378. An AAV Vector-Mediated Micro-Dystrophin Expression in Relatively Small Percentage of Dystrophin-Deficient mdx Myofibers Still Improved the mdx Phenotype through Compensatory Hypertrophy
Authors: Madoka Ikemoto, Madoka Yoshimura, Miki Sakamoto, Yasushi Mochizuki, Katsutoshi Yuasa, Toshifumi Yokota, Yuko Miyagoe-Suzuki, Shin'ichi Takeda
Journal: Molecular Therapy (2004)

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[Background] Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene. An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, though it has a limitation in insertion size up to 4.9 kb. Therefore, a full-length dystrophin cDNA (14 kb) cannot be incorporated into an AAV vector. We previously generated micro-dystrophin transgenic mdx mice. Micro-dystrophin CS1 transgenic mdx mice showed almost complete amelioration of dystrophic phenotypes (BBRC. 293: 1265, 2002).

[Objective] We constructed an AAV vector expressing micro-dystrophin ΔCS1, and introduced it into skeletal muscles of mdx mice and examined whether the dystrophic process had been ameliorated or not.

[Method] To incorporate micro-dystrophin CS1 cDNA (4.9 kb) into an AAV vector, we deleted 5′- and 3′-UTRs and exons 71-78 (alternative splicing regions), resulting 3.8 kb ΔCS1 cDNA. We produced type 2 AAV vector expressing ΔCS1 under the control of muscle specific MCK promoter to avoid immune response against transgene product (Gene Ther. 9: 1576, 2002), designated AAV2-MCKΔCS1. The vector was injected into anterior tibial (TA) muscles of 10-day-old and 5-week-old mdx mice. Mdx muscles show no obvious changes of degeneration at 10-day, whereas 5-week-old mdx muscles exhibit active cycles of muscle degeneration/regeneration.

[Result] When the AAV2-MCKΔCS1 was injected at 5 weeks of age, dystrophin-positive fibers were 51.5 ± 17.3% at 24 weeks after the injection. The ratio of centrally nucleated fibers in ΔCS1-positive fibers was significantly reduced compared with that of ΔCS1-negative fibers, indicating protective function of ΔCS1 against muscle degeneration. Furthermore, AAV-injected muscles revealed complete recovery of the specific tetanic force.

When injected at 10-day-old, ΔCS1-positive fibers was 16.5 ± 7.0% at 24 weeks. Most of ΔCS1-positive fibers were peripherally nucleated. Surprisingly, there was no statistical difference in specific tetanic force between AAV-injected mdx muscles and B10 muscles.

To clarify the mechanism of physiological recovery due to small numbers of ΔCS1-positive fibers, we examined the relationship between myofiber hypertrophy and force generation. We found positive correlation between the muscle weight and the force generation when injected at 10-day-old. To confirm whether increased muscle weight reflected myofiber hypertrophy, we measured cross section areas (CSAs) of individual fibers. Fiber CSAs were remarkably larger in ΔCS1-positive mdx fibers compared with ΔCS1-nagative, non-treated mdx fibers, and even B10 fibers. Thus, selective hypertrophy of ΔCS1-positive fibers seemed to greatly assist contractile force generation.

[Conclusion] The AAV2-MCKΔCS1 could be a powerful tool for the gene therapy of DMD. For clinical application of this strategy to DMD patients, experiments using a bigger animal model, e.g. canine X-linked muscular dystrophy will be very important.