Studies on Expanded C-Termini Microdystrophin Gene Transfer Using rAAV9 Vectors in Skeletal Muscle of Dystrophin mdx Mice

Duchenne Muscular Dystrophy (DMD) is a severe inherited muscle-wasting disease and is caused by mutations in members of a multi-component protein structure called the dystrophin protein complex (DPC). Previous studies have attempted to transfer terminally truncated microdystrophins, however the role of C-terminal domain (CT) is yet to be delineated. CT domain of dystrophin recruits dystrophin associated protein complex (DPC) to form a transmembrane link and perhaps act as mediator of signalling between extracellular matrix and cytoskeleton in muscle fiber. In this study, codon-optimised mouse micro-dystrophin cDNAs have been expanded at the C-terminal region (variants; JC/CC1, CC2, and CC3) allowing for specific/functional micro-dissection of particular dystrophin regions (e.g., dystrophin regional association/localisation studies of syntrophin, dystrobrevin and nNOS binding). These variants have been engineered in AAV plasmid vectors under SPc5/12 optimal muscle-specific promoter, Kozak sequences and domain configurations. We have detected the localization of microdystrophin and part of the DPC at the sarcolemma of the skeletal muscle from mdx mice by western blotting and immunohistochemistry. Recently reported AAV 9 vector expressing C-terminal extended microdystrophin variants are currently subjected to comparative evaluation in mdx mice by intramuscular injection. Complementation of dystrophin deficiency, transgene stability in muscle tissues and host immune modulation will be monitored with the objective of developing safe and effective muscle gene therapy protocols appropriate to move forward towards clinical trials. The current study will present the role of C-terminal domain of dystrophin protein to restore/assist the function of dystrophin in DMD.