Virginia Arechavala-Gomeza

Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study

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.

Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study

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.

Muscle histology vs MRI in Duchenne muscular dystrophy

Objective: There are currently no effective treatments to halt the muscle breakdown in Duchenne muscular dystrophy (DMD), although genetic-based clinical trials are being piloted. Most of these trials have as an endpoint the restoration of dystrophin in muscle fibers, hence requiring sufficiently well-preserved muscle of recruited patients. The choice of the muscles to be studied and the role of noninvasive methods to assess muscle preservation therefore require further evaluation. Methods: We studied the degree of muscle involvement in the lower leg muscles of 34 patients with DMD >8 years, using muscle MRI. In a subgroup of 15 patients we correlated the muscle MRI findings with the histology of open extensor digitorum brevis (EDB) muscle biopsies. Muscle MRI involvement was assigned using a scale 0–4 (normal–severe). Results: In all patients we documented a gradient of involvement of the lower leg muscles: the posterior compartment (gastrocnemius > soleus) was most severely affected; the anterior compartment (tibialis anterior/posterior, popliteus, extensor digitorum longus) least affected. Muscle MRI showed EDB involvement that correlated with the patients age (p = 0.055). We show a correlation between the MRI and EDB histopathologic changes, with MRI 3–4 grades associated with a more severe fibro-adipose tissue replacement. The EDB was sufficiently preserved for bulk and signal intensity in 18/22 wheelchair users aged 10–16.6 years. Conclusion: This study provides a detailed correlation between muscle histology and MRI changes in DMD and demonstrates the value of this imaging technique as a reliable tool for the selection of muscles in patients recruited into clinical trials.

Restoration of the Dystrophin-associated Glycoprotein Complex After Exon Skipping Therapy in Duchenne Muscular Dystrophy

We previously conducted a proof of principle; dose escalation study in Duchenne muscular dystrophy (DMD) patients using the morpholino splice-switching oligonucleotide AVI-4658 (eteplirsen) that induces skipping of dystrophin exon 51 in patients with relevant deletions, restores the open reading frame and induces dystrophin protein expression after intramuscular (i.m.) injection. We now show that this dystrophin expression was accompanied by an elevated expression of α-sarcoglycan, β-dystroglycan (BDG) and--in relevant cases--neuronal nitric oxide synthase (nNOS) at the sarcolemma, each of which is a component of a different subcomplex of the dystrophin-associated glycoprotein complex (DAPC). As expected, nNOS expression was relocalized to the sarcolemma in Duchenne patients in whom the dystrophin deletion left the nNOS-binding domain (exons 42-45) intact, whereas this did not occur in patients with deletions that involved this domain. Our results indicate that the novel internally deleted and shorter dystrophin induced by skipping exon 51 in patients with amenable deletions, can also restore the dystrophin-associated complex, further suggesting preserved functionality of the newly translated dystrophin.

Dystrophin quantification and clinical correlations in Becker muscular dystrophy: implications for clinical trials.

Duchenne muscular dystrophy is caused by mutations in the DMD gene that disrupt the open reading frame and prevent the full translation of its protein product, dystrophin. Restoration of the open reading frame and dystrophin production can be achieved by exon skipping using antisense oligonucleotides targeted to splicing elements. This approach aims to transform the Duchenne muscular dystrophy phenotype to that of the milder disorder, Becker muscular dystrophy, typically caused by in-frame dystrophin deletions that allow the production of an internally deleted but partially functional dystrophin. There is ongoing debate regarding the functional properties of the different internally deleted dystrophins produced by exon skipping for different mutations; more insight would be valuable to improve and better predict the outcome of exon skipping clinical trials. To this end, we have characterized the clinical phenotype of 17 patients with Becker muscular dystrophy harbouring in-frame deletions relevant to on-going or planned exon skipping clinical trials for Duchenne muscular dystrophy and correlated it to the levels of dystrophin, and dystrophin-associated protein expression. The cohort of 17 patients, selected exclusively on the basis of their genotype, included 4 asymptomatic, 12 mild and 1 severe patient. All patients had dystrophin levels of >40% of control and significantly higher dystrophin (P = 0.013), β-dystroglycan (P = 0.025) and neuronal nitric oxide synthase (P = 0.034) expression was observed in asymptomatic individuals versus symptomatic patients with Becker muscular dystrophy. Furthermore, grouping the patients by deletion, patients with Becker muscular dystrophy with deletions with an end-point of exon 51 (the skipping of which could rescue the largest group of Duchenne muscular dystrophy deletions) showed significantly higher dystrophin levels (P = 0.034) than those with deletions ending with exon 53. This is the first quantitative study on both dystrophin and dystrophin-associated protein expression in patients with Becker muscular dystrophy with deletions relevant for on-going exon skipping trials in Duchenne muscular dystrophy. Taken together, our results indicate that all varieties of internally deleted dystrophin assessed in this study have the functional capability to provide a substantial clinical benefit to patients with Duchenne muscular dystrophy.

