919. Gene Therapy for Duchenne Muscular Dystrophy Using a Gutted Adenovirus Expressing Utrophin

Duchenne muscular dystrophy (DMD) is characterized by a mutation in the gene encoding a sarcolemmal protein, dystrophin (dys) and the deficiency of dys leads to progressive loss of muscle fibers. Gutted adenovirus (AdV) vectors are less immunogenic than their predecessors and have the capacity of carrying large DNA inserts such as the full-length dys cDNA (13 kb). The use of gutted AdV vectors encoding full-length dys leads to significant improvement of the dystrophic phenotype of the mdx mouse, an animal model of DMD. However, dys behaves as a neoantigen in genetic dys deficiency states such as the mdx and it causes deleterious immune responses in the treated muscles. An alternative approach would be to use utrophin (utr), a functional homologue of dys, which is normally present only at the sarcolemma of the neuromuscular and myotendinous junctions. Therefore, utr is not expected to behave as a neoantigen in dys-deficient states. In fact, transgenic mdx mice expressing extrasynaptic utr in muscle fibers have shown functional and histological improvement. Previous findings have also shown utrophin, when expressed via a first generation adenovirus to be sufficient and adequate in replacing dystrophin at the sarcolemma and preventing the symptoms of DMD. Thus, in an attempt to minimize the vector immunogenicity and to harness the therapeutic potential of utr, we created a fully deleted AdV encoding full-length utrophin. The vector was administered to cohorts of mdx neonates (2-4 day old) and adults (5 to 7 week old) at doses of 1.45×1010 and 4.35×1010 virus particles respectively into the tibialis anterior (TA) muscles. In neonates at 10 days post-injection (pi), the mean number of extrasynaptic utr+fibers in vector vs control-injected TA was 1596 vs 114, corresponding to 58 % transduction level. At 60 days pi, the level of transduction was found to be near 52% corresponding to approximately 794 utr+fibers. The levels in adults at 10 days pi were 685 and 112 for vector and control injected TAs respectively, corresponding to a 23% transduction level. At the 10 day timepoint, there was a 14- and 6- fold increase in utr expression over control levels in neonates and adults. In adults at 30 days pi, the mean number of utr+fibers was 770 corresponding to 35% transduction level. However, that number dropped substantially to an average of 80 fibers at 60 days pi and remained between 80 and 120 up to the 180 days pi. At no time point were anti-utrophin antibodies detected. The decline of the initial high utr transduction of the TA muscles associated with time may be due to the fact that the initial extrasynaptic utr level is not sufficiently high to prevent the natural progression of the dystrophic phenotype. Further augmentation of the initial utr transduction should be helpful in order to completely and functionally compensate for dys. Duchenne muscular dystrophy (DMD) is characterized by a mutation in the gene encoding a sarcolemmal protein, dystrophin (dys) and the deficiency of dys leads to progressive loss of muscle fibers. Gutted adenovirus (AdV) vectors are less immunogenic than their predecessors and have the capacity of carrying large DNA inserts such as the full-length dys cDNA (13 kb). The use of gutted AdV vectors encoding full-length dys leads to significant improvement of the dystrophic phenotype of the mdx mouse, an animal model of DMD. However, dys behaves as a neoantigen in genetic dys deficiency states such as the mdx and it causes deleterious immune responses in the treated muscles. An alternative approach would be to use utrophin (utr), a functional homologue of dys, which is normally present only at the sarcolemma of the neuromuscular and myotendinous junctions. Therefore, utr is not expected to behave as a neoantigen in dys-deficient states. In fact, transgenic mdx mice expressing extrasynaptic utr in muscle fibers have shown functional and histological improvement. Previous findings have also shown utrophin, when expressed via a first generation adenovirus to be sufficient and adequate in replacing dystrophin at the sarcolemma and preventing the symptoms of DMD. Thus, in an attempt to minimize the vector immunogenicity and to harness the therapeutic potential of utr, we created a fully deleted AdV encoding full-length utrophin. The vector was administered to cohorts of mdx neonates (2-4 day old) and adults (5 to 7 week old) at doses of 1.45×1010 and 4.35×1010 virus particles respectively into the tibialis anterior (TA) muscles. In neonates at 10 days post-injection (pi), the mean number of extrasynaptic utr+fibers in vector vs control-injected TA was 1596 vs 114, corresponding to 58 % transduction level. At 60 days pi, the level of transduction was found to be near 52% corresponding to approximately 794 utr+fibers. The levels in adults at 10 days pi were 685 and 112 for vector and control injected TAs respectively, corresponding to a 23% transduction level. At the 10 day timepoint, there was a 14- and 6- fold increase in utr expression over control levels in neonates and adults. In adults at 30 days pi, the mean number of utr+fibers was 770 corresponding to 35% transduction level. However, that number dropped substantially to an average of 80 fibers at 60 days pi and remained between 80 and 120 up to the 180 days pi. At no time point were anti-utrophin antibodies detected. The decline of the initial high utr transduction of the TA muscles associated with time may be due to the fact that the initial extrasynaptic utr level is not sufficiently high to prevent the natural progression of the dystrophic phenotype. Further augmentation of the initial utr transduction should be helpful in order to completely and functionally compensate for dys.

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