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• W2007123925 abstract "The administration of antisense oligonucleotides (AOs) to skip one or more exons in mutated forms of the DMD gene and so restore the reading frame of the transcript is one of the most promising approaches to treat Duchenne muscular dystrophy (DMD). At present, preclinical studies demonstrating the efficacy and safety of long-term AO administration have not been conducted. Furthermore, it is essential to determine the minimal effective dose and frequency of administration. In this study, two different low doses (LDs) of phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 in the mdx dystrophic mouse were administered for up to 12 months. Mice treated for 50 weeks showed a substantial dose-related amelioration of the pathology, particularly in the diaphragm. Moreover, the generalized physical activity was profoundly enhanced compared to untreated mdx mice showing that widespread, albeit partial, dystrophin expression restores the normal activity in mdx mice. Our results show for the first time that a chronic long-term administration of LDs of unmodified PMO, equivalent to doses in use in DMD boys, is safe, significantly ameliorates the muscular dystrophic phenotype and improves the activity of dystrophin-deficient mice, thus encouraging the further clinical translation of this approach in humans. The administration of antisense oligonucleotides (AOs) to skip one or more exons in mutated forms of the DMD gene and so restore the reading frame of the transcript is one of the most promising approaches to treat Duchenne muscular dystrophy (DMD). At present, preclinical studies demonstrating the efficacy and safety of long-term AO administration have not been conducted. Furthermore, it is essential to determine the minimal effective dose and frequency of administration. In this study, two different low doses (LDs) of phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 in the mdx dystrophic mouse were administered for up to 12 months. Mice treated for 50 weeks showed a substantial dose-related amelioration of the pathology, particularly in the diaphragm. Moreover, the generalized physical activity was profoundly enhanced compared to untreated mdx mice showing that widespread, albeit partial, dystrophin expression restores the normal activity in mdx mice. Our results show for the first time that a chronic long-term administration of LDs of unmodified PMO, equivalent to doses in use in DMD boys, is safe, significantly ameliorates the muscular dystrophic phenotype and improves the activity of dystrophin-deficient mice, thus encouraging the further clinical translation of this approach in humans. Duchenne muscular dystrophy (DMD) is an X-linked inherited neuromuscular disease caused by mutations in the dystrophin gene, which cause a deficiency in production of the dystrophin protein leading to an increase in muscle fragility. Mutations generating shorter but in-frame, and therefore still partially functional proteins, lead to a milder myopathy, sometimes with a very late onset, known as Becker muscular dystrophy.1England 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,2Monaco 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 mdx mouse, which carries a nonsense point mutation in the exon 23, also does not produce dystrophin in its muscles, except for rare revertant dystrophin-positive fibers in skeletal and cardiac muscles.3Bulfield G Siller WG Wight PA Moore KJ X chromosome-linked muscular dystrophy (mdx) in the mouse.Proc Natl Acad Sci USA. 1984; 81: 1189-1192Crossref PubMed Scopus (1400) Google Scholar,4Hoffman EP Morgan JE Watkins SC Partridge TA Somatic reversion/suppression of the mouse mdx phenotype in vivo.J Neurol Sci. 1990; 99: 9-25Abstract Full Text PDF PubMed Scopus (232) Google Scholar Exclusion of exon 23 from the mature mRNA (exon skipping) in the mdx mouse leads to the restoration of dystrophin expression and has been achieved by using different antisense oligonucleotides (AOs) chemistries, among which the most promising are the phosphorothioate-linked 2′-O-methyl RNA (2OMePS) and the phosphorodiamidate morpholino oligomer (PMO).5Alter 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,6Gebski 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,7Heemskerk H de Winter C van Kuik P Heuvelmans N Sabatelli P Rimessi P et al.Preclinical PK and PD studies on 2'-O-methyl-phosphorothioate RNA antisense oligonucleotides in the mdx mouse model.Mol Ther. 2010; 18: 1210-1217Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar,8Lu 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,9Lu 