1. ¹Department of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA
2. ²Division of Molecular Pharmaceutics, University of North Carolina School of Pharmacy, Chapel Hill, NC, USA
3. ³Cardiovascular Institute, University of Pittsburgh, Pittsburgh, PA, USA
4. ⁴Department of Pathology, University of North Carolina School of Medicine, Chapel Hill, NC, USA

Correspondence: Dr X Xiao, Division of Molecular Pharmaceutics, University of North Carolina School of Pharmacy, 303B Beard Hall CB 7360, Chapel Hill, NC 27599, USA. E-mail: xxiao@email.unc.edu

Received 21 December 2007; Revised 7 February 2008; Accepted 13 February 2008; Published online 24 April 2008.

Abstract

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder lacking a curative treatment. We wish to use the dystrophin-deficient golden retriever muscular dystrophy (GRMD) dog, a canine model of DMD, to investigate adeno-associated virus (AAV) vector-mediated minidystrophin gene therapy. The dog model is useful in evaluating vector dose requirement and immunological consequences owing to its large size and outbred nature. In this study, we have cloned and constructed a canine minidystrophin gene vector. Owing to limited availability of the GRMD dogs, here we first examined the functions and therapeutic effects of the canine minidystrophin in the mdx mouse model. We observed efficient minigene expression without cellular immune responses in mdx mice after AAV1-cMinidys vector intramuscular injection. We also observed restoration of the missing dystrophin-associated protein complex (DPC) onto the sarcolemma, including sarcoglycans and dystrobrevin, and a partial restoration of -syntrophin and neural nitric oxide synthase (nNOS). In addition, minidystrophin treatment ameliorated dystrophic pathology, such as fibrosis and myofiber central nucleation (CN). CN remained minimal (<2% ) after AAV injection in the neonatal mdx mice and was reduced from more than 75% to about 25% after AAV injection in adult mdx mice. Finally, in vivo cell membrane leakage test with Evans blue dye showed that the canine minidystrophin could effectively protect the myofiber plasma membrane integrity. Our results, thus, demonstrated the functionality and therapeutic potential of the canine minidystrophin and paved its way for further testing in the GRMD dog model.