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• W2061596420 abstract "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. Anti-sense oligonucleotides (AOs) have been used to manipulate splicing of the dystrophin pre-messenger RNA to induce specific exon skipping in vitro and in vivo, in animal models of muscular dystrophy and in cells from Duchenne muscular dystrophy (DMD) patients. Disease-causing mutations in the dystrophin gene can be eliminated by removal of exons that contain non-sense mutations, or exons that flank frame-shifting deletions, to produce an in-frame transcript (for a review, see refs.1Wilton SD Fletcher S RNA splicing manipulation: strategies to modify gene expression for a variety of therapeutic outcomes.Curr Gene Ther. 2005; 5: 467-483Crossref PubMed Scopus (32) Google Scholar,2Wilton SD Fletcher S Antisense oligonucleotides in the treatment of Duchenne muscular dystrophy: where are we now?.Neuromuscul Disord. 2005; 15: 399-402Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar,3van Deutekom JC Gene therapy: the ‘pro-sense‘ approach to Duchenne muscular dystrophy.Eur J Hum Genet. 2005; 13: 518-519Crossref PubMed Scopus (12) Google Scholar,4van Deutekom JC van Ommen GJ Advances in Duchenne muscular dystrophy gene therapy.Nat Rev Genet. 2003; 4: 774-783Crossref PubMed Scopus (176) Google Scholar). DMD is a systemic disease (for a review, see refs. 5Emery AE Clinical and molecular studies in Duchenne muscular dystrophy.Prog Clin Biol Res. 1989; 306: 15-28PubMed Google Scholar,6Emery AE Muscular dystrophy into the new millennium.Neuromuscul Disord. 2002; 12: 343-349Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and for dystrophin exon skipping to be of benefit in ameliorating human disease, it will be necessary to achieve systemic distribution of the anti-sense agent. The dystrophin gene is prone to deletion in two regions in particular,7Den Dunnen JT Grootscholten PM Bakker E Blonden LA Ginjaar HB Wapenaar MC et al.Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications.Am J Hum Genet. 1989; 45: 835-847PubMed Google Scholar but all types of mutation have been described across the dystrophin gene, including deletions, duplications, inversions, non-sense mutations, and splicing errors.8Muntoni F Torelli S Ferlini A Dystrophin and mutations: one gene, several proteins, multiple phenotypes.Lancet Neurol. 2003; 2: 731-740Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar,9Fajkusova L Lukas Z Tvrdikova M Kuhrova V Hajek J Fajkus J Novel dystrophin mutations revealed by analysis of dystrophin mRNA: alternative splicing suppresses the phenotypic effect of a nonsense mutation.Neuromuscul Disord. 2001; 11: 133-138Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,10Roberts RG Gardner RJ Bobrow M Searching for the 1 in 2,400,000: a review of dystrophin gene point mutations.Hum Mutat. 1994; 4: 1-11Crossref PubMed Scopus (127) Google Scholar The milder allelic disorder, Becker muscular dystrophy, is usually caused by in-frame deletions in the dystrophin gene.11Monaco AP Bertelson CJ Liechti-Gallati S Moser H Kunkel LM An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus.Genomics. 1988; 2: 90-95Crossref PubMed Scopus (953) Google Scholar The dystrophin transcripts of patients with this disorder12Comi GP Prelle A Bresolin N Moggio M Bardoni A Gallanti A et al.Clinical variability in Becker muscular dystrophy. Genetic, biochemical and immunohistochemical correlates.Brain. 1994; 117 (Pt 1):: 1-14Crossref PubMed Scopus (104) Google Scholar,13England SB Nicholson LV Johnson MA Forrest SM Love DR Zubrzycka-Gaarn EE et al.Very mild muscular dystrophy associated with the deletion of 46% of dystrophin.Nature. 1990; 343: 180-182Crossref PubMed Scopus (493) Google Scholar,14Chelly J Gilgenkrantz H Lambert M Hamard G Chafey P Recan D et al.Effect of dystrophin gene deletions on mRNA levels and processing in Duchenne and Becker muscular dystrophies.Cell. 1990; 63: 1239-1248Abstract Full Text PDF PubMed Scopus (129) Google Scholar,15Bosone I Bortolotto S Mongini T Doriguzzi C Chiado-Piat L Ugo I et al.Late onset and very mild course of Xp21 Becker type muscular dystrophy.Clin Neuropathol. 2001; 20: 196-199PubMed Google Scholar,16Mirabella M Galluzzi G Manfredi G Bertini E Ricci E De Leo R et al.Giant dystrophin deletion associated with congenital cataract and mild muscular dystrophy.Neurology. 1998; 51: 592-595Crossref PubMed Scopus (33) Google Scholar and the transcripts responsible for the rare, dystrophin-positive revertant fibers in dystrophic muscle17Lu QL Morris GE Wilton SD Ly T Artem'yeva OV Strong P et al.Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion.J Cell Biol. 2000; 148: 985-996Crossref PubMed Scopus (179) Google Scholar,18Wilton SD Dye DE Blechynden LM Laing NG Revertant fibres: a possible genetic therapy for Duchenne muscular dystrophy?.Neuromuscul Disord. 1997; 7: 329-335Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar,19Sherratt TG Vulliamy T Dubowitz V Sewry CA Strong PN Exon skipping and translation in patients with frameshift deletions in the dystrophin gene.Am J Hum Genet. 1993; 53: 1007-1015PubMed Google Scholar,20Thanh LT Nguyen TM Helliwell TR Morris GE Characterization of revertant muscle fibers in Duchenne muscular dystrophy, using exon-specific monoclonal antibodies against dystrophin.Am J Hum Genet. 1995; 56: 725-731PubMed Google Scholar,21Wallgren-Pettersson C Jasani B Rosser LG Lazarou LP Nicholson LV Clarke A Immunohistological evidence for second or somatic mutations as the underlying cause of dystrophin expression by isolated fibres in Xp21 muscular dystrophy of Duchenne-type severity.J Neurol Sci. 1993; 118: 56-63Abstract Full Text PDF PubMed Scopus (17) Google Scholar indicate potential exon combinations that may be most appropriate for ameliorating DMD. Transcript manipulation allows for tissue-specific dystrophin expression under endogenous regulation, and of all the currently proposed treatments to address DMD, selected exon exclusion is the only option that has natural precedents. Oligonucleotide analogues administered to animal models of muscular dystrophy and evaluated in human myogenic cells have shown variable ability to remove the target exons.22Aartsma-Rus A Kaman WE Bremmer-Bout M Janson AA den Dunnen JT van Ommen GJ et al.Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells.Gene Ther. 2004; 11: 1391-1398Crossref PubMed Scopus (122) Google Scholar,23Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Wilton SD Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.J Gene Med. 2006; 8: 207-216Crossref PubMed Scopus (160) Google Scholar,24Gebski BL Errington SJ Johnsen RD Fletcher S Wilton SD Terminal antisense oligonucleotide modifications can enhance induced exon skipping.Neuromuscul Disord. 2005; 15: 622-629Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,25Gebski BL Mann CJ Fletcher S Wilton SD Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle.Hum Mol Genet. 2003; 12: 1801-1811Crossref PubMed Scopus (169) Google Scholar We have previously reported that a phosphorodiamidate morpholino oligomer (PMO) appeared superior at excluding exon 23 from the dystrophin transcript in vitro25Gebski BL Mann CJ Fletcher S Wilton SD Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle.Hum Mol Genet. 2003; 12: 1801-1811Crossref PubMed Scopus (169) Google Scholar and in vivo23Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Wilton SD Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.J Gene Med. 2006; 8: 207-216Crossref PubMed Scopus (160) Google Scholar,25Gebski BL Mann CJ Fletcher S Wilton SD Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle.Hum Mol Genet. 2003; 12: 1801-1811Crossref PubMed Scopus (169) Google Scholar in the mdx mouse model of muscular dystrophy, compared with a 2′-O-methyl phosphorothioate AO of identical sequence. In this study, we report that enhanced PMO uptake, mediated by a cell-penetrating peptide (CPP) delivery vector,26Youngblood DS Hatlevig SA Hassinger JN Iversen PL Moulton HM Stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells.Bioconjug Chem. 2007; 18: 50-60Crossref PubMed Scopus (143) Google Scholar results in systemic dystrophin expression and reduced dystrophic pathology in mdx mice at a significantly lower dose than used in other studies.23Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Wilton SD Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.J Gene Med. 2006; 8: 207-216Crossref PubMed Scopus (160) Google Scholar,27Alter J Lou F Rabinowitz A Yin H Rosenfeld J Wilton SD et al.Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.Nat Med. 2006; 12: 175-177Crossref PubMed Scopus (433) Google Scholar We propose that as a result of the enhanced uptake of the PMO conjugated to the CPP (PMO-pep), safety and efficacy can be optimized because of the lower dosages required for induced dystrophin expression. Dose evaluation. Neonatal mdxmice, 1–2 days old, were injected intraperitoneally (ip) with a single dose of 1, 2, 5, 10, or 25 mg/kg PMO-pep in normal saline. The mice were killed 2 weeks later. Tissue sections were analyzed for dystrophin expression by immunofluorescence (Figure 1). Dystrophin expression in diaphragm increased with PMO-pep dosage, reaching levels comparable to those of normal C57BL/10ScSn mice after a single treatment of 10 mg/kg. Long-term evaluation. In an attempt to develop a treatment regimen that would induce systemic dystrophin expression at the lowest possible dosage, three litters of mdx mice pups were given four once-weekly ip injections of 1, 2 (data not shown), or 5 mg/kg, beginning at 1 day of age, and analyzed for dystrophin expression by reverse-transcription–polymerase chain reaction (RT-PCR) at 6 weeks of age. The full-length transcript is represented by an amplicon of 901 base pairs, and the in-frame transcript excluding exon 23 is represented by the 688-base-pair product. Treatment with PMO-pep at a dosage of 1 mg/kg/week induced low levels of exon 23 skipping and dystrophin expression in diaphragm, as demonstrated by immunofluorescence; however, dystrophin expression was not detected in other tissues. Dystrophin levels were increased in diaphragm in mdxmice treated with 2 mg/kg/week PMO-pep, but only very low levels of expression were observed in distal muscles (data not shown). Figure 2a shows representative levels of native and induced dystrophin transcripts from an mdxmouse 3 weeks after the final treatment with 5 mg/kg PMO-pep. The amplicon excluding exon 23 (688 base pairs) appears as a major product in all tested tissues from treated mice, with the exception of cardiac muscle. The full-length transcript is present in all tissues except the PCR negative control and the diaphragm sample. In the latter case, exon skipping efficiency appeared to be 100%, as no full-length product was observed. No shortened transcript was detected in muscle from saline-injected mice. The 542-base-pair amplicon represents an out-of-frame transcript missing exons 22 and 23 and has been previously reported.28Mann CJ Honeyman K Cheng AJ Ly T Lloyd F Fletcher S et al.Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse.Proc Natl Acad Sci USA. 2001; 98: 42-47Crossref PubMed Scopus (353) Google Scholar,29Mann CJ Honeyman K McClorey G Fletcher S Wilton SD Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy.J Gene Med. 2002; 4: 644-654Crossref PubMed Scopus (130) Google Scholar Western blotting on samples from treated mdx mice (Figure 2b) confirms that four once-weekly injections of PMO-pep at 5 mg/kg per treatment induced normal levels of dystrophin in diaphragm (110%) and levels of 8 and 3% in tibialis and gluteal muscles, respectively, as determined by densitometry and comparison with C57BL/10ScSn tibialis muscle, 6 weeks after the initial treatment commencing at 1 day of age (3 weeks after the final treatment). No dystrophin was detected in cardiac muscle or in saline-injected controls. Persistence of the induced dystrophin expression was evaluated in mice at 6, 8, 12, and 26 weeks of age after four once-weekly treatments with 5 mg/kg PMO-pep and compared with results for sham-treated mdxand C57BL/10ScSn mice (8 weeks old). Dystrophin staining and muscle architecture appeared near-normal in diaphragm from treated mice aged 6, 8, and 12 weeks compared with C57BL/10ScSn mice (Figure 2c). Although some dystrophin was present in the diaphragm from the treated mice aged 26 weeks (i.e., 23 weeks after the final treatment at 3 weeks of age), the staining appeared discontinuous in some areas and muscle architecture shows some disruption. At 6 weeks of age, tissue samples from the treated animals show dystrophin expression and near-normal muscle architecture, except for the heart, which has no detectable dystrophin. Sections of quadriceps from these mice, labeled with NCL-DYS2 and Alexafluor 488, and Hoechst to visualize nuclei, demonstrated normalized pathology and central nucleation (data not shown). In mice of 8 and 12 weeks of age, dystrophin