ABSTRACT. miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMDJ dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMDJ TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.

Recombinant adeno-associated virus (rAAV)-mediated gene transfer is an attractive approach to the treatment of Duchenne muscular dystrophy (DMD). We investigated the muscle transduction profiles and immune responses associated with the administration of rAAV2 and rAAV8 in normal and canine X-linked muscular dystrophy in Japan (CXMD(J)) dogs. rAAV2 or rAAV8 encoding the lacZ gene was injected into the skeletal muscles of normal dogs. Two weeks after the injection, we detected a larger number of beta-galactosidase-positive fibers in rAAV8-transduced canine skeletal muscle than in rAAV2-transduced muscle. Although immunohistochemical analysis using anti-CD4 and anti-CD8 antibodies revealed less T-cell response to rAAV8 than to rAAV2, beta-galactosidase expression in rAAV8-injected muscle lasted for <4 weeks with intramuscular transduction. Canine bone marrow-derived dendritic cells (DCs) were activated by both rAAV2 and rAAV8, implying that innate immunity might be involved in both cases. Intravenous administration of rAAV8-lacZ into the hind limb in normal dogs and rAAV8-microdystrophin into the hind limb in CXMD(J) dogs resulted in improved transgene expression in the skeletal muscles lasting over a period of 8 weeks, but with a declining trend. The limb perfusion transduction protocol with adequate immune modulation would further enhance the rAAV8-mediated transduction strategy and lead to therapeutic benefits in DMD gene therapy.

Abstract:The purpose of this study was to develop a strain of canine X-linked muscular dystrophy (CXMD), a model of Duchenne muscular dystrophy, in Japan. A female beagle was artificially inseminated with frozen-thawed spermatozoa derived from an affected golden retriever. Subsequently, two carrier female dogs (G1 carriers) and four normal male littermates were produced. Thereafter, the two G1 carriers were mated with beagle sires. As a result, each bitch whelped three times, and out of 54 pups, 17 affected male descendants, and 11 carrier female descendants (G2 carriers) were detected. One G2 carrier was then mated with a beagle sire and 15 pups in two whelpings were produced, including five affected males and four carrier females (G3 carriers). A total of 10 female beagles were artificially inseminated to evaluate the fertility of the frozen-thawed spermatozoa from the two affected dogs. The whelping rates of the two affected dogs were 4/5 and the litter sizes were 5.0 ± 1.41 and 6.0 ± 0.82, respectively. These results indicate that a canine X-linked muscular dystrophy colony has been established in Japan. We called them CXMD J .

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscular disorder caused by a defect in the DMD gene. AAV vector-mediated micro-dystrophin cDNA transfer is an attractive approach to treatment of DMD. To establish effective gene transfer into skeletal muscle, we examined the transduction efficiency of an AAV vector in skeletal muscles of dystrophin-deficient mdx mice. When an AAV vector encoding the LacZ gene driven by a CMV promoter (AAVCMVLacZ) was introduced, ␤-galactosidase expression markedly decreased in mdx muscle 4 weeks after injection due to immune responses against the transgene product.We also injected AAV-CMVLacZ into skeletal muscles of mini-dystrophin-transgenic mdx mice (CVBA3'), which show ameliorated phenotypes without overt signs of muscle