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.

The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.

Stargardt disease is an inherited retinal disease caused by biallelic mutations in the ABCA4 gene, many of which affect ABCA4 splicing. In this study, nine antisense oligonucleotides (AONs) were designed to correct pseudoexon (PE) inclusion caused by a recurrent deep-intronic variant in ABCA4 (c.769-784C>T). First, the ability of AONs to skip the PE from the final ABCA4 mRNA transcript was assessed in two cellular models carrying the c.769-784C>T variant: a midigene assay using HEK293T cells and patient-derived fibroblasts. Based on the splicing-correcting ability of each individual AON, the three most efficacious AONs targeting independent regions of the PE were selected for a final assessment in photoreceptor precursor cells (PPCs). The final analysis in the PPC model confirmed high efficacy of AON2, -5, and -7 in promoting PE exclusion. Among the three AONs, AON2 is chosen as the lead candidate for further optimization, hereby showcasing the high potential of AONs to correct aberrant splicing events driven by deep-intronic variants.

Nucleic acid therapeutics (NATs), including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), messenger RNA (mRNA) therapeutics and programmable editing technologies, are reshaping the therapeutic landscape for inherited metabolic and genetic disease. Unlike conventional small molecules or biologics, NATs enable sequence‐directed modulation of target gene expression and therefore provide unprecedented flexibility for targeting pathogenic mechanisms previously considered undruggable. Recent clinical successes have established proof of concept for multiple NAT modalities across neurological, hepatic, muscular and metabolic disorders. However, broader implementation remains constrained by challenges in tissue‐specific delivery, biomarker validation, natural‐history infrastructure and evidence generation in ultra‐rare populations. In parallel, increasing platform reproducibility is beginning to challenge traditional product‐based regulatory paradigms. This review examines the principal classes of NATs and proposes a practical framework for modality selection based on disease biology, tissue accessibility, durability, reversibility and clinical context. We discuss key translational bottlenecks beyond delivery, including biomarker–outcome relationships, small‐population trial design and the limitations of conventional evidentiary frameworks in rare disease. A central theme of this review is the growing transition from isolated precision medicines towards increasingly programmable therapeutic platforms. We explore how advances in extrahepatic delivery, computational oligonucleotide design, artificial intelligence‐assisted optimisation and programmable editing technologies may progressively support more scalable and individualised therapeutic development. Finally, we discuss emerging regulatory frameworks that may better accommodate platform‐level predictability while maintaining rigorous evaluation of sequence‐specific and disease‐specific risks.