Search

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 1012 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients.

Objective 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. Methods Traditional 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. Results Treated 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. Interpretation Treated 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.

There is no cure for dysferlinopathies, a group of related disorders caused by absent or mutant dysferlin (DYSF). Lack of dysferlin leads to progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement and fibrosis leading to deteriorating muscle weakness. Dysferlin has multiple functions including T-tubule formation and membrane repair. Pre-clinical studies show that only delivery of full-length DYSF corrects the histopathological defects in DYSF−/− mice. As DYSF is too large to be accommodated with canonical packaging, we developed a unique dual vector system using AAV to deliver and express DYSF in muscle. This two vector system (AAV.DYSF.DV) packaged in the rh.74 serotype is defined by a 1 kb region of homology between the two vectors. We compared the efficiency and safety of DYSF.DV delivery in dysferlin deficient mice and in non-human primates (NHPs) in preparation for clinical trials. Dysf−/− mice were treated by intramuscular injection, isolated limb perfusion, and systemic injection with AAV.DYSF.DV. IM injections were performed in the flexor digitorum brevis (FDB) muscle to assess expression and restoration of membrane repair capacity using ascending vector doses (6e9–6e10vg). The FDB was extracted after 10 wks and an ex vivo laser injury membrane repair assay was performed. DYSF.DV restored repair capacity in a dose-dependent manner comparable to WT findings. DYSF.DV was also efficacious when delivered through arterial circulation to hind limbs or systemically via the tail vein. A systemic dose of 6e12vg resulted in widespread gene expression exceeding 50% of muscle fibers; restoring functional deficits in the diaphragm. In NHPs, DYSF.DV delivery was analyzed for gene expression and toxicity using FLAG tag. There was no evidence of toxicity in DYSF-null mice or NHPs showing overexpression of dysferlin. Findings showed clear evidence that functional dysferlin was translated without toxicity laying the foundation for clinical trial.

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene leading to absent or mutant protein. Dysferlin protein has been implicated in multiple functional roles specifically in membrane stabilization/repair, t-tubule formation and vesicle trafficking. The loss of dysferlin causes a progressive dystrophy characterized by chronic muscle fiber loss, fat replacements and fibrosis resulting in deteriorating muscle weakness. Efforts made in the gene therapy arena with dysferlin or surrogate gene replacement have shown some efficacy in restoring membrane repair, however, only delivery of full-length DYSF has been able to correct the underlying histopathology. Therefore, there is a strong rationale to develop therapies that deliver the entire DYSF cDNA. A potential issue with this is the size of the DYSF gene (6.5 kb) which is too large for canonical AAV packaging. To circumvent this, we have developed and previously shown efficacy with a unique dual vector system using AAV to deliver and express DYSF specifically in muscle cells. This two vector system (AAV.DYSF.DV) packaged in the rh. 74 serotype is defined by a 1 kb region of homology between the two vectors. Following delivery to muscle, this overlap serves as a substrate for recombination/repair to generate the full-length gene. Our previous work studied the efficacy of this treatment strategy through intramuscular and regional vascular delivery routes. However, as generalized muscle weakness is common in dysferlinopathies, therapies targeting all muscle groups are warranted to maximize clinical efficacy. In this update, we have treated dysferlin-deficient mice systemically by intravenous injection to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at 3 and 6 months post-treatment for dysferlin expression, restoration of membrane repair capability, diaphragm specific force measurements and muscle histology. Additional animals are awaiting MRI analysis following 1 year of treatment. A single systemic dose of 6×1012 vector genomes (3×1012 vg of each vector) resulted in widespread gene expression exceeding 30% of muscle fibers. Treated muscles showed a significant decrease in central nucleation and collagen deposition. Membrane repair ability was improved toward wild-type and force deficits in the diaphragm were restored to wildtype levels. The mice showed no evidence of local or systemic toxicity, further confirming previous safety data. This study, in conjunction with our previous work, lays the foundation for clinical trial.