Jacqueline van der Spuy

The photoreceptor outer segment is a highly specialized primary cilium that is essential for phototransduction and vision. Biallelic pathogenic variants in the cilia-associated gene CEP290 cause non-syndromic Leber congenital amaurosis 10 (LCA10) and syndromic diseases, where the retina is also affected. While RNA antisense oligonucleotides and gene editing are potential treatment options for the common deep intronic variant c.2991+1655A>G in CEP290, there is a need for variant-independent approaches that could be applied to a broader spectrum of ciliopathies. Here, we generated several distinct human models of CEP290-related retinal disease and investigated the effects of the flavonoid eupatilin as a potential treatment. Eupatilin improved cilium formation and length in CEP290 LCA10 patient-derived fibroblasts, in gene-edited CEP290 knockout (CEP290 KO) RPE1 cells, and in both CEP290 LCA10 and CEP290 KO iPSCs-derived retinal organoids. Furthermore, eupatilin reduced rhodopsin retention in the outer nuclear layer of CEP290 LCA10 retinal organoids. Eupatilin altered gene transcription in retinal organoids by modulating the expression of rhodopsin and by targeting cilia and synaptic plasticity pathways. This work sheds light on the mechanism of action of eupatilin and supports its potential as a variant-independent approach for CEP290-associated ciliopathies.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and β, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.

Inherited retinal disorders (IRDs) affect millions of people worldwide and are a major cause of irreversible blindness. Therapies based on drugs, gene augmentation or transplantation approaches have been widely investigated and proposed. Among gene therapies for retinal degenerative diseases, the fast-evolving genome-editing CRISPR/Cas technology has emerged as a new potential treatment. The CRISPR/Cas system has been developed as a powerful genome-editing tool in ophthalmic studies and has been applied not only to gain proof of principle for gene therapies in vivo, but has also been extensively used in basic research to model diseases-in-a-dish. Indeed, the CRISPR/Cas technology has been exploited to genetically modify human induced pluripotent stem cells (iPSCs) to model retinal disorders in vitro, to test in vitro drugs and therapies and to provide a cell source for autologous transplantation. In this review, we will focus on the technological advances in iPSC-based cellular reprogramming and gene editing technologies to create human in vitro models that accurately recapitulate IRD mechanisms towards the development of treatments for retinal degenerative diseases.

Photoreceptors are sensitive neuronal cells with great metabolic demands, as they are responsible for carrying out visual phototransduction, a complex and multistep process that requires the exquisite coordination of a large number of signalling protein components. Therefore, the viability of photoreceptors relies on mechanisms that ensure a well-balanced and functional proteome that maintains the protein homeostasis, or proteostasis, of the cell. This review explores how the different isoforms of Hsp90, including the cytosolic Hsp90α/β, the mitochondrial TRAP1, and the ER-specific GRP94, are involved in the different proteostatic mechanisms of photoreceptors, and elaborates on Hsp90 function when retinal homeostasis is disturbed. In addition, several studies have shown that chemical manipulation of Hsp90 has significant consequences, both in healthy and degenerating retinae, and this can be partially attributed to the fact that Hsp90 interacts with important photoreceptor-associated client proteins. Here, the interaction of Hsp90 with the retina-specific client proteins PDE6 and GRK1 will be further discussed, providing additional insights for the role of Hsp90 in retinal disease.

Summary: Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.