Description
Introduction
Retinitis pigmentosa (RP) affects approximately 1:4000 people, and 40% of RP cases remain genetically unsolved. We recently discovered complex structural variants (SVs) as the cause of RP17, and our lead hypothesis for the mechanism of disease is the RP17 SVs alter topological associated domain structure and result in ectopic contact of a retinal enhancer with GDPD1. To test this hypothesis, we generated retinal organoid models.
Methods
Patient-derived iPSCs were CRISPR corrected to remove the SV, with synthetic single-stranded DNA templates mediating homology-directed repair. The RP17 patient-derived iPSCs and isogenic control iPSCs were differentiated into retinal organoids alongside an additional control line. ROs were analysed by bulk RNAseq and immunohistochemistry.
Results
RP17 ROs developed normally compared to controls, with lamination and brush borders. Unbiased RNAseq analysis of dysregulated transcripts revealed upregulation of GDPD1 in RP17 ROs compared to the isogenic and independent control ROs. Strikingly, the levels of GDPD1 transcript were reduced to normal levels in the isogenic ROs. Other dysregulated transcripts were also identified as a potential downstream consequence of over expression of GDPD1. Preliminary immunohistochemical analysis indicates a potential cilia phenotype in RP17 ROs, that requires further validation.
Conclusions
RO modelling of RP17 supports the hypothesis that the SVs cause upregulation of GDPD1 by 3D remodeling and retinal enhancer hijacking. The isogenic line, alongside its parental RP17 patient line, provide a robust model with a controlled genetic background to elucidate SV-driven disease mechanisms in RP17.
Lay Abstract
Retinitis pigmentosa (RP) is the most common inherited retinal disease affecting light sensing cells called rod photoreceptors. RP causes night blindness, tunnel vision, and many patients become legally blind. One sub-type of RP, RP17, is caused by complex structural changes of the chromosome, and not a mutation in a gene. Now we have discovered the genetic cause, the challenge is to work out how these structural changes cause RP so that we can think about ways we might develop treatments.
We have made mini retinas in a dish, called retinal organoids, from RP17 patient stem cells. We have also used molecular scissors to correct the structural changes of the chromosome back to a normal chromosome in these patient stem cells, and made retinal organoids. We have found that a gene called GDPD1 on this part of the chromosome hijacks the ON switch of a retina gene, as a consequence of the RP17 structural changes. These retinal organoid models have enabled us to pinpoint how the RP17 structural changes alter expression of genes in the retina, and we can now use these models to further our understanding of why this leads to death of light sensing cells and testing potential therapies.
| Lay Title | Using patient cells to study disease mechanisms in Retinitis Pigmentosa type 17 |
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| Role | PhD Student |