title: A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy.
abstract: 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 (DeltaCT) with an eGFP coding sequence and removing much of the dystrophin central rod domain (DeltaH2-R19). In a transgenic mdx(4Cv) 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 mdx(4Cv) muscles. When myogenic and other stem cells from these mice were transplanted into mdx(4Cv) recipients, donor cells can be readily identified in skeletal muscle by direct green fluorescence or by using antibodies against GFP or dystrophin. In mdx(4Cv) 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 mdx(4Cv) mice are highly useful for developing autologous cell therapies for DMD.
authors.affiliation: Department of Neurology, University of Washington School of Medicine, Seattle, 98195-7720, USA.
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: En
authors.initials: E
authors.lastname: Kimura
authors.forename: Rainer
authors.initials: R
authors.lastname: Ng
authors.forename: Brent M
authors.initials: BM
authors.lastname: Fall
authors.forename: Leonard
authors.initials: L
authors.lastname: Meuse
authors.forename: Morayma
authors.initials: M
authors.lastname: Reyes
authors.forename: John A
authors.initials: JA
authors.lastname: Faulkner
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Chamberlain
mesh_terms: D000818:Animals; D016189:Dystrophin / Q000096:biosynthesis / Q000235:genetics*; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000473:pathology; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D012508:Sarcolemma / Q000378:metabolism; D033581:Stem Cell Transplantation
chemical_list: D016189:Dystrophin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins