An in-depth analysis of the genetics of DMD and disease modifiers such as the NF-kB pathway in patients with Duchenne muscular dystrophy.
Duchenne muscular dystrophy (DMD) is among the most severe forms of inherited muscular dystrophies and the most common hereditary neuromuscular disease, affecting approximately 1 in 5000 live male births.1-3
DMD is a recessive X-linked disorder that arises from the absence of functional dystrophin protein in the skeletal muscle membrane, which is driven by mutations in the DMD gene located on chromosome Xp21.1,3
Dystrophin, a member of the spectrin superfamily of cytoskeletal proteins, is expressed mainly in skeletal and cardiac muscle, and to a lesser extent in the brain. In healthy muscles, dystrophin is found on the intracellular surface of the sarcolemma, along with the sarcomeres. Along with the other proteins that form the dystrophin-associated protein complex (DAPC), dystrophin is important for maintenance of membrane stability in striated muscle cells.4,5
Loss-of-function mutations in DMD or in the genes encoding other proteins in the DAPC, triggers instability of the plasma membrane, calcium ion influx, and myofiber loss, leading to repeated cycles of myonecrosis and regeneration associated with inflammation and repair, ulti- mately rendering muscles more vulnerable to mechanical injury.5,6
DMD is usually diagnosed in children between 2 and 5 years of age, with the appearance of motor developmental delay and an abnormal “waddling” gait, calf muscle pseudohypertrophy, and weakened proximal muscles.1,2
As the patient ages, progressive muscle degeneration occurs, resulting in loss of ambulation when the child is aged between 8 and 12 years.1,2
In addition, respiratory insufficiency, musculoskeletal deformities, and cardiomyopathy also progress with age.1
Cognitive impairment and behavior problems are also present, although these effects are not progressive.1
Respiratory and cardiac complications in patients with DMD lead to premature death at 20 to 30 years of age.2,3
Genetics of DMD and Disease Modifiers
Mutations in the DMD gene form the basis of dystrophin dysfunction and loss in DMD. Dystrophin dysfunction is profound in most patients with DMD, who typically have less than 5% of the normal level of the dystrophin protein.3
The DMD gene is the largest gene in the human genome, with one of the highest spontaneous mutation rates.1,2
DMD, which contains 79 exons and 2.5 Mb of DNA, encodes the 427-kDa dystrophin protein.3 Dystrophin protein has an N-terminal actin-binding domain, a central rod domain, and a C-terminal domain that binds to the DAPC at the plasma membrane.1
The rod domain is composed of 24 spectrin-like repeats, interspersed with 4 hinge domains.1
Many different DMD mutations have been reported in patients with DMD; de novo DMD mutations occur often, in an estimated 12% to 33% of patients.1
Most mutations (70% to 80%) are deletions or duplications; 69% of patients with DMD carry large deletions
in DMD, whereas 11% have large duplications.1,3
Point mutations are seen in 20% to 30% of patients, whereas 10% have nonsense mutations, 7% have missense mutations or small indels, and 3% have intronic or other mutations.1,3
A model of DMD pathogenesis is predicated on the hypothesis that deletions that disrupt the reading frame are likely to result in a more severe DMD phenotype, whereas those that maintain the reading frame result in milder DMD phenotypes or Becker muscular dystrophy; this model has been validated in about 91% of patients with DMD.1
This reading-frame hypothesis has clinical significance not only due to its implications for the phenotypic variability of DMD presentation but also because some clinical interventions that have gained recent regulatory approval or are under development incorporate a strategy called exon skipping. These interventions involve the use of synthetic antisense oligonucleotides that cause an exon adjacent to a deletion mutation to be spliced out during mRNA processing. Although the resulting dystrophin protein is slightly shortened, the change prevents disruption of the reading frame, thus avoiding the worse fate of a drastically truncated or unstable protein.1,7-9
The phenotypic variability in DMD is thought to stem not only from the wide range of mutations but also from genetic pathways interacting with and modifying the effects of the primary DMD mutation. Environmental influences such as exercise and nutri- tion are thought to affect phenotypic variability as well. DMD disease modifiers identified using combinations of transcriptome and genome profiling include SPP1 (which encodes osteopontin), LTBP4 (encoding latent transforming growth factor β–binding protein 4), Anxa6 (encoding annexin A6), THBS1 (encoding throm- bospondin-1), ACTN3 (encoding α-actinin 3), and more recently, CD40 (encoding the immune costimulatory receptor CD40, an activator of the nuclear factor κB [NF-κB] pathway).10-12
