Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic <i>ABCA4</i> Variant c.4539+2001G>A in Stargardt Disease
Deep-sequencing of the <i>ABCA4</i> locus has revealed that ~10% of autosomal recessive Stargardt disease (STGD1) cases are caused by deep-intronic mutations. One of the most recurrent deep-intronic variants in the Belgian and Dutch STGD1 population is the c.4539+2001G>A mutation....
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doaj-3daa0a50a48649dca6964225249e8d972020-11-24T21:20:55ZengMDPI AGGenes2073-44252019-06-0110645210.3390/genes10060452genes10060452Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic <i>ABCA4</i> Variant c.4539+2001G>A in Stargardt DiseaseAlejandro Garanto0Lonneke Duijkers1Tomasz Z. Tomkiewicz2Rob W. J. Collin3Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525GA Nijmegen, The NetherlandsDepartment of Human Genetics, Radboud University Medical Center, 6525GA Nijmegen, The NetherlandsDepartment of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525GA Nijmegen, The NetherlandsDepartment of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525GA Nijmegen, The NetherlandsDeep-sequencing of the <i>ABCA4</i> locus has revealed that ~10% of autosomal recessive Stargardt disease (STGD1) cases are caused by deep-intronic mutations. One of the most recurrent deep-intronic variants in the Belgian and Dutch STGD1 population is the c.4539+2001G>A mutation. This variant introduces a 345-nt pseudoexon to the <i>ABCA4</i> mRNA transcript in a retina-specific manner. Antisense oligonucleotides (AONs) are short sequences of RNA that can modulate splicing. In this work, we designed 26 different AONs to perform a thorough screening to identify the most effective AONs to correct splicing defects associated with c.4539+2001G>A. All AONs were tested in patient-derived induced pluripotent stem cells (iPSCs) that were differentiated to photoreceptor precursor cells (PPCs). AON efficacy was assessed through RNA analysis and was based on correction efficacy, and AONs were grouped and their properties assessed. We (a) identified nine AONs with significant correction efficacies (>50%), (b) confirmed that a single nucleotide mismatch was sufficient to significantly decrease AON efficacy, and (c) found potential correlations between efficacy and some of the parameters analyzed. Overall, our results show that AON-based splicing modulation holds great potential for treating Stargardt disease caused by splicing defects in <i>ABCA4</i>.https://www.mdpi.com/2073-4425/10/6/452antisense oligonucleotidesStargardt diseaseinherited retinal diseasessplicing modulationRNA therapyABCA4iPSC-derived photoreceptor precursor cells |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Alejandro Garanto Lonneke Duijkers Tomasz Z. Tomkiewicz Rob W. J. Collin |
spellingShingle |
Alejandro Garanto Lonneke Duijkers Tomasz Z. Tomkiewicz Rob W. J. Collin Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic <i>ABCA4</i> Variant c.4539+2001G>A in Stargardt Disease Genes antisense oligonucleotides Stargardt disease inherited retinal diseases splicing modulation RNA therapy ABCA4 iPSC-derived photoreceptor precursor cells |
author_facet |
Alejandro Garanto Lonneke Duijkers Tomasz Z. Tomkiewicz Rob W. J. Collin |
author_sort |
Alejandro Garanto |
title |
Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic <i>ABCA4</i> Variant c.4539+2001G>A in Stargardt Disease |
title_short |
Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic <i>ABCA4</i> Variant c.4539+2001G>A in Stargardt Disease |
title_full |
Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic <i>ABCA4</i> Variant c.4539+2001G>A in Stargardt Disease |
title_fullStr |
Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic <i>ABCA4</i> Variant c.4539+2001G>A in Stargardt Disease |
title_full_unstemmed |
Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic <i>ABCA4</i> Variant c.4539+2001G>A in Stargardt Disease |
title_sort |
antisense oligonucleotide screening to optimize the rescue of the splicing defect caused by the recurrent deep-intronic <i>abca4</i> variant c.4539+2001g>a in stargardt disease |
publisher |
MDPI AG |
series |
Genes |
issn |
2073-4425 |
publishDate |
2019-06-01 |
description |
Deep-sequencing of the <i>ABCA4</i> locus has revealed that ~10% of autosomal recessive Stargardt disease (STGD1) cases are caused by deep-intronic mutations. One of the most recurrent deep-intronic variants in the Belgian and Dutch STGD1 population is the c.4539+2001G>A mutation. This variant introduces a 345-nt pseudoexon to the <i>ABCA4</i> mRNA transcript in a retina-specific manner. Antisense oligonucleotides (AONs) are short sequences of RNA that can modulate splicing. In this work, we designed 26 different AONs to perform a thorough screening to identify the most effective AONs to correct splicing defects associated with c.4539+2001G>A. All AONs were tested in patient-derived induced pluripotent stem cells (iPSCs) that were differentiated to photoreceptor precursor cells (PPCs). AON efficacy was assessed through RNA analysis and was based on correction efficacy, and AONs were grouped and their properties assessed. We (a) identified nine AONs with significant correction efficacies (>50%), (b) confirmed that a single nucleotide mismatch was sufficient to significantly decrease AON efficacy, and (c) found potential correlations between efficacy and some of the parameters analyzed. Overall, our results show that AON-based splicing modulation holds great potential for treating Stargardt disease caused by splicing defects in <i>ABCA4</i>. |
topic |
antisense oligonucleotides Stargardt disease inherited retinal diseases splicing modulation RNA therapy ABCA4 iPSC-derived photoreceptor precursor cells |
url |
https://www.mdpi.com/2073-4425/10/6/452 |
work_keys_str_mv |
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