Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4.

Tomasz Z Tomkiewicz,Rob W J Collin,Nuria Suárez-Herrera,Alejandro Garanto,Frans P M Cremers

doi:10.3390/ijms22094621

Abstract

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.

Highlights

Stargardt disease (STGD1, OMIM: 248200) is characterized by progressive degeneration of the retina
Fifteen variants disrupting ATP-binding cassette subfamily A member 4 (ABCA4) splicing were selected for rescue studies using
For the near-exonic variants, the antisense oligonucleotide (AON) were designed to block the aberrant splice site that was created by each variant, preventing accessibility of the spliceosome

Summary

Introduction

Stargardt disease (STGD1, OMIM: 248200) is characterized by progressive degeneration of the retina. ABCA4 is a transmembrane protein located in the rims of the outer segment discs in rods and lamellae in cones [8] It functions as a flippase transporter and facilitates the removal of potentially toxic retinoic acid derivatives generated during the visual cycle from the lumen to the photoreceptor cell cytosol [9]. A2PE is hydrolyzed to N-retinylidene-N-retinyl-ethanolamine (A2E) upon phagocytosis of photoreceptor outer segments by the retinal pigmented epithelium (RPE) cells. This increases the oxidative damage triggered by the A2E as part of lipofuscin accumulation in the RPE cell, causing cellular damage and photoreceptor cell death and progressive loss of vision [10,11,12]

Methods

Results

Conclusion