Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1
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-...
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| Format: | Article |
| Language: | English |
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Elsevier
2016-01-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253117301130 |
| _version_ | 1819154238786240512 |
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| author | Tobias Bonifert Irene Gonzalez Menendez Florian Battke Yvonne Theurer Matthis Synofzik Ludger Schöls Bernd Wissinger |
| author_facet | Tobias Bonifert Irene Gonzalez Menendez Florian Battke Yvonne Theurer Matthis Synofzik Ludger Schöls Bernd Wissinger |
| author_sort | Tobias Bonifert |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-22T15:17:55Z |
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| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-12-22T15:17:55Z |
| publishDate | 2016-01-01 |
| publisher | Elsevier |
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| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-4e6fe53fc26147ffbee3823a07bc026a2022-12-21T18:21:41ZengElsevierMolecular Therapy: Nucleic Acids2162-25312016-01-015C10.1038/mtna.2016.93Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1Tobias Bonifert0Irene Gonzalez Menendez1Florian Battke2Yvonne Theurer3Matthis Synofzik4Ludger Schöls5Bernd Wissinger6Molecular Genetics Laboratory, Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, GermanyMolecular Genetics Laboratory, Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, GermanyCeGaT GmbH, Tübingen, GermanyDepartment of Neurodegenerative Diseases and Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, GermanyDepartment of Neurodegenerative Diseases and Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, GermanyDepartment of Neurodegenerative Diseases and Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, GermanyMolecular Genetics Laboratory, Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, GermanyInherited 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.http://www.sciencedirect.com/science/article/pii/S2162253117301130antisense oligonucleotidesdeep intronic mutationOPA1optic neuropathiessplice correction |
| spellingShingle | Tobias Bonifert Irene Gonzalez Menendez Florian Battke Yvonne Theurer Matthis Synofzik Ludger Schöls Bernd Wissinger Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1 Molecular Therapy: Nucleic Acids antisense oligonucleotides deep intronic mutation OPA1 optic neuropathies splice correction |
| title | Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1 |
| title_full | Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1 |
| title_fullStr | Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1 |
| title_full_unstemmed | Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1 |
| title_short | Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1 |
| title_sort | antisense oligonucleotide mediated splice correction of a deep intronic mutation in opa1 |
| topic | antisense oligonucleotides deep intronic mutation OPA1 optic neuropathies splice correction |
| url | http://www.sciencedirect.com/science/article/pii/S2162253117301130 |
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