FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2072578275> ?p ?o ?g. }

• W2072578275 endingPage "15" @default.
• W2072578275 startingPage "9" @default.
• W2072578275 abstract "Duchenne muscular dystrophy (DMD) is associated with mutations in the dystrophin gene that disrupt the open reading frame whereas the milder Becker's form is associated with mutations which leave an in-frame mRNA transcript that can be translated into a protein that includes the N- and C- terminal functional domains. It has been shown that by excluding specific exons at, or adjacent to, frame-shifting mutations, open reading frame can be restored to an out-of-frame mRNA, leading to the production of a partially functional Becker-like dystrophin protein. Such targeted exclusion can be achieved by administration of oligonucleotides that are complementary to sequences that are crucial to normal splicing of the exon into the transcript. This principle has been validated in mouse and canine models of DMD with a number of variants of oligonucleotide analogue chemistries and by transduction with adeno-associated virus (AAV)-small nuclear RNA (snRNA) reagents encoding the antisense sequence. Two different oligonucleotide agents are now being investigated in human trials for splicing out of exon 51 with some early indications of success at the biochemical level. Duchenne muscular dystrophy (DMD) is associated with mutations in the dystrophin gene that disrupt the open reading frame whereas the milder Becker's form is associated with mutations which leave an in-frame mRNA transcript that can be translated into a protein that includes the N- and C- terminal functional domains. It has been shown that by excluding specific exons at, or adjacent to, frame-shifting mutations, open reading frame can be restored to an out-of-frame mRNA, leading to the production of a partially functional Becker-like dystrophin protein. Such targeted exclusion can be achieved by administration of oligonucleotides that are complementary to sequences that are crucial to normal splicing of the exon into the transcript. This principle has been validated in mouse and canine models of DMD with a number of variants of oligonucleotide analogue chemistries and by transduction with adeno-associated virus (AAV)-small nuclear RNA (snRNA) reagents encoding the antisense sequence. Two different oligonucleotide agents are now being investigated in human trials for splicing out of exon 51 with some early indications of success at the biochemical level. From the moment of its identification, the Duchenne muscular dystrophy (DMD) gene, was clearly going to test the ingenuity of would-be gene therapists. The need to achieve body-wide distribution of the largest known gene is compounded by its structural role as the keystone of a transmembrane cell–surface protein complex; removing the possibility, even with a fully functional protein, of the amplifying effect of an enzyme and implying the need for near-normal molar concentrations to approach normal function. Strange then, that one of the more promising strategies for treating DMD, the skipping of mutated sites, is actually facilitated by the large size and modular structure of dystrophin: its major functional binding sites being separated by a long stretch of rod-like “spacer” that carries no essential function and is the site of the more common DMD mutations. Use of antisense oligonucleotides to modulate splicing of the dystrophin gene so as to restore a translatable mRNA transcript was mooted some years ago on the basis of in vitro data1Dunckley MG Manoharan M Villiet P Eperon IC Dickson G Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides.Hum Mol Genet. 1998; 7: 1083-1090Crossref PubMed Scopus (215) Google Scholar,2Takeshima Y Nishio H Sakamoto H Nakamura H Matsuo M Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.J Clin Invest. 1995; 95: 515-520Crossref PubMed Google Scholar but firm evidence for practical utility of this approach awaited studies in the mdx mouse model of DMD.3Alter 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,4Lu 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 (356) Google Scholar,5Lu 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,6Mann 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 (352) Google Scholar These, in turn, set in train a concerted effort to advance the technology toward human trials, as summarized in the following accounts of work presented and discussed at a meeting held in the Banbury Center at Cold Spring Harbor from the 14th to the 17th of October 2008. Progressive advances in exon skipping for DMD have been related to the application of new antisense oligomer chemistries and their modification for improved delivery (Figure 1). The most widely used chemistry is the 2′O-methylphosphorothioate-modified (2′OMePS) antisense oligoribonucleotide (AON). This modification provides resistance to nuclease degradation while retaining negative charge to facilitate effective delivery in cell culture systems by most delivery reagents.7Aartsma-Rus A Bremmer-Bout M Janson AA den Dunnen JT van Ommen GJ van Deutekom JC Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy.Neuromuscul Disord. 