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. 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