Limb-girdle muscular dystrophy (LGMD) type 2D is characterized by skeletal muscle weakness and results from mutations occurring in the alpha-sarcoglycan gene. Localized in the sarcolemma, the sarcoglycans (alpha, beta, gamma, delta) are a subcomplex of the dystrophin-associated proteins (DAP). Alpha-sarcoglycan (alpha-SG) deficiency is the most common form of sarcoglycan-LGMD and no therapeutic treatments are currently available. The alpha-SG mouse model provides an opportunity to test translational treatment approaches. Prior studies have suggested that AAV-mediated alpha-SG gene transfer could not sustain expression because of transgene toxicity, potentially precluding clinical gene transfer for LGMD2D. In this paper, Dr. Rodino-Klapac and colleagues describe in vivo studies comparing alpha-SG gene expression from either the ubiquitously expressed cytomegalovirus (CMV) promoter, or muscle specific promoters that included desmin, muscle creatine kinase (MCK), and its further modification, truncated MCK (tMCK) in the alpha-SG KO mouse in the context of rAAV gene delivery. The tibialis anterior (TA) muscle of 4-6 week old alpha-SG KO mice were injected with rAAV1.alpha-SG (CMV, MCK, DES promoter) at low (3 x 109 vg) and high (3 x 1010 vg) doses. Sustained gene expression was observed irrespective of promoters at 6 and 12 weeks post gene transfer. Quantitation of alpha-SG gene expression by fiber counts yielded similar levels of myofiber transduction for CMV and MCK at the 6-week high dose, 61.4% versus 64.4% respectively. However, alpha-SG expression using the DES promoter was significantly lower at the high dose with 34% of the myofibres transduced. Similar levels of expression were seen at the 12 week time point for MCK and DES, while CMV exhibited a 25% reduction in expression. These data demonstrate sustained and robust gene expression for as long as 3 months using AAV1, with no reduction in expression using the muscle specific MCK promoter. This is well beyond the time at which transgene cytotoxic effects were previously reported using rAAV2 CMV.alpha-SG. Mononuclear cell analysis showed no evidence of infiltrating B or T cell subsets or macrophages. These findings enhance the possibility for gene therapy as a potential treatment option for LGMD2D.
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