Abstract
Purpose :
We developed the first human RDH12 retinal organoid (RO) models, derived from human induced pluripotent stem cells (hiPSC), obtained from skin biopsies of one patient with autosomal recessive RDH12-Leber congenital amaurosis (RDH12 AR), homozygous for c.619A>G p.(Asn207Asp), one with mild late-onset autosomal dominant RDH12-retinitis pigmentosa (RDH12 AD), carrying the heterozygous variant c.759del p.(Phe254Leufs*24), and one unrelated unaffected individual (WT). While few differences between WT and mutants were observed at later stages of photoreceptor differentiation, we studied the ultrastructure and transcriptomic signature for any intracellular and molecular pathophysiology associated with RDH12-related retinopathies.
Methods :
Transmission electron microscopy was performed on wild-type, RDH12 AR and RDH12 AD mature RO (n=6 regions per line). Bulk RNA was sequenced in mature WT (n=8 replicates, with two RO per replicate, from three differentiations), RDH12 AD (n=12 replicates, from two differentiations, from two RDH12 AD clones) and RDH12 AR RO (n=4 replicates, from two differentiations). RDH12 protein localisation was studied by immunostaining at week-18 and week 44 (n=3 per line).
Results :
WT RO developed 62 ±5 photoreceptors per 100 µm with 10.093 ±6.477 µm segment, while RDH12 AD RO developed less (33 ±4, p<0.005) and shorter photoreceptors (5.514 ±3.419 µm, p<0.005), and RDH12 AR photoreceptors (41 ±10) are shorter (5.890 ±4.445 µm, p<0.005) than WT. Pro-apoptotic genes (AIFM2, TP53AIP1, CASP3, MOAP1) are dysregulated in both RDH12 mutants. RDH12 expression was downregulated in RDH12 AD, but RDH12 protein was localised in photoreceptors; while RDH12 expression is unchanged in RDH12 AR but the protein was absent in RO. Both RDH12 AD and AR showed downregulation of genes associated with retinal dystrophies, including congenital stationary night blindness (LRIT3 and RLBP1). Vitamin A metabolism was affected in RDH12 AD; while phospholipid transport was dysregulated in RDH12 AR.
Conclusions :
Our study unravels pathophysiological mechanisms associated with autosomal dominant and recessive RDH12-retinopathies. Further RDH12 RO models derived from patients with identical or different disease-causing variants will precise the different and common pathways affected in RDH12 patients for the identification of targets for therapeutic approaches.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.