Abstract
Purpose :
ABCA4 c.5461-10T>C is the third most common Stargardt disease type 1 (STGD1)-causing variant, for which no treatment is available. It leads to exclusion of exons 39 and 40, resulting in out-of-frame ABCA4 transcripts and a severely reduced production of wildtype ABCA4 protein. Here, we investigated the restoration of wildtype splicing by QR-1011, an antisense oligonucleotide (AON), as a therapeutic approach using in vitro cell models.
Methods :
The effect of target-specific AONs was investigated by transfection (n=3) of HEK293 cells using a splice-predictive midigene model carrying the mutation. Isoform-specific digital PCR (dPCR) was used for absolute quantification of correct transcript as a percentage of all measured ABCA4 transcripts. Subsequently, QR-1011, the best performing AON, was studied in 3 dimensional CRISPR-Cas9 edited or patient-derived human retinal organoids (ROs), both homozygous for c.5461-10T>C. ROs (n=6 or 8 per group) were treated once with either QR-1011 or a control AON and harvested after 8 weeks. The quantification of transcripts by dPCR was accompanied by immunohistochemistry (IHC) and Western Blot (WB) analysis to identify rescue of wildtype ABCA4 protein expression post-AON treatment. Statistical analysis was performed with either two-tailed Student’s t-test or one-way ANOVA with Dunnett’s multiple comparisons test.
Results :
In midigene-transfected cells, QR-1011 achieved 51,8±2,4% (mean±SEM; p<0,0001 vs. control) of correct transcript and was selected as best splice-modulating AON. In CRISPR-Cas9 edited ROs, QR-1011 corrected 37,9±2,1% (p<0,0001) of the full-length ABCA4 isoform after 8-weeks, while patient-derived ROs contained 46,04±7,1% (p<0,0001) of wildtype transcript after similar treatment. By contrast, in both control groups, only 3% of wildtype ABCA4 transcripts were detected. Furthermore, IHC and WB analysis of treated ROs revealed rescue of wildtype ABCA4 protein, which was localized in outer segments of photoreceptor cells, as opposed to control samples where no protein was observed.
Conclusions :
QR-1011 treatment resulted in splice correction in both midigene and organoid models. Moreover, when administered to ROs, QR-1011 restored the production of ABCA4 protein. These results show the ability of QR-1011 to correct aberrant splicing caused by the c.5461-10T>C mutation in ABCA4 and highlight its therapeutic potential for STGD1.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.