Duchenne muscular dystrophy (DMD) is a rare, X-linked, fatal, degenerative neuromuscular disease caused by dystrophin gene (DMD) mutations. Estimated incidence worldwide is 1 in 5000 live male births.^1,2 The DMD gene (OMIM 300377) encodes for dystrophin, a 427-kDa cytoskeletal protein required for sarcolemmal stability. Protein loss leads to susceptibility to repeated cycles of necrosis and regeneration as well as diminished regenerative muscle capacity, resulting in fat and connective tissue replacement (fibrosis).¹ DMD is progressive, beginning with loss of ambulation between age 9 and 14 years, followed by respiratory complications and cardiac function decline, and ending in death.^3-5

Duchenne muscular dystrophy (DMD) is a rare, X-linked neuromuscular disease caused by pathogenic variants in the DMD gene that result in the absence of functional dystrophin, beginning at birth and leading to progressive impaired motor function, loss of ambulation and life-threatening cardiorespiratory complications. Delandistrogene moxeparvovec, an adeno-associated rh74-viral vector-based gene therapy, addresses absent functional dystrophin in DMD. Here the phase 3 EMBARK study aimed to assess the efficacy and safety of delandistrogene moxeparvovec in patients with DMD. Ambulatory males with DMD, ≥4 years to <8 years of age, were randomized and stratified by age group and North Star Ambulatory Assessment (NSAA) score to single-administration intravenous delandistrogene moxeparvovec (1.33 × 10¹⁴ vector genomes per kilogram; n = 63) or placebo (n = 62). At week 52, the primary endpoint, change from baseline in NSAA score, was not met (least squares mean 2.57 (delandistrogene moxeparvovec) versus 1.92 (placebo) points; between-group difference, 0.65; 95% confidence interval (CI), −0.45, 1.74; P = 0.2441). Secondary efficacy endpoints included mean micro-dystrophin expression at week 12: 34.29% (treated) versus 0.00% (placebo). Other secondary efficacy endpoints at week 52 (between-group differences (95% CI)) included: Time to Rise (−0.64 (−1.06, −0.23)), 10-meter Walk/Run (−0.42 (−0.71, −0.13)), stride velocity 95th centile (0.10 (0.00, 0.19)), 100-meter Walk/Run (−3.29 (−8.28, 1.70)), time to ascend 4 steps (–0.36 (−0.71, −0.01)), PROMIS Mobility and Upper Extremity (0.05 (−0.08, 0.19); −0.04 (−0.24, 0.17)) and number of NSAA skills gained/improved (0.19 (−0.67, 1.06)). In total, 674 adverse events were recorded with delandistrogene moxeparvovec and 514 with placebo. There were no deaths, discontinuations or clinically significant complement-mediated adverse events; 7 patients (11.1%) experienced 10 treatment-related serious adverse events. Delandistrogene moxeparvovec did not lead to a significant improvement in NSAA score at week 52. Some of the secondary endpoints numerically favored treatment, although no statistical significance can be claimed. Safety was manageable and consistent with previous delandistrogene moxeparvovec trials. ClinicalTrials.gov: NCT05096221

Given the increasing number of gene transfer therapy studies either completed or underway, there is growing attention to the importance of preexisting adaptive immunity to the viral vectors used. The recombinant viral vectors developed for gene transfer therapy share structural features with naturally occurring wild-type virus. Antibodies generated against viral vectors obtained through a previous exposure to wild-type virus can potentially compromise transgene expression by blocking transduction, thereby limiting the therapeutic efficacy of the gene transfer therapy; they may also pose potential safety concerns. Therefore, systemic gene transfer delivery requires testing patients for preexisting antibodies. Two different assays have been used: (1) binding assays that focus on total antibodies (both neutralizing and non-neutralizing) and (2) neutralizing assays that detect neutralizing antibodies. In this review we focus on adeno-associated virus-based gene therapies, describing the immune response that occurs to naturally occurring adeno-associated viruses, the implications for patients with this exposure, the assays used to detect preexisting immune responses, and strategies to circumvent preexisting adaptive immunity to expand the patient base that could benefit from such therapies.

Introduction: Delandistrogene moxeparvovec (SRP-9001) is an investigational gene transfer therapy designed for targeted expression of SRP-9001 dystrophin protein, a shortened dystrophin retaining key functional domains of the wild-type protein.Methods: This Phase 2, double-blind, two-part (48 weeks per part) crossover study (SRP-9001-102 [Study 102]; NCT03769116) evaluated delandistrogene moxeparvovec in patients, aged ≥4 to <8 years with Duchenne muscular dystrophy. Primary endpoints (Part 1) were change from baseline (CFBL) in SRP-9001 dystrophin expression (Week 12), by Western blot, and in North Star Ambulatory Assessment (NSAA) score (Week 48). Safety assessments included treatment-related adverse events (TRAEs). Patients were randomized and stratified by age to placebo (n = 21) or delandistrogene moxeparvovec (n = 20) and crossed over for Part 2.Results: SRP-9001 dystrophin expression was achieved in all patients: mean CFBL to Week 12 was 23.82% and 39.64% normal in Parts 1 and 2, respectively. In Part 1, CFBL to Week 48 in NSAA score (least-squares mean, LSM [standard error]) was +1.7 (0.6) with treatment versus +0.9 (0.6) for placebo; p = 0.37. Disparity in baseline motor function between groups likely confounded these results. In 4- to 5-year-olds with matched baseline motor function, CFBL to Week 48 in NSAA scores was significantly different (+2.5 points; p = 0.0172), but not significantly different in 6-to-7-year-olds with imbalanced baseline motor function (−0.7 points; p = 0.5384). For patients treated with delandistrogene moxeparvovec in Part 2, CFBL to Week 48 in NSAA score was +1.3 (2.7), whereas for those treated in Part 1, NSAA scores were maintained. As all patients in Part 2 were exposed to treatment, results were compared with a propensity-score-weighted external control (EC) cohort. The LSM difference in NSAA score between the Part 2 treated group and EC cohort was statistically significant (+2.0 points; p = 0.0009). The most common TRAEs were vomiting, decreased appetite, and nausea. Most occurred within the first 90 days and all resolved.Discussion: Results indicate robust expression of SRP-9001 dystrophin and overall stabilization in NSAA up to 2 years post-treatment. Differences in NSAA between groups in Part 1 were not significant for the overall population, likely because cohorts were stratified only by age, and other critical prognostic factors were not well matched at baseline.

Introduction: The development of adeno-associated virus (AAV) vectors as safe vehicles for in vivo delivery of therapeutic genes has been a major milestone in the advancement of gene therapy, enabling a promising strategy for ameliorating a wide range of diseases, including Duchenne muscular dystrophy (DMD). Areas covered: Based on experience with development of a gene transfer therapy agent for DMD, we discuss ways in which gene therapy for rare disease challenges traditional clinical development paradigms, and recommend a step-wise approach for design and evaluation to support broader applicability of gene therapy. Expert opinion: The gene therapy development approach should intentionally design the therapeutic construct and the clinical study to systematically evaluate agent delivery, safety, and efficacy. Rigorous preclinical work is essential for establishing an effective gene delivery platform and determining the efficacious dose. Clinical studies should thoroughly evaluate transduction, on-target transgene expression at the tissue and cellular level, and functional efficacy.

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 rAAVrh74.MHCK7.micro-dystrophin at high vector titers, supporting initiation of a Phase I/II safety study in boys with DMD.