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,10Malerba A Thorogood FC Dickson G Graham IR Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice.Hum Gene Ther. 2009; 20: 955-965Crossref PubMed Scopus (51) Google Scholar The more prolonged resistance to endonucleases and the higher affinity to the sequence target make the PMO particularly suitable for in vivo applications.11Popplewell LJ Trollet C Dickson G Graham IR Design of phosphorodiamidate morpholino oligomers (PMOs) for the induction of exon skipping of the human DMD gene.Mol Ther. 2009; 17: 554-561Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar,12Amantana A Iversen PL Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers.Curr Opin Pharmacol. 2005; 5: 550-555Crossref PubMed Scopus (124) Google Scholar Furthermore, the PMO can be readily linked to cell-penetrating peptides or octaguanidine groups improving the efficiency of systemic delivery in skeletal13Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Steinhaus JP et al.Morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse.Mol Ther. 2007; 15: 1587-1592Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar,14Moulton HM Fletcher S Neuman BW McClorey G Stein DA Abes S et al.Cell-penetrating peptide-morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo.Biochem Soc Trans. 2007; 35: 826-828Crossref PubMed Scopus (73) Google Scholar,15Jearawiriyapaisarn N Moulton HM Buckley B Roberts J Sazani P Fucharoen S et al.Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice.Mol Ther. 2008; 16: 1624-1629Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar,16Morcos PA Li Y Jiang S Vivo-Morpholinos: a non-peptide transporter delivers Morpholinos into a wide array of mouse tissues.BioTechniques. 2008; 45 (616, 618 passim): 613-614Crossref PubMed Scopus (148) Google Scholar,17Yin H Moulton HM Seow Y Boyd C Boutilier J Iverson P et al.Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function.Hum Mol Genet. 2008; 17: 3909-3918Crossref PubMed Scopus (173) Google Scholar,18Moulton HM Wu B Jearawiriyapaisarn N Sazani P Lu QL Kole R Peptide-morpholino conjugate: a promising therapeutic for Duchenne muscular dystrophy.Ann N Y Acad Sci. 2009; 1175: 55-60Crossref PubMed Scopus (34) Google Scholar,19Wu B Li Y Morcos PA Doran TJ Lu P Lu QL Octa-guanidine morpholino restores dystrophin expression in cardiac and skeletal muscles and ameliorates pathology in dystrophic mdx mice.Mol Ther. 2009; 17: 864-871Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar,20Yin H Moulton HM Betts C Seow Y Boutilier J Iverson PL et al.A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deficient mdx mice.Hum Mol Genet. 2009; 18: 4405-4414Crossref PubMed Scopus (120) Google Scholar and cardiac muscles.19Wu B Li Y Morcos PA Doran TJ Lu P Lu QL Octa-guanidine morpholino restores dystrophin expression in cardiac and skeletal muscles and ameliorates pathology in dystrophic mdx mice.Mol Ther. 2009; 17: 864-871Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar,21Wu B Moulton HM Iversen PL Jiang J Li J Li J et al.Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer.Proc Natl Acad Sci USA. 2008; 105: 14814-14819Crossref PubMed Scopus (201) Google Scholar,22Jearawiriyapaisarn N Moulton HM Sazani P Kole R Willis MS Long-term improvement in mdx cardiomyopathy after therapy with peptide-conjugated morpholino oligomers.Cardiovasc Res. 2010; 85: 444-453Crossref PubMed Scopus (74) Google Scholar At present, safety for these latter modified compounds has not been demonstrated in the human.23Moulton HM Moulton JD Morpholinos and their peptide conjugates: Therapeutic promise and challenge for Duchenne muscular dystrophy.Biochim Biophys Acta. 2010; 1798: 2296-2303Crossref PubMed Scopus (160) Google Scholar Currently only unmodified AOs are in use in clinical trials. Two independent clinical trials in the Netherlands and UK have already demonstrated safety and efficiency of 2OMePS and PMO AOs targeting the exon 51 after intramuscular injection in DMD patients24van Deutekom JC Janson AA Ginjaar IB Frankhuizen WS Aartsma-Rus A Bremmer-Bout M et al.Local dystrophin restoration with antisense oligonucleotide PRO051.N Engl J Med. 2007; 357: 2677-2686Crossref PubMed Scopus (700) Google Scholar,25Kinali M Arechavala-Gomeza V Feng L Cirak S Hunt D Adkin C et al.Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study.Lancet Neurol. 2009; 8: 918-928Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar,26Sazani P Weller DL Shrewsbury SB Safety pharmacology and genotoxicity evaluation of AVI-4658.Int J Toxicol. 