Title: 128th ENMC International Workshop on ‘Preclinical optimization and Phase I/II Clinical Trials Using Antisense Oligonucleotides in Duchenne Muscular Dystrophy’ 22–24 October 2004, Naarden, The Netherlands
Abstract: Twenty-six participants including parents, scientists, industry representatives and clinicians from Australia, Belgium, England, France, The Netherlands, and USA attended the 128th ENMC workshop on the topic of 'Preclinical optimization and Phase I/II Clinical Trials Using Antisense Oligonucleotides in Duchenne Muscular Dystrophy'. The meeting was held in Naarden, The Netherlands, during the weekend of the 22nd–24th October 2004. The aim of the meeting was for the two European consortia (one in The Netherlands/Belgium and the other in the UK) that are preparing for a clinical trial on antisense oligonucleotides (AON) in Duchenne Muscular Dystrophy (DMD) to meet and compare their respective protocols. In addition, the meeting was attended by experts on AON in DMD from other countries and by experts on the use of AON in other fields of medicine. Representatives of four companies, ISIS Pharmaceuticals, Prosensa, Afforce Healthcare and Transgene attended the workshop as well, together with a parents representative. DMD is a common and severe form of muscular dystrophy, caused by intragenic mutations in the dystrophin gene. The majority of these mutations are out-of-frame deletions (and duplications); in-frame deletions/duplications characterize the milder allelic variant Becker muscular dystrophy (BMD). Recent laboratory studies have shown that the addition of AON to cultured patient muscle cells, and their injection into muscles of a mouse model for the disease (the dystrophin deficient mdx mouse) can induce skipping of exons and restore the reading frame in these cell lines and animal models [1Wilton S.D. Dye D.E. Laing N.G. Dystrophin gene transcripts skipping the mdx mutation.Muscle Nerve. 1997; 20: 728-734Crossref PubMed Scopus (45) Google Scholar, 2Dunckley M.G. Manoharan M. Villiet P. Eperon I.C. 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, 3Wilton S.D. Lloyd F. Carville K. et al.Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides.Neuromuscul Disord. 1999; 9: 330-338Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 4Mann C.J. Honeyman K. Cheng A.J. 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 (351) Google Scholar, 5Takeshima Y. Wada H. Yagi M. et al.Oligonucleotides against a splicing enhancer sequence led to dystrophin production in muscle cells from a Duchenne muscular dystrophy patient.Brain Dev. 2001; 23: 788-790Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 6van Deutekom J.C. Bremmer-Bout M. Janson A.A. et al.Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells.Hum Mol Genet. 2001; 10: 1547-1554Crossref PubMed Scopus (256) Google Scholar, 7Aartsma-Rus A. Bremmer-Bout M. Janson A.A. et al.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, 8Dickson G. Hill V. Graham I.R. Screening for antisense modulation of dystrophin pre-mRNA splicing.Neuromuscul Disord. 2002; 12: S67-S70Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 9Aartsma-Rus A. 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 (221) Google Scholar, 10Lu Q.L. Mann C.J. Lou F. 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, 11Wells K.E. Fletcher S. Mann C.J. Wilton S.D. Wells D.J. Enhanced in vivo delivery of antisense oligonucleotides to restore dystrophin expression in adult mdx mouse muscle.Fed Eur Biochem Soc Lett. 2003; 552: 145-149Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 12Aartsma-Rus A. Kaman W.E. Bremmer-Bout M. 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 (121) Google Scholar, 13Aartsma-Rus A. Janson A.A. Kaman W.E. 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, 14Graham I.R. Hill V.J. Manoharan M. Inamati G.B. Dickson G. Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays.J Gene Med. 2004; 6: 1149-1158Crossref PubMed Scopus (22) Google Scholar]. While the limitation of this approach is its temporary nature, and the need for the direct intramuscular injection of the AON, its efficacy in terms of restoring dystrophin expression and assembly of the dystrophin associated glycoprotein complex have improved considerably in the recent past. In the mdx mouse, this was also followed by improved functional properties of the dystrophic muscle [[10]Lu Q.L. Mann C.J. Lou F. 