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Article

Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic RPGR Variant Assessment for Therapy Suitability

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Eye Genetics Research Unit, Children’s Medical Research Institute, Sydney Children’s Hospitals Network, Save Sight Institute, University of Sydney, Westmead, Sydney 2145, Australia
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Department of Clinical Genetics, Western Sydney Genetics Program, Sydney Children’s Hospitals Network, Westmead, Sydney 2145, Australia
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Specialty of Genomic Medicine, Faculty of Medicine and Health, University of Sydney, Westmead, Sydney 2145, Australia
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Sydney Genome Diagnostics, Western Sydney Genetics Program, Sydney Children’s Hospitals Network, Westmead, Sydney 2145, Australia
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Specialty of Ophthalmology, Faculty of Medicine and Health, University of Sydney, Sydney 2006, Australia
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Rare Diseases Functional Genomics Laboratory, Sydney Children’s Hospitals Network and Children’s Medical Research Institute, Westmead, Sydney 2145, Australia
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Specialty of Child and Adolescent Health, University of Sydney, Westmead, Sydney 2145, Australia
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Stem Cell Medicine Group, Children’s Medical Research Institute, University of Sydney, Westmead, Sydney 2145, Australia
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School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney 2006, Australia
*
Author to whom correspondence should be addressed.
Academic Editor: Livia S. Carvalho
J. Pers. Med. 2022, 12(3), 502; https://doi.org/10.3390/jpm12030502
Received: 24 January 2022 / Revised: 16 March 2022 / Accepted: 17 March 2022 / Published: 21 March 2022
(This article belongs to the Special Issue Emerging Gene Therapy Treatments for Inherited Retinal Diseases)
The RPGR gene encodes Retinitis Pigmentosa GTPase Regulator, a known interactor with ciliary proteins, which is involved in maintaining healthy photoreceptor cells. Variants in RPGR are the main contributor to X-linked rod-cone dystrophy (RCD), and RPGR gene therapy approaches are in clinical trials. Hence, elucidation of the pathogenicity of novel RPGR variants is important for a patient therapy opportunity. Here, we describe a novel intronic RPGR variant, c.1415 − 9A>G, in a patient with RCD, which was classified as a variant of uncertain significance according to current clinical diagnostic criteria. The variant lay several base pairs intronic to the canonical splice acceptor site, raising suspicion of an RPGR RNA splicing abnormality and consequent protein dysfunction. To investigate disease causation in an appropriate disease model, induced pluripotent stem cells were generated from patient fibroblasts and differentiated to retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-RO). Abnormal RNA splicing of RPGR was demonstrated in patient fibroblasts, iPSC-RPE and iPSC-ROs, leading to a predicted frameshift and premature stop codon. Decreased RPGR expression was demonstrated in these cell types, with a striking loss of RPGR localization at the ciliary transitional zone, critically in the photoreceptor cilium of the patient iPSC-ROs. Mislocalisation of rhodopsin staining was present in the patient’s iPSC-RO rod photoreceptor cells, along with an abnormality of L/M opsin staining affecting cone photoreceptor cells and increased photoreceptor apoptosis. Additionally, patient iPSC-ROs displayed an increase in F-actin expression that was consistent with an abnormal actin regulation phenotype. Collectively, these studies indicate that the splicing abnormality caused by the c.1415 − 9A>G variant has an impact on RPGR function. This work has enabled the reclassification of this variant to pathogenic, allowing the consideration of patients with this variant having access to gene therapy clinical trials. In addition, we have identified biomarkers of disease suitable for the interrogation of other RPGR variants of uncertain significance. View Full-Text
Keywords: RPGR; inherited retinal disease; rod-cone dystrophy; ciliopathy; iPSC; retinal organoids; retinal pigment epithelium RPGR; inherited retinal disease; rod-cone dystrophy; ciliopathy; iPSC; retinal organoids; retinal pigment epithelium
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MDPI and ACS Style

Chahine Karam, F.; Loi, T.H.; Ma, A.; Nash, B.M.; Grigg, J.R.; Parekh, D.; Riley, L.G.; Farnsworth, E.; Bennetts, B.; Gonzalez-Cordero, A.; Jamieson, R.V. Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic RPGR Variant Assessment for Therapy Suitability. J. Pers. Med. 2022, 12, 502. https://doi.org/10.3390/jpm12030502

AMA Style

Chahine Karam F, Loi TH, Ma A, Nash BM, Grigg JR, Parekh D, Riley LG, Farnsworth E, Bennetts B, Gonzalez-Cordero A, Jamieson RV. Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic RPGR Variant Assessment for Therapy Suitability. Journal of Personalized Medicine. 2022; 12(3):502. https://doi.org/10.3390/jpm12030502

Chicago/Turabian Style

Chahine Karam, Fidelle, To H. Loi, Alan Ma, Benjamin M. Nash, John R. Grigg, Darshan Parekh, Lisa G. Riley, Elizabeth Farnsworth, Bruce Bennetts, Anai Gonzalez-Cordero, and Robyn V. Jamieson. 2022. "Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic RPGR Variant Assessment for Therapy Suitability" Journal of Personalized Medicine 12, no. 3: 502. https://doi.org/10.3390/jpm12030502

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