2010; 29: 143-156Crossref PubMed Scopus (36) Google Scholar and their systemic delivery has been investigated in further clinical trials (ref. 27Goemans NM Buyse G Tulinius M Verschuuren JJG de Kimpe SJ van Deutekom JCT T.O.4 A phase I/IIa study on antisense compound PRO051 in patients with Duchenne muscular dystrophy.Neuromuscular Disorders. 2009; 19: 659-660Abstract Full Text Full Text PDF Google Scholar and NTC00844597). We have recently demonstrated that the choice of an effective dosing regimen for PMO administration is a key parameter in reducing the amount of naked PMO necessary for systemic delivery and that even a low dosage such as 4 weekly injections of 5 mg/kg PMO induces a significant increase in dystrophin expression.10Malerba A Thorogood FC Dickson G Graham IR Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice.Hum Gene Ther. 2009; 20: 955-965Crossref PubMed Scopus (51) Google Scholar Due to the nature of the AO action, repeated chronic administration of AO is necessary to guarantee a continuous production of dystrophin. However, at present, no preclinical studies have been published about the effect of long-term systemic PMO administration in the mdx mouse and the efficacy and the safety of such a long treatment has not been proven. In the present study, we show that systemic delivery of low, clinically applicable, doses of PMO in mdx mice for up to 1 year is safe and ameliorates the pathology of skeletal muscles. Importantly, restored dystrophin expression partially recovered muscle functionality and limb strength. In mice treated for 1 year, the partial, body-wide dystrophin expression resulted in motor activity and movement behavior of mdx that was indistinguishable from normal wild-type C57BL10 mice. These encouraging results support the feasibility of a long-term PMO treatment in humans as a therapy for DMD. Six-week-old mdx mice were injected with PMO diluted in sterile saline via the tail vein. Two doses of PMO were tested in this study: 5 mg/kg/injection and 50 mg/kg/injection that we referred as low dose (LD) and high dose (HD) because respectively lower and higher compared to the highest dose currently under clinical trial in human DMD patients (20 mg/kg, NTC00844597). A regimen of 4 weekly injections (1 cycle) and 6 weeks of no treatment was adopted in each group of treated mice. The cycle of 4 weekly injections was repeated two or five times and animals were analyzed 6 weeks after the last injection (Supplementary Figure S1). After 20 weeks (two treatment cycles), dystrophin was widely expressed in all the muscles analyzed of treated mice achieving 23–27% of dystrophin-positive fibers in tibialis anterior (TA) and ~30% in quadriceps (Figure 1a). By prolonging the treatment to 50 weeks, the number of dystrophin-positive fibers remained unchanged in triceps (42 and 48% for LD and HD, respectively, n = 4–8) whereas they generally increased in the other muscles tested compared to the two cycle time point. In particular, vastus lateralis and gastrocnemius of HD-treated mice contained >80% dystrophin-positive fibers whereas thinner muscles like soleus and EDL generally expressed smaller percentages of dystrophin-positive fibers (Figure 1a). After either 20 or 50 weeks of treatment both dose regimens gave rise to approximately the same number of dystrophin-positive fibers in most muscles even though a stronger signal was observed after immunofluorescence for dystrophin in muscles treated for 20 weeks with the HD (Figure 1b,c). A semiquantitative intensity analysis corroborated these observations confirming that the averaged dystrophin intensity in TA fibers after 20 weeks of HD treatment (70 ± 3% of the intensity of control C57BL10) was higher than in LD-treated muscles (44 ± 2% of control) (n = 4–5, P < 0.0001) (Figure 1d). In TA of 50 weeks treated mice, patches of dystrophin negative fibers were substantially reduced but not entirely absent even though an extended general fluorescent signal, clearly detectable above the threshold set up on the untreated muscles might be the result of a low dystrophin expression in the majority of fibers (Figure 1c). Accordingly, after 50 weeks of treatment a similar averaged level of intensity in TA sections stained for dystrophin was measured for the two doses (60 ± 1.6 and 62 ± 1.5% for LD and HD compared to C57BL10 fiber intensity, n = 3–5, P = 0.35, Figure 1d). However, when fluorescent intensity was measured specifically in the dystrophin-positive fibers of these muscles, the myofibers of HD-treated mice gave rise to similar amount of dystrophin compared to C57BL10 fibers (1,230 ± 30 compared to 1,205 ± 35 intensity units, n = 5, P = 0.16), whereas the fibers of LD-treated mice