Rescue of Aberrant Splicing Caused by a Novel Complex Deep-intronic ABCA4 Allele

<b>Background/Objectives:</b> Stargardt disease (STGD1) is an autosomal recessive disorder caused by pathogenic variants in <i>ABCA4</i> that affects the retina and is characterised by progressive central vision loss. The onset of disease manifestations varies from childhood to early adulthood. <b>Methods:</b> Whole exome (WES), whole gene, and whole genome sequencing (WGS) were performed for a patient with STGD1. <b>Results:</b> WES revealed a heterozygous pathogenic missense variant in <i>ABCA4</i>, but no second pathogenic variant was found. <i>ABCA4</i> whole-gene sequencing, subsequent WGS, and segregation analysis identified a complex deep-intronic allele (NM_000350.2(ABCA4):c.[1555-5882C>A;1555-5784C>G]) <i>in trans</i> to the missense variant. Minigene assays combined with nanopore sequencing were performed to characterise this deep-intronic complex allele in more detail. Surprisingly, the reference minigene revealed the existence of two pseudoexons in intron 11 of the <i>ABCA4</i> gene that are included in low-abundance (<1%) transcripts. Both pseudoexons could be confirmed in cDNA derived from wildtype retinal organoids. Despite mild splicing predictions, the variant minigene revealed that the complex deep-intronic allele substantially increased the abundance of transcripts that included the pseudoexon overlapping with the variants. Two antisense oligonucleotides (AONs) were designed to rescue the aberrant splicing events. Both AONs increased the proportion of correctly spliced transcripts, and one of them rescued correct splicing to reference levels. <b>Conclusions:</b> Minigene assays combined with nanopore sequencing proved instrumental in identifying low-abundance transcripts including pseudoexons from wildtype <i>ABCA4</i> intron 11, one of which was substantially increased by the complex allele.