Gene therapy has generated excitement as a treatment or even a potential cure for inherited diseases. Among them: Duchenne muscular dystrophy (DMD), a rare neuromuscular disorder characterized by progressive muscle weakness and degeneration. 

The most common of muscular dystrophies, DMD affects 1 in 3500 to 5000 live newborn boys, has an early childhood onset, and the most severe clinical symptoms.1,2 Though DMD is predominant in males, a minority of female carriers are symptomatic, with high variability in age at onset and disease severity.3 

Early signs of DMD include delayed motor development. The clinical signs of weakness appear between 2 and 3 years of age and usually begin in the proximal muscles before the distal limb muscles.4 Affected children may have a waddling gait, enlarged calves, and difficulty walking. Other features include cardiomyopathy, respiratory, and orthopedic complications. Varying degrees of cognitive impairment and developmental delay may be present. Patients with DMD lose ambulation by the age of 12 and die in their late teens or 20s from respiratory and cardiac complications.5 

Given the very limited number of DMD treatments available, any developments in the pipeline are welcomed. Because DMD is a genetic disease, the most promising advancements for the condition involve gene therapies. 

The Role of Dystrophin 

DMD is an X-lined disorder caused by recessive mutations in the DMD gene, leading to loss of the protein dystrophin. DMD is the largest known human gene, spanning 2.3 Mb of genomic DNA, and consists of 79 exons.6,7 About 68% of DMD mutations are from deletions of 1 or more exons; approximately 11%, from duplications; and 20%, from small mutations.7,8 Becker muscular dystrophy (BMD), caused by DMD mutations that lead to a reduced amount or size of the dystrophin protein, has a later onset and milder symptoms than DMD.9 

Dystrophin is a 427 kDa cytoplasmic protein, located primarily in skeletal and cardiac muscles. This vital component of the dystrophin-associated glycoprotein complex (DGC) connects the internal cytoskeleton of the muscle fibers to the extracellular matrix. The DGC includes the dystroglycans, sarcoglycans, integrins, and caveolin; mutations in any of these components lead to muscular dystrophies.10 Dystrophin provides structural stability to the sarcolemma of skeletal muscle, protecting it from contraction-induced mechanical injury, and acts as a scaffold for various signaling pathways.8 In the absence of dystrophin, the DGC is destabilized, leading to progressive muscle fiber damage and premature death.8,11 Activation of aberrant intracellular signaling pathways from loss of dystrophin or DGC components contributes to the pathogenesis.9,12 

Currently Approved Therapies 

No known cure for DMD exists at present, but current treatments help control symptoms. FDA-approved therapies include deflazacort (Emflaza; PTC Therapeutics) and eteplirsen (Exondys 51; Sarepta Therapeutics). Ataluren (Translarna; PTC Therapeutics) is approved by the European Medicines Agency for eligible patients with DMD but did not receive FDA approval. 
Treatment with the prednisone or deflazacort is recommended for children with DMD aged 4 years and older. The anti-inflammatory and immunosuppressive properties of these glucocorticoids may slow the decline of muscle strength and function and possibly the onset of cardiomyopathy in patients with DMD. 

A 52-week phase 3 study involving 196 boys with DMD compared the effectiveness of daily doses of either deflazacort (1 arm used 0.9 mg/kg; another, 1.2 mg/kg) or prednisone (0.75 mg/kg daily) against placebo. At 12 weeks of this 4-arm study, both prednisone and deflazacort improved muscle function compared with placebo, but deflazacort was associated with less weight gain than prednisone.13 Increased muscle strength persisted through week 52. Common adverse events (AEs) in all treatment groups were cushingoid appearance, erythema, hirsutism, weight gain, headache, and nasopharyngitis. Approved in February 2017 to treat patients aged 5 years and older, deflazacort is the first FDA-approved corticosteroid for DMD.14 

Another approach is the injection of antisense oligonucleotides such as eteplirsen, which excise selected exons (at or near a frameshift or nonsense mutation) during premessenger RNA splicing, thus correcting the disrupted reading frame and producing shortened functional dystrophin. 

