title: Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1.
abstract: 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.
authors.affiliation: Molecular Genetics Laboratory, Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany.|Graduate School of Cellular and Molecular Neuroscience, University of Tübingen, Tübingen, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Bonifert
authors.affiliation: Molecular Genetics Laboratory, Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany.
authors.forename: Irene
authors.initials: I
authors.lastname: Gonzalez Menendez
authors.affiliation: CeGaT GmbH, Tübingen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Battke
authors.affiliation: Department of Neurodegenerative Diseases and Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany.|German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Theurer
authors.affiliation: Department of Neurodegenerative Diseases and Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany.|German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
authors.forename: Matthis
authors.initials: M
authors.lastname: Synofzik
authors.affiliation: Department of Neurodegenerative Diseases and Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany.|German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
authors.forename: Ludger
authors.initials: L
authors.lastname: Schöls
authors.affiliation: Molecular Genetics Laboratory, Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Wissinger