The goal of the research proposed for this award is to develop strategies for gene therapy in patients with Duchenne muscular dystrophy (DMD). The ideal gene transfer therapy for patients with DMD would be systemic, safe, efficient and targeted to tissues that normally express dystrophin. A promising candidate carrier for dystrophin gene delivery is a replication-deficient viral vector. To date, the retroviral and adenoviral vectors have proved safe and are therefore potentially useful for human gene transfer. However, these vectors can accept DNA inserts of approximately one-half the length of the recombinant dystrophin cDNA. We propose to accommodate the insert size limitation of these vectors by truncating the dystrophin cDNA driven by the muscle creatine kinase promoter. The construction of truncated recombinant dystrophin cDNA molecules will be achieved by engineering deletions of varying sizes in the spectrin-repeat region of dystrophin. The characterization of dystrophin deletions in patients with mild phenotypes has revealed deletions in this region. A polymerase chain reaction-based strategy will be used to precisely delete integral numbers of spectrin-like repeats. It may also be possible to truncate the promoter region and the 3' untranslated region without adversely affecting dystrophin function. After construction, these recombinant genes will be used to generate retroviral and adenoviral vectors that will first be tested for expression in cell culture. The primary aim of these studies will be the production of viral vectors with stable inserts, high viral titer, and expression of functional dystrophin. Dystrophin expression, which will be detected by immunocytochemistry and Western analysis, from truncated, recombinant dystrophin constructs with different deletions may yield important information about the relationship between specific regions of dystrophin and its expression. A second goal of the expression studies in cell culture is to develop a better understanding of the factors that affect gene uptake and stable expression in muscle cells using these viral vectors. Promising constructs for gene therapy will be used to generate transgenic mice on the mdx mouse background; the mdx mouse is a biochemical and genetic model for DMD. These studies will allow demonstration of the tissue-specific expression of dystrophin and the effect of the recombinant, truncated dystrophin gene on the levels of dystrophin-associated proteins in skeletal muscle in vivo. In vivo studies are also planned that will demonstrate the efficacy of dystrophin gene transfer into the mdx mouse using retroviral and adenoviral vectors.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Clinical Investigator Award (CIA) (K08)
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NST-2 Subcommittee (NST)
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Nichols, Paul L
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University of Pittsburgh
Schools of Medicine
United States
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