Abstract
Purpose :
Achieving a clearcut genetic diagnosis in the inherited retinal dystrophies (IRDs) is critical for patients and families, especially when access to clinical trials or therapy, or development of new treatments is at stake. Genetic variants of uncertain significance (VUS) may be present using American College of Medical Genetics and Genomics (ACMG) criteria. To address this problem, we used patient-derived and genome-edited induced pluripotent stem cells (iPSCs) differentiated to retinal organoids (iPSC-RO) or retinal pigment epithelium (iPSC-RPE), to assess biomarkers, RNA sequence and/or transcriptomic analysis, to add to criteria for VUS classification, and in preparation for novel therapy assessment.
Methods :
iPSC patient and control lines, formed from fibroblasts or blood cells, were created and CRISPR/Cas9 guides were designed along with specific oligonucleotides to mediate homology directed repair to generate isogenic control and/or variant iPSC lines of the novel genetic changes under investigation in RPGRIP1, RPGR and RPE65. The variant and control iPSC lines were differentiated to iPSC-RO and/or iPSC-RPE. Gene and protein expression studies were undertaken using transcriptomics, qRT-PCR, and immunofluorescence, to assess transcriptomic variations and protein interactions and localisations.
Results :
iPSC-RO and/or -RPE with VUS in RPGRIP1, RPGR and RPE65 demonstrated abnormal phenotypic, protein expression or localisation, or transcriptomic/RNA sequence abnormalities, compared with isogenic controls. Mutant retinal organoids from genes affecting photoreceptor ciliary/outer segment function, had reduced or mislocalised protein expression of the disease gene under investigation. Abnormal trafficking of key proteins in the photoreceptors was also evident. Transcriptomic assessment identified key signature molecules and pathways correlating with abnormal phenotype, compared with control lines.
Conclusions :
Investigation of novel VUS in IRD genes, RPGRIP1, RPGR and RPE65, in association with isogenic controls and known disease variants in iPSC-RO and -RPE, has identified decreased and abnormal gene and protein expression as markers of disease pathogenicity. This approach is valuable for variant classification and novel therapy testing, providing genetic information and future therapy opportunities in the IRDs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.