expression in tibialis anterior is limited to small groups of fibers. Dystrophin expression in colon was maintained at 12 weeks, but it declined by 26 weeks. To demonstrate that uptake of the PMO was not limited to neonatal mice, animals aged 1 day, 4 weeks, and 1 year were given four once-weekly treatments of PMO-pep at a dosage of 5 mg/kg and analyzed 8 weeks after the first treatment (i.e., at ages 8, 12, and 60 weeks). Dystrophin staining on representative diaphragm sections from all treated mice showed similar intensity to that seen in the C57BL/10ScSn sample (Figure 3a). No dystrophin was observed in the section from a sham-treated mdxmouse. The accumulation of scar tissue in mice aged 12 weeks and older is revealed by Picro Mallory trichrome staining (Figure 3b), and hematoxylin and eosin staining (Figure 3c) shows the marked infiltration of mononuclear cells. Treatment of neonatal mdx mice with PMO-pep (5 mg/kg/week) appears to abrogate the onset of the dystrophic pathology in the diaphragm. There is no evidence of infiltrating mononuclear cells, and connective tissue and muscle fibers appear normal. The same treatment regimen in older mice, commencing at 4 weeks and at 1 year of age, did not abolish the pre-treatment dystrophic pathology and disrupted muscle architecture in diaphragm, but it did result in substantial dystrophin expression (Figure 3a) and a marked reduction in mononuclear cells (Figure 3c). The functionality of the induced dystrophin is demonstrated by the uniformity of the muscle fibers in the hematoxylin and eosin–stained section of diaphragm in animals treated as a neonates. Growth rates of treated mice were monitored and compared with those of the untreated mdxand C57BL/10ScSn mice (Figure 4). There was no apparent difference in growth rates between mice in the various groups, until the age at which mdx mice begin to show evidence of muscle degeneration and regeneration (14–18 days). From this time, mdx mice are smaller than C57BL10/ScSn mice (P = 0.007); however, they continued to gain weight as adults, when the C57BL10/ScSn mice showed slower weight gain, and by approximately 50 days of age, the mice were of similar size. Mice treated with four once-weekly doses of PMO-pep at 5 mg/kg, from 2 to 22 days of age, had a slightly lower mean weight than untreated mdxmice; however, this was not significant and the mice were of similar size at 50 days of age. To monitor the effect of the treatment and the general health of the mice, liver transaminases, alkaline phosphatase, and creatinine were measured in serum samples from PMO-pep-treated and sham-treated mdx mice, and from C57BL10/ScSn mice aged 3–12 weeks. All parameters except alkaline phosphatase were elevated in mdxmice compared with C57BL10/ScSn mice; however, there was no significant difference between the PMO-pep-treated and sham-treated mdx mice (P > 0.05), indicating that the PMO- pep regimen at this dose is unlikely to be causing additional tissue damage (data not shown). In mdxmice aged 3–12 weeks that received four once-weekly injections of PMO-pep at 5 mg/kg per treatment, mean serum creatine kinase levels were approximately 50% of those of sham-treated age-matched controls; however, these levels were higher than those in C57BL10/ScSn mice (data not shown). A number of approaches, including gene or cell replacement, homologous gene up-regulation, and non-sense mutation suppression are being investigated to address the dystrophin deficiency in DMD (for a review, see refs. 4van Deutekom JC van Ommen GJ Advances in Duchenne muscular dystrophy gene therapy.Nat Rev Genet. 2003; 4: 774-783Crossref PubMed Scopus (176) Google Scholar, 30Bogdanovich S Perkins KJ Krag TO Khurana TS Therapeutics for Duchenne muscular dystrophy: current approaches and future directions.J Mol Med. 2004; 82: 102-115Crossref PubMed Scopus (90) Google Scholar). To date, the most significant improvements in DMD prognosis have come from steroid treatment (for a review, see refs. 31Biggar WD Klamut HJ Demacio PC Stevens DJ Ray PN Duchenne muscular dystrophy: current knowledge, treatment, and future prospects.Clin Orthop Relat Res. 2002; 401: 88-106Crossref PubMed Scopus (45) Google Scholar, 32Muntoni F Fisher I Morgan JE Abraham D Steroids in Duchenne muscular dystrophy: from clinical trials to genomic research.Neuromuscul Disord. 