Preclinical assessment of splicing modulation therapy for ABCA4 variant c.768G>T in Stargardt disease.

Abstract

Background: Stargardt disease type 1 (STGD1) is a progressive retinal disorder caused by bi-allelic variants in the ABCA4 gene. A recurrent variant at the exon-intron junction of exon 6, c.768G>T, causes a 35-nt elongation of exon 6 that leads to premature termination of protein synthesis.

Methods: To correct this aberrant splicing, twenty-five 2'-O-methoxyethyl antisense oligonucleotides (AONs) were designed, spanning the entire exon elongation.

Results: Testing of these AONs in patient-derived photoreceptor precursor cells and retinal organoids allow the selection of a lead candidate AON (A7 21-mer) that rescues on average 52% and 50% expression of wild-type ABCA4 transcript and protein, respectively. In situ hybridization and probe-based ELISA demonstrate its distribution and stability in vitro and in vivo. No major safety concerns regarding off-targets, immunostimulation and toxicity are observed in transcriptomics analysis, cytokine stimulation assays in human primary immune cells, and cytotoxicity assays.

Conclusions: Additional optimization and in vivo studies will be performed to further investigate the lead candidate. Considering the high prevalence of this variant, a substantial number of patients are likely to benefit from a successful further development and implementation of this therapy.