Abstract
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.
Highlights
Stargardt disease (STGD1; MIM:248200) is an autosomal recessive condition affecting the retina, and was first described in 1909 by the German ophthalmologist Karl Stargardt [1]
The objectives of this study were to (1) perform an in-depth screening of Antisense oligonucleotides (AONs) targeting the pseudoexon introduced by the recurrent c.4539+2001G>A deep-intronic variant in ATP-binding cassette transporter type 4 subfamily A (ABCA4), (2) identify the best AON(s) to correct the pre-mRNA splicing defect caused by this mutation using patient-derived photoreceptor precursor cells (PPCs), and (3) identify potential correlations between AON characteristics and their efficacy that can provide new insights into a better AON design
Our results showed that two AONs (AON1 and AON4) were able to restore correct ABCA4 splicing by skipping the pseudoexon in a mutation-dependent manner
Summary
Stargardt disease (STGD1; MIM:248200) is an autosomal recessive condition affecting the retina, and was first described in 1909 by the German ophthalmologist Karl Stargardt [1]. The clinical hallmark of STGD1 is progressive bilateral impairment of central vision. Impairment in visual acuity and progressive bilateral atrophy of photoreceptors and the retinal pigment epithelium (RPE) are accompanied by the accumulation of toxic fluorescent deposits of lipofuscin in the macula [2,3]. The ABCA4 protein belongs to the superfamily of membrane-bound ATP-binding cassette transporters [4]. It translocates the visual cycle metabolites, all-trans-retinal and N-retinylidene-phosphatidyl ethanolamine (N-retinylidene-PE), from the lumen to the cytoplasmic side of photoreceptor disc membranes [5]. The decrease in ABCA4 activity causes an accumulation of toxic retinal derivatives, which eventually results in RPE and photoreceptor
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