About 13% of patients with DMD have mutations amenable to skipping exon 51.15 Treating these patients with the exon 51–skipping drug eteplirsen leads to increased dystrophin in muscle.16 Twelve DMD patients aged 7 to 13 years with confirmed exon 51– amenable mutations were treated with eteplirsen (30 or 50 mg/kg a week) or placebo for 24 weeks. The placebo group was switched to 30 or 50 mg/kg eteplirsen during the subsequent 24-week extension phase. At week 48, eteplirsen increased dystrophin expression by 52% and 43% in the 30 and 50 mg/kg cohorts, respectively, with no AEs.16 This study led to accelerated FDA approval of eteplirsen in September 2016 for patients with a confirmed exon 51–amenable DMD gene mutation.17 

The approval was contentious due to the study’s methodological limitations, including the small number of patients, reliance on dystrophin in muscle biopsy for outcome, and speculation around whether the level of new dystrophin expression was sufficient to produce a clinical benefit.18 An open-label extension phase with 3 years of follow-up showed increased walking performance with eteplirsen compared with historical controls.19 A phase 3 trial (NCT02255552) is underway to determine whether eteplirsen improves motor function in patients compared with an untreated control group. 

Two other exon-skipping drugs, SRP-4045 and SRP-4053 for patients with DMD mutations amenable to exon 45 or exon 53 skipping, respectively, are being evaluated in a placebo-controlled phase 3 trial (NCT02500381). 

Another agent that showed promise, ataluren, failed to receive FDA approval after review of data, including the results of 2 clinical trials (NCT00847379 and NCT01826487).20,21 The advisory panel concluded that additional well-controlled studies were needed to provide substantial evidence of ataluren’s effectiveness as therapy.22 

The small-molecule compound was developed for the treatment of patients with DMD who harbor stop mutations in the DMD gene. Ataluren allows ribosomal read-through of the stop mutations, bypassing the mutation so production of functional dystrophin continues. 

More recent preliminary data from the first international drug registry for patients with DMD showed long-term clinical benefit with ataluren compared with published natural history. Of 216 patients with DMD caused by a nonsense mutation and a median age of 9.8 years, those on ataluren had loss of ambulation at a median age of 16.5 years—up to 5 years later than seen with natural disease progression in untreated children.23 

PTC Therapeutics is recruiting patients for a 144-week placebo-controlled phase 3 trial to characterize the long-term effects of ataluren-mediated dystrophin restoration on disease progression, with expected completion by the end of 2021 (NCT03179631). 

Gene Therapies in Development 

Considerable progress has been made in gene therapies for DMD.24 Advantages of this approach include longer duration per dose and the potential to cover more patients with a single agent. Disadvantages include the possibility of immune reactions and the inability to incorporate the large full-length dystrophin into a vector.25 Most gene therapy trials using adeno-associated viral (AAV) vectors have been safety studies with direct muscle injections.26 Targeting the cardiac and diaphragm muscles is critical for patients with DMD, who typically die from cardiorespiratory complications, but these muscles are not easily accessible for direct intramuscular delivery. The use of systemic AAV delivery and muscle-specific promoters to target all the muscles, along with shortened dystrophin constructs (mini- or microdystrophin) to enable packaging into the vector, may solve these problems.25-27 

Current gene therapy trials for DMD employ either of 2 main strategies: restoring dystrophin expression or increasing the expression of a surrogate protein that compensates for dystrophin loss. 

For restoring dystrophin expression, preclinical data from murine and canine DMD models support the use of systemic AAV microdystrophin gene therapy to treat DMD.27 Three clinical trials using this approach were recently initiated. 

