Summary Background We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with ...Duchenne muscular dystrophy. Method We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5–15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597. Findings 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1–10·6) to 16·4% (10·8–22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts. Interpretation The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy. Funding UK Medical Research Council; AVI BioPharma.
Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in the dystrophin gene that disrupt the open reading frame, while in frame mutations result in Becker muscular dystrophy ...(BMD). Ullrich congenital muscular dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring the outcome of therapeutic interventions and disease progression. We quantified miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but unchanged in UCMD compared to controls. Milder DMD patients had higher levels of dystromirs than more severely affected patients. Patients with low forced vital capacity (FVC) values, indicating respiratory muscle weakness, had low levels of serum miR-1 and miR-133b. There was no significant difference in the level of the dystromirs in BMD compared to controls. We also assessed the effect of dystrophin restoration on the expression of the five dystromirs in serum of DMD patients treated systemically for 12 weeks with antisense oligomer eteplirsen that induces skipping of exon 51 in the dystrophin gene. The dystromirs were also analysed in muscle biopsies of DMD patients included in a single dose intramuscular eteplirsen clinical trial. Our analysis detected a trend towards normalization of these miRNA between the pre- and post-treatment samples of the systemic trial, which however failed to reach statistical significance. This could possibly be due to the small number of patients and the short duration of these clinical trials. Although longer term studies are needed to clarify the relationship between dystrophin restoration following therapeutic intervention and the level of circulating miRNAs, our results indicate that miR-1 and miR-133 can be considered as exploratory biomarkers for monitoring the progression of muscle weakness and indirectly the remaining muscle mass in DMD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary Background Mutations that disrupt the open reading frame and prevent full translation of DMD , the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular ...dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. Methods We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. Findings Two patients received 0·09 mg AVI-4658 in 900 μL (0·9%) saline and five patients received 0·9 mg AVI-4658 in 900 μL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44–79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26·4%), and the mean intensity was 17% (range 11–21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0·002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot. Interpretation Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD. Funding UK Department of Health.
•We report the relative frequency, clinical and genetic spectrum of CMD in the UK.•We describe clinical and molecular data for 249 unrelated CMD patients.•MCD1A is the prevalent CMD in the UK ...followed by dystroglycanopathies and Ullrich CMD.•We report 363 mutations in CMD genes, 160 of which are novel.
Congenital muscular dystrophies (CMDs) are clinically and genetically heterogeneous conditions; some fatal in the first few years of life and with central nervous system involvement, whereas others present a milder course. We provide a comprehensive report of the relative frequency and clinical and genetic spectrum of CMD in the UK. Genetic analysis of CMD genes in the UK is centralised in London and Newcastle. Between 2001 and 2013, a genetically confirmed diagnosis of CMD was obtained for 249 unrelated individuals referred to these services. The most common CMD subtype was laminin-α2 related CMD (also known as MDC1A, 37.4%), followed by dystroglycanopathies (26.5%), Ullrich-CMD (15.7%), SEPN1 (11.65%) and LMNA (8.8%) gene related CMDs. The most common dystroglycanopathy phenotype was muscle–eye–brain-like disease. Fifteen patients carried mutations in the recently discovered ISPD, GMPPB and B3GALNT2 genes. Pathogenic allelic mutations in one of the CMD genes were also found in 169 unrelated patients with milder phenotypes, such as limb girdle muscular dystrophy and Bethlem myopathy. In all, we identified 362 mutations, 160 of which were novel. Our results provide one of the most comprehensive reports on genetics and clinical features of CMD subtypes and should help diagnosis and counselling of families with this group of conditions.
Background
Spinal muscular atrophy (SMA) is caused by genetic defects in the survival motor neuron 1 (SMN1) gene that lead to SMN deficiency. Different SMN‐restoring therapies substantially prolong ...survival and function in transgenic mice of SMA. However, these therapies do not entirely prevent muscle atrophy and restore function completely. To further improve the outcome, we explored the potential of a combinatorial therapy by modulating SMN production and muscle‐enhancing approach as a novel therapeutic strategy for SMA.
Methods
The experiments were performed in a mouse model of severe SMA. A previously reported 25‐mer morpholino antisense oligomer PMO25 was used to restore SMN expression. The adeno‐associated virus‐mediated expression of myostatin propeptide was used to block the myostatin pathway. Newborn SMA mice were treated with a single subcutaneous injection of 40 μg/g (therapeutic dose) or 10 μg/g (low‐dose) PMO25 on its own or together with systemic delivery of a single dose of adeno‐associated virus‐mediated expression of myostatin propeptide. The multiple effects of myostatin inhibition on survival, skeletal muscle phenotype, motor function, neuromuscular junction maturation, and proprioceptive afferences were evaluated.