2002; 12 (Suppl. 1): S162-S165Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and nocturnal mechanically assisted ventilation.33Eagle M Baudouin SV Chandler C Giddings DR Bullock R Bushby K Survival in Duchenne muscular dystrophy: improvements in life expectancy since 1967 and the impact of home nocturnal ventilation.Neuromuscul Disord. 2002; 12: 926-929Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar It is not unreasonable to assume that if reduced respiratory stress can substantially improve the life of a DMD patient, benefits from any treatment that partially restores dystrophin expression are likely to be significant, particularly when used in conjunction with the current management practices. Targeted exon skipping will not be applicable to all DMD patients, in particular those affected by large genomic deletions or the loss of essential coding domains. The efficient application of specific exon skipping to bypass dystrophin mutations could result in a shortened dystrophin of at least partial function, and it has been estimated that up to 75% of DMD patients could benefit from exon removal to overcome dystrophin mutations.34Aartsma-Rus A Janson AA Kaman WE Bremmer-Bout M van Ommen GJ den Dunnen JT et al.Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense.Am J Hum Genet. 2004; 74: 83-92Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar Although genomic deletions including large or essential protein-coding domains may be amenable to AO-mediated messenger RNA reading frame restoration, the resultant protein could be functionally compromised. The utility of AO-induced exon skipping in restoring dystrophin expression in experimental systems has been demonstrated.23Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Wilton SD Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.J Gene Med. 2006; 8: 207-216Crossref PubMed Scopus (160) Google Scholar,25Gebski BL Mann CJ Fletcher S Wilton SD Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle.Hum Mol Genet. 2003; 12: 1801-1811Crossref PubMed Scopus (169) Google Scholar,28Mann CJ Honeyman K Cheng AJ Ly T Lloyd F Fletcher S et al.Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse.Proc Natl Acad Sci USA. 2001; 98: 42-47Crossref PubMed Scopus (353) Google Scholar,29Mann CJ Honeyman K McClorey G Fletcher S Wilton SD Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy.J Gene Med. 2002; 4: 644-654Crossref PubMed Scopus (130) Google Scholar,35Aartsma-Rus A Janson AA Kaman WE Bremmer-Bout M den Dunnen JT Baas F et al.Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients.Hum Mol Genet. 2003; 12: 907-914Crossref PubMed Scopus (223) Google Scholar,36Lu QL Mann CJ Lou F Bou-Gharios G Morris GE Xue SA et al.Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse.Nat Med. 2003; 9: 1009-1014Crossref PubMed Scopus (332) Google Scholar,37Lu QL Rabinowitz A Chen YC Yokota T Yin H Alter J et al.Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles.Proc Natl Acad Sci USA. 2005; 102: 198-203Crossref PubMed Scopus (357) Google Scholar The current challenge is to achieve sustained dystrophin expression accompanied by minimal adverse effects in most, if not all, tissues affected by the absence of dystrophin. Ideally, anti-sense compounds for induced exon skipping should be directed to the most amenable target motif, show efficacy at low doses, have sustained action, and be easily delivered as a simple formulation. Previously, we evaluated different anti-sense chemistries in vivo and observed that PMOs induced substantial shortened dystrophin transcript 2 weeks after a single intramuscular injection.23Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Wilton SD Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.J Gene Med. 2006; 8: 207-216Crossref PubMed Scopus (160) Google Scholar It is generally assumed that if AO-induced exon skipping is to provide the greatest benefit to DMD patients, it will be necessary to commence treatment before irreversible muscle loss has occurred. Although intramuscular injection of AOs is useful in evaluating the efficacy of compounds in vivo, any treatment for DMD will need to be effective after systemic delivery. Because skeletal muscle constitutes approximately 30% of the total body mass and because dystrophin isoforms are also expressed in a variety of tissues, DMD can be addressed only by systemic dystrophin restoration. PMO anti-sense compounds have been shown to be effective in removing target exons from the dystrophin pre-messenger RNA,23Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Wilton SD Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.J Gene Med. 2006; 8: 207-216Crossref PubMed Scopus (160) Google Scholar,25Gebski BL Mann CJ Fletcher S Wilton SD Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle.Hum Mol Genet. 