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

Abstract

SummaryBackgroundMutations 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.MethodsWe 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.FindingsTwo 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.InterpretationIntramuscular 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.FundingUK Department of Health.

Longitudinal effect of eteplirsen versus historical control on ambulation in Duchenne muscular dystrophy

“…This is consistent with the safety profile and tolerability of eteplirsen and other PMO‐based oligomers seen in animals41, 42, 43 and in other human studies 44. This lack of toxicity is attributed to PMO chemistry, which is charge‐neutral, largely unmetabolized, and not linked to immune activation,10, 45 platelet activation, or hepatotoxicity.…”

“…Eteplirsen has been previously demonstrated to reliably induce the production of functional dystrophin in patients with DMD,1, 10, 11, 12 and in so doing, significantly slowed the rate of progression of this devastating disease as demonstrated here by comparison of longitudinal 6MWT results to untreated, matched historical controls over 36 months. This was evidenced by a slower decline in walking ability in eteplirsen‐treated patients compared to age‐matched historical controls amenable to exon 51 skipping, with a clinically relevant 75m difference by 24 months, and a statistically significant and clinically relevant difference of 151m by 36 months.…”

“…This is supported by the finding that internally deleted dystrophin proteins occur in patients with Becker muscular dystrophy, a dystrophinopathy allelic to DMD, but with a less severe phenotype 9. The ability of eteplirsen to induce expression of dystrophin in DMD patients was demonstrated by an observed increase of dystrophin‐positive fibers in skeletal muscle, observed increases in dystrophin intensity in this1 and other studies,10, 11 and by the observed restoration of the dystroglycan complex, as evidenced by localization of neuronal nitric oxide synthase and B‐dystroglycan to the sarcolemma 12…”

Physiological and tissue-specific vectors for treatment of inherited diseases

“…It can be used to block aberrant splice sites restoring normal splicing, to promote alternative splice sites to benefit expression of beneficial isoforms or to produce nonfunctional mRNAs to block expression of a dominant-negative mutation. 130 The delivery of the antisense oligonucleotides can be carried out by viral 131 or nonviral 132,133 vectors.…”

Anticancer Gene Transfer for Cancer Gene Therapy

“…Generally while the non-viral physical methods are cost effective and less invasive than a viral approach the efficiency of delivery is extremely low and so far there only few examples of clinical use of these methods. Most significantly exon skipping approaches using oligonucleotides have been used successfully in the clinic for Duchenne Muscular Dystrophy (van Deutekom et al, 2007;Kinali et al, 2009;Cirak et al, 2011) and antisense oligonucleotides for Spinal Muscular Atrophy are in clinical development at present (Passini et al, 2011). Another method of non-viral gene delivery utilizes advanced nano-chemistry and nanoparticles that are analyzed extensively in another chapter of this book and constitute an attractive approach when compared only with the more efficient viral systems.…”