Chronic Systemic Therapy With Low-dose Morpholino Oligomers Ameliorates the Pathology and Normalizes Locomotor Behavior in mdx Mice

The administration of antisense oligonucleotides (AOs) to skip one or more exons in mutated forms of the DMD gene and so restore the reading frame of the transcript is one of the most promising approaches to treat Duchenne muscular dystrophy (DMD). At present, preclinical studies demonstrating the efficacy and safety of long-term AO administration have not been conducted. Furthermore, it is essential to determine the minimal effective dose and frequency of administration. In this study, two different low doses (LDs) of phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 in the mdx dystrophic mouse were administered for up to 12 months. Mice treated for 50 weeks showed a substantial dose-related amelioration of the pathology, particularly in the diaphragm. Moreover, the generalized physical activity was profoundly enhanced compared to untreated mdx mice showing that widespread, albeit partial, dystrophin expression restores the normal activity in mdx mice. Our results show for the first time that a chronic long-term administration of LDs of unmodified PMO, equivalent to doses in use in DMD boys, is safe, significantly ameliorates the muscular dystrophic phenotype and improves the activity of dystrophin-deficient mice, thus encouraging the further clinical translation of this approach in humans.

Antisense Oligonucleotide-Mediated Exon Skipping for Duchenne Muscular Dystrophy: Progress and Challenges.

Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF and the consequent production of a shorter but functional dystrophin protein. This approach may lead to an effective disease modifying treatment for DMD and progress towards clinical application has been rapid. Less than a decade has passed between the first studies published in 1998 describing the use of AOs to modify the DMD gene in mice and the results of the first intramuscular proof of concept clinical trials. Whilst phase II and III trials are now underway, the heterogeneity of DMD mutations, efficient systemic delivery and targeting of AOs to cardiac muscle remain significant challenges. Here we review the current status of AO-mediated therapy for DMD, discussing the preclinical, clinical and regulatory hurdles and their possible solutions to expedite the translation of AO-mediated exon skipping therapy to clinic.

A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient Cavalier King Charles Spaniels is amenable to exon 51 skipping

Background Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion “hot spot” is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD “hot spot”. Methodology/Principal Findings Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5′ donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. Conclusions/Significance Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD.

Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: Implication for clinical trials

Duchenne muscular dystrophy (DMD) is characterised by the absence of dystrophin in muscle biopsies, although residual dystrophin can be present, either as dystrophin-positive (revertant) fibres or traces. As restoration of dystrophin expression is the end point of clinical trials, such residual dystrophin is a key factor in recruitment of patients and may also confound the analysis of dystrophin restoration in treated patients, if, as previously observed in the mdx mouse, revertant fibres increase with age. In 62% of the diagnostic biopsies reports of 65 DMD patients studied, traces or revertants were recorded with no correlation between traces or revertants, the patients performance, or corticosteroids response. In nine of these patients, there was no increase in traces or revertants in biopsies taken a mean of 8.23 years (5.8-10.4 years) after the original diagnostic biopsy. This information should help in the design and execution of clinical trials focused on dystrophin restoration strategies.

Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: Implications for future clinical trials.

Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dystrophin is expressed. AO-induced skipping of exon 51 of the DMD gene, which could treat 13% of DMD patients, has now progressed to clinical trials. We describe here the methodical, cooperative comparison, in vitro (in DMD cells) and in vivo (in a transgenic mouse expressing human dystrophin), of 24 AOs of the phosphorodiamidate morpholino oligomer (PMO) chemistry designed to target exon 53 of the DMD gene, skipping of which could be potentially applicable to 8% of patients. A number of the PMOs tested should be considered worthy of development for clinical trial.