Abstract
Purpose :
Stargardt disease (STGD1) is a progressive retinal disorder that initially affects central vision, and often leads to complete blindness. STGD1 is caused by bi-allelic mutations in the ABCA4 gene, which encodes an ABC-transporter that is exclusively expressed in the retina. Dysfunction of ABCA4 leads to the accumulation of toxic by-products of the visual cycle in the retina, leading to retinal cell death. To date, more than 2,200 different ABCA4 variants have been described, a significant proportion of which affects pre-mRNA splicing. A variant at the exon-intron boundary of exon 6, c.768G>T, is a prevalent ABCA4 variant that causes a 35-nt elongation of exon 6 leading to nonsense-mediated decay. The aim of this study is to develop a therapeutic strategy based on the use of antisense oligonucleotides (AON) to correct this splicing defect.
Methods :
Twenty five antisense AONs spanning the entire exon elongation were designed. Screening of these AONs in patient-derived photoreceptor cells allowed us to select the ten most potent AONs, which were further tested in >200-day old patient-derived retinal organoids. The efficacy to correct splicing and thereby rescue ABCA4 expression in retinal organoids was assessed at RNA and protein level. In addition, the safety aspects of the lead AON were rigorously evaluated by means of various assays, in vitro and in vivo.
Results :
Testing of AONs in photoreceptor precursor cells and retinal organoids indicated a potential lead AON, namely AON 7 21-mer. This AON performed the best in rescuing splicing defect caused by ABCA4 c.768G>T. Treatment with this AON rescued up to 65% and 40% expression of wild-type ABCA4 transcript and protein, respectively, a major improvement compared to the absence of any ABCA4 expression in untreated patient-derived retinal organoids. No major safety concerns were observed with regards to potential off-targets, stability, immunostimulatory profile and cytotoxicity.
Conclusions :
AON 7 21-mer was selected as a lead AON following efficacy and safety assessments. Follow up in vivo toxicology studies will be performed to further evaluate the safety of this AON. Considering the high prevalence of this variant, a substantial amount of patients can benefit from this therapy.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.