Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy

<p>Abstract</p> <p>Background</p> <p>Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs) targeting specific exons the DMD reading frame is restored and partially functional dystrophins are produced. Following proof of concept in cultured muscle cells from patients with various deletions and point mutations, we now focus on single and multiple exon duplications. These mutations are in principle ideal targets for this approach since the specific skipping of duplicated exons would generate original, full-length transcripts.</p> <p>Methods</p> <p>Cultured muscle cells from DMD patients carrying duplications were transfected with AONs targeting the duplicated exons, and the dystrophin RNA and protein were analyzed.</p> <p>Results</p> <p>For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62), by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons.</p> <p>Conclusion</p> <p>The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.</p

Exon skipping in myotube cultures from a patient with an exon 52–62 duplication

<p><b>Copyright information:</b></p><p>Taken from "Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy"</p><p>http://www.biomedcentral.com/1471-2350/8/43</p><p>BMC Medical Genetics 2007;8():43-43.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC1931584.</p><p></p> A-F. RT-PCR analysis. After treatment (+) specific skipping of the original and the duplicated exons 52 and 62 was induced in patient myotubes, while these skips were not found before treatment (-) (A-D). Since the duplicated exons are located between exon 63 and 64, the patient appears to have an exon 63 deletion using primers flanking this exon (D). Skipping of the original exons 62, 63, and the duplicated exon 52 could be detected at low levels in untreated myotube cultures (E). After AON treatment, these levels increased and, in addition, single exon 62 skipping was induced. Primers specific for the duplication (B and E) generated only non specific fragments in the control sample, as confirmed by sequencing analysis (C). Using primers flanking the duplication (F) normally spliced transcripts were detected both in untreated and treated patient RNA, albeit at lower levels than the control. The expected fragment of ~1900 bp containing the duplication was not observed (upper marker band is 2 kb). In-frame and out-of-frame transcripts are shown in green and red, respectively. Duplicated exons are shaded in blue. M is 100 bp DNA size marker, -RT is negative control. . Western blot analysis. No dystrophin could be detected in protein isolated from untreated (NT) myotubes, or from myotubes isolated 1, 4 and 8 days after AON treatment. A clear dystrophin signal (dy4) is present in protein isolated from unaffected control myotube cultures (C) (diluted 1:15 to prevent overexposure). Myosin (MF20) signals could be detected in both treated and untreated patient samples, indicating that the differentiation stage of the myotubes was sufficient to allow dystrophin synthesis

Guidelines for Antisense Oligonucleotide Design and Insight Into Splice-modulating Mechanisms

Antisense oligonucleotides (AONs) can interfere with mRNA processing through RNase H–mediated degradation, translational arrest, or modulation of splicing. The antisense approach relies on AONs to efficiently bind to target sequences and depends on AON length, sequence content, secondary structure, thermodynamic properties, and target accessibility. We here performed a retrospective analysis of a series of 156 AONs (104 effective, 52 ineffective) previously designed and evaluated for splice modulation of the dystrophin transcript. This showed that the guanine-cytosine content and the binding energies of AON-target and AON–AON complexes were significantly higher for effective AONs. Effective AONs were also located significantly closer to the acceptor splice site (SS). All analyzed AONs are exon-internal and may act through steric hindrance of Ser-Arg-rich (SR) proteins to exonic splicing enhancer (ESE) sites. Indeed, effective AONs were significantly enriched for ESEs predicted by ESE software programs, except for predicted binding sites of SR protein Tra2β, which were significantly enriched in ineffective AONs. These findings compile guidelines for development of AONs and provide more insight into the mechanism of antisense-mediated exon skipping. On the basis of only four parameters, we could correctly classify 79% of all AONs as effective or ineffective, suggesting these parameters can be used to more optimally design splice-modulating AONs