Duchennes Muscular Dystrophy (DMD) is an X-linked recessive disorder caused by a deficiency of the protein dystrophin (Dys). Absence of this important cytoskeletal protein leads to progressive muscle wasting and death. One approach for treating this lethal disease is replacement therapy through myoblast transplantation or gene therapy. This project will attempt to develop the delivery vehicles necessary to efficiently transfer Dys minigenes to skeletal muscle in animal models. Gene therapy for DMD is associated with several experimental challenges. The gene delivery vehicle must 1) accommodate athe normal minigene, which in the cause of a full length human Dys cDNA is 14 kb, and 2) efficiently, stable, and safely target a large number of skeletal muscle fibers in vivo. We propose to develop recombinant adenoviruses as vehicles for DMD gene therapy. Preliminary studies indicate that recombinant adenoviruses of the Ad5 serotype are tropic for skeletal muscle myotubes under the right experimental conditions in vivo. Current vectors are limited for DMD gene therapy because 1) they cannot accommodate anything longer than 8 kb, and 2) they are not truly replication defective and, as a result, elicit cellular immune responses in vivo which lead to loss of transgene expression through destruction of the target cell and inflammation. Our studies will utilize the mouse as an experimental model for developing and evaluating adenoviral vectors for gene therapy because of the simplicity of studying immunology and the existence of murine models of DMD. Experiments in this proposal will utilize reporter genes and non-DMD mice. Our initial focus will be to define the immunological responses of the host to the virus and virus and virus infected cells. Experience in other systems should predict that class I restricted cytotoxic T lymphocytes to viral antigens expressed in transduced cells are important immune effectors while class II restricted T helper and B cell responses to input viral proteins, which generate neutralizing antibody, result in difficulties in achieving gene transfer on repeat administration of virus. A significant effort will be made to further cripple the virus by deleting essential genes to minimize viral protein expression and the resulting immune responses and to provide more space within the vector to accommodate Dys minigenes.
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