Modelling and rescue of RP2 Retinitis Pigmentosa using iPSC Derived Retinal Organoids

Abstract

Mutations in RP2 lead to a severe form of X-linked retinitis pigmentosa (XLRP). RP2 functions as a GTPase activating protein (GAP) for the small GTPase ARL3, which is essential for cilia function and for photoreceptor development and maintenance. The mechanisms of RP2 associated retinal degeneration in humans are poorly understood, and genetically engineered animal models of RP2 XLRP present with differing retinal phenotypes and slow degeneration suggesting potential species differences. Here, we developed CRISPR gene edited isogenic RP2 knock-out (RP2 KO) induced pluripotent stem cells (iPSC) and RP2 patient derived iPSC to produce 3D retinal organoids as a human retinal disease model. The isogenic RP2 KO retinal organoids and two unrelated RP2 patient iPSC lines produced retinal organoids with a defined photoreceptor cell layer (ONL) containing rod and cone photoreceptors. Strikingly the RP2 KO and RP2 patient derived organoids showed a thinning of the ONL by 180 days (D180) of culture, which was associated with a spike in cell death in the ONL at D150 of differentiation.RNA sequencing confirmed induction of cell death pathways in the RP2 null organoids at this stage. Photoreceptor cell death and ONL thinning was attributed to cells undergoing terminal rod cell differentiation characterized by reduced RHO expression and fewer rhodopsin immunoreactive photoreceptors. Gene augmentation with a human RP2 transgene in an AAV2/5 vector efficiently transduced RP2 KO organoids and led to high levels of RP2 expression in both rods and cones. Importantly, the viral transduction significantly increased ONL thickness and restored rhodopsin expression, suggesting rescue of the RP2 degeneration phenotype. These data show that 3D retinal organoids can be used to model molecular defects associated with inherited retinal disease, photoreceptor cell death and also to test potential therapies targeted to prevent photoreceptor degeneration.

Restoration of functional PAX6 in aniridia patient iPSC-derived ocular tissue models using repurposed nonsense suppression drugs

“…It is expressed early in ocular morphogenesis, during the establishment of the eye field and optic vesicle, and has multiple roles in the development and maintenance of retinal progenitor cells, lens, cornea and iris 13 . They have been used to accelerate therapeutic development in several retinal and corneal eye disorders [19][20][21] . This is the first study to generate iPSCs from an aniridia patient carrying a heterozygous PAX6 nonsense mutation with a UGAtype PTC and establish patient-specific iPSC-derived optic cups and limbal epithelium stem cell (LESC) models that mimic the haploinsufficiency state.…”

Limitations and Promise of Retinal Tissue From Human Pluripotent Stem Cells for Developing Therapies of Blindness

“…CRISPR-Cas-9 gene correction in retinal organoids has been tested successfully in several human RD models ( Deng et al, 2018 ; Huang et al, 2019 ; Lane et al, 2020 ) as well as in vivo in mice (with up to 45% efficiency of repair of dominant-negative Rho mutation to wild type allele in photoreceptors) ( Li et al, 2018 ). CRISPR-Cas-9-based repair may be especially productive and needed for RP diseases, which are caused by dominant-negative mutations, and may potentially work together with retinal tissue replacement (discussed above).…”