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]. Additional significant development will be necessary to improve the delivery aspects of AON before the antisense approach could be regarded as a realistic therapeutic option in DMD. Development of systemic delivery systems for AON are being pursued by a number of groups; in addition there is a wealth of expertise on the use of systemic delivery of AON in other pathologies. It has been calculated that approximately 70% of DMD patients with intragenic deletions could be theoretically helped by this approach. For a recent review of the literature see [7Aartsma-Rus A. Bremmer-Bout M. Janson A.A. et al.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, 15van Deutekom J.C. van Ommen G.J. Advances in Duchenne muscular dystrophy gene therapy.Nat Rev Genet. 2003; 4: 774-783Crossref PubMed Scopus (175) Google Scholar]. The meeting started with a brief outline of the work planned by the two consortia. Regarding the UK project, funded by the Department of Health, Francesco Muntoni (London) explained that this comprises a preclinical and a clinical part. Regarding the preclinical studies, these involve testing novel AON targeted against exon–intron boundaries of exons 51 and 53; testing the efficacy of exon skipping in DMD patient myoblast cultures; in developing systemic delivery strategies. Regarding the clinical part, the work will focus initially on the characterization of the histological and muscle magnetic resonance imaging (MRI) of the state of preservation of a number of distal muscles (radial; tibialis anterior; extensor digitorum brevis) to decide the best target for the intramuscular injection. The age range of patients selected will likely be between the age of 14 and 18 and all patients studied will have previously had a muscle biopsy in which dystrophin expression has been studied. The specific deletion that will be targeted has not been decided yet, but it is likely to be either exon 51; or exon 53, or two groups of patients, one with a deletion which could be rescued by skipping exon 51 and the second group by exon 53. For a list of the out-of-frame deletions that could be targeted using a similar approach see Table 1. The plan is to administer a combination of AON and carrier polymer (similar to that used in recent animal studies) to three groups of patients. The first three patients will receive a relatively small dose of AON+polymer in the target muscle following direct injection. A muscle biopsy will be performed after 1 month. In the second group, the protocol will be identical, although a higher dose of AON will be used. In the third group, the best tolerated and most effective dosage will be used; however, after 1 month from the first administration, a second AON administration will be performed. A muscle biopsy will be performed 1 month after this second injection.Table 1Overview of the DMD-causing mutations, correctable by skipping one of the exons that will be targeted as part of the work performed in the two consortiaSkippable exonDMD patients potentially benefiting (exons deleted or duplicated)5145–50; 47–50; 48–50; 49–50; 50; 52; 52–63535–52; 47–52; 48–52; 49–52; 50–52; 52 Open table in a new tab The protocol from the Dutch/Belgian consortium, set up and managed by Prosensa BV (NL), was presented by Gert-Jan van Ommen (Leiden). Both the preclinical and clinical development is funded by Prosensa, the Dutch Government, Dutch Parent Project and UPPMD. The Consortium has already identified the most effective AON sequence, directed at an exon-internal, putative splicing enhancer element within exon 51. This AON will be administered to a group of four patients (age range comprised between 8 and 16 years, carrying out-of-frame deletions which would benefit from this approach (Table 1). The patients will be selected on the basis of a positive outcome, i.e. specific exon 51 skipping and dystrophin production, in cultured muscle cells isolated from muscle biopsies previously taken from these patients. The protocol involves administering a single dose AON (h51AON23) by multiple injections within a 1 cm2 area of the tibialis anterior. A biopsy will be taken on day 28, for analysis on RNA and protein level. All biopsies are to be performed by the same investigator, using a well-established procedure involving a small-beaked conchotome. Several safety parameters will be evaluated during and after the treatment of the patients. The next item discussed was the strategy that the different groups were following in order to identify effective AON. Judith van Deutekom (Leiden) described how AON were selected: two AONs were designed per exon, mainly targeting (partly) open structures, and, if possible, directed to purine-rich sequences that may represent putative splicing enhancer elements (ESE). While initially gel mobility shift assays were performed with the different AONs to determine which one binds with most favorable affinity, nowadays a software package (ESEfinder) is used to predict binding sites for the different splicing factors (SR proteins) in more detail. The more specific targeting of these sites typically yielded effective AONs in 67% of molecules tested. Only rarely the design of additional AONs was required. Following this strategy van Deutekom has already identified effective AONs for the skipping of 36 of the 79 dystrophin exons [[13]Aartsma-Rus A. Janson A.A. Kaman W.E. 