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Genes (Basel). 2019 May 10;10(5). pii: E363. doi: 10.3390/genes10050363.

Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa.

Author information

1
Center for Medical Genetics Ghent, Ghent University and Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium. sarah.naessens@ugent.be.
2
Center for Medical Genetics Ghent, Ghent University and Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium. laurien.ruysschaert@ugent.be.
3
Center for Medical Genetics Ghent, Ghent University and Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium. steve.lefever@ugent.be.
4
Cancer Research Institute Ghent (CRIG), Ghent University, 9000 Ghent, Belgium. steve.lefever@ugent.be.
5
Bioinformatics Institute Ghent (BIG), Ghent University, 9000 Ghent, Belgium. steve.lefever@ugent.be.
6
Center for Medical Genetics Ghent, Ghent University and Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium. frauke.coppieters@ugent.be.
7
Center for Medical Genetics Ghent, Ghent University and Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium. Elfride.DeBaere@ugent.be.

Abstract

The recurrent missense variant in Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3), c.166G>A, p.(Gly56Arg) or G56R, underlies 1%-2% of cases with autosomal dominant retinitis pigmentosa (adRP), a frequent, genetically heterogeneous inherited retinal disease (IRD). The mutant NR2E3 protein has a presumed dominant negative effect (DNE) by competition for dimer formation with Cone-Rod Homeobox (CRX) but with abolishment of DNA binding, acting as a repressor in trans. Both the frequency and DNE of G56R make it an interesting target for allele-specific knock-down of the mutant allele using antisense oligonucleotides (AONs), an emerging therapeutic strategy for IRD. Here, we designed gapmer AONs with or without a locked nucleic acid modification at the site of the mutation, which were analyzed for potential off-target effects. Next, we overexpressed wild type (WT) or mutant NR2E3 in RPE-1 cells, followed by AON treatment. Transcript and protein levels of WT and mutant NR2E3 were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot respectively. All AONs showed a general knock-down of mutant and WT NR2E3 on RNA and protein level, showing the accessibility of the region for AON-induced knockdown. Further modifications are needed however to increase allele-specificity. In conclusion, we propose the first proof-of-concept for AON-mediated silencing of a single nucleotide variation with a dominant negative effect as a therapeutic approach for NR2E3-associated adRP.

KEYWORDS:

G56R; NR2E3; allele-specific knockdown; autosomal dominant; gapmer antisense oligonucleotides; putative dominant negative effect; retinitis pigmentosa

PMID:
31083481
DOI:
10.3390/genes10050363
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