Human Molecular Genetics Advance Access originally published online on April 4, 2006
Human Molecular Genetics 2006 15(10):1610-1622; doi:10.1093/hmg/ddl082
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A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy
1Department of Neurology and 2Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, Seattle, WA 98195-7720, USA and 3Department of Biomedical Engineering and 4Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109-2007, USA
* To whom correspondence should be addressed at: K243b HSB, University of Washington School of Medicine, 1959 NE Pacific Street, Seattle, WA 98195-7720, USA. Fax: +1 2066168272; Email: jsc5{at}u.washington.edu
Received February 16, 2006; Accepted March 25, 2006
A promising approach for treating Duchenne muscular dystrophy (DMD) is by autologous cell transplantation of myogenic stem cells transduced with a therapeutic expression cassette. Development of this method has been hampered by a low frequency of cellular engraftment, the difficulty of tracing transplanted cells, the rapid loss of autologous cells carrying marker genes that are unable to halt muscle necrosis and the difficulty of stable transfer of a large dystrophin gene into myogenic stem cells. We engineered a 5.7 kb miniDys–GFP fusion gene by replacing the dystrophin C-terminal domain (
CT) with an eGFP coding sequence and removing much of the dystrophin central rod domain (H2-R19). In a transgenic mdx4Cv mouse expressing the miniDys–GFP fusion protein under the control of a skeletal muscle-specific promoter, the green fusion protein localized on the sarcolemma, where it assembled the dystrophin–glycoprotein complex and completely prevented the development of dystrophy in transgenic mdx4Cv muscles. When myogenic and other stem cells from these mice were transplanted into mdx4Cv recipients, donor cells can be readily identified in skeletal muscle by direct green fluorescence or by using antibodies against GFP or dystrophin. In mdx4Cv mice reconstituted with bone marrow cells from the transgenic mice, we monitored engraftment in various muscle groups and found the number of miniDys–GFP+ fibers increased with time. We suggest that these transgenic mdx4Cv mice are highly useful for developing autologous cell therapies for DMD.
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