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]. Ian Graham (London) explained the strategy he and George Dickson (London) have been following regarding exon 23 of the mdx dystrophin gene. In particular, he pointed out that their group has been successful using systematic constructed hybridization antisense arrays to identify open regions of the pre-mRNA [8Dickson G. Hill V. Graham I.R. Screening for antisense modulation of dystrophin pre-mRNA splicing.Neuromuscul Disord. 2002; 12: S67-S70Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 14Graham I.R. Hill V.J. Manoharan M. Inamati G.B. Dickson G. Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays.J Gene Med. 2004; 6: 1149-1158Crossref PubMed Scopus (22) Google Scholar]. This strategy will now be used to identify AON directed against the exon–intron boundaries of exons 51 and 53. Steve Wilton (Perth) illustrated his strategy for the identification of successful AON. He also has experience with targeting exon–intron boundaries, both of mdx mouse and of the human gene [3Wilton S.D. Lloyd F. Carville K. et al.Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides.Neuromuscul Disord. 1999; 9: 330-338Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 4Mann C.J. Honeyman K. Cheng A.J. 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 (351) Google Scholar, 16Errington S.J. Mann C.J. Fletcher S. Wilton S.D. Target selection for antisense oligonucleotide induced exon skipping in the dystrophin gene.J Gene Med. 2003; 5: 518-527Crossref PubMed Scopus (37) Google Scholar, 17Mann C.J. Honeyman K. McClorey G. Fletcher S. Wilton S.D. 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]. In the human, Steve Wilton has mostly concentrated on skipping located in the 5′ deletion hot spot. He follows a similar strategy to the one initially used by van Deutekom, with a pre-screening using bandshift assays, followed by verification in muscle cultures. In addition to the predictive approach, he also uses an empirical approach, where overlapping AON against the exon–intron boundaries of interest are used in cultured cells to verify their efficacy. A number of presentations outlined the relative efficacy and stability of different modifications to the AON backbone (Ian Graham, Aartsma-Rus, Steve Wilton) and a recent review on this can be found in Jason et al. [[18]Jason T.L. Koropatnick J. Berg R.W. Toxicology of antisense therapeutics.Toxicol Appl Pharmacol. 2004; 201: 66-83Crossref PubMed Scopus (136) Google Scholar]. Phosphorothioate AON have been extensively used in man with a wealth of general data related to its safety in the human, although fully 2-O-methylated PS AONs have not been studied yet. Regarding other backbone modifications such as, for example, morpholino or PNA AON [12Aartsma-Rus A. Kaman W.E. Bremmer-Bout M. 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 (121) Google Scholar, 19Gebski B.L. Mann C.J. Fletcher S. Wilton S.D. Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle.Hum Mol Genet. 2003; 12: 1801-1811Crossref PubMed Scopus (167) Google Scholar] some data on safety in the human are available [[18]Jason T.L. Koropatnick J. Berg R.W. Toxicology of antisense therapeutics.Toxicol Appl Pharmacol. 2004; 201: 66-83Crossref PubMed Scopus (136) Google Scholar]. Furthermore, in addition to the efficiency the backbone should have a high specificity (this is not the case for the LNA AON). While many participants are clearly interested in pursuing this aspect of preclinical research, the planned trials will likely use the 2-O-methylated phosphorothioated AON, unless data on a clear advantage of different modifications, previously tested in the human, are generated in the interim. Regarding the use of carrier polymers, Terry Partridge (London) presented the collaborative work performed with Qi-Long Lu (London UK/Charlotte, USA) on the improved efficacy of distribution of transfection, in vivo but not in vitro, using the pluronic polymer F127. The data presented showed that with the use of this polymer, the number of fibres transfected following a single or repeated injections in the tibialis anterior of mdx mouse improved significantly, with more than 70% of the fibres eventually producing different levels of dystrophin [[10]Lu Q.L. Mann C.J. Lou F. 