Abstract

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression to skeletal muscles. To overcome limited packaging capacity of AAV vectors (< 5kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence-optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin associated protein complex which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we have extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain extended microdystrophin showed efficient dystrophin expression in TA muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevinat the sarcolemma and significantly improved the muscle resistance to lengthening (eccentric) contractions-induced muscle damage in the mdx mice compared to MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins substantially improves their efficiency on restoring muscle function in DMD patients.