Screening for antisense modulation of dystrophin pre-mRNA splicing

Dickson, George; Hill, Vanessa J.; Graham, Ian R.

doi:10.1016/s0960-8966(02)00085-8

Cited by 23 publications

(17 citation statements)

References 47 publications

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“…In the last few years, experiments in several laboratories have demonstrated the principle that sequence-specific AOs can induce targeted exon skipping to reestablish the reading frame of dystrophin mRNA in myogenic cell cultures (6,7,12,20). More recently, we demonstrated in mdx mice that 2OMeAOs delivered by intramuscular injection is able to induce dystrophin expression at normal levels in a large numbers of fibers and that this is accompanied by functional improvement (11).…”

Section: Discussionmentioning

confidence: 92%

Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles

Lü

Rabinowitz

Chen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

365

291

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Antisense oligonucleotide-mediated alternative splicing has great potential for treatment of Duchenne muscular dystrophy (DMD) caused by mutations within nonessential regions of the dystrophin gene. We have recently shown in the dystrophic mdx mouse that exon 23, bearing a nonsense mutation, can be skipped after intramuscular injection of a specific 2 -O-methyl phosphorothioate antisense oligoribonucleotide (2OMeAO). This skipping created a shortened, but in-frame, transcript that is translated to produce near-normal levels of dystrophin expression. This expression, in turn, led to improved muscle function. However, because DMD affects muscles body-wide, effective treatment requires dystrophin induction ideally in all muscles. Here, we show that systemic delivery of specific 2OMeAOs, together with the triblock copolymer F127, induced dystrophin expression in all skeletal muscles but not in cardiac muscle of the mdx dystrophic mice. The highest dystrophin expression was detected in diaphragm, gastrocnemius, and intercostal muscles. Large numbers of fibers with near-normal level of dystrophin were observed in focal areas. Three injections of 2OMeAOs at weekly intervals enhanced the levels of dystrophin. Dystrophin mRNA lacking the targeted exon 23 remained detectable 2 weeks after injection. No evidence of tissue damage was detected after 2OMeAO and F127 treatment either by serum analysis or histological examination of liver, kidney, lung, and muscles. The simplicity and safety of the antisense protocol provide a realistic prospect for treatment of the majority of DMD mutations. We conclude that a significant therapeutic effect may be achieved by further optimization in dose and regime of administration of antisense oligonucleotide.exon skipping ͉ muscular dystrophy

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Section: Discussionmentioning

confidence: 92%

Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles

Lü

Rabinowitz

Chen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

365

291

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“…This application uses sequence-specific AONs for targeted exon skipping to correct the reading frame of mutated dystrophin mRNA. [3][4][5][6][7][8] DMD presents an unparalleled prospect for gene correction by AONs therapy for two main reasons. First, the gene consists of 79 exons spanning >2.3 million base pairs.…”

Section: Introductionmentioning

confidence: 99%

Octa-guanidine Morpholino Restores Dystrophin Expression in Cardiac and Skeletal Muscles and Ameliorates Pathology in Dystrophic mdx Mice

et al. 2009

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Steric-block antisense oligonucleotides (AONs) are able to target RNAs for destruction and splicing alteration. Reading frame restoration of the dystrophin transcript can be achieved by AON-mediated exon skipping in the dystrophic mdx mouse model. However, simple, unmodified AONs exhibit inefficient delivery systemically, leading to dystrophin induction with high variability in skeletal muscles and barely detectable in cardiac muscle. Here, we examined a Morpholino oligomer conjugated with a dendrimeric octaguanidine (Vivo-Morpholino) and demonstrated that the delivery moiety significantly improved dystrophin production in both skeletal and cardiac muscles in mdx mice in vivo. Single intravenous (IV) injections of 6 mg/kg Vivo-MorpholinoE23 (Vivo-ME23) generated dystrophin expression in skeletal muscles at the levels higher than the injection of 300 mg/kg unmodified ME23. Repeated injections at biweekly intervals achieved near 100% of fibers expressing dystrophin in skeletal muscles bodywide without eliciting a detectable immune response. Dystrophin protein was restored to approximately 50 and 10% of normal levels in skeletal and cardiac muscles, respectively. Vivo-Morpholinos showed no signs of toxicity with the effective dosages and regime, thus offering realistic prospects for the treatment of a majority of Duchenne muscular dystrophy (DMD) patients and many other diseases by targeting RNAs.

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“…Most DMD mutations occur within the rod domain, which spans more than half the length of the protein, but seems to have limited functional importance (4,5). Antisense therapy uses specific oligomers to remove the mutated or additional exon(s) that disrupt the reading frame, thus restoring the expression of shortened forms of dystrophin protein retaining critical functions (6)(7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Moulton

Iversen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

223

258

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Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cellpenetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.M utations in the dystrophin gene underlie two forms of muscular dystrophy: Duchenne and Becker muscular dystrophy (DMD and BMD). DMD is caused mainly by nonsense and frame-shift mutations with little or no production of functional dystrophin protein, leading to disease onset in early childhood with lethal consequences. BMD is caused by mutations that typically create shortened but in-frame transcripts with production of partially functional dystrophin, leading to variable and often overt symptoms (1-3). Most DMD mutations occur within the rod domain, which spans more than half the length of the protein, but seems to have limited functional importance (4, 5). Antisense therapy uses specific oligomers to remove the mutated or additional exon(s) that disrupt the reading frame, thus restoring the expression of shortened forms of dystrophin protein retaining critical functions (6-11).We previously demonstrated that i.m. delivery of a specific 2Ј-O-methyl phosphorothioate antisense oligonucleotide (2ЈOMeAON) was able to skip targeted dystrophin exon 23 in mdx mouse, a model of DMD (9). This mouse carries a nonsense point mutation within exon 23 and lacks dystrophin expression (except in a few rare revertant fibers) in all muscles, including the heart (12, 13). Skipping the mutated exon 23 restored both the reading frame and dystrophin expression, with functional improvement of the treated muscles (14) [supporting information (SI) Fig. S1a]. Recently we showed that a phosphorodiamidate morpholino oligomer (PMO), E23ϩ7-18 targeting the junction of exon 23 and intron 23 of mouse dystrophin (referred to as PMOE23 hereafter), was able to induce up to functional levels of dystrophin expression in some skeletal muscles by regular i.v. injections in mdx mice (15). However, dystrophin expression induced by both 2ЈOMeAON and PMO required high doses and was highly variable between muscles and myofibers in terms of observed efficacy. Of greater conc...

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Screening for antisense modulation of dystrophin pre-mRNA splicing

Cited by 23 publications

References 47 publications

Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles

Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles

Octa-guanidine Morpholino Restores Dystrophin Expression in Cardiac and Skeletal Muscles and Ameliorates Pathology in Dystrophic mdx Mice

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

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