2002; 12: S71-S77Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar The potential of this chemistry for treating DMD was initially demonstrated in dystrophic mdx mice5Lu 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,6Mann 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 (352) Google Scholar and more recently by intramuscular injection in DMD patients.8van 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 (699) Google Scholar However, for systemic delivery, 2′OMePS showed limited efficiency in the mdx mouse. Three intravenous (i.v.) injections of 2 mg 2′OMePS/mouse (~60–80 mg/kg) at weekly intervals did induce detectable dystrophin expression in all skeletal muscles, but only in sparse focal patches of muscle fibers within each muscle and never at >5% of normal levels. Disappointingly too, little or no dystrophin expression was seen in cardiac muscle. No toxicity to liver or kidney was observed. Thus, assuming that the preclinical model recapitulates precisely the efficiency and pharmacokinetics of administration to DMD boys, 2′OMePS appear safe but it is uncertain whether their systemic use would induce sufficient dystrophin expression to have a therapeutic impact in DMD boys.4Lu 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 (356) Google Scholar More recently, phosphorodiamidate morpholino oligomers (PMO) have been explored for exon skipping in the dystrophin gene. In PMO, the phosphodiester bond is replaced by phosphorodiamidate linkage and the ribose replaced by a morpholino moiety (Figure 1). PMOs are charge-neutral and refractory to biological degradation. This chemistry has long been used for translational blockade in zebrafish; penetrating the cells of the developing fishes relatively easily.9Nasevicius A Ekker SC Effective targeted gene “knockdown” in zebrafish.Nat Genet. 2000; 26: 216-220Crossref PubMed Scopus (2123) Google Scholar It has also been applied to cultured mammalian cells10Bruno IG Jin W Cote GJ Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements.Hum Mol Genet. 2004; 13: 2409-2420Crossref PubMed Scopus (70) Google Scholar where its delivery appears to be impeded by its nonionic nature. In response to this problem, “scrape-loading” (creating pores in the membrane) and “leashing” (complexing PMO with negatively charged complementary DNA sequences) were then developed to enhance delivery by use of commercially available delivery reagents, such as polyethyleneimine and lipofectin.11Gebski 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 (168) Google Scholar However, on direct injection into muscles the leash adjunct proved toxic and was therefore not tested by i.v. administration. Despite the poor entry of unmodified PMO into cells in tissue culture, it was later found to enter muscle fibers better than 2′OMePS in vivo in the dystrophic mdx mouse. A single intramuscular injection of 10 µg PMO induced significantly higher levels of dystrophin expression than the same PMO complexed with leash and lipofectin.3Alter 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 Furthermore, regular weekly i.v. injections of PMO targeting mouse dystrophin exon 23 induced up to 50% of normal levels of dystrophin in body-wide skeletal muscles in the mdx mice, with improved muscle pathology, decreased serum levels of muscle creatine kinase and partial restoration of normalized muscle strength. Even after systemic administration for 1 year, no toxicity has been detected in muscles or other organs. A more recent investigation at higher dosages12Heemskerk HA de Winter CL de Kimpe SJ van Kuik-Romeijn P Heuvelmans N Platenburg GJ et al.In vivo comparison of 2'-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping.J Gene Med. 2009; 11: 257-266Crossref PubMed Scopus (153) Google Scholar confirmed that PMO produced higher levels of exon 23 skipping than 2′OMePS and thus appears to be a promising antisense oligomer chemistry for the treatment of DMD patients.3Alter 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 Although both 2′OMePS and PMO induce exon skipping systemically, it was disappointing to find that dystrophin expression was highly variable within and between muscles, even after repeated i.v. injections.3Alter 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,4Lu 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 (356) Google Scholar,12Heemskerk HA de Winter CL de Kimpe SJ van Kuik-Romeijn P Heuvelmans N Platenburg GJ et al.In vivo comparison of 2'-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping.J Gene Med. 2009; 11: 257-266Crossref PubMed Scopus (153) Google Scholar Why this is so, is not clearly understood, but may be due to the reliance on passive diffusion for entry into muscle fibers. For PMO, the lack of