Abstract
Although an increasing number of disease genes have been identified, the exact cellular mechanisms of retinitis pigmentosa (RP) remain largely unclear. Retinal organoids (ROs) derived from the induced pluripotent stem cells (iPSCs) of patients provide a potential but unvalidated platform for deciphering disease mechanisms and an advantageous tool for preclinical testing of new treatments. Notably, early-onset RP has been extensively recapitulated by patient-iPSC-derived ROs. However, it remains a challenge to model late-onset disease in a dish due to its chronicity, complexity, and instability. Here, we generated ROs from late-onset RP proband-derived iPSCs harboring a PDE6B mutation. Transcriptome analysis revealed a remarkably distinct gene expression profile in the patient ROs at differentiation day (D) 230. Changes in the expression genes regulating cGMP hydrolysis prompted the elevation of cGMP levels, which was verified by a cGMP enzyme-linked immunosorbent assay (ELISA) in patient ROs. Furthermore, significantly higher cGMP levels in patient ROs than in control ROs at D193 and D230 might lead to impaired formation of synaptic connections and the connecting cilium in photoreceptor cells. In this study, we established the first late-onset RP model with a consistent phenotype using an in vitro cell culture system and provided new insights into the PDE6B-related mechanism of RP.
Highlights
Retinitis pigmentosa (RP) has a prevalence of approximately one in 4000, affecting approximately 1.5 million individuals in total worldwide (Hartong et al, 2006)
To generate induced pluripotent stem cells (iPSCs), Episomal iPSC Reprogramming Plasmids (Cat. #SC900A-1; System Biosciences, United States) expressing four Yamanaka factors (Oct4, Sox2, Lin28, Klf4, and L-Myc), p53shRNA, and a miR-302/367 cluster were transformed into mononuclear cells using 4D Nucleofector (LONZA). iPSC colonies appeared after approximately 25 days
A proband patient with night blindness was diagnosed with RP in his late 40s (Figure 1A)
Summary
Retinitis pigmentosa (RP) has a prevalence of approximately one in 4000, affecting approximately 1.5 million individuals in total worldwide (Hartong et al, 2006). RP, a hereditary retinal degenerative disease, is characterized by irreversible loss of photoreceptor cells. In the progressive stage of this disease, the cone photoreceptor system might be affected, which eventually causes daytime vision impairment or complete visual loss (Hartong et al, 2006). Mutations in the gene encoding the beta subunit of rod cGMP-phosphodiesterase type 6 (PDE6B) account for 4 to 5% of autosomal recessive RP (Danciger et al, 1995). PDE6B mutation was identified as disease-causing gene in 2.4% patients from a large cohort of 1095 patients with RP (Khateb et al, 2019). Prevalent mouse models harboring Pde6b mutations, rd, and rd have been widely used for deciphering pathogenesis and examining novel therapeutics for RP (Bowes et al, 1990; Barhoum et al, 2008)
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