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Abstract
SummaryRP2 mutations cause a severe form of X-linked retinitis pigmentosa (XLRP). The mechanism of RP2-associated retinal degeneration in humans is unclear, and animal models of RP2 XLRP do not recapitulate this severe phenotype. Here, we developed gene-edited isogenic RP2 knockout (RP2 KO) induced pluripotent stem cells (iPSCs) and RP2 patient-derived iPSC to produce 3D retinal organoids as a human retinal disease model. Strikingly, the RP2 KO and RP2 patient-derived organoids showed a peak in rod photoreceptor cell death at day 150 (D150) with subsequent thinning of the organoid outer nuclear layer (ONL) by D180 of culture. Adeno-associated virus-mediated gene augmentation with human RP2 rescued the degeneration phenotype of the RP2 KO organoids, to prevent ONL thinning and restore rhodopsin expression. Notably, these data show that 3D retinal organoids can be used to model photoreceptor degeneration and test potential therapies to prevent photoreceptor cell death.
Highlights
The reprogramming of patient-derived cells into induced pluripotent stem cells has enabled the derivation and differentiation of a range of somatic cell types and has revolutionized our ability to study inherited disease (Takahashi et al, 2007)
RP2 Knockout and RP2 Patient induced pluripotent stem cells (iPSCs) Develop Mature retinal organoids (ROs) Fibroblasts from two unrelated individuals (R120X-A and R120X-B) carrying the nonsense mutation c.358C > T; p.R120X were reprogrammed into iPSCs by nucleofection (Okita et al, 2011; Schwarz et al, 2015)
Scattered rhodopsin-positive cells were first detectable in control organoids in the recoverin-positive outer nuclear layer (ONL) from day 150 (D150) increased in number over time as the ROs matured up to day 180 (D180) (Figure S1A)
Summary
The reprogramming of patient-derived cells into induced pluripotent stem cells (iPSCs) has enabled the derivation and differentiation of a range of somatic cell types and has revolutionized our ability to study inherited disease (Takahashi et al, 2007). Unlike previous models in 2D, these 3D structures contain photoreceptors with morphologically identifiable features; including, inner segments rich in mitochondria, rudimentary outer segments with connecting cilia, and synaptic pedicles, in addition to bipolar, Muller glia, ganglion and amacrine cells, synaptic layers, and an outer limiting membrane (OLM), arranged in retinal layers (reviewed in Capowski et al, 2019) These advanced models have proven to have many translational research applications, including transplantation studies (Gonzalez-Cordero et al, 2017; Shirai et al, 2016), retinal disease modeling, and testing the efficacy of potential therapies in human photoreceptor cells (Deng et al, 2018; Parfitt et al, 2016; Schwarz et al, 2017; Sharma et al, 2017). The human phenotype is relatively severe with some patients experiencing macular atrophy in childhood (Jayasundera et al, 2010), highlighting the necessity for human retinal models of disease
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