Jerry Mendell, MD, of Nationwide Children’s Hospital in Columbus, Ohio, conducted a phase 1/2a gene therapy clinical trial (NCT03375164) of the investigative therapy rAAVrh74.MHCK7 micro-dystrophin. AAV micro-dystrophin was infused (2 x 1014 vector genomes/kg in 10 mL/kg) via peripheral arm vein to target all muscles in the body. MHCK7 promoter was chosen based on preclinical data showing that it drives robust microdystrophin expression in the heart. In June 2018, Sarepta Therapeutics announced positive interim results.28 Preliminary results from the first 3 children dosed showed 76.2% microdystrophin expression in day 90 muscle biopsies, 87% decrease in serum creatine kinase (a biomarker for muscle damage) at day 60, and no serious AEs.28,29 

“I have been waiting my entire 49-year career to find a therapy that dramatically reduces CK levels and creates significant levels of dystrophin,” Mendell said. “Although the data are early and preliminary, these results, if they persist and are confirmed in additional patients, will represent an unprecedented advancement in the treatment of DMD.” 

The second study, a phase 1b trial (NCT03362502) by Pfizer, is evaluating the safety and tolerability of PF-06939926 in patients with DMD. In March 2018, the first patient received a single intravenous infusion of PF-06939926.30 Early data are expected in the first half of 2019. 

SGT-001 (AAV9 micro-dystrophin) is also being investigated. In a phase 1/2 study (NCT03368742) by Solid Biosciences, the drug’s safety, tolerability and efficacy will be evaluated in children and adolescents with DMD. Initial data from a prespecified interim analysis are expected in the second half of 2019.31 

Notable differences between these 3 trials include the AAV serotype and dose, promoter, micro-dystrophin configuration, patient age, and gene mutation. The Sarepta trial involves AAV-rh74, whereas AAV-9 is used in the Pfizer and Solid trials. All 3 trials include a muscle-specific promoter: MHCK7 (Sarepta), MCK (Pfizer), or CK8 (Solid). The Pfizer and Solid trials are dose-escalation studies and open to patients with all DMD mutations, whereas the Sarepta trial is a single-dose study and just for patients with frameshift or nonsense mutation between exons 18 to 58.24 

Increasing the expression of surrogate genes to substitute for the loss of dystrophin can potentially treat DMD regardless of the type or location of mutation and may have utility in other muscular dystrophies. Two notable clinical trials using surrogate gene therapy have been recently launched. 

In an ongoing phase 1/2a clinical trial (NCT03333590) by Sarepta, the gene therapy rAAVrh74.MCK.GALGT2 will be delivered via the femoral artery to the muscles of both legs of patients with DMD. The primary objective is to assess the safety of intravascular administration of AAVrh74 carrying the GALGT2 gene under the control of muscle-specific MCK promoter. The gene encodes the protein β-1,4-N-acetylgalactosaminyltransferase (GalNAc transferase) which can cause glycosylation of specific proteins including α-dystroglycan, a component of the DGC. In the mdx mouse model of DMD, viral transfer of GALGT2 results in expression of GalNAc transferase across the entire muscle membrane, along with upregulation of proteins such as utrophin, and functional improvement equivalent to that seen with microdystrophin.32 

Finally, rAAV1.CMV.huFollistatin344 (follistatin) is a secretory  protein and potent inhibitor of the myostatin signaling pathway.33 Preclinical studies with intramuscular delivery of the FS344 isoform demonstrated safety and efficacy in enhancing muscle mass.34,35 A phase 1/2a trial in patients with BMD (NCT01519349) showed that intramuscular delivery of FS344 into the quadriceps improved walking performance in 4 of the 6 patients without AEs.36 This prompted the initiation of a similar phase 1/2 trial for patients with DMD aged 7 years and older (NCT02354781). The vector will be distributed more widely than in the BMD study via multiple injections to gluteal muscles, quadriceps, and tibialis anterior muscles. 
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