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]. Similar efficiency was also achieved in older and more fibrotic muscle. In this experiment there was restoration of the dystrophin-associated glycoprotein complex and of the force generated by the treated mdx muscle. However, only partial protection against the damage following eccentric exercise was achieved [[10]Lu Q.L. Mann C.J. Lou F. 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]. Recent data from the same investigators using AON plus the same pluronic polymer showed that the intravenous administration of these reagents in mdx mice was followed by low but detectable levels of skipping in a number of muscles, although not in all [[20]Lu Q.L. Rabinowitz A. Chen Y.C. 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 (355) Google Scholar]. Art Levin, from ISIS Pharmaceuticals (USA), presented the preclinical and clinical data on safety and efficacy of AON from a number of human clinical trials. He explained that AON have on the whole a good safety profile from data available on human trials. Toxicity to AON can, however, be observed, and this can be both hybridization dependent, and hybridization independent. The first are typically secondary to an exaggerated pharmacological effect of the administered AON (and in this context none is expected for dystrophin); there is also the possibility of hybridization to unidentified targets, although this is extremely unlikely. The more unpredictable toxicity arises from a hybridization independent mechanism. This can be due to specific motifs that have (currently) undefined receptors; or to specific motifs that define a receptor interaction. The best known is the immune reaction against the CpG motif, which is activated via the Toll-like receptor 9. Other parameters relevant for the AON toxicity relate to their bio-distribution. Kidney has the highest concentration following either subcutaneous (SC) or intravenous (IV) administration. Following the administration of very high doses, it is possible to observe histological changes in proximal tubules in animal models. These changes are reversible. However, at the far lower doses used in clinical trials this does not appear to be of practical clinical significance and detailed studies in more than 1000 patients have failed to show any abnormality in renal functional analysis. Liver is the organ with the second highest concentration of AON. In rodents, administration of AON often results in hepatic changes, with mononuclear infiltrates, Kupfer cell infiltrates and often elevation of ALT and AST. In the human there is usually no observed toxic liver effect; however, at very high doses some transient change in transaminase levels can be observed. Regarding other tissues, splenomegaly can be often observed in rodents; however, this side effect has not been noticed in humans or primates. Regarding the sequence specific toxicity, this is highly dependent on sequence. AON, especially those with phosphorothioate modification, stimulate immune responses if CpG motifs are present. CpG-dependent immune stimulation is Th1-dependent. Palindromic sequences can also be immune-stimulating and should also be avoided. There can also be other not fully predictable effects on immune response dependent on sequence recognition by receptors of innate immunity. In rodents, this is often a prominent side effect, but much less so in humans and primates. On the whole clinical manifestations of the administration of AON in human clinical trials have been limited to the local side effects following SC injection (on the whole i.v. administration seems to be much better tolerated) and generalized side effects such as fever and chills that similar to the response to interferon administration, respond well to paracetamol. More than 4000 patients with different disorders have been treated so far using systemic delivery of first generation AON (phosphorothioate backbone), and approximately 500 following local administration. The typical dosage used ranged from 0.5 mg/kg every other day for 1 month in Crohn's disease, to 200 mg twice weekly for 3 months in rheumatoid arthritis, to higher dosages of 2 mg/kg day in other protocols dealing