Cargando…

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

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 ha...

Descripción completa

Detalles Bibliográficos
Autores principales: Naessens, Sarah, Ruysschaert, Laurien, Lefever, Steve, Coppieters, Frauke, De Baere, Elfride
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562693/
https://www.ncbi.nlm.nih.gov/pubmed/31083481
http://dx.doi.org/10.3390/genes10050363
_version_ 1783426361179766784
author Naessens, Sarah
Ruysschaert, Laurien
Lefever, Steve
Coppieters, Frauke
De Baere, Elfride
author_facet Naessens, Sarah
Ruysschaert, Laurien
Lefever, Steve
Coppieters, Frauke
De Baere, Elfride
author_sort Naessens, Sarah
collection PubMed
description 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.
format Online
Article
Text
id pubmed-6562693
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-65626932019-06-17 Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa Naessens, Sarah Ruysschaert, Laurien Lefever, Steve Coppieters, Frauke De Baere, Elfride Genes (Basel) Article 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. MDPI 2019-05-10 /pmc/articles/PMC6562693/ /pubmed/31083481 http://dx.doi.org/10.3390/genes10050363 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Naessens, Sarah
Ruysschaert, Laurien
Lefever, Steve
Coppieters, Frauke
De Baere, Elfride
Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa
title Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa
title_full Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa
title_fullStr Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa
title_full_unstemmed Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa
title_short Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa
title_sort antisense oligonucleotide-based downregulation of the g56r pathogenic variant causing nr2e3-associated autosomal dominant retinitis pigmentosa
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562693/
https://www.ncbi.nlm.nih.gov/pubmed/31083481
http://dx.doi.org/10.3390/genes10050363
work_keys_str_mv AT naessenssarah antisenseoligonucleotidebaseddownregulationoftheg56rpathogenicvariantcausingnr2e3associatedautosomaldominantretinitispigmentosa
AT ruysschaertlaurien antisenseoligonucleotidebaseddownregulationoftheg56rpathogenicvariantcausingnr2e3associatedautosomaldominantretinitispigmentosa
AT lefeversteve antisenseoligonucleotidebaseddownregulationoftheg56rpathogenicvariantcausingnr2e3associatedautosomaldominantretinitispigmentosa
AT coppietersfrauke antisenseoligonucleotidebaseddownregulationoftheg56rpathogenicvariantcausingnr2e3associatedautosomaldominantretinitispigmentosa
AT debaereelfride antisenseoligonucleotidebaseddownregulationoftheg56rpathogenicvariantcausingnr2e3associatedautosomaldominantretinitispigmentosa