506. β-Sarcoglycan Gene Transfer Prevents Muscle Fibrosis and Inflammation in an Aged LGMD2E Mouse Model

Abstract

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the β-sarcoglycan (SGCB) gene causing loss of a structural protein component of the dystrophin-associated protein complex (DAPC) located at the sarcolemma. This loss results in histopathological features including chronic muscle fiber necrosis, inflammation, fat replacement and fibrosis, accompanied by deteriorating muscle strength and function. To date, no effective therapy exists to treat this debilitating disease. A suitable model for translational studies is the murine model of LGMD2E that completely lacks β-sarcoglycan (Sgcb-null mouse), and has clinical-pathological features in skeletal and cardiac muscle that replicate the human disease. Considering that a major question unanswered by gene replacement therapy is the potential efficacy of gene replacement once significant degrees of connective tissue have infiltrated dystrophic muscle, the studies described here in the β-sarcoglycan knock out mouse have particular relevance for planning future clinical trials. On the clinical side, fibrosis presents a major impasse for functional recovery. In this study, we directly addressed this question using a codon optimized human β-sarcoglycan gene (hSGCB) driven by a muscle specific tMCK promoter and the AAVrh.74 serotype (scAAVrh74.tMCK.hSGCB). We first showed restoration of expression 12 weeks post treatment following a direct intramuscular injection which was accompanied by improvements in histological parameters. Furthermore, following a clinically relevant isolated limb vascular delivery (5X10^11 vg) we found that >90% of muscle fibers expressed β-sarcoglycan in lower limb muscles. Histopathology showed a decrease in central nucleation and normalization of muscle fiber size. Immunohistochemical staining for various immune components including CD3, CD4, CD8, and macrophages showed a reduction in the numbers of immune cells following treatment. The restoration of β-sarcoglycan expression and improvement in overall histology also correlated with improvement in functional outcomes assessed by absolute and specific force generation and resistance to contraction induced injury. To measure fibrosis, quantification of picrosirius stained sections revealed a reduction of collagen deposition in TA (from 40.72 ± 1.40% to 21.22 ± 1.09%, p 80% of muscle fibers were transduced which was accompanied by a significant reduction of fibrosis similar to what was achieved following vascular delivery in younger mice. This pre-clinical study addresses the potential for gene replacement to reverse the debilitating fibrosis, typical of many of the muscular dystrophies providing momentum for movement to a clinical gene replacement for LGMD2E.