charge may present less of an impediment to cell surface contact thus allowing more efficient entry than 2′OMePS into muscle fibers, particularly those with leaky membranes as seen in dystrophic muscles. Such dependence on muscle damage for effective delivery of AONs, would have the advantage of limiting the amount of AON entering untargeted and undamaged nonmuscle cells, thus diminishing possible side effects. However, for long-term effective treatment of DMD, it would carry the disadvantage that muscle fibers rescued by PMO-induced exon skipping would have to re-enter a myopathic state to permit further PMO entry. Such a requirement for recurring cycles of rescue and degeneration in treated muscles could severely limit the value of antisense therapy for DMD patients. The requirement of muscle damage for effective delivery and AON induced dystrophin expression is further demonstrated by the relative lack of dystophin expression in cardiac muscle of mdx mice after systemic injection of either 2′OMePS AON or PMO.3Alter 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,4Lu 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 (356) Google Scholar,12Heemskerk HA de Winter CL de Kimpe SJ van Kuik-Romeijn P Heuvelmans N Platenburg GJ et al.In vivo comparison of 2'-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping.J Gene Med. 2009; 11: 257-266Crossref PubMed Scopus (153) Google Scholar Cardiac muscles in the mice are less affected than skeletal muscle by the dystrophic process and neither conspicuous pathological change nor functional impairment are seen until late stages. Consistently, only trace amounts of dystrophin are detected in cardiac muscle even after repeated injections into mdx mice of either 20′ MePS or PMO AON3Alter 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,4Lu 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 (356) Google Scholar,12Heemskerk HA de Winter CL de Kimpe SJ van Kuik-Romeijn P Heuvelmans N Platenburg GJ et al.In vivo comparison of 2'-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping.J Gene Med. 2009; 11: 257-266Crossref PubMed Scopus (153) Google Scholar even with doses of PMO that induce high levels of dystrophin in skeletal muscles. However, direct injection of AON or adeno-associated virus (AAV)-mediated AON delivery induced effective dystrophin expression in cardiac muscles, suggesting that efficiency of delivery rather than of exon-skipping is the critical factor in this organ.13Denti MA Incitti T Sthandier O Nicoletti C De Angelis FG Rizzuto E et al.Long-term benefit of adeno-associated virus/antisense-mediated exon skipping in dystrophic mice.Hum Gene Ther. 2008; 19: 601-608Crossref PubMed Scopus (56) Google Scholar One way of enhancing intracellular delivery is to employ cell-penetrating peptides or polymers to provide active transport of AON into muscle fibers. Earlier studies showed that conjugation to an arginine-rich peptide significantly improved PMO-mediated antiviral activity14Abes S Moulton HM Clair P Prevot P Youngblood DS Wu RP et al.Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents.J Control Release. 2006; 116: 304-313Crossref PubMed Scopus (167) Google Scholar as well as delivery of PMO for dystrophin exon skipping in cell cultures and on intramuscular injection into muscles.15Yin H Lu Q Wood M Effective exon skipping and restoration of dystrophin expression by peptide nucleic acid antisense oligonucleotides in mdx mice.Mol Ther. 2008; 16: 38-45Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar More recently, Jearawiriyapaisarn et al.16Jearawiriyapaisarn 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 (202) Google Scholar used a transgenic mouse that expresses enhanced green fluorescent protein as a positive readout for the efficiency of exon exclusion to evaluate the potency, functional biodistribution, and toxicity of PMOs conjugated to various arginine-rich cell-penetrating peptides containing 6-aminohexanoic acid (X) and/or β-alanine. The greatest restoration of enhanced green fluorescent protein expression in both skeletal and cardiac muscles was observed with PMO tagged with a peptide of (RXRRBR)2XB (PPMO). When applied to the dystrophic mdx mice model of DMD, a single i.v. injection of 30 mg/kg of PPMO restored dystrophin in all skeletal muscles to almost normal levels17Wu 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 (200) Google Scholar that were maintained by regular biweekly administration over 12 weeks and accompanied by improvement in muscle strength and pathology, with significant lowering of serum creatine kinase levels. Most importantly, i.v. injections of PPMO elicited near-normal levels of dystrophin in cardiac muscle (Figure 2) and prevented dobutamine-induced cardiac failure. Efficient exon skipping was also achieved in smooth muscles in other organs such as the esophagus. Treatment with the PPMO did not cause detectable toxicity. Recently, this PPMO has been shown to considerably ameliorate