To achieve dystrophin replacement gene therapy for Duchenne Muscular Dystrophy (DMD), the dystrophin protein must be truncated to microdystrophin due to the limited packaging capacity of adeno-associated viruses (AAV). RGX-202 is a recombinant AAV8 vector with an optimized human mirocystrophin transgene and a promoter designed to increase expression in muscle (Spc5-12). The protein coding sequence has been codon optimized; depleted of CpG dinucleotides; and includes 4 rods, 3 hinges, and an extended coding region of the carboxyl terminal (CT) domain containing both the alpha-syntrophin and alpha-dystrobrevin binding sites. Here, we present data supporting potential benefits of the added CT domain on microdystrophin expression levels, Dystrophin Associated Protein Complex (DAPC) recruitment at the sarcolemma, and a novel finding of satellite cell correction in the mdx mouse model.
RGX-202 transgene expression was compared to shorter versions of microdystrophins containing different lengths of CT domain via in vitro infection. Western blot showed RGX-202 had the strongest protein expression level. Different versions of microdystrophin vectors were further evaluated in mdx mice, a muscular dystrophic mouse model, via intravenous administration. RGX-202 again had the highest microdystrophin expression in skeletal muscles and recruited more DAPC components as compared to its counterparts with shorter CT-domain. Furthermore, we observed that the numbers of Pax7+ satellite cells were decreased in RGX-202 treated muscle as measured by RNAScope, suggesting amelioration of muscle regeneration by the treatment.
In conclusion, increased microdystrophin protein levels both in vitro and in vivo were observed with the addition of the CT domain in RGX-202. In mdx mouse, treatment with RGX-202 was shown to restore the DAPC at the sarcolemma and prevent the pathological cycle of degeneration / regeneration that ultimately results in muscle wasting. This initial characterization provides a rationale for the further development of RGX-202 as a microdystrophin gene therapy treatment for DMD.