Stargardt disease-associated in-frame exon 17 skipping in ABCA4 results in partial ABCA4 function

Abstract

Purpose : ABCA4, the gene implicated in Stargardt disease (STGD1), contains 17 exons with multiples of three nucleotides. The effect of variants leading to in-frame exon skipping is unknown. In Usher syndrome-associated genes, antisense oligonucleotides (AONs) have been used to induce skipping of in-frame exons carrying severe variants in order to ameliorate their effect. Upon the identification of a STGD1 proband carrying an exon 17 canonical splice site variant, we investigated the activity of ABCA4 lacking 22 amino acids encoded by exon 17, and searched for AONs that induce exon 17 skipping.

Methods : A French STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, (that is predicted to result in in-frame skipping of exon 17) and a null variant (c.735T>G, p.(Tyr245*)). We studied the clinical characteristics of this proband using multi modal imaging and complete ophthalmological examination. We performed in vitro splice assays in HEK293T cells with a wild-type (WT) and c.2653+1G>A mutant midigene, and studied the subcellular localization of mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17. We also studied the stability of ABCA4 lacking Asp864-Gly885 and performed all-trans-retinal-activated ATPase activity studies. We tested 40 AONs to induce exon 17 skipping and examined their effect in WERI-Rb-1 cells and human retinal organoids (ROs).

Results : The proband showed a late onset (age 43 for first suspected symptoms) of STGD1, suggesting that c.2653+1G>A does not have a fully deleterious effect. In a splice assay c.2653+1G>A led to the deletion of exon 17 in ABCA4 RNA. ABCA4 lacking Asp864-Gly885 encoded by exon 17 was stable and retained 57% all-trans-retinal-activated ATPase activity compared to WT ABCA4. Asp864-Gly885 was within an unstructured linker region between transmembrane 6 and nucleotide-binding domain-1 within the ABCA4 structure. To reduce the pathogenicity of severe ABCA4 variants in exon 17, we designed AONs to induce exon 17 skipping. The best AON achieved 9% of exon 17 skipping in WT WERI-Rb-1 cells and 55% in WT ROs.

Conclusions : An in-frame exon 17 deletion does not result in a null ABCA4 allele and, when in trans with a null allele, does not lead to a severe STGD1 phenotype. By applying AON technology, severe variants in exon 17 potentially can be treated by exon skipping which increases the residual ABCA4 activity in STGD1 patients.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.