the severe pathology in the dystrophin–utrophin double null mouse.18Goyenvalle A Babbs A Powell D Kole R Fletcher S Wilton SD et al.Prevention of dystrophic pathology in severely affected dystrophin/utrophin-deficient mice by morpholino-oligomer-mediated exon-skipping.Mol Ther. 2010; 18: 198-205Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Together, these findings illustrate the theoretical feasibility of using PPMO to rescue dystrophin expression in both skeletal and cardiac muscles of DMD patients. However, use of peptides to enhance delivery raises the possibility of an immune response that may prevent repeated administration or cause rejection of targeted tissues or both, especially because DMD patients would require regular life-long administration. Although no immune response was observed in the above study17Wu 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 (200) Google Scholar or in previous reports with similar peptides in animal models,14Abes S Moulton HM Clair P Prevot P Youngblood DS Wu RP et al.Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents.J Control Release. 2006; 116: 304-313Crossref PubMed Scopus (167) Google Scholar,19Fletcher 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 immunogenicity varies considerably between species, arguing for longer-term studies in a range of animal models. But final verification can come only from clinical trials. It is, therefore, important to develop nonpeptide alternatives to enhance delivery of oligomers. The known sequence and structure of the peptide used in the PPMO provides a basis for modeling such nonpeptide polymers as delivery vehicles with similar or improved function. With this in mind, Wu et al. exploited a nonlinear, nonpeptidic dendrimer as a transporter for delivery of PMO. This consists of eight guanidinium head groups bonded to a trifunctional triazine as a core scaffold, which is then conjugated to PMO targeting exon 23 (ref. 6Mann 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 (352) Google Scholar) (termed Vivo-PMO).20Wu 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 The study demonstrated that the Vivo-PMO targeting mouse dystrophin exon 23 (Vivo-PMOE23) is highly effective for exon skipping and dystrophin induction in mdx mice. A single i.v. injection of 6 mg/kg Vivo-PMOE23 generated dystrophin expression in skeletal muscles at levels equivalent to the injection of 300 mg/kg unmodified PMOE23. Repeated injections of 6 mg/kg Vivo-PMOE23 achieved ~50% and 10% wild-type levels of dystrophin expression in body-wide skeletal muscles and in cardiac muscle respectively, without eliciting a detectable immune response. Vivo-PMOs showed no signs of toxicity at the effective dosage regime that reduced the serum levels of creatine kinase significantly.20Wu 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 These results thus offer prospects for the development of new nonpeptide delivery moieties with improved function and low toxicity. Although antisense-mediated exon skipping clinical trials currently conducted in United Kingdom and Netherlands targeting exon 51 show promising results,8van 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 (699) Google Scholar,21Kinali 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 (572) Google Scholar such single exon skipping covers only a proportion of DMD patients. Even if antisense oligos against most exons in the DMD gene become available, approximately half of DMD patients will require multiexon skipping by targeting of more than one exon, depending, not on the size but on the type of mutation (e.g., deletion, duplication, point mutation, etc.) and the “phase” of the mutated exon and its neighboring exons. For example, to treat a patient with deletion of exon 7, one needs to target at least two exons (e.g., both exon 6 and exon 8) to put the mutation back in frame (Figure 1). In fact, canine X-linked muscular dystrophy harbors such mutation22Sharp NJ Kornegay JN Van Camp SD Herbstreith MH Secore SL Kettle S et al.An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy.Genomics. 1992; 13: 115-121Crossref PubMed Scopus (287) Google Scholar (i.e., a splice site mutation in intron 6 that excludes exon 7 from the mRNA transcript (Figure 3)) and is therefore, a good model for testing the efficacy and efficiency of double-exon skipping.23Shimatsu Y Yoshimura M Yuasa K Urasawa N Tomohiro M Nakura M et al.Major clinical and histopathological characteristics of canine X-linked muscular dystrophy in Japan, CXMDJ.Acta Myol. 2005; 24: 145-154PubMed Google Scholar The dystrophic dog has several further advantages over the mdx mouse. First, it provides the prospect of more detailed analyses of clinical condition, such as clinical grading, magnetic resonance imaging, three-dimensional-echocardiography, and electrocardiogram.23Shimatsu Y Yoshimura M Yuasa K Urasawa N Tomohiro M Nakura M et al.Major clinical and histopathological characteristics of canine X-linked