Abstract
Purpose :
Copy number variants in wild-type rhodopsin (RHO) have been reported as a novel cause of autosomal dominant retinitis pigmentosa (adRP) in a 68-year-old male (first reported in ARVO 2019). The current study focuses on (i) modeling the disease phenotype using 3D retinal organoids (RO), and (ii) attenuating the overexpression of wild-type RHO using Photoregulin3 (PR3), a small molecule inhibitor of upstream regulator NR2E3.
Methods :
Induced pluripotent stem cells (iPSCs) reprogrammed (n=3 clones/subject) from the patient with adRP associated with four copies of RHO and an unaffected first-degree relative with two copies of RHO (control) were differentiated into RO. RO collected at various time-points of differentiation days (D) - D120 (rod photoreceptor birth), D200 (early maturation), and D300 (late maturation) were assessed for morphology (Bright-field, Transmission electron microscopy; TEM), RHO expression (quantitative real-time PCR; qRT-PCR, bulk RNA-seq), protein localization and quantification (Immunofluorescence; IF and western blotting; WB). Patient RO of D300-350 was treated with varying doses of PR3 (0.5, 0.25, and 0.1 µM) for one week and assessed for RHO levels and localization (qRT-PCR and IF).
Results :
Morphological evaluation of patient-specific iPSC-RO displayed delayed maturation or absence of outer segments (Bright-field/TEM), ~6-10-fold increased RHO expression (qRT-PCR, bulk RNA-seq), impaired localization of Rhodopsin (Rho) protein within the cell body as opposed to being present in the outer segments (IF), and ~4-fold elevated Rho (WB) at all the time points compared to control. PR3-treated patient RO showed a substantial decrease in the RHO mRNA levels (10- to 30-fold) in a dose-dependent manner compared to the vehicle-treated RO, along with a partial rescue in the transport of Rho from cell bodies to the inner and outer segments. All data are represented as Mean±SEM (n=3 independent experiments).
Conclusions :
Tight regulation of RHO is essential for rod photoreceptor function and maintenance. This study provides a proof-of-principle for personalized medicine by utilizing (a) patient-specific iPSC to model disease and (b) investigated a small molecule approach as a clinically viable strategy to target and modify the disease progression in patients with adRP associated with RHO-overexpression.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.