S.D. Wilton

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.

Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.

For the majority of Duchenne muscular dystrophy (DMD) mutations, antisense oligonucleotide (AON)-mediated exon skipping has the potential to restore a functional protein. Here we show that weekly intravenous injections of morpholino phosphorodiamidate (morpholino) AONs induce expression of functional levels of dystrophin in body-wide skeletal muscles of the dystrophic mdx mouse, with resulting improvement in muscle function. Although the level of dystrophin expression achieved varies considerably between muscles, antisense therapy may provide a realistic hope for the treatment of a majority of individuals with DMD.

Regulation of eukaryotic gene expression by the untranslated gene regions and other non-coding elements

There is now compelling evidence that the complexity of higher organisms correlates with the relative amount of non-coding RNA rather than the number of protein-coding genes. Previously dismissed as “junk DNA”, it is the non-coding regions of the genome that are responsible for regulation, facilitating complex temporal and spatial gene expression through the combinatorial effect of numerous mechanisms and interactions working together to fine-tune gene expression. The major regions involved in regulation of a particular gene are the 5′ and 3′ untranslated regions and introns. In addition, pervasive transcription of complex genomes produces a variety of non-coding transcripts that interact with these regions and contribute to regulation. This review discusses recent insights into the regulatory roles of the untranslated gene regions and non-coding RNAs in the control of complex gene expression, as well as the implications of this in terms of organism complexity and evolution.

Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD

Manipulation of pre-mRNA splicing by antisense oligonucleotides (AOs) offers considerable potential for a number of genetic disorders. One of these is Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene typically result in premature termination of translation that causes a loss of functional protein. AOs can induce exon skipping such that the mutation is by-passed and the reading frame restored, producing an internally deleted protein similar to that found in the milder Becker muscular dystrophy. To date, this approach has been applied to the mdx mouse model in vitro and in vivo and in human myoblast cultures. Here, we report the application of AO-directed exon skipping to induce dystrophin expression in vitro in a canine model of DMD, golden retriever muscular dystrophy (GRMD). The efficacy of 2′-O-methyl phosphorothioate (2OMe), phosphorodiamidate morpholino oligomers (PMOs) and peptide-linked PMOs (PMO-Pep) to induce dystrophin expression was assessed. The 2OMe chemistry was only effective for short-term induction of corrected transcript and could not induce detectable dystrophin protein. The PMO chemistry generally induced limited exon skipping at only high concentrations; however, a low level of dystrophin protein was produced in treated cells. Use of the PMO-Pep, applied here for the first time to a DMD model, was able to induce high and sustained levels of exon skipping and induced the highest level of dystrophin expression with no apparent adverse effects upon the cells. The induction of dystrophin in the GRMD model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials.

Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.

Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterised by an absence of functional protein, while Becker muscular dystrophy is usually caused by in‐frame deletions allowing synthesis of some functional protein. Treatment options are limited, and we are investigating the potential of transcript manipulation to overcome disease‐causing mutations. Antisense oligonucleotides have been used to induce specific exon removal during processing of the dystrophin primary transcript and thereby by‐pass protein‐truncating mutations. The antisense oligonucleotide chemistry most widely used to alter pre‐mRNA processing is 2′‐O‐methyl‐modified bases on a phosphorothioate backbone.

Current Status of Pharmaceutical and Genetic Therapeutic Approaches to Treat DMD

Duchenne muscular dystrophy (DMD) is a genetic disease affecting about one in every 3,500 boys. This X-linked pathology is due to the absence of dystrophin in muscle fibers. This lack of dystrophin leads to the progressive muscle degeneration that is often responsible for the death of the DMD patients during the third decade of their life. There are currently no curative treatments for this disease but different therapeutic approaches are being studied. Gene therapy consists of introducing a transgene coding for full-length or a truncated version of dystrophin complementary DNA (cDNA) in muscles, whereas pharmaceutical therapy includes the use of chemical/biochemical substances to restore dystrophin expression or alleviate the DMD phenotype. Over the past years, many potential drugs were explored. This led to several clinical trials for gentamicin and ataluren (PTC124) allowing stop codon read-through. An alternative approach is to induce the expression of an internally deleted, partially functional dystrophin protein through exon skipping. The vectors and the methods used in gene therapy have been continually improving in order to obtain greater encapsidation capacity and better transduction efficiency. The most promising experimental approaches using pharmaceutical and gene therapies are reviewed in this article.

Homozygosity for a nonsense mutation in the alpha-tropomyosin slow gene TPM3 in a patient with severe infantile nemaline myopathy.

The nemaline myopathies are muscle disorders of variable severity and age of onset, with characteristic nemaline bodies in the sarcoplasm. Genes for dominant (NEM1) and recessive (NEM2A) nemaline myopathy have been localised to chromosomes one and two, respectively. A missense mutation in the alpha-tropomyosin gene (TPM3) has been associated with NEM1 in one family. Probands from 76 other nemaline myopathy families have now been screened for TPM3 mutations. One proband, who was not noted to have any weakness neonatally, but who died at 21 months of age, was shown to be homozygous for a single strand conformation polymorphism (SSCP) in skeletal-muscle-specific exon 1 of TPM3. Sequencing revealed homozygosity for a nonsense mutation at codon 31 (CAG to TAG). The patient should have no functioning alpha-tropomyosin slow protein. The nemaline bodies in this patient were exclusively in type one fibres, consistent with the expression of TPM3 only in type one fibres.

The FSHD Atrophic Myotube Phenotype Is Caused by DUX4 Expression

Background Facioscapulohumeral muscular dystrophy (FSHD) is linked to deletions in 4q35 within the D4Z4 repeat array in which we identified the double homeobox 4 (DUX4) gene. We found stable DUX4 mRNAs only derived from the most distal D4Z4 unit and unexpectedly extended to the flanking pLAM region that provided an intron and a polyadenylation signal. DUX4 encodes a transcription factor expressed in FSHD but not control primary myoblasts or muscle biopsies. The DUX4 protein initiates a large transcription deregulation cascade leading to muscle atrophy and oxidative stress, which are FSHD key features. Methodology/Principal Findings We now show that transfection of myoblasts with a DUX4 expression vector leads to atrophic myotube formation associated with the induction of E3 ubiquitin ligases (MuRF1 and Atrogin1/MAFbx) typical of muscle atrophy. DUX4 induces expression of downstream targets deregulated in FSHD such as mu-crystallin and TP53. We developed specific siRNAs and antisense oligonucleotides (AOs) targeting the DUX4 mRNA. Addition of these antisense agents to primary FSHD myoblast cultures suppressed DUX4 protein expression and affected expression of the above-mentioned markers. Conclusions/Significance These results constitute a proof of concept for the development of therapeutic approaches for FSHD targeting DUX4 expression.

Prevention of Dystrophic Pathology in Severely Affected Dystrophin/Utrophin-deficient Mice by Morpholino-oligomer-mediated Exon-skipping

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon-skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of dystrophin expression in the mdx mouse model. Here, we report the efficiency of the PPMO-mediated exon-skipping approach in the utrophin/dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of dystrophin pre-mRNA in dKO mice induce a near-normal level of dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These findings suggest great potential for PPMOs in systemic treatment of the DMD phenotype.