Current State of the Art
In vitro studies
We have identified a set of most effective AONs with which the skipping of 38 out of 79 DMD exons can be induced [1-3]. This set belongs to a large collection of over 150 AONs that we have designed over the last 7 years. AON-design has primarily been based on a partial overlap with predicted open secondary structures in exonic target RNA, with a success rate of 67% [3]. Moreover, we have recently determined that the most effective AONs show significantly higher SF2/ASF-, SC35- and SRp40-values (as predicted by ESEfinder), and are mostly located closer to the 3' splice site when compared to less effective AONs. This suggested that effective exon-internal AONs primarily act by blocking SR binding sites (which often correspond to open structures).
We have demonstrated the therapeutic applicability of AON-induced exon skipping in cultured muscle cells from 12 DMD patients affected by different deletions, duplications and point mutations [2,4-6]. With dystrophin expression to almost normal levels in up to 80% of cells, the treatment was highly efficient. By using combinations of AONs we have shown that two or even more exons can be skipped simultaneously. This so-called double-or multi exon skipping not only further increases the therapeutic applicability to over 90% of DMD patients, but also renders this therapy significantly less mutation-specific [2]. Unfortunately, further studies revealed that skipping larger stretches of exons is less straightforward [7]
We have also tested different AON chemistries (2’OMePS, LNA, PNA, morpholino's) for efficiency, specificity, and toxicity [8]. The 2’O-methyl-phosphorothioate RNA molecules (2’OMePS) were most efficient and specific, and due to the fact that they are currently the best-studied molecules, closest to being applied in clinical trial studies.
In vivo studies
In normal mice with healthy muscle fibers, AONs can be delivered using a drug carrier like polyethylenimine (PEI). Following local, intramuscular injections of AONs (at a dose of 3.6 nmol) linked to PEI, we obtained skipping levels up to 3% [9]. The exon skipping levels accumulated up to 10 to 12 days and then slowly decreased to low levels at week 4 post-injection. We observed a correlation of this long-term effect with the intramuscular persistence of the AON. This suggests that in future clinical applications, daily administrations may not be necessary.
In the mdx mouse, a DMD animal model carrying a nonsense mutation in exon 23 [10], the muscle fiber membranes are impaired due to dystrophin deficiency. This allows a significantly higher uptake of AONs when compared to healthy mice. Even without the use of a drug carrier, we have obtained relatively high levels of exon 23 skipping (up to 20%) following intramuscular injections of an AON in exon 23 [9].
In two other recent studies in mdx mice [11,12], Dr. Lu and colleagues demonstrated promising proof-of-principle for therapeutic exon skipping in vivo. They initially applied local injections of an exon 23 AON into the gastrocnemius muscles, using the "drug carrier" F127 [11]. Exon 23 skipping was induced at high levels, leading to close to normal dystrophin expression in up to 20% of fibers. This significantly improved local muscle function in treated mice. In a follow-up study [12], the same AON formulation was systemically delivered. Limited numbers of dystrophin-positive fibers (1% to 5%) were detected in all muscle groups analyzed including the diaphragm. Remarkably, there was no therapeutic effect observed in the heart. Recently, systemic studies with a morpholino counterpart of this AON have been performed [13]. This resulted in higher numbers of dystrophin-positive fibers (10-50%), but again no dystrophin could be observed in the heart.
Finally, using our transgenic mouse model (hDMD), carrying an integrated copy of the full-length 2.3 Mb human DMD gene, we have recently set up human-sequence specific exon skipping in vivo [9]. AONs targeting human exons 44, 46 and 49, delivered intramuscularly, showed specific skipping of the human (and not the murine) exons in hDMD mice. We are currently engineering transgenic hDMD/mdx models carrying human-specific deletions. These models will be very valuable for the pre-clinical development of human-sequence specific AONs in muscle tissue.
First clinical proof-of-concept study
In collaboration with Prosensa B.V. we have recently successfully finished a "first-in-man” study, based on local intramuscular injections of an exon 51 skipping AON in 4 DMD patients [14].
A comparable study where different doses of a morpholino AON targeting exon 51 will be injected locally has initiated in the UK.
In collaboration with Prosensa BV we are currently preparing for larger Phase I/II clinical studies based on systemic administration.
1
Aartsma-Rus
A, Bremmer-Bout M, Janson A et al: Targeted exon skipping as a potential gene correction therapy for Duchenne muscular
dystrophy. Neuromuscul Disord 2002; 12 Suppl: S71-S77
Abstract
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2
Aartsma-Rus
A, Janson AA, Kaman WE et al: Antisense-induced multiexon
skipping for Duchenne muscular dystrophy makes more sense.
Am J Hum Genet 2004; 74: 83-92.
Abstract
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3
Aartsma-Rus A, de Winter CL, Janson AM, et
al: Functional analysis of 114 exon-internal AONs for targeted DMD exon
skipping: indication for steric hindrance of SR protein binding sites. Oligonucleotides
2005; 15: 284 -297
Abstract
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4
van Deutekom JC, Bremmer-Bout M, Janson AA et al:
Antisense-induced exon skipping restores dystrophin
expression in DMD patient derived muscle cells. Hum Mol Genet
2001; 10: 1547-1554.
Abstract
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5
Aartsma-Rus A, Janson AA, Kaman WE et al:
Therapeutic antisense-induced exon skipping in cultured muscle
cells from six different DMD patients. Hum Mol Genet
2003; 12: 907-914.
Abstract
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6 Aartsma-Rus A, Janson AM, van Ommen G-J et al.:
Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy.
BMC Med Genet 2007; 8: 43
Abstract
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7
Aartsma-Rus A, Kaman WE, Weij R, et al.: Exploring the frontiers of therapeutic exon skipping for Duchenne Muscular Dystrophy by double targeting within one or multiple exons.
Molecular Therapy 2006; 14:401-407
Abstract
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8
Aartsma-Rus
A, Kaman WE, Bremmer-Bout M et al: Comparative analysis of antisense
oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells.
Gene Ther 2004; 11: 1391-1398
Abstract
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9
Bremmer-Bout M, Aartsma-Rus A, de Meijer EJ et al:
Targeted exon skipping in transgenic hDMD mice:
A model for direct preclinical screening of human-specific
antisense oligonucleotides. Mol Ther 2004; 10:
232-240.
Abstract
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10 Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ: The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 1989; 244: 1578-1580 (top▲)
11 Lu QL, Mann CJ, Lou F et al: Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat Med 2003; 9(8): 1009-1014. (top▲)
12 Lu QL, Rabinowitz A, Chen YC et al: Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc Natl Acad Sci U S A 2005; 102(1): 198-203.(top▲)
13 Alter J, Lou F, Rabinowitz A et al: Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nat Med; 2006 12: 175-177. (top▲)
14 Van Deutekom JCT, Janson AM, Ginjaar IB et
al: Local dystrophin restoration with antisense oligonucleotide
PRO051.
N Engl J Med; 2007; 357: 2677-89
Abstract
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