Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic ABCA4 Variant c.4539+2001G>A in Stargardt Disease

Genes

卷:10

RobW. J. Collin¹  TomaszZ. Tomkiewicz¹  Alejandro Garanto¹  Lonneke Duijkers² 

[1] Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525GA Nijmegen, The Netherlands;

[2] Department of Human Genetics, Radboud University Medical Center, 6525GA Nijmegen, The Netherlands;

关键词: antisense oligonucleotides;    Stargardt disease;    inherited retinal diseases;    splicing modulation;    RNA therapy;    ABCA4;    iPSC-derived photoreceptor precursor cells;   

DOI  :  10.3390/genes10060452

来源: DOAJ

【 摘 要 】

Deep-sequencing of the ABCA4 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 ABCA4 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 ABCA4.

【 授权许可】

Unknown