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.

Hybrid splicing minigene and antisense oligonucleotides as efficient tools to determine functional protein/RNA interactions

“…However, for in cellulo assays, the type of chemical modification is important. Both parameters—the length and chemical modification of 2′OMe-PS—determine its efficacy to inhibit MBNL/RNA complex formation, consistent with previously published results that showed the applicability of 2′OMe-PS AONs for blocking many different cis -regulatory RNA elements, including intron-exon junctions or mutation-induced alternative splice sites 34 – 37 . Hence, the 2′OMe-PS type was chosen for further experiments.…”

Identification and Rescue of Splice Defects Caused by Two Neighboring Deep-Intronic ABCA4 Mutations Underlying Stargardt Disease

“…Previous IRD-associated intronic variants have created new splice acceptor or donor sites that allowed the insertion of a PE. 22,30,[42][43][44][45][46][47][48][49][50] To our knowledge we are the first to report on the insertion of a PE that is not due to this mechanism but possibly because of the creation of new ESE motifs in IRDs. Previously, the loss of an ESE and the creation of a splicing suppressor has been reported in the OPN1LW/OPN1MW gene array due to the coding variant c.532A>G (p.Ile178Val), which is associated with exon 3 exclusion and a congenital color vision defect (MIM: 303800 and 303900).…”

“…In addition, AONbased splice correction has been demonstrated in cell lines from individuals carrying deep-intronic variants in USH2A (MIM: 608400) 29 and OPA1 (MIM: 605290). 30 In this study, we employed induced pluripotent stem cell (iPSC) technology to identify the splicing defect caused by the two neighboring ABCA4 variants (M1 and M2) in intron 30 and describe that the inclusion of the identified pseudoexon (PE) can be mitigated by administration of AONs to photoreceptor precursor cells derived from individuals with STGD1.…”

ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease

“…We observed sizeable exon elongations in significantly occurring RNA products due to c.160+5G>C, c. 768G>T, c.1937+13T>G, c.4538A>G, and c.4538A>C variants. Splice modulation using antisense oligonucleotides (AONs) may be an option to redirect the splicing back to the natural splice sites. AONs have already demonstrated their therapeutic potential for several eye disease-associated genes, such as CEP290 (Collin et al 2012;Gerard et al 2012;Garanto et al 2016;Parfitt et al 2016), USH2A (Slijkerman et al 2016), or OPA1 (Bonifert et al 2016), and have a great potential as therapeutic molecules for inherited retinal diseases.…”