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.

Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy

“…Surprisingly, sarcolemmal nNOS expression is not altered in mdx mice that express C-terminal domain-truncated dystrophins (15,16). Furthermore, even in the presence of an intact C-terminal domain, nNOS is not detected at the sarcolemma (21,25,31). Taken together, these findings indicate that the dystrophin C-terminal domain may, at most, play a nominal role in nNOS recruitment.…”

Microtubule binding distinguishes dystrophin from utrophin

“…To determine whether overexpression of utrophin could rescue microtubule disorganization in dystrophin-deficient mouse extensor digitorum longus (EDL) muscle fibers, we compared mdx mice transgenically overexpressing utrophin (Fiona-mdx), nearly full-length dystrophin (Dys-TG-mdx), or a miniaturized dystrophin (mini-Dys-TG-mdx) lacking spectrin-like repeats 4 through 19 (33,41,42). Western blotting of quadriceps muscle lysates verified that all three transgenic proteins are overexpressed above WT levels (Fig.…”

Prosurvival Factors Improve Functional Engraftment of Myogenically Converted Dermal Cells into Dystrophic Skeletal Muscle

“…Donor cells were derived from the miniDys-GFP/mdx 4cv (miniDys-GFP) mouse line that contains a mini-dystrophin-eGFP fusion protein cDNA driven by human alpha-skeletal actin regulatory elements on the mdx 4cv background [28]. Host mice were 4-8 weeks old, and donor and host were syngeneic.…”

“…Whole muscle mononuclear (WMM) cells were isolated as described [28] from 4-weekold miniDys-GFP mice and were transplanted immediately, without culturing.…”