Duchenne muscular dystrophy is initiated by dystrophin deficiency, but downstream pathophysiological pathways such as membrane instability, NFĸB activation, mitochondrial dysfunction, and induction ...of TGFβ fibrosis pathways are thought to drive the disability. Dystrophin replacement strategies are hopeful for addressing upstream dystrophin deficiency; however, all methods to date use semi-functional dystrophin proteins that are likely to trigger downstream pathways. Thus, combination therapies that can target multiple downstream pathways are important in treating DMD, even for dystrophin-replacement strategies. We sought to define blood pharmacodynamic biomarkers of drug response in the mdx mouse model of Duchenne muscular dystrophy using a series of repurposed drugs. Four-week-old mdx mice were treated for four weeks with four different drugs singly and in combination: vehicle, prednisolone, vamorolone, rituximab, β-aminoisobutyric acid (BAIBA) (11 treatment groups; n = 6/group). Blood was collected via cardiac puncture at study termination, and proteomic profiling was carried out using SOMAscan aptamer panels (1,310 proteins assayed). Prednisolone was tested alone and in combination with other drugs. It was found to have a good concordance of prednisolone-responsive biomarkers (56 increased by prednisolone, 39 decreased) focused on NFκB and TGFβ cascades. Vamorolone shared 45 (80%) of increased biomarkers and 13 (33%) of decreased biomarkers with prednisolone. Comparison of published human corticosteroid-responsive biomarkers to our mdx data showed 14% (3/22) concordance between mouse and human. Rituximab showed fewer drug-associated biomarkers, with the most significant being human IgG. On the other hand, BAIBA treatment (high and low dose) showed a drug-associated increase in 40 serum proteins and decreased 5 serum proteins. Our results suggest that a biomarker approach could be employed for assessing drug combinations in both mouse and human studies.
We aimed to perform an observational study of age at loss of independent ambulation (LoA) and side-effect profiles associated with different glucocorticoid corticosteroid (GC) regimens in Duchenne ...muscular dystrophy (DMD).
We studied 340 participants in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS). LoA was defined as continuous wheelchair use. Effects of prednisone or prednisolone (PRED)/deflazacort (DFZ), administration frequency, and dose were analyzed by time-varying Cox regression. Side-effect frequencies were compared using χ(2) test.
Participants treated ≥1 year while ambulatory (n = 252/340) showed a 3-year median delay in LoA (p < 0.001). Fourteen different regimens were observed. Nondaily treatment was common for PRED (37%) and rare for DFZ (3%). DFZ was associated with later LoA than PRED (hazard ratio 0.294 ± 0.053 vs 0.490 ± 0.08, p = 0.003; 2-year difference in median LoA with daily administration, p < 0.001). Average dose was lower for daily PRED (0.56 mg/kg/d, 75% of recommended) than daily DFZ (0.75 mg/kg/d, 83% of recommended, p < 0.001). DFZ showed higher frequencies of growth delay (p < 0.001), cushingoid appearance (p = 0.002), and cataracts (p < 0.001), but not weight gain.
Use of DFZ was associated with later LoA and increased frequency of side effects. Differences in standards of care and dosing complicate interpretation of this finding, but stratification by PRED/DFZ might be considered in clinical trials. This study emphasizes the necessity of a randomized, blinded trial of GC regimens in DMD.
This study provides Class IV evidence that GCs are effective in delaying LoA in patients with DMD.
We sought to determine the mechanisms underlying failure of muscle regeneration that is observed in dystrophic muscle through hypothesis generation using muscle profiling data (human dystrophy and ...murine regeneration). We found that transforming growth factor β-centered networks strongly associated with pathological fibrosis and failed regeneration were also induced during normal regeneration but at distinct time points. We hypothesized that asynchronously regenerating microenvironments are an underlying driver of fibrosis and failed regeneration. We validated this hypothesis using an experimental model of focal asynchronous bouts of muscle regeneration in wild-type (WT) mice. A chronic inflammatory state and reduced mitochondrial oxidative capacity are observed in bouts separated by 4 d, whereas a chronic profibrotic state was seen in bouts separated by 10 d. Treatment of asynchronously remodeling WT muscle with either prednisone or VBP15 mitigated the molecular phenotype. Our asynchronous regeneration model for pathological fibrosis and muscle wasting in the muscular dystrophies is likely generalizable to tissue failure in chronic inflammatory states in other regenerative tissues.