with malignancies. Fewer patients (∼300) have been treated so far using new generation AON (uniform phosphorothioated backbone with flanking 2′ methoxyethoxy wing) delivered systemically at doses comprised between 0.5 and 9 mg/kg per week for up to 3 weeks. It was noted that the local (intramuscular, i.m.) administration of a small dose of AON as proposed in both DMD trials should be safe. However, most of the expertise resides with AON with already well known modifications. It looks, therefore, realistic for the scope of these phase I trials, to only consider the use of known and tested chemical AON modifications. Gerard Platenburg, from Prosensa (The Netherlands), clarified the various necessary steps to move a project from the pre-clinical phase to the clinical arena. He also explained various regulatory aspects related to clinical trials and in particular to the manufacturing of clinical grade AON for clinical trials. In particular, it was stressed that the same batch used for the toxicity analysis will be used for the clinical trials. Finbarr Cotter (London) presented his experience over 10 years in human clinical trials using AON. He first reiterated some of the aspects related to the bio-distribution of AON (they do not cross the blood brain barrier, nor target the testis). He then described the rationale of a number of cancer trials in which he participated. In a number of cancers the anti-apoptotic molecule Bcl2 is abnormally up-regulated; Bcl2 has, therefore, been the target of AON aimed at its down-regulation [21Webb A. Cunningham D. Cotter F. et al.BCL-2 antisense therapy in patients with non-Hodgkin lymphoma.Lancet. 1997; 349: 1137-1141Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 22Waters J.S. Webb A. Cunningham D. et al.Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma.J Clin Oncol. 2000; 18: 1812-1823Crossref PubMed Scopus (443) Google Scholar, 23Cotter F.E. Antisense therapy of hematologic malignancies.Semin Hematol. 1999; 36: 9-14PubMed Google Scholar, 24Cotter F.E. Antisense therapy for lymphomas.Hematol Oncol. 1997; 15: 3-11Crossref PubMed Scopus (31) Google Scholar, 25Cotter F.E. Waters J. Cunningham D. Human Bcl-2 antisense therapy for lymphomas.Biochim Biophys Acta. 1999; 1489: 97-106Crossref PubMed Scopus (54) Google Scholar]. He described the typical stages necessary to take a molecule from phases I to III trials. In phase I, trials in cancer patients in whom high Bcl2 levels were known to occur, a dose escalation at 100% increments is performed until some toxicity using multiple parameters is identified. The dose is then decreased to the immediately lower dose, at which no significant toxicity was observed. Among side effects frequently observed, skin reactions are a common problem following subcutaneous (s.c.) administration. On the whole IV is better tolerated. For doses of phosphorothioate AON higher than 5 mg/kg per day, thrombocytopenia and hyperglycemia were transiently observed. Relatively common but less severe observed side effects of high dosage i.v. or s.c. administration were transient leucopenia, neutropenia and lymphopenia, fever, asthenia and hypotension. Some of these flu-like symptoms respond well to the administration of paracetamol. Serge Braun (Transgene, France) presented in detail the protocol design and optimization of the first gene therapy trial in DMD, performed in France following the collaborative efforts of his group at Transgene, the clinical team of the Institute de Myologie, Hôpital Pitié-Salpêtrière, Paris (Prof. Micheal Fardeau; Dr Normal Romero), funded by AFM. The scope of this trial was to assess the safety of the administration of a full-length dystrophin containing plasmid into a single muscle of a group of patients with DMD (three) or BMD (six). The results of this first study have been recently published [[26]Romero N.B. Benveniste O. Payan C. et al.Current protocol of a research phase I clinical trial of full-length dystrophin plasmid DNA in Duchenne/Becker muscular dystrophies. Part II: clinical protocol.Neuromuscul Disord. 