256. An Ideal Therapeutic Agent for Duchenne Muscular Dystrophy Involving a Gutted Adenovirus Expressing Full-Length Utrophin

Abstract

Duchenne muscular dystrophy (DMD) is characterized by necrosis and progressive loss of muscle fibers. DMD patients have a mutation in the gene encoding dystrophin (dys), a large sarcolemmal membrane-associated cytoskeletal protein. Gene replacement therapy using fully deleted adenoviral vectors shows great potential for the eventual treatment of DMD.These vectors are less immunogenic than their predecessors and have the capacity to carry the full-length dys cDNA (13 kb). The introduction of fully deleted adenoviral (AdV) vectors encoding full-length dys into muscles leads to significant improvement of the dystrophic phenotype of the mdx mouse, an animal model of DMD. However, the introduction of dys, a neoantigen, into mdx muscle causes immune responses resulting in increased appearance of inflammatory infiltrates and damage to the muscle. An alternative approach is to use utrophin (utr), a functional homologue of dys, normally present only at the neuromuscular junction. This approach of simply increasing the expression of a protein that is already present in dystrophic muscle would prevent any immune response to the transgene product. Thus, we have created a fully deleted AdV encoding full-length murine utr cDNA regulated by a powerful hybrid promoter consisting of the chicken b-actin promoter and CMV enhancer. Cells infected with the purified recombinant AdV showed strong utr expression levels as determined by Western blot analysis. Furthermore, during in vivo studies, high transduction levels were obtained in the tibialis anterior (TA) muscles of mdx mice. The vector was administered to cohorts of neonates (2-4 day old) and adults (5 to 7 week old) at doses of 1.45 |[times]| 1010 and 4.35 |[times]| 1010 virus particles respectively. In the first group of neonates (n=5) at 10 days post-injection the mean number of sarcolemmal utr-positive fibers in injected vs control-injected TA was 1596 +/- 297 vs 114 +/- 76, which corresponds to |[sim]|58 % transduction level. The mean number of utr-positive fibers in the adults (n=7) was 685 +/- 505 and 112 +/- 80 for viral and control injected TAs respectively, which corresponds to |[sim]|23 % transduction level. The numbers obtained correspond to an approximate 14 and 6 fold increase in utr expression over control levels in neonates and adults. These results were further validated by consistent utr levels observed by Western blots using muscle sections. Subsequent in vivo evaluations will be performed at 30, 60, 90, 180, and 360 days post-injection. The injected muscles will be examined for utr expression, the restoration of the dys-associated protein complex, and the reversal of the physiological dystrophic phenotype. Considering our preliminary data thus far, helper-dependent, fully deleted (gutted) AdV expressing full length utr promises to be the ideal agent for gene replacement therapy in dys-deficiency states.