Exon-Skipping in Duchenne Muscular Dystrophy Takeda, Shin'ichi; Clemens, Paula R; Hoffman, Eric P
Journal of neuromuscular diseases,
11/2021, Letnik:
8, Številka:
s2
Journal Article
Recenzirano
Odprti dostop
Duchenne muscular dystrophy (DMD) is a devastating, rare disease. While clinically described in the 19th century, the genetic foundation of DMD was not discovered until more than 100 years later. ...This genetic understanding opened the door to the development of genetic treatments for DMD. Over the course of the last 30 years, the research that supports this development has moved into the realm of clinical trials and regulatory drug approvals. Exon skipping to therapeutically restore the frame of an out-of-frame dystrophin mutation has taken center stage in drug development for DMD. The research reviewed here focuses on the clinical development of exon skipping for the treatment of DMD. In addition to the generation of clinical treatments that are being used for patient care, this research sets the stage for future therapeutic development with a focus on increasing efficacy while providing safety and addressing the multi-systemic aspects of DMD.
Becker muscular dystrophy is caused by mutations in the DMD gene that permit significant residual dystrophin protein expression in patient muscle. This is in contrast to DMD gene mutations in ...Duchenne muscular dystrophy where little or no dystrophin is produced (typically < 3% normal levels). Clinically, Becker muscular dystrophy is extremely variable, from slightly milder than DMD, to asymptomatic hyperCKemia at old age. The factors driving clinical variability in Becker muscular dystrophy have now been studied in some depth, and the findings are likely highly relevant to anticipated clinical findings in exon skipping therapy in DMD. The specific mutations in Becker dystrophy play an important role, and clinical variability is less with high frequency mutations (deletions exons 45-47, 45-48). The percentage of dystrophin content in patient muscle is not well-correlated with clinical findings. Muscle MRI findings (degree of fibrofatty replacement) are very well-correlated with the degree of patient disability, regardless of mutation or muscle dystrophin content. Taken together, data to date suggest that the main determinant driving clinical disability in Becker dystrophy patients is the degree of fibrofatty replacement in muscle. Thus, as with DMD, DMD gene mutations and resulting dystrophin protein abnormalities initiate the disease process, but downstream tissue pathophysiology plays a dominant role in disease progression. Factors influencing the age-dependent rate of fibrofatty replacement of muscles are responsible for much of the clinical variability seen in Becker dystrophy, as well as Duchenne dystrophy. These fibrosis-related factors include genetic modifiers, degree of muscle inflammation, and induction of microRNAs in muscle that bind to dystrophin mRNA and down-regulate dystrophin protein content in patient muscle. Studies to date regarding clinical variability in Becker dystrophy suggest that exon skipping therapy in DMD may show variable efficacy from patient to patient.
Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of ...frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45–55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45–55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45–55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45–55 skipping in a dystrophin-deficient animal model.
Glucocorticoid treatment is recommended as a standard of care in Duchenne muscular dystrophy; however, few studies have assessed the long-term benefits of this treatment. We examined the long-term ...effects of glucocorticoids on milestone-related disease progression across the lifespan and survival in patients with Duchenne muscular dystrophy.
For this prospective cohort study, we enrolled male patients aged 2–28 years with Duchenne muscular dystrophy at 20 centres in nine countries. Patients were followed up for 10 years. We compared no glucocorticoid treatment or cumulative treatment duration of less than 1 month versus treatment of 1 year or longer with regard to progression of nine disease-related and clinically meaningful mobility and upper limb milestones. We used Kaplan-Meier analyses to compare glucocorticoid treatment groups for time to stand from supine of 5 s or longer and 10 s or longer, and loss of stand from supine, four-stair climb, ambulation, full overhead reach, hand-to-mouth function, and hand function. Risk of death was also assessed. This study is registered with ClinicalTrials.gov, number NCT00468832.
