RLBP1 gene replacement studies identify two CRALBP isoforms that are differentially expressed in murine retina and human iPSC-derived retinal models

Abstract

Purpose : RLBP1 variants are associated with a spectrum of inherited retinal dystrophies (IRDs): retinitis punctata albescens (RPA) characterised by night blindness in childhood and legal blindness from 40 years of age; Bothnia dystrophy (BD), characterised by an early involvement of the macula; Newfoundland rod-cone dystrophy (NFRCD), characterised by night blindness from infancy and severe visual loss from 20 years. We generated patient-specific induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) models of the three clinical forms to model RLBP1-associated IRDs. Furthermore, we tested the pertinence of these models for proof-of-concept gene replacement studies in parallel to Rlbp1^-/- mice.

Methods : We generated an AAV2/5 vector expressing RLBP1 under control of a ubiquitous promoter, and assayed RLBP1 and CRALBP expression. We performed subretinal injections of Rlbp1^-/- mice and assayed visual cycle kinetics and visual function following photobleaching and dark-adaptation, in comparison to control mice. We generated and characterised morphologically (immunofluorescence studies, electron microscopy, retinoid levels) and functionally (phagocytosis and secretion assays) iPSC-derived RPE models from patients with RPA, BD and NFRCD, in comparison to control RPE. We transduced these models and evaluated CRALBP expression and retinoid levels post-treatment.

Results : We showed that the AAV2/5-CAG-RLBP1 vector expressed a functional CRALBP protein that improved visual cycle kinetics and outer retinal function in Rlbp1^-/- mice. Unexpectedly, this vector produced two distinct CRALBP isoforms. We determined the origin of these isoforms and showed that they are naturally and differentially expressed both in the mouse retina, as well as in the human retina using iPSC-derived retinal organoids and RPE. Lastly, we showed that iPSC-derived RPE from RLBP1 patients displayed altered phenotype and functionality, which correlated with clinical severity, compared to control RPE. Furthermore, these differences were improved following AAV2/5-CAG-RLBP1 transduction demonstrating the pertinence of these models for proof-of-concept studies.

Conclusions : Taken together, this study provides novel insights into CRALBP expression and RLBP1-associated pathophysiology, and raises important considerations for successful RLBP1 gene supplementation therapy.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.