Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1
| Autor: | Bernd Wissinger, Florian Battke, Matthis Synofzik, Yvonne Theurer, Tobias Bonifert, Ludger Schöls, Irene Gonzalez Menendez |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Population Mutant splice correction optic neuropathies Biology OPA1 03 medical and health sciences Western blot Drug Discovery deep intronic mutation medicine splice ddc:610 education Gene Genetics education.field_of_study medicine.diagnostic_test Oligonucleotide lcsh:RM1-950 Transfection Molecular biology 030104 developmental biology lcsh:Therapeutics. Pharmacology RNA splicing Molecular Medicine Original Article antisense oligonucleotides |
| Zdroj: | Molecular Therapy. Nucleic Acids Molecular Therapy: Nucleic Acids, Vol 5, Iss C (2016) Molecular Therapy / Nucleic Acids 5(11), e390 (2016). doi:10.1038/mtna.2016.93 |
| ISSN: | 2162-2531 |
| DOI: | 10.1038/mtna.2016.93 |
| Popis: | Inherited optic neuropathies (ION) present an important cause of blindness in the European working-age population. Recently we reported the discovery of four independent families with deep intronic mutations in the main inherited optic neuropathies gene OPA1. These deep intronic mutations cause mis-splicing of the OPA1 pre-messenger-RNA transcripts by creating cryptic acceptor splice sites. As a rescue strategy we sought to prevent mis-splicing of the mutant pre-messenger-RNA by applying 2'O-methyl-antisense oligonucleotides (AONs) with a full-length phosphorothioate backbone that target the cryptic acceptor splice sites and the predicted novel branch point created by the deep intronic mutations, respectively. Transfection of patient-derived primary fibroblasts with these AONs induced correct splicing of the mutant pre-messenger-RNA in a time and concentration dependent mode of action, as detected by pyrosequencing of informative heterozygous variants. The treatment showed strong rescue effects (~55%) using the cryptic acceptor splice sites targeting AON and moderate rescue (~16%) using the branch point targeting AON. The highest efficacy of Splice correction could be observed 4 days after treatment however, significant effects were still seen 14 days post-transfection. Western blot analysis revealed increased amounts of OPA1 protein with maximum amounts at ~3 days post-treatment. In summary, we provide the first mutation-specific in vitro rescue strategy for OPA1 deficiency using synthetic AONs. |
| Databáze: | OpenAIRE |
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