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Kevin Gregory-Evans, Anat Yanai, Cheryl Y Gregory-Evans; Nonsense suppression rescues cell death and photoreceptor protein expression in CDH23 patient iPSC-derived retinal organoids. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4761.
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© ARVO (1962-2015); The Authors (2016-present)
Most animal models of Usher syndrome do not display a retinal phenotype making the condition difficult to study. However, the generation of patient iPSC-derived retinal organoids now provides a platform to study retinal pathogenesis and test promising drug-candidates. Here we tested a nonsense suppression approach in retinal organoids from a patient with a nonsense mutation in the CDH23 gene.
A peripheral blood sample was obtained from an Usher syndrome patient with Arg1437X nonsense mutation in CDH23 and from a normal control. The buffy coat fractions containing mononucleocytes were reprogrammed into iPSCs using Oct3/4, Sox2, Klf4, and c-Myc factors at the Centre for Commercialization of Regenerative Medicine (CCRM) in Toronto, Canada. Two genetically verified clones were obtained from each sample, which were then subjected to the standard Sasai 3D-differentiation protocol. From day 12 of differentiation onwards, organoid spheres were treated daily with 0.5 or 1.0 µg/ml Ataluren® until day 150. Efficacy of nonsense suppression was assessed by morphology, immunohistochemistry, western blotting and TUNEL staining.
Differentiation of cells from the control sample progressed as expected expressing PAX6 at 21 days and developing neuro-epithelium at day 40. However, in the affected CDH23 patient organoids only 31% developed neuroepithelium at day 40. After 60 days of differentiation, TUNEL staining revealed that 58% of the cells were apoptotic and all cells were dead by day 150. This provided a hard-endpoint to determine whether nonsense suppression drugs could improve survival. When developing retinal organoids were treated with 1 µg/ml Ataluren, 78% developed neuroepithelium at day 40 and at day 60 only 12% had TUNEL-positive cells. At day 120 Cadherin 23 protein was detected by western blotting and at 150 days we demonstrated expression of photoreceptor-specific proteins including recoverin, rod arrestin and S-opsin compared to untreated organoids which did not survive.
These data suggest that Ataluren nonsense suppression could provide therapeutic benefit to Usher syndrome patients carrying nonsense mutations. Furthermore, iPSC-derived retinal organoids provide a platform to model diseases of the outer retina, especially when animal models are lacking.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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