243. Delivery of Igf-I and Dystrophin to Dystrophic mdx Muscle

Abstract

Duchenne muscular dystrophy is among the most common genetic diseases and is caused by mutations in the dystrophin gene. Dystrophic muscles display an extensive degeneration and regeneration process, whereby muscle fibers progressively lose their self-renewal potential and are gradually replaced by adipose and fibrotic tissue. Gene replacement therapy using truncated versions of dystrophin have been shown to protect dystrophic muscles from contraction-induced injury and partially reverse muscle pathology. An alternative approach involves the activation of satellite cells to maintain the regenerative potential of dystrophic muscle. Igf-I, an important mediator of cell growth and differentiation, has been shown to increase muscle mass and strength and to enhance muscle repair mechanism in dystrophic mdx muscles (Barton et al., 2002). However, Igf-1 is unable to restore mechanical integrity to muscle fibers lacking dystrophin. To determine if the beneficial effect of Igf-I is synergistic with the protective effect of dystrophin in ameliorating dystrophic pathology, we compared the effects of delivering Igf-I alone versus co-delivering both Igf-I and dystrophin to adult, dystrophic mdx mouse muscles. We generated recombinant adeno-associated viral vectors pseudotyped with the serotype 6 capsid protein that carry expression cassettes in which the muscle-specific creatine kinase promoter/enhancer drove either the micro-dystrophin (AAV-udys) or the Igf-I cDNA (AAV-Igf-I). Tibialis anterior muscles of mdx mice were injected with each vector separately, together or with buffer control and then analyzed four months post injection. Immunohistochemical analysis demonstrated persistent expression of dystrophin that reached an average of 40% of the total muscle cross sectional area. We also observed persistent expression of Igf-I mRNA at levels 200-400 fold greater than endogenous mdx Igf-I levels in AAV-Igf-I and AAV-udys co-injected muscles. In contrast, injection of AAV-Igf-I alone resulted in a 4-fold decline of Igf-I mRNA levels in the four months following injection into dystrophic mdx muscles. Functional measurements demonstrated that AAV-udys injected animals were partially protected from contraction-induced injury after two lengthening contractions, whereas animals injected with AAV-Igf-I alone were as susceptible as mdx animals to muscle damage. AAV-Igf-I treated animals, on the other hand, showed an increase in muscle mass, which was not seen after AAV-udys only treatment. In contrast, co-injection of AAV-Igf-I and AAV-udys resulted in increased muscle mass and muscle strength, and in protection from contraction-induced injury. These results suggest that the combination of AAV-Igf-I and AAV-udys acted synergistically and was beneficial for the animal.