A minor allele in the 5ʹ-untranslated region of CD40 was associated with earlier loss of ambulation, a clinically meaningful measure of DMD severity.12
SPP1, LTBP4, CD40, and THBS1 have been implicated in several interconnected pathways that modulate inflammatory response to muscle damage, regeneration, and fibrosis, whereas a common truncation of the ACTN3 gene reduces muscle power and sprint performance.10,11
These modifiers may be of clinical interest in DMD treatment, as actionable targets, to improve the function of the dystrophin pathway in patients with DMD.11
Current and Emerging Pharmacological Management of DMD Currently, there is no medical cure for DMD, and the disease has a poor prognosis.3
DMD treatment is centered on glucocorticoid therapy, prevention of contractures, and medical care of cardiomyopathy and respiratory compromise.3
Glucocorticoid steroids, such as prednisone or deflazacort, are the mainstay of DMD treatment; however, tolerance to chronic use, toxicity with long-term use, and heterogeneous treatment response are some critical disadvantages of glucocorticoid therapy in DMD4 despite improvement in muscle function and reduced need for scoliosis surgery, among other improved clinical outcomes.1,2
Current and emerging treatments for DMD rely on 2 major strategies: They either target the primary defect, such as therapies that seek to restore dystrophin expression and function, or they seek to mitigate the secondary and downstream consequences of dystrophin loss, such as treatments that target NF-κB–mediated inflammation, fibrosis, or muscle wasting (TABLE 1).1,2,7,13-20
The NF-κB Pathway in Dystrophic Pathogenesis
NF-κB is a multifunctional, ubiquitously expressed transcription factor with pleiotropic functions in cell survival, apoptosis, growth, and differentiation. NF-κB is also involved in modulating cellular responses to stress, as well as immune/inflamma- tory responses and skeletal and muscle development.17,21
During canonical NF-κB activation, the p65 transactivation subunit of the NF-κB p50/p65 heterodimer is tightly regulated by the inhibitory protein IκB. Upon activation of the NF-κB pathway, the upstream inhibitor of NF-κB kinase kinase (IKK) is phosphorylated, and in turn it phosphorylates IκB.2,21
Subsequent proteasomal degradation of IκB unmasks a nuclear localization site on p65, allowing NF-κB to translocate to the nucleus, bind to target genes and activate their expression.22
The activation of the NF-κB inflammatory pathway mediates the infiltration of immune cells into dystrophic muscles; indeed, immunoreactivity for activated NF-κB was reported in all regenerating fibers and signif- icant proportions (20%-40%) of necrotic fibers in DMD muscles.23
The key role of this inflammatory pathway in DMD has been demonstrated in preclinical cellular and animal model studies. For instance, in the mdx mouse, a model of DMD lacking dystrophin, skeletal muscle–specific activation of NF-κB was shown to precede the onset of dystrophic damage.23 NF-κB–mediated overexpression of inflammatory cytokines has been reported in dystrophic muscles; for instance, the levels of tumor necrosis factor-α were approximately 1000 times higher in serum samples from patients with DMD than in those from controls.23
Mouse model studies also showed that p65 haplo- insufficiency was associated with increased cell proliferation and myogenic differentia- tion; moreover, pharmacologic inhibition of the IKK/NF-κB pathway enhanced myogenic differentiation, improving muscle regeneration.23-25
NF-κB activation in skeletal muscle in DMD is a consequence of the loss of sarcolemmal dystrophin. This activation has been shown to be an early event in DMD pathology, even occurring in the muscles of infants with DMD prior to the onset of fibrosis or clinical manifestations.17
Suppression of NF-κB activation (even by only 50%) in animal models of DMD, via pharmacological inhi- bition or genetic manipulation, improves histology, function, and regeneration of muscle. This suggests that inhibition of this inflammatory pathway may help mitigate secondary muscle-directed effects in patients with DMD.17,24
Two agents that target NF-κB– mediated inflammation, vamorolone and edasalonexent, are currently in advanced-phase clinical trials.
