Stargardt disease is a progressive retinal disorder caused by biallelic mutations in the ABCA4 gene that encodes the ATPbinding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease.

Inherited retinal diseases (IRDs) are a group of diseases whose common landmark is progressive photoreceptor loss. The development of gene-specific therapies for IRDs is hampered by their wide genetic heterogeneity. Mitochondrial dysfunction is proving to constitute one of the key pathogenic events in IRDs; hence, approaches that enhance mitochondrial activities have a promising therapeutic potential for these conditions. We previously reported that miR-181a/b downregulation boosts mitochondrial turnover in models of primary retinal mitochondrial diseases. Here, we show that miR-181a/b silencing has a beneficial effect also in IRDs. In particular, the injection in the subretinal space of an adeno-associated viral vector (AAV) that harbors a miR-181a/b inhibitor (sponge) sequence (AAV2/8-GFP-Sponge-miR-181a/b) improves retinal morphology and visual function both in models of autosomal dominant (RHO-P347S) and of autosomal recessive (rd10) retinitis pigmentosa. Moreover, we demonstrate that miR-181a/b downregulation modulates the level of the mitochondrial fission-related protein Drp1 and rescues the mitochondrial fragmentation in RHO-P347S photoreceptors. Overall, these data support the potential use of miR-181a/b downregulation as an innovative mutation-independent therapeutic strategy for IRDs, which can be effective both to delay disease progression and to aid gene-specific therapeutic approaches.

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The c.5461-10T>C p.[Thr1821Aspfs*6,Thr1821Valfs*13] variant has been identified as the most common severe Stargardt disease type 1 (STGD1)-associated variant in ABCA4. STGD1 is the most recurrent hereditary form of maculopathy and so far, no treatment is available for STGD1. In STGD1 patients homozygous for this variant, the onset of the disease typically is in childhood and patients are legally blind by early adulthood. The variant leads to exon skipping and generates out-of-frame ABCA4 transcripts that prevent the translation of functional ABCA4 protein. We applied antisense oligonucleotides (AONs) to restore the wild-type RNA splicing in ABCA4 c.5461-10T>C. The effect of AONs was investigated in vitro using an ABCA4 midigene model and 3D human retinal organoids (ROs) homozygous for the ABCA4 c.5461-10T>C variant. The mRNA in untreated ROs contained only disease-associated isoforms, whereas the organoids treated with the lead AON sequence showed 53% splicing correction and restoration of ABCA4 protein. Collectively, these data identified the lead candidate QR-1011 as a potent splice-correcting AON to be further developed as therapeutic intervention for patients harboring the severe ABCA4 c.5461-10T>C variant.