440 patients were enrolled during two recruitment periods (2006–09 and 2012–16). Time to all disease progression milestone events was significantly longer in patients treated with glucocorticoids for 1 year or longer than in patients treated for less than 1 month or never treated (log-rank p<0·0001). Glucocorticoid treatment for 1 year or longer was associated with increased median age at loss of mobility milestones by 2·1–4·4 years and upper limb milestones by 2·8–8·0 years compared with treatment for less than 1 month. Deflazacort was associated with increased median age at loss of three milestones by 2·1–2·7 years in comparison with prednisone or prednisolone (log-rank p<0·012). 45 patients died during the 10-year follow-up. 39 (87%) of these deaths were attributable to Duchenne-related causes in patients with known duration of glucocorticoids usage. 28 (9%) deaths occurred in 311 patients treated with glucocorticoids for 1 year or longer compared with 11 (19%) deaths in 58 patients with no history of glucocorticoid use (odds ratio 0·47, 95% CI 0·22–1·00; p=0·0501).
In patients with Duchenne muscular dystrophy, glucocorticoid treatment is associated with reduced risk of losing clinically meaningful mobility and upper limb disease progression milestones across the lifespan as well as reduced risk of death.
US Department of Education/National Institute on Disability and Rehabilitation Research; US Department of Defense; National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases; and Parent Project Muscular Dystrophy.
Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) ...through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.
Objective
Muscle inflammation is a feature in myositis and Duchenne muscular dystrophy (DMD). Autoimmune mechanisms are thought to contribute to muscle weakness in patients with myositis. However, a ...lack of correlation between the extent of inflammatory cell infiltration and muscle weakness indicates that nonimmune pathologic mechanisms may play a role. The present study focused on 2 microRNA (miRNA) sets previously identified as being elevated in the muscle of patients with DMD—an “inflammatory” miRNA set that is dampened with glucocorticoids, and a “dystrophin‐targeting” miRNA set that inhibits dystrophin translation—to test the hypothesis that these miRNAs are similarly dysregulated in the muscle of patients with myositis, and could contribute to muscle weakness and disease severity.
Methods
A major histocompatibility complex class I–transgenic mouse model of myositis was utilized to study gene and miRNA expression and histologic features in the muscle tissue, with the findings validated in human muscle biopsy tissue from 6 patients with myositis. Mice were classified as having mild or severe myositis based on transgene expression, body weight, histologic disease severity, and muscle strength/weakness.
Results
In mice with severe myositis, muscle tissue showed mononuclear cell infiltration along with elevated expression of type I interferon and NF‐κB–regulated genes, including Tlr7 (3.8‐fold increase, P < 0.05). Furthermore, mice with severe myositis showed elevated expression of inflammatory miRNAs (miR‐146a, miR‐142‐3p, miR‐142‐5p, miR‐455‐3p, and miR‐455‐5p; ~3–40‐fold increase, P < 0.05) and dystrophin‐targeting miRNAs (miR‐146a, miR‐146b, miR‐31, and miR‐223; ~3–38‐fold increase, P < 0.05). Bioinformatics analyses of chromatin immunoprecipitation sequencing (ChIP‐seq) data identified at least one NF‐κB consensus element within the promoter/enhancer regions of these miRNAs. Western blotting and immunofluorescence analyses of the muscle tissue from mice with severe myositis demonstrated reduced levels of dystrophin. In addition, elevated levels of NF‐κB–regulated genes, TLR7, and miRNAs along with reduced dystrophin levels were observed in muscle biopsy tissue from patients with histologically severe myositis.
Conclusion
These data demonstrate that an acquired dystrophin deficiency may occur through NF‐κB–regulated miRNAs in myositis, thereby suggesting a unifying theme in which muscle injury, inflammation, and weakness are perpetuated both in myositis and in DMD.
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