muscular dystrophy in Japan, CXMDJ.Acta Myol. 2005; 24: 145-154PubMed Google Scholar Second, the canine X-linked muscular dystrophy model, is closer in clinical phenotype than the mdx mouse model to human DMD. Indeed, it shows, if anything, a more severe progression than DMD; this, in combination with its similarity in body weight, makes it especially useful for physiological and toxicological studies.23Shimatsu Y Yoshimura M Yuasa K Urasawa N Tomohiro M Nakura M et al.Major clinical and histopathological characteristics of canine X-linked muscular dystrophy in Japan, CXMDJ.Acta Myol. 2005; 24: 145-154PubMed Google Scholar Finally, there may be some advantage in the fact that many target sites for exon skipping show identity of DNA (mRNA) sequences between dog and human. Drug regulation authorities such as US Food and Drug Administration are inclined to regard antisense oligonucleotides (AOs) of different sequences as different drugs; thus, to target the range of mutations encountered in DMD patients, many AO sequences will need to be designed, tested, and approved. Between man and mouse targeting homologous sites has little predictive value, perhaps due to minor sequence differences.24Mitrpant C Adams AM Meloni PL Muntoni F Fletcher S Wilton SD Rational design of antisense oligomers to induce dystrophin exon skipping.Mol Ther. 2009; 17: 1418-1426Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Dogs and humans however, share considerable sequence identity; for most exons in the DMD gene one can design a single 20–25mer antisense sequence that is applicable to both and comparison of targeting efficiencies between these species should be explored further. Overall, the dog experiments provide a promising message for DMD patients. McClorey and colleagues transfected cultured myotubes from dystrophic dogs in vitro with a cocktail of antisense oligos targeting exons 6 and 8, noting restoration of reading frame in mRNA.25McClorey G Moulton HM Iversen PL Fletcher S Wilton SD Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD.Gene Ther. 2006; 13: 1373-1381Crossref PubMed Scopus (177) Google Scholar Recently, we sought to test efficacy and toxicity of i.v. PMO induced exon skipping in vivo in the DMD dog model.26Yokota T Lu QL Partridge T Kobayashi M Nakamura A Takeda S et al.Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs.Ann Neurol. 2009; 65: 667-676Crossref PubMed Scopus (325) Google Scholar We identified a cocktail that, by either intramuscular injection or systemic i.v. delivery, resulted in extensive dystrophin expression to therapeutic levels. Weekly or biweekly systemic i.v. injections, over the course of 5–22 weeks, with a three-morpholino cocktail (120–200 mg/kg in total of three oligos/injection) targeting exon 6 and exon 8, induced therapeutic levels of dystrophin expression throughout the body, to an average of 26% of normal levels. Expression of dystrophin was associated with significant functional and clinical stabilization, being accompanied by reduced inflammation as observed histologically and by magnetic resonance imaging, improved or stabilized clinical symptoms and timed running tests. Histology and blood tests indicated no evidence of toxicity. Dystrophin expression was also detected in cardiac muscles by immunohistochemistry but, as in the mdx mouse,3Alter 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,4Lu 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 (356) Google Scholar less than in skeletal muscles and concentrated in small patches. Recently, we have found that an i.v. injection of peptide-conjugated morpholinos (PPMOs) at 12 mg/kg elicited increased dystrophin expression in the canine heart, as detected by western blotting (Yokota et al., data not shown). An unexpected observation in the dog study was that, in tissue culture, either of the two antisense oligonucleotide components of the cocktail directed against exon 6 were able, alone, to efficiently induce the desired exon 5–10 splicing in the absence of the sequence against exon 8. By contrast, they did not do this in vivo. In addition, excision of exon 8 by the exon 6-specific sequences alone occurred only in the context of the mutant exon 7 splice site (i.e., it did not occur in wild-type dog cells). Similarly, AO adminis" @default.
• W2072578275 created "2016-06-24" @default.
• W2072578275 creator A5020667221 @default.
• W2072578275 creator A5022775705 @default.
• W2072578275 creator A5033841379 @default.
• W2072578275 creator A5055891535 @default.
• W2072578275 creator A5074650312 @default.
• W2072578275 creator A5089234882 @default.
• W2072578275 date "2011-01-01" @default.
• W2072578275 modified "2023-10-15" @default.
• W2072578275 title "The Status of Exon Skipping as a Therapeutic Approach to Duchenne Muscular Dystrophy" @default.
• W2072578275 cites W1969582765 @default.
• W2072578275 cites W1973902775 @default.
• W2072578275 cites W1975297154 @default.
• W2072578275 cites W1982537592 @default.