2003; 12: 1801-1811Crossref PubMed Scopus (169) Google Scholar,27Alter J Lou F Rabinowitz A Yin H Rosenfeld J Wilton SD et al.Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.Nat Med. 2006; 12: 175-177Crossref PubMed Scopus (433) Google Scholar are reputedly non-toxic,38Iversen PL Arora V Acker AJ Mason DH Devi GR Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a phase I safety study in humans.Clin Cancer Res. 2003; 9: 2510-2519PubMed Google Scholar and have minimal non-anti-sense effects.39Iversen PL Phosphorodiamidate morpholino oligomers: favorable properties for sequence-specific gene inactivation.Curr Opin Mol Ther. 2001; 3: 235-238PubMed Google Scholar,40Summerton J Morpholino antisense oligomers: the case for an RNase H-independent structural type.Biochim Biophys Acta. 1999; 1489: 141-158Crossref PubMed Scopus (565) Google Scholar We now report that systemic PMO distribution, efficient intracellular uptake of the oligomer, and targeted exon exclusion are greatly enhanced by using a CPP conjugated to the PMO. Dystrophin expression in diaphragm and limb muscles was demonstrated using RT-PCR, western blotting, and immunofluorescence. Normalized dystrophin expression in diaphragm and marked expression in smooth muscle of the gut after ip delivery of the PMO-pep could have been mediated, in part, by diffusion from the abdominal cavity. However, exon 23 skipping and uniform dystrophin expression in distal muscles, albeit at lower levels, can have resulted only from systemic distribution of the oligomer. The dosages of PMO-pep (125 μg/adult mouse, once weekly) are markedly lower than those used previously for an unconjugated PMO (625 μg/adult mouse, thrice weekly).23Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Wilton SD Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.J Gene Med. 2006; 8: 207-216Crossref PubMed Scopus (160) Google Scholar Using a substantially higher dosage, Alter et al.27Alter J Lou F Rabinowitz A Yin H Rosenfeld J Wilton SD et al.Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.Nat Med. 2006; 12: 175-177Crossref PubMed Scopus (433) Google Scholar reported dystrophin expression after seven intravenous injections of unconjugated PMO at a dosage of 2,000 μg/mouse once weekly in adult mice. At the dosages used in our current report, the peptide vector and PMO do not appear to cause any obvious adverse effects that could be detected by routine observation and blood biochemistry analysis. Serum transaminases are normally elevated when muscle damage is present, and we show that there was no further elevation of these parameters in mice treated with PMO-pep, indicating that the administration of the compound at the dosages used here was unlikely to have had adverse effects on the liver. Use of the ip delivery route permits treatment of neonatal animals and allowed us to demonstrate that PMO-induced dystrophin can largely prevent onset of the dystrophic process that normally begins at approximately 14 days of age.41Muntoni F Mateddu A Marchei F Clerk A Serra G Muscular weakness in the mdx mouse.J Neurol Sci. 1993; 120: 71-77Abstract Full Text PDF PubMed Scopus (72) Google Scholar Treatment of older mice restored dystrophin expression in diaphragm and reduced the number of mononuclear cells invading the tissue; however, as expected, the dystrophic pathology was not reversed, as the muscle damage and dystrophic phenotype were already well established. The progression of muscle wasting in DMD is relentless, and" @default.
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• W2061596420 date "2007-09-01" @default.
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• W2061596420 title "Morpholino Oligomer–Mediated Exon Skipping Averts the Onset of Dystrophic Pathology in the mdx Mouse" @default.
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