Potential off-targets investigation of a lead antisense oligonucleotides targeting ABCA4 c.768G>T in retinal organoids

Stargardt disease type 1 (STGD1) is a progressive retinal disorder caused by bi-allelic variants in the ABCA4 gene. More than 2,400 unique ABCA4 variants have been described, a significant proportion of which affects pre-mRNA splicing. 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. Testing of antisense oligonucleotides (AON) in photoreceptor precursor cells and retinal organoids allowed the selection of a lead candidate AON that performed the best in rescuing the splicing defect caused by ABCA4 c.768G>T. Total transcriptomics analysis was performed to investigate the potential off-targets of this AON. Overall design: Retinal organoids were treated with 10 uM of the lead candidate AON or its sense oligonucleotidesfor 10 days. RNA was isolated from these materials and subsequently subjected to total RNA-seq with rRNA depletion. Two independent biological replicates were included in the analysis