Dataset Information

Morpholino Oligomer-Induced Dystrophin Isoforms to Map the Functional Domains in the Dystrophin Protein.

ABSTRACT: Dystrophin plays a crucial role in maintaining sarcolemma stability during muscle contractions, and mutations that prevent the expression of a functional protein cause Duchenne muscular dystrophy (DMD). Antisense oligonucleotide-mediated manipulation of pre-messenger RNA splicing to bypass Duchenne-causing mutations and restore functional dystrophin expression has entered the clinic for the most common DMD mutations. The rationale of "exon skipping" is based upon genotype-phenotype correlations observed in Becker muscular dystrophy, a milder allelic disorder generally characterized by in-frame deletions and internally truncated but semi-functional dystrophin isoforms. However, there is a lack of genotype-phenotype correlations downstream of DMD exon 55, as deletions in this region are rare and most single exon deletions would disrupt the reading frame. Consequently, the amenability of mutations in this region of the DMD gene to exon skipping strategies remains unknown. Here, we induced "Becker muscular dystrophy-like" in-frame dystrophin isoforms in vivo by intraperitoneal injection of peptide-conjugated phosphorodiamidate morpholino oligomers targeting selected exons. The dystrophin isoform encoded by the transcript lacking exons 56+57 appears to be more functional than that encoded by the 58+59-deleted transcript, as determined by higher dystrophin expression, stabilized ?-dystroglycan, and less severe dystrophic pathology, indicating some potential for the strategy to address Duchenne-causing mutations affecting these exons.

SUBMITTER: Li D

PROVIDER: S-EPMC7516190 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Morpholino Oligomer-Induced Dystrophin Isoforms to Map the Functional Domains in the Dystrophin Protein.

Li Dunhui D Adams Abbie M AM Johnsen Russell D RD Fletcher Susan S Wilton Steve D SD

Molecular therapy. Nucleic acids 20200821

Dystrophin plays a crucial role in maintaining sarcolemma stability during muscle contractions, and mutations that prevent the expression of a functional protein cause Duchenne muscular dystrophy (DMD). Antisense oligonucleotide-mediated manipulation of pre-messenger RNA splicing to bypass Duchenne-causing mutations and restore functional dystrophin expression has entered the clinic for the most common <i>DMD</i> mutations. The rationale of "exon skipping" is based upon genotype-phenotype correl ...[more]

PMID: 33230432

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Project description:Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded 'revertant' dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Project description: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 ?-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.

Project description:BackgroundMutations 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 microL (0.9%) saline and five patients received 0.9 mg AVI-4658 in 900 microL 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.

Dataset Information

Morpholino Oligomer-Induced Dystrophin Isoforms to Map the Functional Domains in the Dystrophin Protein.

Publications

Morpholino Oligomer-Induced Dystrophin Isoforms to Map the Functional Domains in the Dystrophin Protein.

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