expressed significantly less dystrophin compared to the HD treatment (1,095 ± 40 intensity units) [n = 3–5, P < 0.0001 (Figure 1e)]. Western blot analysis confirmed the results obtained by dystrophin intensity study showing that after 20 weeks of PMO administration, TA muscles of HD-treated mice expressed generally more dystrophin compared to the muscles of LD-treated mice. However, the difference was not significant (20 ± 3 and 43 ± 13% of dystrophin expressed by C57 for LD and HD, respectively, n = 4–5, P = 0.17). A similar result was found for total quadriceps muscles (32 ± 5 and 52 ± 14% for LD and HD, respectively, n = 4–5, P = 0.27) (Figure 2a,c). After 50 weeks of PMO treatment, no obvious difference in the levels of dystrophin expression was found between the LD and HD doses in TA (61 ± 6 and 71 ± 7%, respectively, n = 5, P = 0.32), total quadriceps (44 ± 11 and 50 ± 8%, n = 5, P = 0.7), and Tri (34 ± 9 and 48 ± 6%, n = 5, P = 0.21) (Figure 2b,c). No dystrophin expression was observed in cardiac muscles of PMO-treated mice (data not shown). These data suggest that the 20 weeks LD and HD PMO administration produced a different amount of dystrophin in mdx skeletal muscles, but this difference tends to decrease with a longer administration.Figure 2Western immunoblotting evaluation of dystrophin expression in skeletal muscles of mdx mice following chronic repeated treatment with intravenous PMO. Mdx mice were treated for a period of 20 or 50 weeks over which respectively two or five cycles of repeated low dose (LD: 5 mg/kg/week) and high dose (HD: 50 mg/kg/week). PMO were administered intravenously. (a) Dystrophin (dys) and α-tubulin (α-tub) in TA and Qa muscles from mdx mice treated for 20 weeks with PMO: three representative samples are shown for each treatment. (b) Dystrophin (dys) and α-tubulin (α-tub) in TA, Qa, and Tri muscles from mdx mice treated for 50 weeks with PMO: three representative samples are shown for each treatment. (c) Densitometric analysis of dystrophin expression from western immunoblot of PMO-treated TA, Qa, and Tri muscles. Forty microgram for mdx, or twenty-five microgram for C57 were loaded. Values for dystrophin densitometry were normalized internally against α-tubulin and presented as a percentage of the level found in control C57BL10 mouse muscle. No statistically significant difference in dystrophin expression between LD- and HD-treated samples was observed at the end of either the 20- or 50-week treatment period. PMO, phosphorodiamidate morpholino oligomer; Qa, total quadriceps; TA, tibialis anterior; Tri, triceps brachii.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The considerable dystrophin restoration observed after 20 weeks of treatment in diaphragm of HD-treated mice, TA, and vastus lateralis was in keeping with a reduced number of centrally nucleated fibers compared with untreated mdx mice (in TA 82.5 ± 1.4% compared to 89 ± 0.5%, in vastus lateralis 74.6 ± 1.9% compared to 91.9 ± 1.3, in diaphragm after HD treatment 57.4 ± 2.9% compared to 67.4 ± 1.8%, n = 4–6, P < 0.0001) (Figure 3a). In muscles presenting a lower number of dystrophin-positive fibers after LD administration, such as the rectus femoris (about 10–15%, not shown in the graph) and in diaphragm (3.8 ± 1.1%), no change in centrally nucleated fiber number was observed (Figure 3a). The extension of the treatment to 50 weeks further reduced the percentage of centrally nucleated fibers compared to age-matched mdx controls (in TA 72.4 ± 1.9 and 74 ± 1.7% for HD and LD, respectively compared to 83.3 ± 3.4% in mdx, n = 8, P = 0.02 for LD, P = 0.012 for HD; in vastus lateralis 68 ± 1.8 and 65.3 ± 1.7% for HD and LD, respectively compared to 84.4 ± 1.2% in mdx, P = 0.0032 for LD and P < 0.0001 for HD; in rectus femoris 67.5 ± 3 and 66.7 ± 2.8% for HD and LD, respectively compared to 84 ± 2.2% in mdx, P < 0.0001 for LD and HD) indicating lower fiber turnover. In TA of HD-treated animals for 50 weeks, weight (84.2 ± 2.4 mg compared to 95.3 ± 4 mg in mdx, n = 8, P = 0.03) (Figure 3b) and cross-sectional area (9.6 ± 0.3 mm2 compared to 10.8 ± 0.5 mm2 in mdx, n = 8, P = 0.048) (Figure 3c) significantly decreased, indicating a reduction of the typical mdx hypertrophy. This was accompanied by a significant increase in average fiber cross-sectional area (327 ± 10 µm2 and 349 ± 11 µm2 for LD and HD compared to 270 ± 7 µm2 in mdx, n = 8, P < 0.0001) (Figure 3d) in all the fibers suggesting a decrease in fiber splitting. Importantly, the cross-sectional area of the dystrophin-positive fibers reached values comparable to the normal fibers (Figure 3d), confirming a protective role