Results
We show that myostatin inhibition acts synergistically with SMN‐restoring antisense therapy in SMA mice treated with the higher therapeutic dose PMO25 (40 μg/g), by increasing not only body weight (21% increase in male mice at Day 40), muscle mass (38% increase), and fibre size (35% increase in tibialis anterior muscle in 3 month female SMA mice), but also motor function and physical performance as measured in hanging wire test (two‐fold increase in time score) and treadmill exercise test (two‐fold increase in running distance). In SMA mice treated with low‐dose PMO25 (10 μg/g), the early application of myostatin inhibition prolongs survival (40% increase), improves neuromuscular junction maturation (50% increase) and innervation (30% increase), and increases both the size of sensory neurons in dorsal root ganglia (60% increase) and the preservation of proprioceptive synapses in the spinal cord (30% increase).
Conclusions
These data suggest that myostatin inhibition, in addition to the well‐known effect on muscle mass, can also positively influence the sensory neural circuits that may enhance motor neurons function. While the availability of the antisense drug Spinraza for SMA and other SMN‐enhancing therapies has provided unprecedented improvement in SMA patients, there are still unmet needs in these patients. Our study provides further rationale for considering myostatin inhibitors as a therapeutic intervention in SMA patients, in combination with SMN‐restoring drugs.
Clinical trials using strategies aimed at inducing dystrophin expression in Duchenne muscular dystrophy (DMD) are underway or at advanced planning stage, including splice switching antisense ...oligonucleotides (AON), drugs to induce read-through of nonsense mutations and viral mediated gene therapy. In all these strategies, different dystrophin proteins, often internally deleted, are produced, similar to those found in patients with the milder DMD allelic variant, Becker muscular dystrophy (BMD). The primary biological endpoint of these trials is to induce functional dystrophin expression. A reliable and reproducible method for quantification of dystrophin protein expression at the sarcolemma is crucial to monitor the biochemical outcome of such treatments. We developed a new high throughput semi quantitative fluorescent immunofluorescence method for quantifying dystrophin expression in transverse sections of skeletal muscle. This technique is completely operator independent as it based on an automated scanning system and an image processing script developed with Definiens software. We applied this new acquisition-analysis method to quantify dystrophin and sarcolemma-related proteins using paediatric control muscles from cases without a neuromuscular disorder as well as DMD and BMD samples. The image analysis script was instructed to recognize myofibres immunostained for spectrin or laminin while dystrophin was quantified in each identified myofibre (from 2,000 to over 20,000 fibres, depending on the size of the biopsy). We were able to simultaneously extrapolate relevant parameters such as mean sarcolemmal dystrophin, mean spectrin and laminin intensity, fibre area and diameter. In this way we assessed dystrophin production in each muscle fibre in samples of DMD, BMD and controls. This new method allows the unbiased quantification of dystrophin in every myofibre within a transverse muscle section and will be of help for translational research projects as a biological outcome in clinical trials in DMD and BMD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Merosin deficient congenital muscular dystrophy 1A (MDC1A) results from mutations in the LAMA2 gene. We report 51 patients with MDC1A and examine the relationship between degree of merosin ...expression, genotype and clinical features. Thirty-three patients had absence of merosin and 13 showed some residual merosin. Compared to the residual merosin group, patients with absent merosin had an earlier presentation (<7 days) ( P = 0.0073), were more likely to lack independent ambulation ( P = 0.0215), or require enteral feeding ( P = 0.0099) and ventilatory support ( P = 0.0354). We identified 33 novel LAMA2 mutations; these were distributed throughout the gene in patients with absent merosin, with minor clusters in exon 27, 14, 25 and 26 (55% of mutations). Patients with residual merosin often carried at least one splice site mutation and less frequently frameshift mutations. This large study identified novel LAMA2 mutations and highlights the role of immunohistochemical studies for merosin status in predicting clinical severity of MDC1A.
