K. Heller

AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models

Dysferlinopathies 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.

622. Systemic β-Sarcoglycan Gene Therapy for Treatment of Cardiac and Skeletal Muscle Deficits in LGMD2E

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the beta-sarcoglycan (SGCB) gene causing loss of a sarcolemmal structural protein component of the dystrophin-associated protein complex (DAPC). This leads to a progressive dystrophy with numerous histopathological features, resulting in deteriorating muscle function. This occurs not only in limb muscle but also in the diaphragm and the heart. Consequences include respiratory failure and cardiomyopathy in 50% or more of LGMD2E patients. SGCB knockout mice share many of the phenotypic deficiencies of LGMD2E patients. In this mouse model we quantified dystrophic histopathology, fibrosis, and functional outcomes in lower limb, upper limb, and torso skeletal muscles, as well as the diaphragm and cardiac muscles. Diaphragms of SGCB-/- mice demonstrated reduced specific force output (116.24 mN/mm2) compared to wild-type (WT) mice (236.67 mN/mm2), and hearts from SGCB-/- mice had lower ejection fraction (58.19%) compared to WT mice (75.04%) as determined by MRI. Additionally, radiographic imaging defined the degree of kyphoscoliosis in SGCB-/- mice. Laser monitoring of open-field cage activity showed a reduction of ~55% in both total ambulation as well as hindlimb vertical rearing in SGCB-/- mice compared to WT. For treatment, we designed a self-complementary AAVrh74 vector containing a codon optimized human SGCB transgene driven by a muscle specific promoter. We next demonstrated efficacy of vector delivery by intramuscular (IM) injection to the tibialis anterior muscle, as well as isolated-limb perfusion (ILP) to the lower limb muscles of SGCB-/- mice. Along with restoration of SGCB expression in treated muscle, we saw histological and functional improvements and a reduction in fibrosis as indicated by reduced collagen deposition. These regional gene delivery studies were followed by systemic delivery of scAAV.hSGCB through the tail vein of SGCB-/- mice to provide a potential rationale for gene delivery in clinical trial that would lead to clinically meaningful results. Tail vein injection of scAAV.hSGCB resulted in nearly 100% transgene expression in numerous muscles throughout the hindlimbs, forelimbs, torso, and the heart, that was accompanied by improvements in histopathology including reduction in central nucleation and increased fiber diameter. Kyphoscoliosis of the spine was also improved, and total ambulation increased in scAAV.hSGCB treated mice by ~22% while hindlimb vertical rearing increased by ~77% in treated mice compared to KO. We also saw complete restoration of diaphragm function following treatment with specific force output improved to 226.07 mN/mm2. In this well-defined mode of LGMD2E, we have demonstrated that systemic delivery of scAAV.hSGCB normalizes histologic and functional outcome measures in limb, diaphragm, and heart. These findings have established a path for AAV mediated gene therapy for LGMD2E that we are currently pursuing.

G.P.94

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon. We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform. We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1. U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Notably, utrophin levels remain unchanged. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD. The efficiency of this treatment in inducing expression of the N-truncated dystrophin in 6 patient cell lines with different 5 ′ mutations is underway, and will be presented as well.

G.O.26

Duchenne Muscular Dystrophy (DMD) is the most common, severe neuromuscular disorder caused by mutations in the DMD gene. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle, that like the dystrophin–glycoprotein complex, links the extracellular matrix (ECM) to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and thus overexpression will not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically to the more severe mouse model of DMD, the mdx/utrn−/− mouse deficient for both dystrophin and utrophin at 2–4days of age. We demonstrated widespread expression of ITGA7 at the sarcolemma to multiple muscle groups following gene transfer eight weeks post injection. The increased expression of ITGA7 significantly extended longevity and reduced kyphosis and joint contractures, common to the mdx/utrn−/− mouse. The additional α 7 expression significantly protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles eight weeks post gene transfer. Taken together, these results show that this therapeutic approach continues to demonstrate promise as a viable treatment for DMD.