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

Literature-aided meta-analysis of microarray data: a compendium study on muscle development and disease-2

Ddition to relevant subclusters. The dataset ids are shown between the tree and the heatmap. The colored bars provide background information on the datasets.<p><b>Copyright information:</b></p><p>Taken from "Literature-aided meta-analysis of microarray data: a compendium study on muscle development and disease"</p><p>http://www.biomedcentral.com/1471-2105/9/291</p><p>BMC Bioinformatics 2008;9():291-291.</p><p>Published online 24 Jun 2008</p><p>PMCID:PMC2459190.</p><p></p

Literature-aided meta-analysis of microarray data: a compendium study on muscle development and disease-0

<p><b>Copyright information:</b></p><p>Taken from "Literature-aided meta-analysis of microarray data: a compendium study on muscle development and disease"</p><p>http://www.biomedcentral.com/1471-2105/9/291</p><p>BMC Bioinformatics 2008;9():291-291.</p><p>Published online 24 Jun 2008</p><p>PMCID:PMC2459190.</p><p></p

Literature-aided meta-analysis of microarray data: a compendium study on muscle development and disease-1

level.<p><b>Copyright information:</b></p><p>Taken from "Literature-aided meta-analysis of microarray data: a compendium study on muscle development and disease"</p><p>http://www.biomedcentral.com/1471-2105/9/291</p><p>BMC Bioinformatics 2008;9():291-291.</p><p>Published online 24 Jun 2008</p><p>PMCID:PMC2459190.</p><p></p

Literature-aided meta-analysis of microarray data: a compendium study on muscle development and disease-3

Ion to relevat subclusters. The dataset ids are shown between the tree and the heatmap. The colored bars provide background information on the datasets.<p><b>Copyright information:</b></p><p>Taken from "Literature-aided meta-analysis of microarray data: a compendium study on muscle development and disease"</p><p>http://www.biomedcentral.com/1471-2105/9/291</p><p>BMC Bioinformatics 2008;9():291-291.</p><p>Published online 24 Jun 2008</p><p>PMCID:PMC2459190.</p><p></p

Preventing Formation of Toxic N-Terminal Huntingtin Fragments Through Antisense Oligonucleotide-Mediated Protein Modification

<p>Huntington’s disease (HD) is a progressive autosomal dominant disorder, caused by a CAG repeat expansion in the HTT gene, which results in expansion of a polyglutamine stretch at the N-terminal end of the huntingtin protein. Several studies have implicated the importance of proteolytic cleavage of mutant huntingtin in the HD pathogenesis and it is generally accepted that N-terminal huntingtin protein fragments are more toxic than full-length huntingtin protein. Important cleavage sites are encoded by exon 12 of HTT. Recent publications have shown the feasibility of reducing huntingtin levels using antisense oligonucleotides, but concerns were raised towards possible unwanted side effects from lowering huntingtin protein levels too much. Our approach reduces mutant huntingtin toxicity by modifying the huntingtin protein without changing overall protein levels. We use 2’O-methyl modified antisense oligonucleotides with a phosphorothioate (PS) backbone to induce skipping of exon 12 in huntingtin pre-mRNA, thereby preventing the formation of toxic N-terminal huntingtin protein fragments. In vitro studies showed successful exon skipping and appearance of a shorter huntingtin protein. Cleavage assays showed reduced formation of the 586 amino acid N-terminal huntingtin fragment in the treated samples. In vivo studies revealed exon skipping after single injection of antisense oligonucleotides in the mouse striatum.</p