Modelling novel variants and disease in retinal disorders using human induced pluripotent stem cells

Abstract

The diagnostic rate of inherited retinal diseases (IRDs) returns a 50-70% success rate, with known and novel disease genes and variants identified. Differentiation of human induced pluripotent stem cells (iPSC) provides an attractive system of modelling IRDs, as retinal organoids (iPSC-ROs) containing photoreceptors and retinal pigment epithelium (iPSC-RPE) can be produced for probing of pathogenicity and disease mechanisms in biologically relevant tissues. Fibroblasts from patients with RPGR or ALPK1-associated IRDs were used to create iPSC-RPE and -ROs. Isogenic control clones were created for ALPK1 using CRIPSR/Cas9 gene editing. Characterisation techniques included SNP array, RT-qPCR, Sanger sequencing, Western blot analysis, ELISA, and immunofluorescence and RNA-Seq. Patient iPSC lines successfully formed iPSC-RPE and -RO. The RPGR model displayed decreased RPGR expression and RPGR ciliary localisation, with diminished CEP290 co-staining at the primary cilia. Opsin protein mislocalisation was noted alongside F-actin accumulation and abnormal apoptosis in the iPSC-ROs. Western blot analysis revealed isoform loss preventing RPGR ciliary localisation. The ALPK1 models displayed ciliary changes as well as increased secretion of IL-6 and ICAM-1, higher NF-κβ translocation, and increased apoptotic photoreceptors. Transcriptomics across both models identified altered markers of cilia trafficking, visual phototransduction, the retinoid cycle and innate immune activation. In assessing variants and disease genes, markers of disease include altered protein and cell localisation, amplified cytokine profiles, and cell death. This work reflects how the genetic landscape, in conjunction with protein expression, can pinpoint pathways of interest as hallmarks of disease, aiding in the characterisation of IRDs.