IMPORTANCE Micro-dystrophin gene transfer shows promise for treating patients with Duchenne muscular dystrophy (DMD) using recombinant adeno-associated virus serotype rh74 (rAAVrh74) and codon-optimized human micro-dystrophin driven by a skeletal and cardiac muscle-specific promoter with enhanced cardiac expression (MHCK7). OBJECTIVE To identify the 1-year safety and tolerability of intravenous rAAVrh74.MHCK7.micro-dystrophin in patients with DMD. DESIGN, SETTING, AND PARTICIPANTS This open-label, phase 1/2a nonrandomized controlled trial was conducted at the Nationwide Children's Hospital in Columbus, Ohio. It began on November 2, 2017, with a planned duration of follow-up of 3 years, ending in March 2021. The first 4 patients who met eligibility criteria were enrolled, consisting of ambulatory male children with DMD without preexisting AAVrh74 antibodies and a stable corticosteroid dose (Ն12 weeks). INTERVENTIONS A single dose of 2.0 × 10 14 vg/kg rAAVrh74.MHCK7.micro-dystrophin was infused through a peripheral limb vein. Daily prednisolone, 1 mg/kg, started 1 day before gene delivery (30-day taper after infusion). MAIN OUTCOMES AND MEASURES Safety was the primary outcome. Secondary outcomes included micro-dystrophin expression by Western blot and immunohistochemistry. Functional outcomes measured by North Star Ambulatory Assessment (NSAA) and serum creatine kinase were exploratory outcomes. RESULTS Four patients were included (mean [SD] age at enrollment, 4.8 [1.0] years). All adverse events (n = 53) were considered mild (33 [62%]) or moderate (20 [38%]), and no serious adverse events occurred. Eighteen adverse events were considered treatment related, the most common of which was vomiting (9 of 18 events [50%]). Three patients had transiently elevated γ-glutamyltransferase, which resolved with corticosteroids. At 12 weeks, immunohistochemistry of gastrocnemius muscle biopsy specimens revealed robust transgene expression in all patients, with a mean of 81.2% of muscle fibers expressing micro-dystrophin with a mean intensity of 96% at the sarcolemma. Western blot showed a mean expression of 74.3% without fat or fibrosis adjustment and 95.8% with adjustment. All patients had confirmed vector transduction and showed functional improvement of NSAA scores and reduced creatine kinase levels (posttreatment vs baseline) that were maintained for 1 year. CONCLUSIONS AND RELEVANCE This trial showed rAAVrh74.MHCK7.micro-dystrophin to be well tolerated and have minimal adverse events; the safe delivery of micro-dystrophin transgene; the robust expression and correct localization of micro-dystrophin protein; and improvements in creatine kinase levels and NSAA scores. These findings suggest that rAAVrh74.MHCK7.micro-dystrophin can provide functional improvement that is greater than that observed under standard of care. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT03375164

Duchenne muscular dystrophy (DMD) is a rare, X-linked, fatal, degenerative neuromuscular disease caused by mutations in the DMD gene. More than 2,000 mutations of the DMD gene are responsible for progressive loss of muscle strength, loss of ambulation, and generally respiratory and cardiac failure by age 30. Recently, gene transfer therapy has received widespread interest as a disease-modifying treatment for all patients with DMD. We designed an adeno-associated virus vector (rAAVrh74) containing a codon-optimized human micro-dystrophin transgene driven by a skeletal and cardiac muscle-specific promoter, MHCK7. To test the efficacy of rAAVrh74.MHCK7.micro-dystrophin, we evaluated systemic injections in mdx (dystrophin-null) mice at low (2 × 10 12 vector genome [vg] total dose, 8 × 10 13 vg/kg), intermediate (6 × 10 12 vg total dose, 2 × 10 14 vg/kg), and high doses (1.2 × 10 13 vg total dose, 6 × 10 14 vg/kg). Three months posttreatment, specific force increased in the diaphragm (DIA) and tibialis anterior muscle, with intermediate and high doses eliciting force outputs at wild-type (WT) levels. Histological improvement included reductions in fibrosis and normalization of myofiber size, specifically in the DIA, where results for low and intermediate doses were not significantly different from the WT. Significant reduction in central nucleation was also observed, although complete normalization to WT was not seen. No vector-associated toxicity was reported either by clinical or organ-specific laboratory assessments or following formal histopathology. The findings in this preclinical study provided proof of principle for safety and efficacy of systemic delivery of at high vector titers, supporting initiation of a Phase I/II safety study in boys with DMD.

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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.