2002; 12: S45-S48Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar]. Serge Braun explained how the protocol was developed, how long it took from the original preparation of the trial to its completion (5 years). Nine patients were sequentially enrolled in the study. All received one or two injections of dystrophin plasmid DNA into the radialis muscle and had a muscle biopsy taken 3 weeks after the last injection. The expression of dystrophin was studied using antibodies directed against the patients deleted dystrophin region, and nested RT-PCR studies. The plasmid DNA was seen at the injection site biopsies in 9/9 patients. Full-length dystrophin expression was restored in 6/9 of patients, although the percentage of labeled fibres was very low. Reassuringly, no side effects were observed; in particular no cellular or humoral anti-dystrophin response was detected. Following these preliminary but encouraging results, the French investigators are currently considering regional intravenous or arterial delivery of the same construct. Experiments in this direction are currently being performed in animal models [27Zhang G. Ludtke J.J. Thioudellet C. et al.Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy.Hum Gene Ther. 2004; 15: 770-782Crossref PubMed Scopus (72) Google Scholar, 28Braun S. Naked plasmid DNA for the treatment of muscular dystrophy.Curr Opin Mol Ther. 2004; 6: 499-505PubMed Google Scholar]. The preliminary data indicate this to be well tolerated and quite effective in the animal model. The next step will be to perform a phases I–II trial in patients with DMD, with the aim of regional plasmid DNA delivery to the forearm muscles. The discussion focused again on AON and dystrophin. Francesco Muntoni presented the figures of patients available in the UK Consortium with deletions who would benefit from skipping exons 51 or 53. He mentioned that in the UK there is a well established network of 14 pediatric neuromuscular centres working together towards the definition of a common assessment protocol that could be used towards future therapeutic trials (The North Star Network, funded by the Muscular Dystrophy Campaign) and that patients followed in any of these centres could be recruited to this phase I trial. He also noted that the choice of the 3′ end of the distal hot spot region as more likely to result in functional protein than that of other regions of the gene. Indeed, a number of reports of asymptomatic cases with deletions of exons 48–51; 48–53 [[29]Melis M.A. Cau M. Muntoni F. et al.Elevation of serum creatine kinase as the only manifestation of an intragenic deletion of the dystrophin gene in three unrelated families.Eur J Paediatr Neurol. 1998; 2: 255-261Abstract Full Text PDF PubMed Scopus (56) Google Scholar]; 48 [[30]Morrone A. Zammarchi E. Scacheri P.C. et al.Asymptomatic dystrophinopathy.Am J Med Genet. 1997; 69: 261-267Crossref PubMed Scopus (33) Google Scholar] and 48–51 and 50–53 [31Koenig M. Beggs A.H. Moyer M. et al.The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion.Am J Hum Genet. 1989; 45: 498-506PubMed Google Scholar, 32Beggs A.H. Hoffman E.P. Snyder J.R. et al.Exploring the molecular basis for variability among patients with Becker muscular dystrophy: dystrophin gene and protein studies.Am J Hum Genet. 1991; 49: 54-67PubMed Google Scholar] are on record. Ieke Ginjaar (Leiden) presented the cases currently followed among the various centres in The Netherlands and in Belgium, who could be recruited as part of the trial. The preferred choice is for patients who could benefit from skipping of exon 51, as these represent the most abundant cohort of DMD patients. The second option would be exon 46 which as a single-mutation cohort would be the largest. They also discussed that unexpectedly some mutations that ought to occur and be represented in the Leiden DMD database (http://www.dmd.nl/) apparently do not occur or are significantly underrepresented, or are identified incidentally in patients ascertained because of elevated serum CK. These are the deletions of 45–51; 50–51; 47–51, 48–51, 51–52. This further reinforces the view that this appears to be a well-dispensable region of the dystrophin protein and, therefore, exon skipping to induce some of the above mentioned scenarios ought to be very effective. Steve Wilton presented his data on targeting patients with deletions towards the 5′ end of the gene. Some of these patients can also have a remarkably mild phenotype and in particular he mentioned a patient from Newcastle with a deletion of exons 3–9 who plays competitive sport. Steve Wilton already has very effective oligonucleotides tested in human muscle cell lines to induce skipping of exons 4; 8; 9; 15; 16; 19 and 20; 31; 33 and 35. Jenny Morgan (London) and Annemieke Aartsma-Rus (Leiden) discussed the available techniques to test AON in either primary muscle cell cultures from patients with DMD or us
Publication Year: 2005
Publication Date: 2005-06-01
Language: en
Type: review
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 33
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