Inherited retinal diseases (IRDs) cause progressive loss of light-sensitive photoreceptors in the eye and can lead to blindness. Gene-based therapies for IRDs have shown remarkable progress in the past decade, but the vast majority of forms remain untreatable. In the era of personalised medicine, induced pluripotent stem cells (iPSCs) emerge as a valuable system for cell replacement and to model IRD because they retain the specific patient genome and can differentiate into any adult cell type. Three-dimensional (3D) iPSCs-derived retina-like tissue called retinal organoid contains all major retina-specific cell types: amacrine, bipolar, horizontal, retinal ganglion cells, Müller glia, as well as rod and cone photoreceptors. Here, we describe the main applications of retinal organoids and provide a comprehensive overview of the state-of-art analysis methods that apply to this model system. Finally, we will discuss the outlook for improvements that would bring the cellular model a step closer to become an established system in research and treatment development of IRDs.

Retinitis Pigmentosa (RP) is a genetically heterogeneous form of inherited retinal disease that leads to progressive visual impairment. One genetic subtype of RP, RP54, has been linked to mutations in PCARE (Photoreceptor Cilium Actin Regulator). We have recently shown that PCARE recruits WASF3 to the tip of a primary cilium, and thereby activates an Arp2/3 complex which results in the remodeling of actin filaments that drives the expansion of the ciliary tip membrane. Based on these findings, and the lack of proper photoreceptor development in mice lacking Pcare, we postulated that PCARE plays an important role in photoreceptor outer segment disk formation. In this study, we aimed to decipher the relationship between predicted structural and function amino acid motifs within PCARE and its function. Our results show that PCARE contains a predicted helical coiled coil domain together with evolutionary conserved binding sites for photoreceptor kinase MAK (type RP62), as well as EVH1 domain binding linear motifs. Upon deletion of the helical domain, PCARE failed to localize to the cilia. Furthermore, upon deletion of the EVH1 domain binding motifs separately or together, co-expression of mutant protein with WASF3 resulted in smaller ciliary tip membrane expansions. Finally, inactivation of the lipid modification on the cysteine residue at amino acid position 3 also caused a moderate decrease in the sizes of ciliary tip expansions. Taken together, our data illustrate the importance of amino acid motifs and domains within PCARE in fulfilling its physiological function.

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