• W2072578275 cites W1990084717 @default.
• W2072578275 cites W1990965764 @default.
• W2072578275 cites W1991945869 @default.
• W2072578275 cites W1996860050 @default.
• W2072578275 cites W1997327853 @default.
• W2072578275 cites W1998756807 @default.
• W2072578275 cites W2005245653 @default.
• W2072578275 cites W2010757394 @default.
• W2072578275 cites W2013949717 @default.
• W2072578275 cites W2018862827 @default.
• W2072578275 cites W2021202438 @default.
• W2072578275 cites W2022736892 @default.
• W2072578275 cites W2022947422 @default.
• W2072578275 cites W2027064784 @default.
• W2072578275 cites W2031129013 @default.
• W2072578275 cites W2031456867 @default.
• W2072578275 cites W2033618457 @default.
• W2072578275 cites W2041137184 @default.
• W2072578275 cites W2042837832 @default.
• W2072578275 cites W2043454383 @default.
• W2072578275 cites W2047689734 @default.
• W2072578275 cites W2048680477 @default.
• W2072578275 cites W2061596420 @default.
• W2072578275 cites W2062442451 @default.
• W2072578275 cites W2066950610 @default.
• W2072578275 cites W2067199762 @default.
• W2072578275 cites W2067671642 @default.
• W2072578275 cites W2072847969 @default.
• W2072578275 cites W2073703550 @default.
• W2072578275 cites W2075254739 @default.
• W2072578275 cites W2078163685 @default.
• W2072578275 cites W2087269855 @default.
• W2072578275 cites W2090849645 @default.
• W2072578275 cites W2091081049 @default.
• W2072578275 cites W2094796370 @default.
• W2072578275 cites W2100682779 @default.
• W2072578275 cites W2103961253 @default.
• W2072578275 cites W2110154453 @default.
• W2072578275 cites W2117345872 @default.
• W2072578275 cites W2122933784 @default.
• W2072578275 cites W2132793803 @default.
• W2072578275 cites W2152060821 @default.
• W2072578275 cites W2158156749 @default.
• W2072578275 cites W4249596337 @default.
• W2072578275 cites W4376453120 @default.
• W2072578275 doi "https://doi.org/10.1038/mt.2010.219" @default.
• W2072578275 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3017449" @default.
• W2072578275 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20978473" @default.
• W2072578275 hasPublicationYear "2011" @default.
• W2072578275 type Work @default.
• W2072578275 sameAs 2072578275 @default.
• W2072578275 citedByCount "92" @default.
• W2072578275 countsByYear W20725782752012 @default.
• W2072578275 countsByYear W20725782752013 @default.
• W2072578275 countsByYear W20725782752014 @default.
• W2072578275 countsByYear W20725782752015 @default.
• W2072578275 countsByYear W20725782752016 @default.
• W2072578275 countsByYear W20725782752017 @default.
• W2072578275 countsByYear W20725782752018 @default.
• W2072578275 countsByYear W20725782752019 @default.
• W2072578275 countsByYear W20725782752020 @default.
• W2072578275 countsByYear W20725782752021 @default.
• W2072578275 countsByYear W20725782752022 @default.
• W2072578275 countsByYear W20725782752023 @default.
• W2072578275 crossrefType "journal-article" @default.
• W2072578275 hasAuthorship W2072578275A5020667221 @default.
• W2072578275 hasAuthorship W2072578275A5022775705 @default.
• W2072578275 hasAuthorship W2072578275A5033841379 @default.
• W2072578275 hasAuthorship W2072578275A5055891535 @default.
• W2072578275 hasAuthorship W2072578275A5074650312 @default.
• W2072578275 hasAuthorship W2072578275A5089234882 @default.
• W2072578275 hasBestOaLocation W20725782751 @default.
• W2072578275 hasConcept C104317684 @default.
• W2072578275 hasConcept C126322002 @default.
• W2072578275 hasConcept C194583182 @default.
• W2072578275 hasConcept C2778776201 @default.
• W2072578275 hasConcept C2778943923 @default.
• W2072578275 hasConcept C2779030066 @default.
• W2072578275 hasConcept C2780394045 @default.
• W2072578275 hasConcept C36823959 @default.
• W2072578275 hasConcept C54355233 @default.
• W2072578275 hasConcept C71924100 @default.
• W2072578275 hasConcept C86803240 @default.

• next