Anti-inflammatory NF-κB–targeted Agents in DMD
Inhibition of NF-κB–mediated inflammatory responses is one of the primary mechanisms underlying the clinical effects of the steroidal agents that are the currently accepted mainstay of DMD treatment.2
However, due to the adverse effects associated with chronic and long-term use of glucocorticoids, and increasing knowledge and availability of agents that can target the NF-κB pathway, devel- opment of inhibitors of this pathway with potential clinical efficacy and acceptable safety profiles is of interest in DMD treatment. Orally bioavailable NF-κB inhibitors, including edasalonexent and the steroid analogue vamorolone, are currently being investigated in clinical studies in patients with DMD (TABLE 2).16,26-28
Edasalonexent is an orally administered, novel, small-molecule inhibitor of NF-κB in which salicylic acid and docosahexaenoic acid (DHA) are covalently conjugated through an ethylenediamine linker that is hydrolyzed intracellularly only by the fatty acid amide hydrolase.17,22
Preclinical studies have demonstrated that salicylic acid prevents NF-κB–mediated muscle atrophy; DHA suppresses NF-κB–mediated inflammation and is metabolized intracellularly to anti-inflammatory eicosanoids, which can also improve muscle fiber regeneration.17
The design of this agent allows for synergistic action of its linked compounds to inhibit NF-κB, likely enabling the higher potency of edasalonexent compared with that of each of its individual components.17,22
Data from a phase 1 clinical program for edasalonexent, which included 3 placebo-controlled studies in adults, were recently reported.17
The data showed that edasalonexent was safe and well tolerated and that it inhibited NF-κB pathway activation, with significant decreases in the expression of genes associated with the NF-κB pathway and proteasome in peripheral mononuclear cells (P = .02 and P = .002, respectively) after 2 weeks of edasalonexent treatment.17
A subsequent phase 1 study evaluated the safety, tolerability, pharmacokinetics, and exploratory pharmacodynamics of 3 doses of edasa- lonexent in pediatric patients (ambulatory males aged between 4 and <8 years) with genetically confirmed DMD.28
In this 1-week, open-label, multiple-dose study with 3 sequential ascending doses (33, 67, and 100 mg/kg/day) of edasalonexent, all doses were well tolerated, with no serious adverse events, no drug discontinuations, and no dose reductions. Moreover, levels of NF-κB–regulated genes and serum proteins were decreased following treatment with edasalonexent for
7 days. Given that the edasalonexent-mediated inhibition of NF-κB is independent of the underlying DMD mutation, the authors noted that this agent might have broad applicability in all patients with DMD and may provide clinical efficacy either as a monotherapy or in combina- tion with dystrophin-restoration therapies. Along these lines, a recent report outlined delivery of NF-κB/p65-directed small hairpin RNA in combination with mini-dystrophin replacement gene therapy on the same adenoviral vector in a mouse DMD model29
; improvement in overall health of the double-knockout mice was observed, suggesting that combining dystrophin-remediation therapies with NF-κB– targeted agents might enable additive therapeutic effects in DMD.29
Vamorolone is a first-in-class steroidal drug with a high degree of chemical and 3-dimensional structural homology to glucocorticoids.16
Although vamorolone also binds glucocorticoid receptors (GRs) and mineralocorticoid receptors with an affinity similar to that of glucocorticoids, vamorolone/GR complexes do not dimerize and exhibit decreased gene transcriptional activity compared with glucocorticoid/GR complexes.16
Due to these key mechanistic differences, vamorolone is thought to exert membrane-stabilizing and anti-inflammatory properties, including inhibition of NF-κB, without the significant immunosuppressive and hormonal effects that are char- acteristic of glucocorticoids. Phase 1 clinical studies have indicated that the typical adverse effects of steroids were greatly attenuated in patients treated with this novel steroid analogue26,30
; for instance, suppression of the adrenal axis was 10-fold lower with vamorolone than with prednisone.26
In a 2-week, open-label, phase 2, multiple ascending-dose study (0.25, 0.75, 2.0, and 6.0 mg/kg/day), vamorolone’s clinical safety, pharmacokinetics, and pharmacody-namic biomarkers were evaluated in 48 boys with DMD (aged 4 to <7 years).16
Vamorolone was safe and well tolerated through the highest dose tested, with a pharmacokinetic profile comparable to that of prednisolone. Moreover, biomarker analysis indicated an improved safety profile compared with glucocorticoids, including reduction of insulin resistance, beneficial changes in bone turnover, and a reduc- tion in adrenal suppression.16
A subsequent study found that the 2.0 mg/kg/day dosage group met the primary efficacy outcome of improved muscle function (time to stand; 24 weeks of vamorolone treatment vs natural history controls) without evidence of most of the adverse effects seen with glucocorticoids.27
The emergence of clinically useful NF-κB–targeted agents with the potential to arrest or reverse inflammatory responses and muscle degeneration in patients with DMD, without the adverse events associated with glucocorticoids, provides a novel option for DMD treatment that is agnostic to the primary DMD mutation. Such agents hold significant promise as monotherapy or in combination with other classes of agents for the treatment of all patients with DMD, a possibility that provides new hope for tackling this currently incurable progressive disease.
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