During the last decade, multiple clinical trials for Duchenne muscular dystrophy (DMD) have focused on the induction of dystrophin expression using different strategies. Many of these trials have ...reported a clear increase in dystrophin protein following treatment. However, the low levels of the induced dystrophin protein have raised questions on its functionality. In our present study, using an unbiased, high-throughput digital image analysis platform, we assessed markers of regeneration and levels of dystrophin associated protein via immunofluorescent analysis of whole muscle sections in 25 DMD boys who received 48-weeks treatment with exon 53 skipping morpholino antisense oligonucleotide (PMO) golodirsen. We demonstrate that the de novo dystrophin induced by exon skipping with PMO golodirsen is capable of conferring a histological benefit in treated patients with an increase in dystrophin associated proteins at the dystrophin positive regions of the sarcolemma in post-treatment biopsies. Although 48 weeks treatment with golodirsen did not result in a significant change in the levels of fetal/developmental myosins for the entire cohort, there was a significant negative correlation between the amount of dystrophin and levels of regeneration observed in different biopsy samples. Our results provide, for the first time, evidence of functionality of induced dystrophin following successful therapeutic intervention in the human.
The primary molecular endpoint for many Duchenne muscular dystrophy (DMD) clinical trials is the induction, or increase in production, of dystrophin protein in striated muscle. For accurate endpoint ...analysis, it is essential to have reliable, robust and objective quantification methodologies capable of detecting subtle changes in dystrophin expression. In this work, we present further development and optimisation of an automated, digital, high-throughput script for quantitative analysis of multiplexed immunofluorescent (IF) whole slide images (WSI) of dystrophin, dystrophin associated proteins (DAPs) and regenerating myofibres (fetal/developmental myosin-positive) in transverse sections of DMD, Becker muscular dystrophy (BMD) and control skeletal muscle biopsies. The script enables extensive automated assessment of myofibre morphometrics, protein quantification by fluorescence intensity and sarcolemmal circumference coverage, colocalisation data for dystrophin and DAPs and regeneration at the single myofibre and whole section level. Analysis revealed significant variation in dystrophin intensity, percentage coverage and amounts of DAPs between differing DMD and BMD samples. Accurate identification of dystrophin via a novel background subtraction method allowed differential assessment of DAP fluorescence intensity within dystrophin positive compared to dystrophin negative sarcolemma regions. This enabled surrogate quantification of molecular functionality of dystrophin in the assembly of the DAP complex. Overall, the digital script is capable of multiparametric and unbiased analysis of markers of myofibre regeneration and dystrophin in relation to key DAPs and enabled better characterisation of the heterogeneity in dystrophin expression patterns seen in BMD and DMD alongside the surrogate assessment of molecular functionality of dystrophin. Both these aspects will be of significant relevance to ongoing and future DMD and other muscular dystrophies clinical trials to help benchmark therapeutic efficacy.
ABSTRACT
In skeletal muscle, excitation–contraction (EC) coupling is the process whereby the voltage‐gated dihydropyridine receptor (DHPR) located on the transverse tubules activates calcium release ...from the sarcoplasmic reticulum by activating ryanodine receptor (RyR1) Ca2+ channels located on the terminal cisternae. This subcellular membrane specialization is necessary for proper intracellular signaling and any alterations in its architecture may lead to neuromuscular disorders. In this study, we present evidence that patients with recessive RYR1‐related congenital myopathies due to primary RyR1 deficiency also exhibit downregulation of the alfa 1 subunit of the DHPR and show disruption of the spatial organization of the EC coupling machinery. We created a cellular RyR1 knockdown model using immortalized human myoblasts transfected with RyR1 siRNA and confirm that knocking down RyR1 concomitantly downregulates not only the DHPR but also the expression of other proteins involved in EC coupling. Unexpectedly, this was paralleled by the upregulation of inositol‐1,4,5‐triphosphate receptors; functionally however, upregulation of the latter Ca2+ channels did not compensate for the lack of RyR1‐mediated Ca2+ release. These results indicate that in some patients, RyR1 deficiency concomitantly alters the expression pattern of several proteins involved in calcium homeostasis and that this may influence the manifestation of these diseases.
We provide evidence in this study that RyR1 deficiency caused by mutations in RYR1 gene is responsible for the collapse of excitation‐contraction coupling by disrupting the physi‐ological colocalization of the dihydropyridine receptor and ryanodine receptor in skeletal muscle. The upregulation of an alternative calcium regulating system via IP3R is identifi‐ed in the condition of RyR1 deficiency and in‐dicates the possible existence of a complex interplay on RyR1 and IP3R signalling cascade in the pathophysiology of RYR1‐related conge‐nital myopathies.
PDF
