Abstract
Purpose :
Doyne honeycomb retinal dystrophy (DHRD) is an autosomal dominant condition, caused by the missense EFEMP1 variant c.1033C>T (p.R345W). Similar to age-related macular dystrophy, DHRD is characterised by the accumulation of drusen deposits beneath the retinal pigment epithelium (RPE), resulting in progressive vision loss from early adulthood. No specific treatment is currently available for DHRD and disease pathogenesis is not fully understood. The purpose of this study was to investigate the molecular mechanisms of DHRD pathogenesis and explore the use of antisense oligonucleotides (ASOs) as a potential therapeutic by utilising DHRD patient-derived and wild-type (WT) isogenic control induced pluripotent stem cell (iPSC)-RPE as a model.
Methods :
Renal epithelial cells from DHRD patients harbouring the R345W mutation were reprogrammed to iPSC lines and WT isogenic controls were generated through CRISPR/Cas9-mediated homology-directed repair. R345W and WT iPSCs were differentiated to RPE and the expression of key RPE and disease-relevant markers, such as EFEMP1, RPE65, TIMP3, MERTK, and APOE, were assessed by reverse transcriptase quantitative PCR (qPCR) and immunocytochemistry. ASOs targeting the c.1033C>T mutation were designed and administered to R345W RPE cells, with subsequent assessment of efficacy via qPCR, targeted next generation sequencing (NGS) and immunocytochemistry.
Results :
Striking phenotypic differences between R345W mutant and isogenic control iPSC-RPE were observed at the transcript and protein level. ASOs demonstrated a significant reduction in EFEMP1 transcript levels, as assessed by qPCR, and were validated by targeted NGS to specifically reduce the expression of the mutant allele by 70-90%. The successful modulation of mutant EFEMP1 expression underscores the potential of ASOs as a therapeutic strategy for DHRD.
Conclusions :
Investigations comparing DHRD patient-derived and WT isogenic control iPSC-RPE lines have elucidated the effects of the c.1033C>T EFEMP1 mutation in iPSC-RPE cells and uncovered potential therapeutic avenues for DHRD. Altogether, these findings offer promising insights for the development of targeted therapies for DHRD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.