Genome editing has emerged as a transformative approach for understanding and treating retinal degenerative diseases. Combining this technology with pluripotent stem cells provides an ideal platform for modeling human development and disease, and investigating emerging therapeutic strategies ultimately aimed towards in vivo correction. This approach enables both functional studies to understand retinal degeneration and the early development of targeted therapies for inherited disease. This review offers a comprehensive overview of genome-editing techniques and the ability to create new clinically relevant models to understand human disease in retinal research, focusing on the use of the CRISPR-Cas9 system in induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), as well as highlighting recent advancements in base and prime editing. Gene editing in various retinal diseases is discussed in context of studies focusing on disease modeling or developing therapeutic strategies. Continued refinement of these techniques will be essential for advancing translational applications in retinal disease treatment.

Macular diseases are one of the leading causes of vision loss in the western world. In the UK alone, almost 1.5 million people suffer from these devastating diseases, which primarily affect the macula, a region in the retina responsible for detailed central vision. In many patients, cellular changes, attributable to ageing or inherited mutations, are associated with the retinal pigment epithelium (RPE), a single layer of cells that sustains and supports the light-sensitive retina. In the absence of functional RPE, the retina becomes damaged and vision deteriorates. Currently, there are no treatments for these diseases. Over the last two decades, induced pluripotent stem cells have revolutionized our study of retinal disorders, enabling researchers to produce previously inaccessible RPE cells in a dish. The ability to recreate these cells from patients has provided new model systems to understand the mechanisms behind the disease and accelerate the development of new therapies to treat sight loss.

Biallelic ARHGEF18 mutations cause human adult-onset retinal degeneration. We now find that Arhgef18 associates with the retinal outer limiting membrane (OLM), an adherens junction between Müller glia and photoreceptors. Arhgef18 knockout in Müller glial cells led to OLM disruption and vision loss by P60. While mice developed morphologically normal retinas, retinal rosettes started to form by P8, and retinas then progressively degenerated with OLM disintegration, retinal thinning, and vascular leakage. ARHGEF18/p114RhoGEF depletion in Müller cells in culture confirmed disruption of junctional recruitment of OLM proteins. Depletion also induced activation of NF-κB and β-catenin signalling, activation of the multifunctional kinase Tank-binding kinase 1 (TBK1) and reduced mitochondrial activity. TBK1 inhibition or directly supporting mitochondrial activity with nicotinamide attenuated NF-κB and β-catenin signalling and rescued mitochondrial activity. Thus, Arhgef18 is essential for OLM maintenance, and its disruption leads to activation of mechanisms that are targetable for possible therapeutic approaches.

SUMMARY: Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. Here we show that p114RhoGEF/ ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating PKA signalling, and is required for PKA-induced actomyosin remodelling, CREB-driven gene expression of proteins required for trophoblast differentiation, and, hence, trophoblast cell-cell fusion. Our data thus indicate that p114RhoGEF links actomyosin dynamics and cell-cell junctions to PKA/CREB signalling, gene expression and cell-cell fusion.

Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by incomplete retinal vascularization that causes blindness. Inactivation of different genes of the Norrin/β-catenin pathway has been linked to 50% of the cases of FEVR. Zhu et al. identified three novel mutations in CTNNA1 (a-catenin) and one in LRP5 (low-density lipoprotein receptor-related protein 5) in FEVR families. CTNNA1 mutations cause overactivation of the β-catenin pathway, actin cytoskeleton remodelling, and disruption of adherens junctions. Endothelial inducible knockout of Ctnna1 or Cadh5 (VE-cadherin) in mice leads to overactivation of Norrin/β-catenin signalling, overproliferation, and retinal angiogenesis defects. Homozygous mice carrying the Ctnna1-F72S point mutation, a mutation identified in FEVR patients, die during embryogenesis. However, heterozygous mice exhibit a retinal vascular phenotype similar to the endothelial-specific conditional Ctnna1 knockout mice, providing further evidence for the pathogenicity of the CTNNA1-F72S mutation identified in patients. This work identifies mutations in CTNNA1 that over-activate the Norrin/β-catenin pathway, causing defective retinal vascular development and a FEVR-like phenotype. Thus, the precise regulation of β-catenin, a central component in merging VE-cadherin and Norrin signalling, is essential to maintain proper vascular development. As other mutant variants of CTNNA1 and LRP5 have been linked to different diseases, the identification of novel variants in FEVR patients will be necessary to understand the exact mechanism of how different protein regions can give disease specificity. Increasing our understanding of how β-catenin regulation contributes to retinal vascularization could lead to the identification of novel drug targets for retinal diseases.