Antisense oligonucleotide-based rescue of complex splicing defects in ABCA4

Abstract

Purpose : The ABCA4 gene, implicated in Stargardt disease (STGD1), has a high percentage of splicing altering pathogenic variants, some of which leading to complex defects. Antisense oligonucleotides (AONs) have shown promising results in targeting splicing altering variants. Here, we selected two regions in ABCA4, containing four variants, leading to complex splicing defects on which we performed AON-based rescue studies.

Methods : Three variants were selected in intron 13, i.e. c.1938-619A>G and c.1938-621G>A that lead to inclusion of a pseudoexon (PE2) on their own or together with a second PE (PE1), located upstream, and c.1938-514A>G that results in the inclusion of PE3 or PE3 and PE1 together (Figure 1). Three novel AONs were designed to target PE2 and PE3, while PE1 was targeted using a previously tested AON1 with adjustments in the sugar moieties (Sangermano et al. Genet Med. 21:1751-1760; 2019). Variant c.6148-84A>T in intron 44 was selected as it resulted in either inclusion of a short PE (PE4) or of a longer PE (PE5) together with exon 44 skipping. All AONs had a phosphorothioate backbone and a 2’-O-methoxyethyl sugar modification. AON efficacy was assessed using in vitro splice assays in HEK293T cells using midigene constructs containing the variants of interest. (Figure 1: Schematic representation of variants in intron 13 (a) and intron 44 (b) causing complex splicing defects. The blue squares represent the PE inclusions. In green, the positions targeted by the AONs used in the study are shown).

Results : Treatment with AON1 for all intron 13 variants successfully promoted PE1 exclusion. AON2 and AON3 efficiently corrected the splicing defects caused by variants c.1938-619A>G and c.1938-621G>A, including the PE1 insertion, while AON4 showed no effect. Aberrant splicing due to c.1938-514A>G was completely corrected by AON3 and partially (~80%) by AON4. Surprisingly, AON2, not targeting PE3 directly, was also able to partially correct PE3 insertion. For intron 44 splicing defects, AON5 showed a partial effect while AON6 was able to completely restore normal splicing.

Conclusions : AON-based splicing modulation is a promising approach to target complex splicing defects in ABCA4. Different variants localized in specific regions can be efficiently targeted by a single AON when the related PEs share part of their sequence.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.