Generating Models of PRPH2-associated retinal dystrophy

Abstract

Purpose : With the long-term goal of advancing therapeutics for PRPH2-associated retinal dystrophies, this project aims at characterizing novel, nonhuman primate (NHP) and patient-derived retinal organoid models harboring pathogenic variants in the D2 loop of peripherin-2.

Methods : For the NHP model, we used the Oregon National Primate Research Center’s Macaque Genotype and Phenotype (mGAP) database of annotated sequence variants in >2500 rhesus monkeys to identify three NHPs with PRPH2 mutations. Multimodal retinal imaging as well as full-field and multifocal electroretinography (ERG) was performed. To generate the retinal organoids, first, whole blood samples were collected and PBMCs were isolated from two patients harboring the c.828+3A>T PRPH2 mutation. PBMCs were reprogrammed using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Reprogrammed human iPSC colonies were maintained in mTeSR™ Plus medium, and after 10 passages, pluripotency was confirmed via RT-PCR and immunocytochemistry to look for the presence of SOX2, OCT4, and NANOG. Sanger sequencing was used to confirm the gene mutation. After confirming the genetic mutation, iPSCs were differentiated into retinal organoids.

Results : Of the three macaques with PRPH2 mutations, one carries the missense variant p.Trp246Leu, and two carry the missense variant p.Thr129Ile, both in the D2 loop, the most common site of pathologic human mutations. Fundus autofluorescence imaging of a 6-year-old female harboring the p.Thr129Ile missense variant showed a striking increase in hyper-reflective foci, which correlated to outer segment abnormalities in optical coherence tomography (OCT) images. Segmentation of the OCT images showed no difference in retinal thickness compared to age-matched controls. Multifocal ERG showed a 50% decrease of the N1P response within the central 3 degrees compared to age-matched controls, providing a quantifiable cone-mediated phenotype. Human iPSCs from two PRPH2 patients harboring the c.828+3A>T mutation were successfully generated.

Conclusions : These models will be useful for evaluating the safety and efficacy of PRPH2-specific therapies.

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