RESUMO
Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the ß-sarcoglycan (SGCB) gene causing loss of functional protein and concomitant loss of dystrophin-associated proteins. The disease phenotype is characterized by muscle weakness and wasting, and dystrophic features including muscle fiber necrosis, inflammation and fibrosis. The Sgcb-null mouse recapitulates the clinical phenotype with significant endomysial fibrosis providing a relevant model to test whether gene replacement will be efficacious. We directly addressed this question using a codon optimized human ß-sarcoglycan gene (hSGCB) driven by a muscle-specific tMCK promoter (scAAVrh74.tMCK.hSGCB). Following isolated limb delivery (5 × 10(11) vector genome (vg)), 91.2% of muscle fibers in the lower limb expressed ß-sarcoglycan, restoring assembly of the sarcoglycan complex and protecting the membrane from Evans blue dye leakage. Histological outcomes were significantly improved including decreased central nucleation, normalization of muscle fiber size, decreased macrophages and inflammatory mononuclear cells, and an average of a 43% reduction in collagen deposition in treated muscle compared with untreated muscle at end point. These measures correlated with improvement of tetanic force and resistance to eccentric contraction. In 6-month-old mice, as indicated by collagen staining, scAAVrh74.tMCK.hSGCB treatment reduced fibrosis by 42%. This study demonstrates the potential for gene replacement to reverse debilitating fibrosis, typical of muscular dystrophy, thereby providing compelling evidence for movement to clinical gene replacement for LGMD2E.
Assuntos
Terapia Genética/métodos , Músculo Esquelético/efeitos dos fármacos , Sarcoglicanopatias/terapia , Sarcoglicanas/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Fibrose/genética , Fibrose/terapia , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/fisiopatologia , Distrofias Musculares/genética , Distrofias Musculares/terapia , Mutação , Sarcoglicanopatias/genética , Sarcoglicanas/metabolismoRESUMO
Duchenne muscular dystrophy is a monogenic disease potentially treatable by gene replacement. Use of recombinant adeno-associated virus (AAV) will ultimately require a vascular approach to broadly transduce muscle cells. We tested the impact of preexisting AAV antibodies on microdystrophin expression following vascular delivery to nonhuman primates. Rhesus macaques were treated by isolated limb perfusion using a fluoroscopically guided catheter. In addition to serostatus stratification, the animals were placed into one of the three immune suppression groups: no immune suppression, prednisone, and triple immune suppression (prednisone, tacrolimus, and mycophenolate mofetil). The animals were analyzed for transgene expression at 3 or 6 months. Microdystrophin expression was visualized in AAV, rhesus serotype 74 sero-negative animals (mean: 48.0 ± 20.8%) that was attenuated in sero-positive animals (19.6 ± 18.7%). Immunosuppression did not affect transgene expression. Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high-level transduction (60.8 ± 18.0%), which is comparable with that of AAV sero-negative animals (53.7 ± 7.6%), whereas non-pheresed sero-positive animals demonstrated significantly lower transduction levels (10.1 ± 6.0%). These data support the hypothesis that removal of AAV binding antibodies by plasmapheresis permits successful and sustained gene transfer in the presence of preexisting immunity (natural infection) to AAV.
Assuntos
Dependovirus/imunologia , Distrofina/genética , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Plasmaferese , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Dependovirus/genética , Genes Reporter , Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde/genética , Humanos , Macaca mulatta , Masculino , Músculo Esquelético/metabolismo , Plasmaferese/métodos , Transdução Genética , TransgenesRESUMO
BACKGROUND: Alpha-sarcoglycan (alpha-SG) deficiency (limb-girdle muscular dystrophy [LGMD] type 2D) is the most common form of sarcoglycan-LGMD. No treatment is currently available. Prior studies suggest that overexpression of alpha-SG via adeno-associated virus (AAV)-mediated gene transfer results in poorly sustained gene expression related to transgene toxicity. These findings potentially preclude gene therapy as a treatment approach for LGMD2D. METHODS: The human alpha-SG gene (halpha-SG) was directly transferred to the tibialis anterior muscle of 4- to 5-week-old alpha-SG KO mice using AAV, type 1. The gene was placed under control of 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). Low (3 x 10(9) vg) and high (3 x 10(10) vg) doses of AAV1.halpha-SG were administered. RESULTS: 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 both MCK promoters (60 to 70%), while 34% of fibers were transduced with the DES promoter. There was a trend toward lower expression at the 12-week time point with the CMV promoter. Western blot analysis revealed alpha-SG overexpression using CMV and both the MCK promoters. CONCLUSION: Our data demonstrate robust and sustained adeno-associated virus type 1 alpha-sarcoglycan gene expression under control of muscle creatine kinase promoters, without evidence of cytotoxicity. These findings support the use of gene therapy as a potential treatment approach for limb-girdle muscular dystrophy type 2D.