for the expressed dystrophin. Finally, the fibrotic tissue in TA muscles, measured as the collagen VI positive area in representative transverse sections of treated muscles, was comparable to that of normal mice (12.7 ± 0.9 and 12 ± 0.7% for LD and HD compared to 9.9 ± 1.6% in wild-type mice, n = 8, P= 0.18 and P = 0.2 for LD and HD, respectively) and significantly lower than the collagen VI found in mdx muscle (16.5 ± 1.1%, Figure 3e). These data demonstrate that the chronic administration of low, clinically applicable doses of PMO ameliorates the histology of dystrophic muscles. In mice treated for 50 weeks, the diaphragm showed the most obvious morphological improvement particularly after HD treatment. In contrast to limb muscles in mdx mouse, the diaphragm undergoes continuous degeneration with a loss of myofibers starting from 6 months of age. The diaphragms of treated mice showed less infiltrate with more closely packed myofibers compared to the diaphragms of untreated mdx mice (Figure 4a). In agreement with these findings, the fibrotic tissue in diaphragms of HD PMO-treated mice occupied significantly less area compared to the tissue of untreated mice as shown by immunostaining for collagen VI (51.7% of the total area in HD compared to 70.2 ± 2.9% in mdx mice, n = 8, P = 0.0018) (Figure 4b,c). Although no significant difference was observed in the number of centrally nucleated fiber between treated and untreated mdx mice (Figure 3a) the number of fibers per unit area of diaphragm in HD-treated mice was comparable to that of C57BL10 mice (1,935 ± 163 fibers/mm2 in HD-treated mice compared to 2,137 ± 168 fibers/mm2 in wild-type mice, n = 8, P = 0.23) (Figure 4d). These data demonstrate that the considerable muscle fiber loss occurring in diaphragms of 13-month-old mdx mice can be delayed by administering clinically applicable doses of PMOs. To examine whether dystrophin expression in muscle of LD- and HD-treated mice was sufficient to improve muscle function, we measured in vivo both the contractile properties of TA muscles and their susceptibility to contraction-induced injury. We found that the 20 weeks administration of PMO followed by dystrophin expression in ~25% of TA muscle fibers, only resulted in a tendency to improve specific force after LD treatment [20.6 ± 0.3 N/cm2 compared to 19 ± 0.4 N/cm2 in mdx, n = 4–6 (P = 0.046)] (Figure 5a). The 50 weeks PMO administration induced a significant increase in specific force in TA of HD-treated mice (19.2 ± 0.5 N/cm2 in HD compared to 17.9 ± 0.2 N/cm2 in mdx mice, n = 8, P = 0.046) (Figure 5a). Interestingly, treating mdx mice for 20 weeks at both doses of PMO induced sufficient levels of dystrophin to statistically improve the muscle resistance to seven eccentric contractions compared to mdx controls (41.6 ± 4.9 and 50.4 ± 5.7% of initial force for LD and HD, respectively compared to 27.2 ± 3.8% in mdx mice, n = 4–6, P < 0.05) (Figure 5b). However, the resistance to eccentric contractions was not improved in TA muscles after the 50 weeks treatments (Figure 5c). The high amount of dystrophin-positive fibers observed in the triceps of mice treated for 20 weeks correlated with a statistically significant 30% increase in forelimb force of treated mice compared to mdx control mice as measured by grip strength test (1.86 ± 0.06 and 1.87 ± 0.11 kilogram-force/kg for LD and HD, respectively compared to 1.37 ± 0.07 kilogram-force/kg in mdx, n = 4–6, P = 0.0015 for LD and P = 0.0046 for HD compared to mdx mice) (Figure 5d). The same type of analysis in 50 weeks-treated mice showed a significant increase in limb muscle strength in HD-treated mice (2.22 ± 0.11 kilogram-force/kg for HD compared to 1.91 ± 0.06 kilogram-force/kg in mdx n = 8, P = 0.03) (Figure 5d). The analysis of creatine kinase concentration in the blood-derived serum of both dose-treated mice for 20 weeks (Figure 5e) confirmed the improvement in sarcolemmal function (2,004 ± 457 and 1,570 ± 478 U/l for LD and HD, respectively compared to 3,494 ± 358 U/l in mdx, n = 4–6, P = 0.0322 and P = 0.0089, respectively for LD and HD). After five cycles of treatment, the levels of creatine kinase concentration in serum were kept low after both the treatments indicating a functional effect of the restored dystrophin (1,700 ± 192 and 1,619 ± 544 U/l for LD and HD compared to 4,155 ± 468 U/l in mdx mice, n = 8, P = 0.0005 and P = 0.005 for LD and HD, respectively) (Figure 5e). This was confirmed by coimmunostaining for dystrophin and mouse immunoglobulin G (IgG) showing that no necrotic fibers containing IgG fibers were present in treated muscles after two or five cycles