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Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse.

ABSTRACT: Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease arising from defects in the dystrophin gene, typically nonsense or frameshift mutations, that preclude the synthesis of a functional protein. A milder, allelic version of the disease, Becker muscular dystrophy, generally arises from in-frame deletions that allow synthesis of a shorter but still semifunctional protein. Therapies to introduce functional dystrophin into dystrophic tissue through either cell or gene replacement have not been successful to date. We report an alternative approach where 2'-O-methyl antisense oligoribonucleotides have been used to modify processing of the dystrophin pre-mRNA in the mdx mouse model of DMD. By targeting 2'-O-methyl antisense oligoribonucleotides to block motifs involved in normal dystrophin pre-mRNA splicing, we induced excision of exon 23, and the mdx nonsense mutation, without disrupting the reading frame. Exon 23 skipping was first optimized in vitro in transfected H-2K(b)-tsA58 mdx myoblasts and then induced in vivo. Immunohistochemical staining demonstrated the synthesis and correct subsarcolemmal localization of dystrophin and gamma-sarcoglycan in the mdx mouse after intramuscular delivery of antisense oligoribonucleotide:liposome complexes. This approach should reduce the severity of DMD by allowing a dystrophic gene transcript to be modified, such that it can be translated into a Becker-dystrophin-like protein.

SUBMITTER: Mann CJ

PROVIDER: S-EPMC14541 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse.

Mann C J CJ Honeyman K K Cheng A J AJ Ly T T Lloyd F F Fletcher S S Morgan J E JE Partridge T A TA Wilton S D SD

Proceedings of the National Academy of Sciences of the United States of America 20010101 1

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease arising from defects in the dystrophin gene, typically nonsense or frameshift mutations, that preclude the synthesis of a functional protein. A milder, allelic version of the disease, Becker muscular dystrophy, generally arises from in-frame deletions that allow synthesis of a shorter but still semifunctional protein. Therapies to introduce functional dystrophin into dystrophic tissue through either cell or gene replacement hav ...[more]

PMID: 11120883

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Project description:BackgroundAntisense oligonucleotides (AOs) can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting.ResultsAlthough many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers.ConclusionThe combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino oligomers, indicating design parameters established with one chemistry may be applied to the other.

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.

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Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse.

Publications

Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse.

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