the efficient endogenous DMA repair systems to correct mutations in mammalian cells led us to develop a new approach to gene therapy. This alternative strategy employs a synthetic oligonucleotide to direct the exchange of the mutant base in the affected gene or allele. The process, termed, gene repair or gene editing, has proven to be efficacious in a number of model systems and disease targets, including muscular dystrophy. The work in DMD, however, has not achieved levels of correction that enable therapeutic benefit. Thus, a primary challenge for this technique is to increase the frequency of repair or to isolate cells that have been corrected and expand them in culture. In this proposal, we aim to carry out preliminary investigations into the feasibility of meeting both challenges using primary muscle cells obtained from mdxScv and normal mice. We will examine the most efficient way of transfecting both a reporter plasmid and an oligonucleotide and achieving high levels of correction in either dividing or differentiating cells.
We aim to develop a dual correction protocol in which a mutant reporter gene eYFP or DsRFP and the dystrophin mutation are simultaneously repaired. We shall select cells displaying functional gene editing activity by the emergence of fluorescence and assess the coordinate repair of the dystrophin mutation at the phenotypic and genotypic levels. Our long term goal is to develop a protocol in which primary muscle cells can be isolated and propagated ex vivo, then eventually transplanted back into the patient. ? ?

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
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National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
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Porter, John D
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University of Delaware
Schools of Arts and Sciences
United States
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Maguire, Katie; Suzuki, Takayuki; DiMatteo, Darlise et al. (2009) Genetic correction of splice site mutation in purified and enriched myoblasts isolated from mdx5cv mice. BMC Mol Biol 10:15