compared to the untreated mdx controls (Supplementary Figure S2a). The dystrophin-associated protein complex showed a dose–response restoration with a uniform and wide expression in the sarcolemma of dystrophin-positive fibers after HD treatment (Supplementary Figure S2b). In particular, the neuronal nitric oxide synthase protein was correctly associated with the sarcolemma of 80–90% of dystrophin-positive fibers (45–50% of the total fibers in TA, Supplementary Figure S2c). Importantly, no difference was observed in neuronal nitric oxide synthase expression between two or five cycles of PMO treatment showing that the force transfer mechanism mediated by dystrophin-neuronal nitric oxide synthase was similarly restored. Taken together, these data suggest that there is a substantial functional improvement at the end of the treatment. Dosages that in young muscles were sufficient to provide resistance to eccentric exercise do not appear sufficient in adult/aged muscles. However, when the force generated by all the muscles of the same limb is considered, long-term PMO-treated mice show a substantial improvement compared to untreated mdx mice. Mice were monitored using open-field behavioral activity cages and 20 different parameters were recorded at the end of the experiment (the complete set of data is available upon request). In 12 out of 20 parameters, 13-month-old-untreated mdx mice showed significant differences in locomotor behavior compared to age- and sex-matched normal mice (Figure 6a). After five cycles, treated mice exhibited a dose-dependent increase in horizontal (active time, total activity, front to back counts) and vertical (rearing time) activities and a substantial decrease in inactive time with a consequent normalization compared to the age-matched C57BL10 mice (Figure 6b–d). In most of the parameters, the HD-treated mdx mice had higher performances compared to C57BL10 with statistically significant changes in “fast activity ” and “fast static counts ” (Figure 6a). However, 13-month-old C57BL10 mice weighed more due to a greater body fat content than age-matched mdx28Gregorevic P Blankinship MJ Allen JM Chamberlain JS Systemic microdystrophin gene delivery improves skeletal muscle structure and function in old dystrophic mdx mice.Mol Ther. 2008; 16: 657-664Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar and this probably reduced the fast activity of wild-type mice. These data suggest that the prolonged administration of PMO produces a beneficial effect in skeletal muscle of treated mice restoring the wild-type activity. No deaths or loss of weight due to PMO administration were detected during the course of the experiment (Supplementary Figure S3). To investigate possible toxic effect due to long-term treatment, the concentrations of kidney and liver enzymes were evaluated in serum. The long-term PMO delivery did not modify the concentration of aspartate aminotransferase, alanine aminotransferase, and gamma glutamyltransferase in serum, which suggests that there are no detrimental effects of PMO in the liver. Creatinine concentration was recorded in the same range as untreated mice indicating no adverse effect of PMO on the kidney (Figure 7a). The histological structure of both organs was also evaluated by hematoxylin and eosin staining and no infiltrate or vacuolization was observed confirming the safety of PMO treatment (Figure 7b). All these data showed that 50 weeks chronic administration of PMO is safe in mdx mice. The choice of an optimal dosing regimen plays a pivotal role in clinical applications of any medicinal compound, including AOs for the treatment of DMD. PMO is one of the most promising AO chemistries due to the high efficiency and safety profile in in vivo applications.5Alter 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,26Sazani P Weller DL Shrewsbury SB Safety pharmacology and genotoxicity evaluation of AVI-4658.Int J Toxicol. 2010; 29: 143-156Crossref PubMed Scopus (36) Google Scholar However, at present, only short-term studies have been performed5Alter 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,26Sazani P Weller DL Shrewsbury SB Safety pharmacology and genotoxicity evaluation of AVI-4658.Int J Toxicol. 2010; 29: 143-156Crossref PubMed Scopus (36) Google Scholar,29Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Wilton SD Dystrophin ex" @default.
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• W2007123925 title "Chronic Systemic Therapy With Low-dose Morpholino Oligomers Ameliorates the Pathology and Normalizes Locomotor Behavior in mdx Mice" @default.
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