Prevention of Dystrophic Pathology in Severely Affected Dystrophin/Utrophin-deficient Mice by Morpholino-oligomer-mediated Exon-skipping
101
Citation
34
Reference
10
Related Paper
Citation Trend
Abstract:
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. 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.Keywords:
Morpholino
Exon skipping
Utrophin
Oligomer
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, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Antisense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, whilst maintaining or restoring the reading frame, and thereby overcome proteintruncating mutations. The mdx mouse has a nonsense mutation in exon 23 of the dystrophin gene that precludes functional dystrophin production, and this model has been used in the development of treatment strategies for dystrophinopathies. A phosphorodiamidate morpholino oligomer has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdx mouse, in vivo and in vitro. A cell-penetrating peptide-conjugated oligomer, targeted to the mouse dystrophin exon 23 donor splice site, was administered to mdx mice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The cell penetrating peptide greatly enhanced uptake of the phosphorodiamidate morpholino oligomer, resulting in widespread dystrophin expression. Treatment of neonatal mdx mice induced dystrophin expression and averted the onset of the dystrophic process that normally begins shortly before 3 weeks of age.
Morpholino
Exon skipping
mdx mouse
Utrophin
Nonsense mutation
Cite
Citations (0)
Morpholino
Utrophin
Cite
Citations (0)
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. 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.
Morpholino
Exon skipping
Utrophin
Oligomer
Cite
Citations (101)
Morpholino
mdx mouse
Exon skipping
Cite
Citations (0)
Morpholino Oligomer–Mediated Exon Skipping Averts the Onset of Dystrophic Pathology in the mdx Mouse
Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterized by an absence of functional protein, whereas Becker muscular dystrophy, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Anti-sense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, while maintaining or restoring the reading frame, and thereby overcome protein-truncating mutations. The mdx mouse has a non-sense mutation in exon 23 of the dystrophin gene that precludes functional dystrophin production, and this model has been used in the development of treatment strategies for dystrophinopathies. A phosphorodiamidate morpholino oligomer (PMO) has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdxmouse, in vivo and in vitro. In this report, a cell-penetrating peptide (CPP)-conjugated oligomer targeted to the mouse dystrophin exon 23 donor splice site was administered to mdxmice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The CPP greatly enhanced uptake of the PMO, resulting in widespread dystrophin expression. Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterized by an absence of functional protein, whereas Becker muscular dystrophy, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Anti-sense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, while maintaining or restoring the reading frame, and thereby overcome protein-truncating mutations. The mdx mouse has a non-sense mutation in exon 23 of the dystrophin gene that precludes functional dystrophin production, and this model has been used in the development of treatment strategies for dystrophinopathies. A phosphorodiamidate morpholino oligomer (PMO) has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdxmouse, in vivo and in vitro. In this report, a cell-penetrating peptide (CPP)-conjugated oligomer targeted to the mouse dystrophin exon 23 donor splice site was administered to mdxmice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The CPP greatly enhanced uptake of the PMO, resulting in widespread dystrophin expression.
Morpholino
Exon skipping
mdx mouse
Utrophin
Cite
Citations (154)
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 cell-penetrating 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.
Morpholino
Exon skipping
mdx mouse
Oligomer
Creatine kinase
Cite
Citations (226)
Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked myopathy involving cycles of muscle cell degeneration, regeneration, and inflammation. DMD results from mutations in the dystrophin gene that result in a consequent failure to translate dystrophin protein. Exon skipping is a promising therapeutic strategy employing antisense oligonucleotides (AO) to exclude exons that disrupt the open reading frame, so as to produce a truncated, partially functional dystrophin protein. Several different AOs, including the phosphorodiamidate morpholino (PMO) and the 2'O-methyl phosphorothioate (2'OMe), have been shown to induce dystrophin expression and are currently being investigated in clinical trials. However, drug uptake and efficacy is inconsistent and highly-variable between and within individual muscles. Our objective was to identify factors within dystrophic muscle responsible for the observed variability in myofiber penetration, exon skipping, and dystrophin expression following systemic AO administration. Specifically, we investigated the role of myofiber regeneration on exon skipping. By treating dystrophin-null mdx mice with a single high-dose of the PMO together with staggered pulses of bromodeoxyuridine (BrdU) we can precisely identify timeframes of myocyte proliferation and relate this to the efficiency of PMO delivery. Initially, we utilized a tagged-PMO to track its efficiency of entry into muscle fibers and determine how long after administration it persists. Intriguingly, we observed that the efficiency of entry of PMO into myofibers is strongly associated with the late stages of differentiation and fusion of satellite cells into regenerating dystrophic mdx myofibers. We also observed a specific co-localization of BrdU-positive myonuclei with dystrophin-positive myofibers when the pulse of BrdU immediately preceded PMO delivery, implying that satellite cell proliferation and fusion into regenerating myofibers play a crucial role in the effectiveness of PMO-mediated exon skipping. Our investigation of the mechanisms involved in PMO uptake and efficacy will provide valuable insights for optimizing protocols for this promising approach to therapy for DMD.
Morpholino
Exon skipping
mdx mouse
Cite
Citations (0)
Morpholino
Exon skipping
mdx mouse
Cite
Citations (483)
Abstract Objective Duchenne muscular dystrophy (DMD) is caused by the inability to produce dystrophin protein at the myofiber membrane. A method to rescue dystrophin production by antisense oligonucleotides, termed exon‐skipping, has been reported for the mdx mouse and in four DMD patients by local intramuscular injection. We sought to test efficacy and toxicity of intravenous oligonucleotide (morpholino)‐induced exon skipping in the DMD dog model. Methods We tested a series of antisense drugs singly and as cocktails, both in primary cell culture, and two in vivo delivery methods (intramuscular injection and systemic intravenous injection). The efficiency and efficacy of multiexon skipping (exons 6–9) were tested at the messenger RNA, protein, histological, and clinical levels. Results Weekly or biweekly systemic intravenous injections with a three‐morpholino cocktail over the course of 5 to 22 weeks induced therapeutic levels of dystrophin expression throughout the body, with an average of about 26% normal levels. This was accompanied by reduced inflammatory signals examined by magnetic resonance imaging and histology, improved or stabilized timed running tests, and clinical symptoms. Blood tests indicated no evidence of toxicity. Interpretation This is the first report of widespread rescue of dystrophin expression to therapeutic levels in the dog model of DMD. This study also provides a proof of concept for systemic multiexon‐skipping therapy. Use of cocktails of morpholino, as shown here, allows broader application of this approach to a greater proportion of DMD patients (90%) and also offers the prospect of selecting deletions that optimize the functionality of the dystrophin protein. Ann Neurol 2009
Morpholino
Exon skipping
mdx mouse
Cite
Citations (374)
The progress of antisense-based therapies using first generation Morpholino oligonucleotides for Duchenne muscular dystrophy (DMD) is expected to partially restore dystrophin expression and may prolong the lifespan of DMD patients. In a recent issue of The Journal of Pathology, a sophisticated study by Vila et al used a dystrophic mouse model of DMD to demonstrate that Morpholino-induced exon skipping induced dystrophin expression in skeletal muscle and stimulated cell mediated and humoral responses to dystrophin. The study highlights the need to further investigate the autoimmune response against de novo synthesised truncated dystrophin protein and its long-term consequences after exon-skipping therapy for DMD. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Exon skipping
Morpholino
mdx mouse
Cite
Citations (2)