Limb-girdle muscular dystrophies are a genetically diverse group of diseases characterized by chronic muscle wasting and weakness. Recessive mutations in ANO5 (TMEM16E) have been directly linked to several clinical phenotypes including limb-girdle muscular dystrophy type 2L and Miyoshi myopathy type 3, although the pathogenic mechanism has remained elusive. ANO5 is a member of the Anoctamin/TMEM16 superfamily that encodes both ion channels and regulators of membrane phospholipid scrambling. The phenotypic overlap of ANO5 myopathies with dysferlin-associated muscular dystrophies has inspired the hypothesis that ANO5, like dysferlin, may be involved in the repair of muscle membranes following injury. Here we show that Ano5-deficient mice have reduced capacity to repair the sarcolemma following laser-induced damage, exhibit delayed regeneration after cardiotoxin injury and suffer from defective myoblast fusion necessary for the proper repair and regeneration of multinucleated myotubes. Together, these data suggest that ANO5 plays an important role in sarcolemmal membrane dynamics. Genbank Mouse Genome Informatics accession no. 3576659.

Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44 ± 4% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9 ± 4% of myofibers in macaques with high preexisting rAAVrh74 immunity (P < 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy.

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosome profiling. Generation of a truncated reading frame upstream of the IRES by exon skipping leads to synthesis of a functional N-truncated isoform in both patient-derived cell lines and in a new DMD mouse model, where expression protects muscle from contraction-induced injury and corrects muscle force to the same level as control mice. These results support a novel therapeutic approach for patients with mutations within the 5’ exons of DMD.

ObjectiveDysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment.MethodsTraditional gene therapy is restricted by the packaging capacity limit of adeno-associated virus (AAV), however, use of a dual vector strategy allows for delivery of over-sized genes, including dysferlin. The two vector system (AAV.DYSF.DV) packages the dysferlin cDNA utilizing AAV serotype rh.74 through the use of two discrete vectors defined by a 1 kb region of homology. Delivery of AAV.DYSF.DV via intramuscular and vascular delivery routes in dysferlin deficient mice and nonhuman primates was compared for efficiency and safety.ResultsTreated muscles were tested for dysferlin expression, overall muscle histology, and ability to repair following injury. High levels of dysferlin overexpression was shown for all muscle groups treated as well as restoration of functional outcome measures (membrane repair ability and diaphragm specific force) to wild-type levels. In primates, strong dysferlin expression was demonstrated with no safety concerns.InterpretationTreated muscles showed high levels of dysferlin expression with functional restoration with no evidence of toxicity or immune response providing proof of principle for translation to dysferlinopathy patients.

Limb-girdle muscular dystrophy type 2E (LGMD2E), resulting from mutations in β-sarcoglycan (SGCB), is a progressive dystrophy with deteriorating muscle function, respiratory failure, and cardiomyopathy in 50% or more of LGMD2E patients. SGCB knockout mice share many of the phenotypic deficiencies of LGMD2E patients. To investigate systemic SGCB gene transfer to treat skeletal and cardiac muscle deficits, we designed a self-complementary AAVrh74 vector containing a codon-optimized human SGCB transgene driven by a muscle-specific promoter. We delivered scAAV.MHCK7.hSGCB through the tail vein of SGCB mice to provide a rationale for a clinical trial that would lead to clinically meaningful results. This led to 98.1% transgene expression across all muscles that was accompanied by improvements in histopathology. Serum creatine kinase (CK) levels were reduced following treatment by 85.5%. Diaphragm force production increased by 94.4%, kyphoscoliosis of the spine was significantly reduced by 48.1%, overall ambulation increased by 57%, and vertical rearing increased dramatically by 132% following treatment. Importantly, no adverse effects were seen in muscle of wild-type mice injected systemically with scAAV.hSGCB. In this well-defined model of LGMD2E, we have demonstrated the efficacy and safety of systemic scAAV.hSGCB delivery, and these findings have established a path for clinically beneficial AAV-mediated gene therapy for LGMD2E.